WO2023076639A1

WO2023076639A1 - Primer, probe and controls for detection and discrimination of covid-19 and other coronaviruses diagnostic assay for the human virus causing covid-19-cov-2(covid-19) and its variants

Info

Publication number: WO2023076639A1
Application number: PCT/US2022/048300
Authority: WO
Inventors: Jung Joo Moon; Sung Woo Moon
Original assignee: Jung Joo Moon; Sung Woo Moon
Priority date: 2021-10-28
Filing date: 2022-10-28
Publication date: 2023-05-04

Abstract

The systems and methods described herein are directed to a testing method to diagnose the COVID-19, which minimize false positive and false negative results and account for new variants. The systems and methods are directed to the development of a combination of primers and probes that has 100% inclusivity with all reported COVID-19 virus strains, while also using a detection algorithm.

Description

PRIMER, PROBE AND CONTROLS FOR DETECTION AND DISCRIMINATION OF COVID-19 AND OTHER CORONAVIRUSES DIAGNOSTIC ASSAY FOR THE HUMAN VIRUS CAUSING COVID-19-COV-2(COVID-19) AND ITS VARIANTS

Cross-Reference Information

This application claims priority from U.S. Provisional Application Serial Number 63/272,687 filed on October 28, 2021, which is hereby incorporated herein by reference in its entirety.

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences of the present invention. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The ASCII text file, entitled “MOON-PCT6 ST25”, as created on October 27, 2022 and is an 8480 byte size file, using Patentin version 3.5 and is incorporated herein by reference in its entirety. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to a diagnostic assay for the virus causing severe acute respiratory syndrome Sars-CoV 2 (COVID-19, COVID-19; COVID-19-CoV-2) in humans ("Sars-CoV disease 2019").

BACKGROUND

Recently, there has been an outbreak of atypical pneumonia in Wuhan province in mainland China, in December, 2019. Coronavirus disease 2019 (CO VID-19, 2019-nCoV) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new vims and disease are unknown before the outbreak began in Wuhan, China, in December 2019, and quickly spread across the globe resulting in the 2019-20 coronavirus pandemic, as defined by the WHO (World Health Organization). SARS- CoV-2 is found to be a positive-sense, single-stranded RNA virus belonging to the genus Betacoronavirus.

In humans, CO VID-19 typically spreads from one person to another via respiratory droplets produced during coughing and sneezing as it contacts respiratory or ocular/nasal/oral mucous membranes or direct contact. Common symptoms include fever, cough, and shortness of breath, and time from exposure to onset of symptoms is generally between two and 14 days. Muscle pain, sputum production and sore throat are less common. While the majority of cases result in mild symptoms, and are sometimes asymptomatic, some progress to severe pneumonia and multi-organ failure. The rate of deaths per number of diagnosed cases (case fatality rate (CFR)) is on average around 3.4%, ranging from 0.2% in those less than 20 to approximately 15% in those over 80 years old. The WHO (World Health Organization) declared the infectivity of COVID-19 as lower than Severe Acute Respiratory Syndrome (SARS-CoV, coronavirus) but higher than the Middle East Respiratory Syndrome (MERS-CoV, MERS coronavirus) on January 24th, 2020. WHO declared the basic reproduction number (RO) of the COVID-19 virus as ranging between 1.4 and 2.92. RO is an indication of the transmissibility of a virus, representing the average number of new infections generated by an infectious person in a totally naive population. For RO > 1, the number infected is likely to increase, as it means that 1 infectious person will transmit disease to more than 1 totally naive person. For SARS-CoV, RO is 4, and for MERS-CoV, RO ranged between 0.4 to 0.9. Thus, on January 30, 2020, the Director-General of the WHO declared that the outbreak of CO VID-19 constitutes a Public Health Emergency of International Concern (PHEIC). Soon afterwards, the WHO characterized it as pandemic on March 11, 2020 and current WHO risk assessment is “very high” on a global level. There are a total of 865,585 cases and 48,816 deaths associated with COVID-19 inUnited States alone (April 24, 2020). Good Gene, InC (South Korea) has developed a molecular assay (real-time RT-PCR, real time Reverse transcriptase Polymerase Chain Reaction) for the rapid and reliable diagnosis of COVID-19, which enables the testing of new ORFlab, RdRP, and N regions of CO VID-19 and human internal control genes within a single tube. However, the molecular assays of CO VID-19 often have high amounts of false positive and false negative results. To further exacerbate this pandemic, new strains of CO VID-19 are developing due to mutations.

Therefore, there is a high need for an accurate testing method to diagnose the COVID-19, which minimize false positive and false negative results and account for new variants. The systems and methods are directed to the development of a combination of primers and probes that has 100% inclusivity' with all reported CO VID- 19 virus strains.

Summary

In an embodiment, a composition comprises a set of locked nucleic acid (LNA)-modified probes and a set of primers, each of the probes comprising two or more nucleotides, wherein the primers comprises the two or more nucleotides targeted for the diagnosis, thereby screening for corona virus disease-2019.

In another embodiment, the probes and primers are configured for the diagnosis and screening of “variants” of corona vims disease-2019.

In another embodiment, the variants comprise Alpha, Beta, Gamma, Delta, Eta, Iota, Kappa, Lambda, Alpha+484, Delta Plus, MU (B.1.621), Epsilon, Zeta, and Theta by WHO classification.

In another embodiment, the said LNA probes comprises: (i) nucleotides around 12-15 base pairs (bp); (ii) melting temperature (Tm) close to 65°C; and (iii) discriminating position in LNA probe is at position 2 rather than 1 or 3.

In another embodiment, the LNA probe comprise combinations of L5F,L18F, T19R, T20N, P26S, Q52R, A67V, D69-70, G75V, T76I, D80A,T95I, D138Y, G142D, D144, E154K, D157-158, R190S, D215G, D242-244, R246I, D246-252, D253G, K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, A570D, D614G, H655Y, Q677P, Q677H, P681H, P681R, A701V, T716I, T859N, F888L, D950N, S982A, T1027I, Q1071H, DI 118H, V1176F mutation hot spots of SARS-CoV-2.

In another embodiment, the LNA probe is used in real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) test for detection of the variants. In another embodiment, the primers target a portion of the S ARS-Cov-2 spike protein in combination as we as an internal control.

In another embodiment, the said primers are used together with said LNA probes with a positive, negative, and/or extraction control.

In another embodiment, the said modified LNA probes are targeting SARS-Cov-2 S (Spike) protein against the wild type of L5F,L18F, T19R, T20N, P26S, Q52R, A67V, D69-70, G75V, T76I, D80A,T95I, D138Y, G142D, D144, E154K, D157-158, R190S, D215G, D242-244, R246I, D246-252, D253G, K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, A570D, D614G, H655Y, Q677P, Q677H, P681H, P681R, A701V, T716I, T859N, F888L, D950N, S982A, T1027I, Q1071H, D1118H, V1176F mutation hot spots of SARS-CoV-2 for highly selective binding to mutation hot spots of the SARS-CoV-2.

In another embodiment, the said modified LNA probes are especially targeting a combination of K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, P681H, P681R in S (Spike) protein.

In another embodiment, the said modified LNA probes are targeting, but not limited to; SARS-Cov-2 S (Spike) protein against the wild type of K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, P681H, P681R in S (Spike) protein for highly selective binding to mutation hot spots of the SARS-CoV-2 such that resulting binding combinations with said modified LNA probes are deemed most useful in differentiating the variants.

In another embodiment, LNA probe is used multiplex real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) test comprising a one-step reaction, two-step reaction, or more than two-step reaction in tube for detection of SARS-Cov-2 variants.

In another embodiment, wherein the set of probes in the same tube are labeled with markers different from each other.

In another embodiment, the said modified LNA probes including markers, wherein the markers comprise fluorescent dye.

In another embodiment, the said modified LNA hydrolysis probes including markers comprising a polynucleotide, a reporter, and a quencher label.

In an embodiment, a method of determining the genotype at a locus of interest in a sample obtained from a subject, the method comprises: a) contacting the sample comprising genetic material with the composition of any of claims 1 to 15; and b) detecting the binding of a set of probes to the wild type of the target material, thereby determining the mutation status at the locus.

In another embodiment, the mutation locus is a single nucleotide.

In another embodiment, the method comprises: a) performing an amplifying step comprising contacting the sample with a set of primers to produce an amplification product including the locus of interest; b) performing a hybridizing step comprising contacting the amplification product of step a) with the composition of any of claims 1 to 17; and c) detecting the hybridizing of a set of probes to the genetic material, thereby determining the genotype at the locus.

In another embodiment, the locus of interest differentiates variants of SARS-CoV-2 and the amplification product is the S protein of SARS-CoV-2.

In another embodiment, the method further comprises: (i) measuring a presence or an absence of fluorescence in a sample; (ii) detecting the fluorescence in real-time; (iii) employing a polymerase enzyme having 5' to 3' exonuclease activity as an amplification; and/or (iv) employing a reverse transcriptase step; (v) wherein the sample is a biological sample, preferably a sample selected from the group consisting of a respiratory sample.

In another embodiment, the reporter marker is selected from the group consisting of fluorescein, LC-Yellow 555, FAM, VIC, HEX, Rhodamine B, Rhodamine 6G, LC-Red 610, LC-Red 640, LC-Red 670, LC-Red 705, Cy3, Cy 3.5, Cy5, Cy5.5; texas red, HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC(fluorescein isothiocyanate), Oregon green, alexa fluor, JOE (6-Carboxy-4',5' Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-X- Rhodamine), TET(Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6- carboxytetramethyl-rhodamine), NED (N-(l -Naphthyl) ethylenediamine, Cyanine dye or thiadicarbocyanine).

In another embodiment, the quencher marker is selected from the group consisting of TAMRA (6- carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher, Iowa black.

In another embodiment, the library of sets of probes, wherein the sets of probes are spatially separated from each other.

In another embodiment, a presence of amplification product using the LNA modified probes and primers from the real-time RT-PCR prove that a particular codon is of wild type, and an absence of amplification product using the probes and primers described from the real-time RT-PCR prove that the particular codon is of variant, mutant type.

In another embodiment, the real-time RT-PCR of the S protein of SARS-Cov-2 uses both modified LNA wildtype and mutant-type probes for the codons of hot spot mutations above mentioned against the wild type of K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, P681H, P681R in S (Spike) protein using sequences mentioned in Table 1.

In another embodiment, the real-time RT-PCR of the S protein of SARS-Cov-2 uses forward and reverse primers for codons of hot spot mutations via the sequences mentioned in Table 1.

In another embodiment, the real-time RT-PCR of the S protein of SARS-Cov-2 uses forward and reverse primers for the codons of hot spot mutations via the sequences in Table 1, in combination with internal control and N primer and probes.

In another embodiment, the real-time RT-PCR of the S protein of SARS-Cov-2 uses forward and reverse primers for the codons of hot spot mutations using the sequences in Table 1 in combination with the internal control and the N primer/probes comprises: a presence of amplification product using the LNA modified probes and primers from the real-time RT-PCR prove that a particular codon is of wild type, and an absence of amplification product using the probes and primers described from the real-time RT-PCR prove that the particular codon is of variant, mutant type.

In another embodiment, the variant, mutant type that World Health Organization, WHO or Communicable disease center, CDC or other organizations define as variants of concern, interest, or Alerts for Further Monitoring.

In another embodiment, the real-time RT-PCR of the S protein of SARS-Cov-2 uses respiratory comprising nasopharyngeal, oropharyungeal swab, nasal swab, throat swab, sputum, tracheal/bronchial secretion, bronchoalveolar lavage, bronchial lavage, pleural fluid, saliva, blood or blood derivatives, urine, feces

In another embodiment, the method further comprises: standard material and negative control, plus minus extraction control run parallel for correct diagnosis, wherein the standard material comprising: In vitro transcript of receptor binding protein (RBD) of Spike protein of SARS-Cov-2 RNA (genomic RNA) retrieved from multiple RNA controls; those with wild type mutations in all hot spots as proven by sequencing and alpha mutant SARS-Cov-2 RNA, beta mutant SARS-Cov-2 RNA, gamma mutant SARS-Cov-2 RNA, delta mutant SARS-Cov-2 RNA, kappa mutant SARS-Cov-2 RNA from BEI, TWIST, or Korean CDC; RBD IVT RNA with mutant type nucleotide codons for: K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, N501Y, F490S, P681H, P681R for diagnosis of SARS-COv-2 presence (NP, N protein) and RNA dependent RNA polymerase (RdRp) IVT RNA; Human beta-actin IVT RNA prepared for internal control QC; and negative and positive extraction controls. In another embodiment, the real-time RT-PCR of the S protein of SARS-Cov-2 result is interpreted by filling out the form per table by a software.

In another embodiment, the real-time RT-PCR of the S protein of SARS-Cov-2 result is used for in vitro diagnostic use of SARS-CoV-2 variants, and to determine what kind of a variant the result is categorized as.

In another embodiment, wherein the real-time RT -PCR is used for the diagnosis and surveillance of CO VID- 19 mutants in community and hospital settings as a point of care or diagnostic test.

In an embodiment, a DNA chip for detecting and genotyping CO VID- 19 Variants and for analyzing their COVID-19 Variants, comprises oligonucleotide probes having base sequences.

In another embodiment, the oligonucleotide probe comprises a base sequence which binds complementarity to a human beta-globin gene.

In another embodiment, the oligonucleotide probe having a base sequence which binds complementarity to an oligonucleotide having a base sequence with the 5' end labeled with Cy5.

In another embodiment, an area of the DNA chip on which the probe is spotted is partitioned into 8 wells.

In another embodiment, a kit for detecting and genotyping COVID-19 Variants and analyzing COVID-19 Variants, comprises the DNA chip, a primer set for amplifying DNAs of CO VID- 19 Variants, and a label for detecting the amplified DNAs binding complementarity to the DNA chip.

In another embodiment, the primer set is a primer set for amplifying nucleic acids of COVID-19.

In another embodiment, the label is selected from a group consisting of Cy5, Cy 3, biotinylated material, EDANS (5-(2'- aminoethyl) amino- 1 -naphthalenesulfonic acid), tetramethylrhodamine (TMR), tetramethylrhodamine isothiocyanate (TMRITC), x-rhodamine and Texas Red.

In another embodiment, the labeling means is Cy5 and labeled dCTP and unlabeled dCTP are reacted at a molar ratio of 1:12.5.

In an embodiment, a method for detecting and genotyping COVID-19 Variants and analyzing COVID-19 Variants, comprises: amplifying DNAs of CO VID-19 Variants by single or multiplex PCR using a primer for amplifying nucleic acids of the COVID-19 Variants; and hybridizing the amplified DNAs on the DNA chip; and detecting the hybridized product.

In another embodiment, the amplification by single or multiplex PCR is carried out using one or more primer set(s) selected from a group consisting of a primer set for amplifying nucleic acids of COVID-19 or other respiratory pathogens.

In another embodiment, the amplification by single or multiplex PCR comprises: mixing the primer set with template DNA, Taq DNA polymerase, dNTP, distilled water and PCR buffer to form a resulting mixture; predenaturing the resulting mixture at 95°C for 10 minutes to form a resulting product; subjecting the resulting product to 40 cycles of denaturation at 94°C for 30 seconds, primer annealing at 58°C for 30 seconds and extension at 72°C for 30 seconds; and subjecting the resulting product to final extension at 72°C for 5 minutes.

In another embodiment, the amplification by multiplex PCR is carried out using a primer set having a combination of base sequences at a molar ratio of l:l:l:l:l: l: l: l.

In another embodiment, the amplification by multiplex PCR is carried out using a primer set having combination of base sequences at a molar ratio of 1 : 1 : 1 : 1 : 1.

In another embodiment, a PCR product by the primer set having combination of base sequences of has a size as desired by an operator. In another embodiment, a PCR product by the primer set having combination of base sequences has a size as desired by an operator.

DRAWINGS

The above and other aspects, features and advantages of the disclosed exemplary embodiments will be more apparent from the following detailed description taken in conjunction with the accompanying drawings.

Figs 1 A-C depict the general schematics of the present invention, as flow charts and functional block diagrams.

Figs 2A-2G and 3 A-J depict examples of chromographs of positive and negative results of different CO VID 19 variants.

Figs 4A-4H depict examples of sigmoidal curves associated with positive, negative, and inconclusive results for presence of different CO VID 19 variants.

Figs 5A-5E depict the designs of LNA chips for variants of CO VID 19 variants and the sequence of the CO VID 19 mutant.

Figs 6A-6Q depict different instances of the wells of the DNA chip array.

Fig. 7 depicts a whistle-shaped device for obtaining samples or specimens for the detection kit.

Fig. 8 depicts negative, positive, and retest results on the whistle-shaped device.

Fig. 9 depicts a user interface used in combination with a detection algorithm and branching decisions applied by the detection algorithm.

Fig. 10 depicts a table of mutations, which is applied and utilized by the detection algorithm.

Figs 11 A-F depict Excel Outputs when the detection algorithm is applied.

DETAILED DESCRIPTION

The systems and methods herein are directed to Coronavirus Disease 2019 (COVID-19) Detection and Genotyping Kit (GOODGENE SARS-CoV-2 delta variant real time reverse transcription polymerase chain reaction assay (GG SARS-CoV-2 Delta Real Time RT-PCR Kit) for the detection of Delta and other variant of CO VID- 19. The systems and methods herein build upon the primers, probes, and control for the detection and discrimination of COVID-19 and other coronavirus diagnostic assays as disclosed in US Patent Application No. 17/31669, entitled “Primer, Probe And Controls For Detection And Discrimination Of Covid- 19 And Other Coronaviruses” and filed on May 10, 2021, which is hereby incorporated in its entirety. The systems and methods herein include: 1) a snapshot aspect; 2) a testing kit aspect (hereinafter “the kit”); 3) sequences for reverse transcription polymerase chain reaction (RT-PCR) aspect; 4) CO VID-19 DNA chip; and 5) variant detection algorithm aspect. The basic schematics are depicted in Fig. 1 A, Fig. IB, and Fig. 1C. The systems and methods herein provide an: (i) in vitro diagnostic (IVD) medical device for identification of WHO delta-variant SARS-CoV-2 (Medical prescription required); (ii) an accurate, simple, quick and cost- effective detection of CO VID-19 and identification of “WHO delta-type variant of SARS-CoV-2 which carries the highest threat of all the type of CO VID-19 and its related variant, such as “kappa-type variant of interest”; a “point of care (POC)” testing; (iii) an indication in Persons Under Investigation (i.e., Individuals suspected of being affected by SARS-CoV-2 by their health care provider or officer in districts); and (iv) qualitative detection of SARS-CoV-2 and delta-type mutation of spike protein from human respiratory samples, including nasopharyngeal or oropharyngeal swabs or washing, sputum, bronchial alveolar lavage fluid and tracheal aspirates.

The systems and methods herein may involve the following features below, which are based on the technical aspect above.

One step, single tube, multiplex (quadplex) real time reverse transcription PCR assay by using hydrolysis probes(Taqman probes) and primers for spike protein (S) of SARS-CoV-2 and IC (beta-actin) which allows detection of CO VID-19 and identification of “WHO delta-type and its related variant of SARS-CoV-2 with high sensitivity and specificity (> 99%).

Qualitative detection of SARS-CoV-2 and analysis of its genotype focused on delta-type mutation of spike protein by real time RT- PCR assay.

Detection of a delta-type variant (lineage B.l.617.2), which carries mutations of L452R and T478K, that broke out in India and is defined to be one of “variants of concern (VOCs)”. Delta type is supposed to replicate faster and be more infectious during the early stages of infection. The risk of hospitalization, admission to intensive care unit and death associated with the Delta variant compared to non-VOCs increased by 120% (93-153%), 287% (198-399%) and 137% (50-230%), respectively. Delta type is quickly becoming predominant type all over the world. More than 50% of specimens nowadays is found to be Delta type in many countries worldwide. Another variant related with Delta variant and prevalent in India is Kappa type variant (lineage B.l.617.1) which carries mutation of L452R and E484Q, which is defined as one of “Variants of Interest (VOI)” which carries much lower risk of transmissibility and reinfection as compared to delta type. Another types which need to be discriminated are Lambda type and Epsilon type which carries mutation of L452R but none of T478K or E484Q. Epsilon type is diminishing quickly, The risk of lambda type which is popular in South America remains to be defined.

The present GG COV19 Delta kit of the systems and methods herein is a type of competitive assay: The assay uses a probe which is highly specific to wild type sequence, i.e. wild sequence of codon 452, codon 478 and codon 484 and therefore binds to only wild type sequence codon and generates a strong fluorescence signal when respective sample contains wild type sequence. In contrast, wild type super-selective probe does not bind mutant sequence of codon 452, codon 478 or codon 484 and therefore does not generates a fluorescence signal when sample contains only mutant type sequence, i.e. L452R, T478K or E484Q. Therefore, samples with wild type sequence of S show positive result, whereas, those with variant of S show negative result on the present real time RT-PCR assay.

Following are components of GG SARS-CoV-2-Delta Real-Time RT-PCR kit.

GG SARS-CoV-2 -Delta Kit for each catalog number.

The volume of component varies with each of kit (GG-COV-S-50, 50 tests/kit; GG-COV- S -100, 100 tests/kit, GG-COV- S -200, 200 tests/kit; GG-COV- S -1000, 1,000 tests/kit), which increases in proportion to number of testings to be done.

Table 1. Components Supplied with The Kit and Its Volume

Note) Volume expressed in DI

Note) Actual volume in the kit allows for 10-20% overage to account for pipetting error, etc.

Store the compositions of the systems and methods herein within dark spaces at -15 to 20°C.

The system and methods herein may involve RT-PCR assay aspect. More specifically, the RT-PCR assay aspect comprises: One/two step, single/dual tube, multiplex real time reverse transcription (RT)-PCR assay by using hydrolysis probes (Taqman probes) and primers for spike protein (S) of SARS-CoV-2 and IC (betaactin) and/or which allows for detection of SARS-CoV-2 and identification of “WHO variants of concern SARS-CoV-2” with high sensitivity and specificity (> 99%). The systems and methods herein provide analysis of a combination of hot spot mutations of spike protein (K417T, K417N, L452R, E478K, E484Q, S501Y, D614G, and P681R) which are key markers of VOCs and allow discrimination of each type. The systems and methods herein also allow screening of other variants, such as type Kappa, Lambda, Epsilon, Theta, Zeta, Theta and Eta/Iota as labeled as “variants of interest” (VOI) by World Health Organization (WHO) or Communicable Disease Center (CDC) of United States (USA). VOCs are broadly divided into 2 types: alpha/beta/gamma type and delta type. Alpha, beta and gamma type is based on mutation of codon 501 (N501Y). Alpha type is a prototype of N501Y (broke out in United Kingdom, so called UK type) which were added by mutation of codon 484 (T478K) and codon 417 to become type beta (broke out in South Africa) and type gamma (broke out in Brazil). Alpha, Beta, Gamma type have been dominant VOCs in Europe, America and Africa until recently. WHO and CDC recommends genotyping study of spike protein to be done to identify VOCs and VOIs. The standard test for genotyping is sequencing assay, but this is not acceptable for big scale test in large population. We herein have developed a real time PCR-based GG assay which is simple, quick, cost-effective test and can be easily applied to point of acre testing in large population.

The systems and methods herein a kind of competitive assay. The competitive assay uses a probe which is highly specific to wild type sequence, i.e. wild type sequence of codons of S and therefore binds to only wild type sequence and generates a strong fluorescence signal only when respective sample contains wild type sequence. In contrast, the wild type super-selective probe does not bind mutant sequence of codons and therefore does not generates a fluorescence signal when sample contains only mutant type sequence, i.e. L452R, T478K, E484K or N501Y. Therefore, samples with wild type codon of S spike show positive result, whereas, those with VOCs of S show negative result on real time PCR assay.

The systems and methods herein also provide software (i.e., variant detection algorithm) for point of care (POC) testing which analyzes result of real time PCR and provides its interpretation in an automatic way, where the specimen or sample: contains SARS-CoV-2; is one of VOCs; and is a Delta/Delta. The user interface for the variant detection algorithm of the systems of methods herein applies a RT-PCR formula to interpret sample in, for example, Tube 1 in Fig. 9. The detection algorithm analyzes data associated with the sample, which determines if there are, but not limited to, the amino acid mutations in Fig. 12. The user interface can output the following, based on the findings of the variant detection algorithm: mutation information (SI and S2 in Fig. 9), IC information, the diagnosis (i.e., “interpretation” in Fig. 9) and future steps (i.e., “next steps” in Fig. 9). The VOCs are associated with enhanced transmissibility or virulence, reduction in neutralization by antibodies, the ability to evade detection, or a decrease in therapeutics or vaccination effectiveness. As of July 2021, four SARS-CoV-2 VOCs have been identified by WHO (WHO type Alpha (lineage B.1.1.7); Beta(B.1.351); and Gamma (P.1) and Delta (B.1.617.2)). All four reported VOCs have mutations in the receptor binding domains (RBD) of S protein which results in increased affinity of the spike protein to its receptors (ACE 2 receptors) enhancing the viral attachment and its subsequent entry into the host cells, which increase transmissibility and reinfection rate and the risk of hospitalization, admission to intensive care unit and death as compared to non-VOCs. Delta type (B.1.617.2) and its related type Kappa (B.1.617.1) broke out in India (so called Indian type) separately from Alpha/Beta/Gamma type. Delta and Kappa types are based on mutation of codon 452 (L452R). The table depicted in Fig. 10 was used by the algorithm in connection with the steps in Fig. 9 to make determinations, where the pound (#)in Fig. 10 denotes differentiations beween delta and delta plus; and the asterisk (*) in Fig. 10 denotes specific areas within the codons.

Delta type caries mutation of codon 478 (T478K) in addition to L452R, which together are supposed to markedly increase infectivity. Kappa-type carries mutation of codon 484 (E484Q) in addition to L452R which carries much less risk than Delta type and therefore classified as Variant of Interest (VOI). Delta-type has dominated over Kappa-type in India and is now quickly becoming a dominant type in all over the world (More than 75% of newly coming VOCs is apparently a Delta type). The codons which are detected and analyzed by the systems and methods may be summarized as follows: DELTA has 452, 478, 681; Delta plus variant has K417N in addition to L452R, E478K; Lambda variant has L452R, F490S. (Delta + 490); and the new variant, MU (B.1.621), a variant under investigation, carries mutations inK417N, E484K, S501Y, D614G, and P681R. (484 501 681 (alpha + delta)).

The systems and methods herein may include a locked nucleic acid (LN A) probe for the RT-PCR aspect. LNA is an artificial sequence, as described further below; as a type of nucleic acid analog of the systems and methods herein that contains a 2'-O, 4'-C methylene bridge. This bridge-locked in the 3'-endo conformation can restrict the flexibility of the ribofuranose ring and locks the structure into a rigid bicyclic formation. This enables strong binding and confers enhanced assay performance and an increased breadth of applications. Specifically for the systems and methodsh herein, the melting temperature is easier to control by using LNA probes instead of regular probes as the melting temperature of the double strand increases by 2~8°C each time a LNA monomer is inserted into oligonucleotide and can particularly useful when detecting sequences that are highly similar with a shorter probe. The LNA probe may have: high accuracy, high sensitivity, high specificity, and sensitive allele-frequency detection, while saving time and money, requiring little optimization, and having great flexibility for the new mutations that come out as COVID-19 progresses (guaranteed 2 week development time for each new mutation). There are high throughput level, applicability, and consistency, which can detect all microRNA without being effected by GC content. The stable results can detect microRNA from hard-to-analyze samples such as FFPE or body fluids, while having multiplex capability with low costs and being easy to use. The kits include a whistle shaped device (see Fig. 7) for obtaining a specimen or sample; N primer/probe; and Internal Control (IC).

Basic KIT

Extended KIT

2 step process->diagnosis of delta, alpha, beta/gamma, eta/iota, lambda, kappa, and MU (B.1.621)

SET 1 : IC, N, 452 and 501 (BASIC)

SET 2-A : IC, 484, 490, 681

SET 2-B : IC, 484, 478, 681

Delta KIT

Delta-2 kit where SET: IC (beta-actin), 452, 478, 484 -> delta, kappa

Delta-3 kit where SET: IC (beta-actin), 452, 490, 417->delta, delta plus, Lambda

The systems and methods herein may involve a whistle shaped device for obtaining a specimen or sample to tested for COVID19 or variants of COVID19. The whistle shaped device (GG CVP-04/05/06) is designed to enable the easy development of customized sandwich lateral flow assays, by combining Latex bead conjugation technologies with an immunochromatography test performed on LFA strips. (See Fig. 7.) The signal intensities can be qualitatively analyzed using the supplied scoring card or, for a quantitative detection, an LFA reader can be used, as also described in US Provisional Patent Application No. 63237076, entitled “Lateral Flow Assay by Using Carboxyl Latex Beads and Biotin-Polystreptavidin for the Detection of CO VID-19 Infection and Diagnostic Kit Using the Lateral Flow Assay” and filed on August 25, 2021, which is hereby incorporated in its entirety.

The "Good Plus CO VID-19 Flow-Antigen test” is a lateral flow assay which can detect as little as O.Olng/ml N (Nucleocapsid) antigen of SARS-Cov-2 in aerosol/saliva (GG CVP-04/05) specimens with higher analytical sensitivity than most if not all the commercially available antigen kits (LOD 10-20 time lower). It shows higher clinical performance (sensitivity 85-95% and specificity 100% in reference to real time PCR assay) that allows ordinary people to detect SARS-Cov-2 within 20 minutes without need of additional instalment. The negative, positive, and retest results are depicted in Fig. 8. The systems and methods herein involve a DNA chip for detecting and genotyping CO VID- 19 Variants and for analyzing their CO VID-19. More specifically, a DNA chip and a kit capable of quickly and accurately detecting or genotyping the highly prevalent and important eleven variants causing CO VID- 19 (SARS-Cov- 2). The presence of, and the genotype and CO VID-19 Variants of the CO VID-19 can be analyzed quickly and accurately from a sample with excellent sensitivity, specificity, reproducibility, and accuracy of the 14 COVID-19-variant causing. Related hot spot mutation hot spots may be automatically identified quickly and accurately from multiple samples, and selection of treatments may be aided.

Variants of the present invention includes an oligonucleotide probe having a number of base sequences selected from SEQ IDs listed. An oligonucleotide probe having a base sequence binds complementarity to a human beta-globin gene. In the DNA chip of the present invention, the oligonucleotide probe having a base sequence binds complementarity to an oligonucleotide having a base sequence of with the 5' end labeled with Cy5.

Preferably, the DNA chip of the present invention, the area on which the probe is spotted is partitioned into 8 wells.

Preferably, a kit for detecting and genotyping CO VID- 19 Variants and analyzing COVID-19 Variants of the present invention includes the DNA chip, a primer set for amplifying DNAs of CO VID- 19 Variants, and a labeling means for detecting the amplified DNAs binding complementarily to the DNA chip.

Preferably, the kit of the present invention includes a primer set for amplifying nucleic acids of any combination of base sequences in the Sequence Tables below.

Preferably, in the kit of the present invention, the labeling means is one or more selected from a group consisting of Cy5, Cy3, biotinylated material, EDANS (5-(2'-aminoethyl)amino-l -naphthalenesulfonic acid), tetramethylrhodamine (TMR), tetramethylrhodamine isothiocyanate (TMRITC), x-rhodamine and Texas Red.

Preferably, in the kit of the present invention, the labeling means is Cy5 and labeled dCTP and unlabeled dCTP are reacted at a molar ratio of 1 : 12.5.

Preferably, a method for detecting and genotyping CO VID-19 Variants and analyzing COVID-19 Variants according to the present invention includes: (a) amplifying DNAs of COVfD-f9 Variants by single or multiplex PCR using a primer for amplifying nucleic acids of the COVID-19 Variants; (b) hybridizing the amplified DNAs on the DNA chip according to any one of claims 1 to 4; and (c) detecting the hybridized product. Preferably, in the method for detecting and genotyping COVID-19 Variants and analyzing COVID-19 Variants according to the present invention, the amplification by single or multiplex PCR is carried out using one or more primer set(s) selected from a group consisting of a primer set for amplifying nucleic acids of COVID-19 variant mutation hot spots.

Preferably, in the method for detecting and genotyping COVID-19 Variants and analyzing COVID-19 Variants according to the present invention, the amplification by single or multiplex PCR includes: (a) mixing the primer set with template DNA, Taq DNA polymerase, dNTP, distilled water and PCR buffer; (b) predenaturing the resulting mixture at 95°C for 10 minutes; (c) subjecting the resulting product to 40 cycles of denaturation at 94°C for 30 seconds, primer annealing at 58°C for 30 seconds and extension at 72°C for 30 seconds; and (d) subjecting the resulting product to final extension at 72°C for 5 minutes.

Preferably, in the method for detecting and genotyping COVID-19 Variants and analyzing COVID-19 Variants according to the present invention, the amplification by multiplex PCR is carried out using a primer set having base sequences of combination of SEQ IDs at a molar ratio of l:l:l: l: l: l: l:l.

Preferably, in the method for detecting and genotyping COVID-19 Variants and analyzing COVID-19 Variants according to the present invention, the amplification by multiplex PCR is carried out using a primer set having base sequences of SEQ IDs, a primer set having base sequences, a primer set having base sequences, a primer set having base sequences and a primer set having base sequences at a molar ratio of 1:1:1:1:1.

Preferably, in the method for detecting and genotyping COVID-19 Variants and analyzing COVID-19 Variants according to the present invention, a PCR product by the primer set having base sequences of number of base sequences selected from SEQ IDs listed has a PCR product by the size desired.

Preferably, in the method for detecting and genotyping COVID-19 Variants and analyzing COVID-19 Variants according to the present invention, a PCR product by the primer set having number of base sequence selected from SEQ IDs listed. The systems and methods herein may determine the test sites for the genes of the 50 CO VID- 19 Variants-related mutation hot spots and human beta-globin gene and devised PCR primers for amplifying them (Example 5), prepared DNA clones for the representative genes of control mutation hot spots and each mutation hot spot (Example 6), established methods for acquiring and storing clinical samples (Example 7), established methods for isolating DNA from the sample (Example 8), established single PCR conditions for the representative genes of the mutation hot spots (Example 9), performed single PCR and sequencing for the clinical sample and integrated the result into a database (Examples 10 and 11), established multiplex PCR conditions for the genes of the 14 mutation hot spots, 5 CO VID-19 Variants-related genes and human beta-globin gene (Example 12), performed multiplex multiplex PCR for human clinical samples to determine the applicability of the method (Example 13), designed probes for analyzing hybridization of the genes of the 14 mutation hot spots, 5 COVID-19 Variants-related genes and human beta-globin gene and manufactured a DNA chip using them (Examples 14 and 15), established analysis conditions by performing analysis of Standard material using the DNA chip (Example 16), and confirmed that detection of infection by the 14 mutation hot spots as well as genotyping and analysis of CO VID-19 Variants is possible for clinical samples using the DNA chip.

In the DNA chip of the systems and methods herein, 5 to 50 probes may be used for the representative genes of the mutation hot spots. As a result, false negative and false positive errors that may occur when one probe is used for each gene may be avoided, and diagnosis sensitivity and specificity may be maximized.

In the DNA chip of the present invention, human beta-globin, actin or glyceraldehydes-3-phosphate dehydrogenase gene may be further included as a reference marker. In case the reference marker is betaglobin, it preferably has a base sequence which corresponds to match sequences retrieved when primer sequence was BLAST searched through GIS AID. (See Excel Matches in Fig. 11) By using the reference marker, hybridization on the DNA chip and the previous procedures of DNA isolation and PCR amplification can be verified and false negative error can be detected.

A method for manufacturing the DNA chip of the present invention comprises: preparing a DNA probe capable of complementarity binding to the nucleic acids of the COVID-19 Variants, with the 5' end of the base sequence bound to amine; binding the DNA probe on an aldehyde-bound solid surface; and reducing the aldehyde remaining without being bound to the DNA probe. The binding between the probe DNA and the aldehyde on the solid surface may be accomplished by Schiff base reaction of the amine and the aldehyde.

The solid may be selected from glass, silicon dioxide, plastic, or ceramic.

A kit comprising the DNA chip of the systems and methods may comprise a primer for amplifying nucleic acids of the CO VID-19 Variants, selected from the base sequences of SEQ ID Nos., and a labeling means, and may further comprise a human beta-globin primer. The labeling means may employ various known labels.

For example, Cy5, Cy3, biotinylated material, EDANS, TMR, TMRITC, x-rhodamine or Texas Red may be used. If Cy5 is used, the labeled product may be directly detected via fluorescence signals using an analyzer such as a confocal laser scanner, without additional reactions. Therefore, it may be effective and sensitive.

Example 1 - The Whistle Shaped Device

The protocol is optimized for SARS-CoV-2 N antigen detection in respiratory swab specimens. Use of other specimens have not been evaluated with this kit and should be inquired separately and is for in vitro diagnostic use only. This product has not been FDA cleared or approved or been authorized by FDA under an Emergency Use Authorization (EUA) and is for investigational use only. The SARS-CoV-2 positive control swabs have been prepared from recombinant viral proteins and do not contain infectious material. The whistle shaped device has been authorized only for the investigational use for the detection of proteins from SARS- CoV-2, not for any other viruses or pathogens. Children aged 13 years old and younger should be tested by a parent or legal guardian. Wear a safety mask or other face-covering when collecting swab specimen from a child or another individual. Wash hands thoroughly for at least 20 seconds before and after handling swab samples. In order to obtain accurate results, the user must follow the instructions for use. Immediately use after opening the test device in the pouch. Keep testing kit and kit components away from children and pets before and after use. Excess blood or mucus on the swab specimen may interfere with test performance and may yield a false-positive result. Avoid touching any bleeding areas of the nasal cavity when collecting specimens. Inadequate or inappropriate sample collection, storage, and transport can result in incorrect results. If specimen storage is necessary, swabs can be placed into the extraction vial for up to four hours. Specimens should not be stored dry. When collecting a nasal swab sample, use only the Nasal Swab provided in the kit. Keep foreign substances and household cleaning products away from the test during the testing process. Contact with foreign substances and household cleaning products may result in an incorrect test result.

Not supplied: Bovine Serum Albumin (BSA)

RT : room temperature (20-24 °C)

A positive control is provided with the batch kit (GG CVP-04): Recombinant SARS-CoV-2 nucleocapsid protein antigen is mixed with buffer. This should be dropped into the whistle-shaped device of the systems and methods herein with a plastic pipette instead of the assay procedure. It is recommended that positive external control swabs are run once with every new lot, shipment, and each new user. The buffer should be stored in room temperature. (20-24 deg C). However, temperatures between 0 to 30 °C (32 to 86 °F) does not affect test function. Keep kit in a parallel surface once the seal is open as spilling of the buffer may effect test results. Individually the concentrations shown should not affect the reaction. However, in combination with additional compounds that are not recommended above a certain concentration, the reaction may be affected. The lysis buffer is used for rapid extraction of vims antigen from samples.

Superior reactions are normally generated using ~100 pL of specimen. Specimen with antigen concentration less than 1 mM can still be used to generate good results provided the maximum conjugation volume is not exceeded. Note that adding less than the required amount of specimen may result in unbound label post conjugation (hook effect).

Your Good Ag COVID-19 test can be stored at RT (room temperature) for up to 12 months. For longer storage, the Good Ag COVID-19 test can be stored at 4°C. The best storage conditions for any particular environment must be determined by additional experimentation.

The Good Ag CO VID-19 Flow Antigen test Kit allows point of care testing (POC). However, we recommend that in GG-CAG-03 with nasopharyngeal swabs, the sample collection be done by a health care provider. The hands-on time for the sample acquisition and buffer mix procedure is about 2-3 minutes and the Good Ag CO VID-19 test is ready to interpret within 20 minutes. Not hazardous waste (Hazardous Components: None in reportable quantities) and refer to MSDS in https://cellgenemedix.com/msds. Collect samples as soon as possible within 5 days of symptom onset. The sample should be treated with lysis buffer as soon as possible after collection. The processed sample in buffer vial may be stored at 2 to 8°C for 2 days, or at -20°C for 3 months, or at -70°C for long term storage. However, storage after 1 hour has not been verified and each storage condition for any particular environment must be determined by additional experimentation. If the sample can‘t be immediately disposed, it should be put into buffer and tightly sealed for storage, usually at 2 to 8°C for 1 day, or -70°C for long term storage. Avoid freezing-thawing repeatedly.

Preparation:

1. Wash hands thoroughly for at least 20 seconds before the test.

2. Unpack the test components from the tray. 3. Remove from the device pouch and the test device place it on a flat, clean surface.

4. Locate the flow device, open the rubber cap and gently peel off the aluminum foil seal, being sure to keep the device upright and place it in the packaging tray.

5. Be careful not to touch the tip.

Aerosol (saliva) collection (G-CVP-04/05):

1. Put the flow device in your mouth and blow 15-20 times, making a sound like “phew” for at least 1 minute. Alternately, in some countries (but not in the Unites States), it is possible to spit 2-3 times into the device (further evaluation needed).

2. Gently remove the flow device from mouth and put the rubber cap back on the device.

Extraction process:

1. Make sure that the rubber cap is on, flick the flow device vigorously for at least 30 seconds.

2. Locate the button and push once for 3 seconds (not more than 5 seconds). Place the flow device in a flat surface.

LFA Device interpretation:

1. Start the timer. Read the result at 15 minutes.

2. The test result should not be read after 20 minutes. Do not move or lift the test device during this time.

3. Interpretation: “Positive” results occurred when two distinct colored lines appear, where one red- colored line next to “C” and one blue-colored line next to “T” indicate COVID-19 positive result. NOTE: The color intensity in the test region will vary depending on the amount of SARS-CoV-2 nucleocapsid protein antigen present in the sample. Any faint colored line(s) in the test region(s), which indicates a positive result. “Negative” results occurred when one red-colored line only next to “C”, which indicates a negative result. “Invalid” results occurred if the red-colored line in the control region “C” is not visible, which indicates an invalid result. Re-run the test one time using the remaining specimen in the extraction vial if an invalid result is obtained during initial testing.

Example 2 - GOODGENE SNAPSHOT SOP

The SNaPshot® Multiplex System investigated up to ten SNP markers simultaneously by using PCR amplification and subsequent dideoxy single-base extension of an unlabeled primer and capillary electrophoresis. After electrophoresis and fluorescence detection, the alleles of a single marker appeared as different colored peaks at roughly the same size in the electropherogram plot. The size of the different allele peaks varied slightly due to differences in molecular weight of the dyes. (See Figures 1 A, IB, and 1C.) The HPLC chromatograph traces can be reproduced and summarized below when performing COVID-19 variant analysis in the systems and methods herein. The variant analysis of the systems and methods herein can involve determining the sequence hot spot (i.e., the nucleotide or nucleic acid position). The sequence hot spots can be the following positions: 417, 462, 477, 484 (wherein there can be two possible mutations as denoted by the superscript 1 and 2), 501, and 614. The salient features of the respective HPLC chromatograph traces have a color associated with the predominant peak in terms of intensity. There can be other peaks present (i.e., miscellaneous peaks) in a chromatograph, but their abundance intensities are substantially lower than the intensity of the predominant peak in said chromatograph. The retention time and relative intensities of the miscellaneous peaks can vary. For example, at the 417 sequence hot spot, the miscellaneous peaks can be more intense in the instances of the wild type than the mutant type. In contrast, at the 462 sequence hot spot, the miscellaneous peaks can be less intense in the instances of the wild type than the mutant type. The miscellaneous peaks can be attributed to the sequence hot spot, in combination with the other nucleotides of the polynucleotide, thereby generating polynucleotides with HPLC chromatographs in the Figures 2A-2G.

By implementing the algorithm of systems and methods herein with the traces depicted in Figures 2A-2G, true positive and true negative outcomes for the different CO VID variants can be performed, based on the highlighted peaks, which are not necessarily the most intense peaks. Stated another way, the miscellaneous and most intense peaks can be analyzed to construct a chromatogram for yielding more accurate determinations of true positive and true negative outcomes. In the actual chromatograms, the diagnostic peaks are highlighted. Instead, in Figures 3A-Fig 3 J, the peaks have an asterisk (*) to denote the peak used to make the determination of positive for a variant or negative for a variant. A peak intensities and retention times of the asterisked, i.e., highlighted, peaks vary or are shifted, thereby improving the definiteness of the determination. The nucleotide associated with the highlighted peaks are indicated below.

•Thawed all reagents on ice black entirely before experiment.

•Used personal protective equipment such as (but not limited to) gloves, eye protection, and lab coats when handling kit reagents while performing this assay and handling materials including samples, reagents, pipettes, and other equipment and reagents.

•Performed all manipulations of samples within a Class II (or higher) biological safety cabinet, and disposed of all specimens in a Class II biological safety cabinet depending on guideline of CDC.

•RNA extraction, preparation of reagents and PCR were performed in separate room as described below.

A. RNA Extraction

1) High purity viral RNA was extracted from upper/lower respiratory tract samples carried in viral transport media according to the manufacturer's recommended procedure using a commercially available viral RNA extraction kit such as QIAamp Viral RNA Mini kit andQIAamp DSP Viral RNA Mini Kit (Qiagen) Exgene Viral DNA and RNA Extraction Kit (GeneAll, Cat No.128-150, 16-748)

2) Verified the purity and quantity of the extracted RNA by using fluorometer before RT-PCR testing. (For example, there are peaks in the UV/Vis at spectrum 260 and 280 nm, and the ratio of absorbances at 260 and 280 nm is >=1.8.

3) Extracted RNA should be used for experiment right away. Short term storage conditions are -20°C; and long term storage conditions are -70°C deep freeze.

B. Reverse transcription

High purity DNA was reverse transcribed from above RNA according to the manufacturer's recommended procedure using a commercially available viral DNA reverse transcription kit such as AnyScript™ Reverse Transcriptase Kit (Biotechrabbit, Hennigsdorf, Germany) or AMV Reverse Transcriptase (Promega, Madison, WI, USA) C. Monoplex PCR

1) Clean area around PCR equipment, pipette, centrifuge with 70% ethanol to decontaminate.

2) Turned on PCR machine and set the PCR conditions as indicated below.

3) Prepared Monoplex PCR Reaction Mix by combining all the components listed in the appropriate table with a total volume of 20 y(’.

4) Mixed thoroughly by pipetting template DNA with the master mix and spin-down rapidly, taking care that no bubbles are formed.

5) Performed single PCR under the conditions as specified in the table above.

6) Checked tire size of PCR product by automated/gel electrophoresis (2% agarose gel) using 5 tl PCR product and 1 6X loading dye to check size of PCR product.

D. SNaPshot sequencing 1) Mixed 4 y(’ of Monoplex PCR product, 2 y(’ of EXOSAP-IT PCR Product Cleanup Reagent Express (#EN0581, thermo fisher), and 4

and RNase-free Water.

2) Mixed thoroughly and incubate at 37°C for 4 minute and 80°C for 1 minute.

3) Prepared Reaction Mix by combining all the components listed in the appropriate table with 1 f t PCR product.

4) Mixed the components by pipetting profusely and performed SNaPshot thermal cycling as below.

5) Post-extension treatment: Combined 5 of yl’ SNaPshot product with l yl’ of SAP (shrimp alkaline phosphatase (#78390500UN, thermo fisher), lU/(z£) and 1

RNase-free water and mix thoroughly.

6) Incubated at 37°C for 60 minutes.

7) Deactivated enzyme by incubating at 75°C for 15 minutes.

8) Add 120 Liz size standard 1 / t (#4324287, thermo fisher) and Hi-Di formamide 1.5 "(’(#4401457. thermo fisher) to 1 X of SNaPshot product post-extension treatment and denature for 3 minutes at 95°C.

9) Loaded the Applied Biosystems 3730 DNA analyzer and started sequencing.

10) Cleaned up by: ddNTP removal of 5

of PCR product, 1

of dH2O, at 37°C for 1 hr and at 75 °C for 15 min incubation.

11) Interpreted results as below using GeneMapper software by Applied Biosystems vs 5.0 as recommended. E. Quality Control (Standard material/Positive control/Negative control)

A pivotal factor in successfully performing and interpreting the result of real time RT-PCR was proper control materials. Each assay needed six types of different controls as listed below.

Upon receipt of control RNA materials, aliquots were made and stored at < -70°C until use.

On use, thawed an aliquot of each control for each assay and held on ice until added to plate. Added each aliquot instead of template RNA to real time RT- PCR reaction. The template controls are listed below.

Positive Template Control (PTC) or Positive Control (provided with the kit): PTC RNAs are prepared from in vitro transcript of receptor binding domain (RED) of spike protein (S).

No Template Control (NTC) or Negative Control: Sterile, RNase-free molecular grade water, which was checked for contamination during extraction and PCR plate set-up.

Upon receipt of control RNA materials, make aliquots and store them at < -70°C until use.

On use, thaw an aliquot of each control for each assay and hold on ice until adding to plate. Add each aliquot instead of template RNA to real time RT- PCR reaction.

(1) Internal Control (IC) (provided with the kit)

Composition: In vitro transcript RNA was prepared from plasmid DNA clone carrying amplicon of human beta-actin gene. One 1 DI (equivalent to 10^A3 copies of in vitro transcript RNA) was used. IC material must show signal of IC (beta-actin) on real time RT-PCR assay (Ct <35) but not S of SARS-CoV-2.

Positive Template Control (PTC) or Positive Control (provided with the kit)

All types of PTC (for the type of mutant sequenced) RNAs prepared from in vitro transcript of receptor binding domain (RED) of spike protein (S) were provided as follows.

(5) No Template Control (NTC) or Negative Control (not provided)

Composition: Sterile, RNase-free molecular grade water was used to check for contamination during extraction and PCR plate set-up.

NTC must not show signal for any of the markers including all codons of interest.

F. Result Interpretation After sequencing was carried out, the result of the Covid-19 variant analysis is put into variant detection algorithm (www.cellgenemedix.com) in the member login area. The variant detection algorithm was used to analyze and interpret the wild type versus mutant type sequence. The updated version of the variant detection algorithm was able to genotype, based on this table.

The following position mutant information was associated with the chromatographs of Fig. 1 and Fig. 2, as pertaining to in vitro diagnostic use; by regimen of health care providers only; and standard precautions where all patient specimens and positive controls should be considered potentially infectious and handled accordingly.

Example; 614 1 position mutant: “COVID-19 Pandemic variant”

501+ 614 position mutant: “CO VID-19 UK (B.1.1.7) variant” (ALPHA)

417+ 484^J+ 501+ 614 position al! mutant: “COVID-19 South Africa (B.1.351) variant” (BETA)

484^J+ 501+ 614 position mutant: “CO VID-19 Brazil (P.l) variant” (GAMMA)

452+ 478 +614 +681 position mutant: “COVID-19 Indian (B.1.617) variant” (DELTA)

452+ 484² (+614)+ 681 position mutant: “Kappa variant”

452+ 484² (+614)+ 417+681 position mutant: “Delta plus variant”

452 (+614) position mutant: “Lambda variant”

484² (+614)+ 501 position mutant: “new variant, MU (B.1.621)”

G. Snapshot Primer

Example 3 - Data Interpretation

Interpretation is done using Table as below.

Table attachment

Example 3 - GG SARS-Cov-2 Variants Real Time RT-PCR SOP (417,452, 478, 484, 501)

A. Basic Theory

■ One step, two-tube, multiplex (pentaplex) real time reverse transcription (RT)-PCR assay by using hydrolysis probes (Taqman probes) and primers for spike protein (S) of SARS-CoV-2 and IC (betaactin) which allows detection of SARS-CoV-2 and identification of “WHO variants of concern S ARS- CoV-2” with high sensitivity and specificity (> 99%).

■ Provides analysis of 5 hot spot mutations of spike protein (K417N, L452R, T478K, E484K, N501Y) which are key markers of VOCs and allow discrimination of each type. The test also allows screening of other variants such as type Delta Plus, Kappa, Lambda, Epsilon, Theta, Zeta, Theta and Eta/Iota as labeled as “variants of interest” (VOI) by World Health Organization (WHO) or Communicable Disease Center (CDC) of United States (USA).

■ VOCs are associated with enhanced transmissibility or virulence, reduction in neutralization by antibodies, the ability to evade detection, or a decrease in therapeutics or vaccination effectiveness. As of July 2021, four SARS-CoV-2 VOCs have been identified by WHO: WHO type Alpha (lineage B, 1.1.7); Beta (B.1.351); and Gamma (P. 1) and Delta (B. 1 ,617.2). All four reported VOCs have mutations in the receptor binding domains (RBD) of S protein which results in increased affinity of the spike protein to its receptors (ACE 2 receptors) enhancing the viral attachment and its subsequent entry into the host cells, which increase transmissibility’ and reinfection rate and the risk of hospitalization, admission to intensive care unit and death as compared to non-VOCs.

■ VOCs are broadly divided into 2 types: alpha/beta/gamma type and delta type.

■ Alpha, beta and gamma type is based on mutation of codon 501 (N501Y). Alpha type is a prototype of N501 Y (broke out in United Kingdom, so called UK type) which were added by mutation of codon 484 (T478K) and codon 417 (K417N) to become type beta (broke out in South Africa) and type gamma (broke out in Brazil). Alpha, Beta, Gamma type have been dominant VOCs in Europe, America and Africa until recently.

■ Delta type (B , 1 ,617,2) and its related type Kappa (B , 1 ,617.1) broke out in India (so called Indian type) separately from Alpha/Beta/Gamma type. Delta and Kappa types are based on mutation of codon 452 (L452R). Delta type caries mutation of codon 478 (T478K) in addition to L452R, which together are supposed to markedly increase infectivity. Kappa-type carries mutation of codon 484 (E484Q) in addition to L452R which carries much less risk than Delta type and therefore classified as Variant of Interest (VOI). Delta-type has dominated over Kappa-type in India and is now quickly becoming a dominant type in all over the world (More than 75% of newly coming VOCs is apparently a Delta type). Delta Plus type is the new recently changed version carries mutation of codon K417N in addition to L452R and T478K, and is estimated to significantly increase infectivity than the delta type.

■ WHO and CDC recommends genotyping study of spike protein to be done to identify VOCs and VOIs. The standard test for genotyping is sequencing assay, but this is not acceptable for big scale test in large population. We herein have developed a real time PCR-based GG assay which is simple, quick, cost-effective test and can be easily applied to point of acre testing in large population

■ The present test is a kind of competitive assay: The assay uses a probe which is highly specific to wild type sequence, i.e. wild type sequence of codon 417, 452, 478, 484 and 501 of S and therefore binds to only wild type sequence and generates a strong fluorescence signal only when respective sample contains wild type sequence. In contrast, the wild type super-selective probe does not bind mutant sequence of codon 452, 478, 484 and 501 and therefore does not generate a fluorescence signal when sample contains only mutant type sequence, i.e. K417N, L452R, T478K, E484K or N501Y. Therefore, samples with wild type codon of 417, 452, 478, 484 and 501 show positive result, whereas, those with VOCs of S show negative result on real time PCR assay. We provide software for point of care (POC) testing which analyzes result of real time PCR and provides its interpretation in an automatic way, e.g.: (1) The specimen contains SARS-CoV-2; (2) It is one of VOCs; (4) It is a Delta type.

■ Following is components of GG SARS-CoV-2 -VOCs Real Time RT-PCR kit type 1 (GG COV19 - Standard A)

GG COV-VOC kit for each catalog number.

The volume of component varies with each of kit (GG-COV-V1-50, 50 tests/kit; GG-COV-V1-100, 100 tests/kit;GG-COV-Vl-200, 200 tests/kit; GG-COV-V1-1000, 1,000 tests/kit), which increases in proportion to number of testing to be done.

B. Table 1. Components Supplied with The Kit and Its Volume

Note) Volume expressed in DI

Note) Actual volume in the kit allows for 10-20% overage to account for pipetting error, etc C. Storage

Store at dark space at -15 to -20°C.

D. Product validity period

6 months from the date of production.

1 month from first unpacking and thawing of the reagents.

Avoid excessive freeze/thaw cycles for reagents.

E. Procedure

Thaw ail reagents completely on ice before testing, wear appropriate personal protective equipment (e.g. gowns, gloves, eye protection) when handling samples, and dispose of all specimens in a Class II biological safety cabinet depending on guideline of CDC. RNA extraction, preparation of reagents and PCR should be performed in separate room as follows:

1. RNA extraction

1) High purity viral RNA is extracted from sample carried in viral transport media according to the manufacturer's recommended procedure using a commercially available viral RNA extraction kit such as QIAamp Viral RNA Mini kit and QIAamp DSP Viral RNA Mini Kit (Qiagen)

2) Verily the purity' and quantity of the extracted viral RNA before RTPCR testing.

2. Real time PCR instruments and software

Any multicolor (4 or more) real PCR instruments can be used with GG SARS-CoV-2 Variants Real time RT-PCR kit.

In particular followings were tested in many samples and showed excellent performance:

1) Applied Biosystems 7500 Real Time PCR System (Thermo Fisher Scientific) with 7500 Software v2.3

2) CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with CFX Manager™ Software V3.1; or CFX Maestro™ Software VI.

3) Rotor-Gene Q 5-plex HRM (Qiagen, Hilden, Germany) with Rotor-Gene Q Series Software 2.1.0. Note) Software which is used for POC testing will be also provided on demand.

3. Real Time Reverse Transcription PCR Master Mix Setup

1) Clean and decontaminate all work surfaces, pipets, centrifuges and other equipment prior to use

2) Turn on the Real Time PCR system and set it as shown below Table 2 and 3

Table 2. Condition of Real Time RT-PCR

Table 3. Fluorescence Detector

3) Prepare a Real Time PCR reaction master mix according to the composition as in the table below. The final reaction volume is 20 pl.

Table 4. Real time RT-PCR Reaction Component

Note) All of above components except clinical sample RNA are provided in the GG SARS-CoV-2-VOCs Real Time RT-PCR kit-2.

4) After mixing the prepared reaction master mix and the sample template RNA or control RNA materials by pipetting well, quickly centrifuge (spin-down) to avoid bubbles

5) Place the tube or plate containing the mixed solution into the Real Time PCR equipment, set up and proceed for the test.

4. Control materials

Important keys in success of performance and interpretation of result of Real Time RT-PCR is proper control materials. Each assay needs eight types of different controls as listed below.

Upon receipt of control RNA materials, make aliquots and store them at < -70° C until use.

On use, thaw an aliquot of each control for each assay and hold on ice until adding to plate. Add each aliquot instead of template RNA to Real Time RT-PCR reaction. (1) Internal Control (IC) (provided with the kit)

Composition: In vitro transcript RNA prepared from plasmid DNA clone carrying amplicon of human bet-actin gene. One 1 DI (equivalent to 10^A3 copies of in vitro transcript RNA) is used. IC is essential. Use through the entire sample processing procedure, excluding the extraction.

IC material must show signal of beta-actin (Cy-3 channel) on real time RT-PCR assay (Ct < 38) but not those of SI, S2, S3, S4 and S5 of SARS-Cov-2.

Positive Template Control (PTC) or Positive Control (provided with the kit)

Six types of PTC (PTC-1, 2, 3, 4, 5 and 6) are provided as follows.

Each of all of 6 PTCs is essential.

Use through the entire sample processing procedure, excluding the extraction.

(2) Positive Template Control type 1 (PTC-1)

Composition: In vitro transcript RNA of S (RED) of S of SARS-CoV-2 (wild type without mutation of any of codon 417, codon 452, codon 478, codon 484 or codon 501, prepared from genomic RNA from SARS-CoV-2, Isolate USA-WA1/2020; NR-52285), each diluted by DEPC-treated water containing carrier RNA and RNase inhibitor (“diluents”) to concentration of 100 copies/Dl and mixed in 1 : 1 (1000 copies RNA of N of SARS-CoV-2 + 1000 copies RNA of wild type S per DI). 2 DI is used. PTC-1 represents clinical sample of SARS CoV-2 which does not carry mutation in any of codon 417, codon 452, codon 478, codon 484 or codon 501 of S (“Non-VOC SARS-CoV-2”).

PTC-1 must show signal of N, S 1 (S for wild type codon 452), S2 (S for wild type codon 478), S3 (S for wild type codon 501), S4 (S for wild type codon 417) and S5 (S for wild type codon 484) (Ct<40) but not that of IC on real time RT-PCR assay.

(3) Positive Template Control type 2 (PTC-2)

Composition: In vitro transcript RNA of N and S of SARS-CoV-2 with mutation of codon 501(N501Y) of S but without mutation of any of codon 417, codon 452, codon 478 and codon 484, prepared from genomic RNA from SARS-CoV-2, Isolate USA/CA_CDC_5574/2020 (Lineage B.1.1.7); NR-52285, BEI Resources, ATCC, USA), each diluted by DEPC-treated water containing carrier RNA and RNase inhibitor (“diluents”) to concentration of 1,000 copies/Dl and mixed in 1 : 1 (1,000 copies RNA /each). Two DI is used.

PTC-2 represents clinical sample of SARS CoV-2 with WHO alpha-type VOCs which carries mutation in codon 501 of S gene of SARS-CoV-2 (N501Y).

PTC-2 must show signal of N, S 1 (S for wild type codon 452), S5 (S for wild type codon 484), S2 (S for wild type codon 478), S4 (S for wild type codon 417) but not that of S3 (S for wild type codon 501) and IC (no sigmoidal amplification).

(4) Positive Template Control type 3 (PTC-3)

Composition: Mixture of in vitro transcript RNA of N and S of S ARS-CoV -2 with mutation of codon 417 (K417N), 501 (N501Y) and 484 (E484K) but without mutation of codon 452 or codon 478, prepared from genomic RNA from SARS-CoV-2, Isolate from South Africa (Lineage B.1.1.7); NR- 43382, BEI Resources, ATCC, USA), each diluted by DEPC-treated water containing carrier RNA and RNase inhibitor to concentration of 1,000 copies/Dl and mixed in 1 : 1 (1,000 copies RNA /each). Two DI is used.

PTC-3 represents clinical sample of SARS CoV-2 with WHO beta-type “variant of concern” which carries mutation in codon 417 (K417N), codon 501 (N501Y) and codon 484 (E484K) of S gene. PTC-3 must show signal of S 1 (S for wild type codon 452) and S2 (S for wild type codon 478) (Ct < 40) but not S4 (S for wild type codon 417), S5 (S for wild type codon 484) and S3 (S for wild type codon 501) (no sigmoidal amplification). (5) Positive Template Control type 4 (PTC-4)

Composition: Mixture of in vitro transcript RNA of N and S of S ARS-CoV -2 with mutation of codon 501(N501Y) and 484 (E484K) but without mutation of codon 417, codon 452 and codon 478 of S, prepared from genomic RNA from SARS-CoV-2, Isolate Twist bioscience, EPI ISL 792683, USA), each diluted by DEPC-treated water containing carrier RN A and RNase inhibitor to concentration of 1,000 copies/Dl and mixed in 1 : 1 (1,000 copies RNA /each). Two DI is used.

PTC-4 represents clinical sample of S ARS CoV-2 with WHO gamma-type “variant of concern” which carries mutation in codon 501 (N501Y) and codon 484 (E484K) of S.

PTC-4 must show signal of SI (S for wild type codon 452), S2 (S for wild type codon 478), S4 (S for wild type codon 417) (Ct < 40) but not that of S5 (S for wild type codon 484) or S3 (S for wild type codon 501) (no sigmoidal amplification).

(6) Positive Template Control type 5 (PTC-5)

Composition: Mixture of in vitro transcript RNA prepared from plasmid DNA clone of N and S of SARS-CoV-2 with mutation of codon 452 (L452R) and codon 478 (T478K), as typically found in delta-type “variant of concern” by WHO label), each diluted by DEPC-treated water containing carrier RNA and RNase inhibitor to concentration of 1,000 copies/Dl and mixed in 1 : 1 (1,000 copies RNA /each). Two DI is used.

PTC-5 represents clinical sample of S ARS CoV-2 with WHO delta type “variant of concern” which carries L452R mutation and T478K of S gene of SARS-CoV-2.

PTC-5 must show signal of S5 (S for wild type codon 484), S3 (S for wild type codon 501), S4 (S for wild type codon 417) (Ct < 40) but not that of S 1 (S for wild type codon 452) or S2 (S for wild type codon 478) (no sigmoidal amplification).

(7) Positive Template Control type 6 (PTC-6)

Composition: Mixture of in vitro transcript RNA prepared from plasmid DNA clone of N and S of SARS-CoV-2 with mutation of codon 417 (K417N), codon 452 (L452R) and codon 478 (T478K), as typically found in delta plus-type “variant of concern” by WHO label), each diluted by DEPC-treated water containing carrier RN A and RNase inhibitor to concentration of 1,000 copies/Dl and mixed in 1 : 1 (1,000 copies RNA /each). Two DI is used.

PTC-5 represents clinical sample of S ARS CoV-2 with WHO delta plus type “variant of concern” which carries K417N, L452R mutation and T478K of S gene of SARS-CoV-2.

PTC-5 must show signal of S5 (S for wild type codon 484) and S3 (S for wild type codon 501) (Ct < 40) but not S 1 (S for wild type codon 452), S2 (S for wild type codon 478) and S4 (S for wild type codon 417) (no sigmoidal amplification)

(8) No Template Control (NTC) or negative control (not provided)

Composition: Sterile, RNase-free molecular grade water

Essential, use with every batch of patient sample through the entire sample processing procedure, excluding the extraction

Used to check for contamination during extraction and PCR plate set-up

NTC should not show signal of any of IC, N, SI, S2, S3, S4 and S5.

(9) Negative extraction control (NEC) (not provided)

Composition: Clinical patient specimen that has previously been tested and reported as SARS-CoV-2 negative. Process same volume of NEC in parallel with new patient samples to be tested.

Used as the negative extraction control for the entire testing system to check adequacy of RNA extraction and for contamination during PCR set-up, inefficient lysis of specimen, improper assay setup. NEC must show signal of IC, but should not show signal of any of N, S 1, S2, S3, S4 or S5 of SARS- CoV-2.

F. Data Analysis and Interpretation

When the run is complete, store and analyze the data according to the device manufacturer's instructions. Ct value of each target of each sample is checked and analysis should be performed for each target individually by using manual threshold value setting. Threshold values should be within the exponential phase of the fluorescence curve and adjusted above the background signal. Usually, threshold is set at 0.05%.

Basic rule)

If the Ct value is less than 40, it is determined as “positive signal”.

If the run shows no amplification or Ct value is greater than or equal to 40, it is determined as “negative signal”.

Data analysis and interpretation is carried out step by steps follows:

A. First Step: Examination and Interpretation of Control Results (See Table 5)

The controls for the Real Time Fluorescent RT-PCR Kit for Detecting VOCs of SARS-CoV-2 are evaluated using the nucleic acid amplification curve and Ct values generated by the RT-PCR system software. The Ct cut-off values are determined using the receiver operator characteristic curves of tested clinical samples. If the results from controls are invalid: Repeat from the RT-PCR step using residual extraction material. If repeat results are not as expected, re-extract and re-test (RT-PCR run) all samples.

The positive control and internal control should provide an amplification curve in the Texas-Red/FAM/ Cy-5 and Cy-3 channel, respectively, that appear to be in a sigmoidal shape.

Table 5. Interpretation of control results

NTC: negative template control

IC: internal control

PTC-1: positive templatecontrol- 1

PTC-2: positive templatecontrol-2

PTC-3 : positive templatecontrol-3

PTC-4: positive templatecontrol-4

PTC-5: positive template control-5

PTC-6: positive template control-6

NEC: negative extraction control

Positive signal: sigmoidal amplification, Ct < 40

ND: no data (no sigmoidal amplification)

1) The negative control (NTC; NC) should show no data (no sigmoidal amplification) in all of the Cy-5, Cy-3, Texas Red, FAM channel. If positive results are obtained for any of channel, the real time RT- PCR run is invalid and suggests contamination. Repeat the RT-PCR step for all patient samples using residual, previously extracted material. If contamination is confirmed, discard working reagent dilutions and remake from fresh stocks. Clean potential DNA contamination from bench surfaces and pipettes in the reagent setup and template addition work areas.

2) The internal control (IC) and the negative extraction control (NEC) should be negative for the S 1 (FAM channel, Tube 1), S2 (Texas Red channel, Tube 1), S3 (Cy-5 channel, Tube 2), S4 (Texas Red channel, Tube 2) and S5 (FAM channel, Tube 2) markers but positive for beta-actin in the Cy-3 channel (IC marker; Ct value<35). If positive results are obtained for any of Cy-5, Texas Red, FAM channel, the extraction run and the RT-PCR run are invalid and the entire process should be repeated for all patient samples using residual specimen.

3) PTC-1 (wild type S, representing “Non-VOC SARS-CoV-2) must show sigmoidal amplification and its Ct should be less than 40 (i.e. positive results) in 4 channels including Cy-5, Texas Red, FAM channel.

Do not go to the next step unless these controls are valid and acceptable.

B. Second Step: Examination and Interpretation of Patient Specimen Results: Assessment of clinical specimen test results should be performed after the positive, internal, negative, and extraction controls have been examined and determined to be valid and acceptable. If the controls are not valid, the patient results must not be interpreted. To be deemed valid, a test must satisfy all the no control requirements noted above. If is the control requirements are not satisfied or only satisfied in part, the run is invalid and patient results must not be interpreted.

If the control results are valid and acceptable, apply the following to interpret patient results:

I. If the run shows sigmoidal amplification and the Ct value for any target is less than 40, it is determined as “positive result”. If the run shows no amplification or Ct is 40 or more than or equal to 40 it is determined as “negative result”.

II. The rule for analysis of S1(FAM channel for codon 452), S2 (Texas Red channel for codon 478), S3 (Cy-5 channel for codon 501), S4 (Texas Red channel for codon 417) and S5 (FAM channel for codon 484):

■ When result shows negative result for any of SI, S2, S3, S4 or S5, the respective codon is defined to be “variant or mutant”. In contrast, when result shows positive result for any of SI, S2, S3, S4 or S5, the respective codon is defined to be “wild type”.

■ When all of SI, S2, S3, S4 and S5 show positive result, it is interpreted as “SARS-Cov-2 present, but none of variant of WHO type alpha, beta, gamma or delta (Non-VOCs)’TII. In case IC is negative, regardless of result of any of S, report as “invalid” and run repeated test.

IV. Additional confirmatory testing may be conducted if “it is clinically indicated” or when detailed sequence information of whole genome of S are necessary (i.e., re-test, or use an alternative method for genotyping of SARS-CoV-2 such as sequencing assay) See Table 6 and figures for interpretation of results

Table 6. Interpretation of patient specimen results

Pos: positive,

Neg: negative,

* None of WHO-alpha, beta, gamma, delta type VOC or kappa, lambda, epsilon type. VOI

** Usually not necessary, but if it is necessary to get precise genomic information or when it is clinically indicated, perform additional study (automated sequencing assay or next generation sequencing).

As depicted in Fig. 13, the algorithm of the system and methods herein applies the decision branches. If the results are not consistent in Fig. 13, retesting is recommended and a certain type may not be ascertained. Table 7 is invoked by the systems and methods herein in case of troubles.

Table 7. Troubleshooting

RT-rPCR: reverse transcription real time PCR.

G. PERFORMANCE

1) Limit of detection (LoD), copies number

* 140D1 in volume when RNA is extracted by QIAamp Viral RNA kit (Qiagen)

2) In silico inclusivity and exclusivity

100% of all the strains of SARS-CoV-2 without mutation of codon 452 of S detected.

100% of all the strains of SARS-CoV-2 with mutation of codon 452 of S excluded.

100% of all the strains of SARS-CoV-2 without mutation of codon 478 of S detected.

100% of all the strains of SARS-CoV-2 with mutation of codon 478 of S excluded.

100% of all the strains of SARS-CoV-2 without mutation of codon 484 of S detected.

100% of all the strains of SARS-CoV-2 with mutation of codon 484 of S excluded.

100% of all the strains of SARS-CoV-2 without mutation of codon 501 of S detected.

100% of all the strains of SARS-CoV-2 with mutation of codon 501 of S excluded.

100% of all the strains of SARS-CoV-2 without mutation of codon 417 of S detected.

100% of all the strains of SARS-CoV-2 with mutation of codon 417 of S excluded.

3) In silico exclusivity

No significant homology with any microorganism of human respiratory tract but shows >95% homology with several bat or pangolin beta coronavirus such as Beta-coronavirus/ hCoV- 19/pangolin/Guangxi/P5L/2017, bat coronavirus RaTG13 (MN996532.1 and hCoV- 19/bat/Yunnan/RmYN02/2019). This can be explained by evolution of SARS-Cov-2. However, cross reactivity due to contamination of human respiratory tract by these bat or pangolin virus are unlikely

4) Analytical sensitivity 100% of all the naso and oro-pharyngeal swab samples containing IVT transcript RNA of S of SARS- CoV-2 (wild type without mutation of any of codon 452, codon 478, codon 484, codon 501 and codon 417, prepared from genomic RNA from SARS-CoV-2, Isolate USA-WA1/2020; NR-52285 of BEI), to concentration of 100 copies/ DI (LOD) showed positive results (sigmoidal amplification with Ct <40) on GG Variants real time RT-PCR assay (analytical sensitivity 100%).

5) Analytical specificity

100% of all the naso and oro-pharyngeal swab samples containing IVT transcript RNA of S of SARS- CoV-2 (mutant type with mutation of any of codon 452, codon 478 codon 484, codon 501 or codon 417) in concentration of from 100 to 10^A9 copies/Dl showed negative result (no amplification) on GG Variants Real Time RT-PCR assay (analytical specificity 100%).

6) Wet test cross reactivity

No cross reactivity with any respiratory organism including Human coronavirus, Influenza A virus (H1N1), Influenza B virus, Respiratory syncytial virus, Human metapneumovirus, Parainfluenza virus (type 1, 2, 3), Rhinovirus, Enterovirus, Mycobacterium tuberculosis, Adenovirus (type 1, 3, 5, 7, 8, 11, 18, 23, 55), Haemophilus influenza, Mycoplasma peumoniae, Streptococcus pneumonia, Streptococcus pyrogenes, Legionale pneumophila, Candida albicans, Psurdomonasaeryginosa, Staphylococcus epidermidis, Streptococcus salivarus, Chlamydiae pneumonia.

7) Interference by common drug applied to nasopharynx, oropharynx or respiratory tract, blood or mucus No interference was found with any of following drugs or blood or mucus:

Zanamivir, Artemether-lumefantrine, Doxycycline hyclate, Quinidine, Lamivudine, Ribavirin, Acetaminophen, Acetylsaicylic acid, Ibuprofen, Mupirocin, Tobramycin, Erythromycin, Ciprofloxacin, Neo-synephrine, Rhinocort, Sodium cromoglycate, Olopatidine hydrochloride, Anbesol (Bezocaine 20%), Stresils. Throat candy (mint), Mucin (bovine submaxillary gland, type I-S), Biotin. Saline nasal spray, Oseltamivir, Daclatasvir, Afrin nasal spray, Homeopathic zicam allergy relief nasal gel.

8) Precison

No inter-observer or inter-lot difference were found.

9) Clinical performance by using matrix of nasopharyngeal and oropharyngeal swabs

Sensitivity and specificity 100%

10) Clinical performance by using matrix of sputum

Sensitivity and specificity 100%

11) Clinical comparative study

Compared with automated sequencing by using ABI 3730 device (200 naso and oro-pharyngeal swabs)

H. WARNINGS & PRECAUTIONS

• For in vitro diagnostic use

• By regimen of health care providers only

• Follow standard precautions. All patient specimens and positive controls should be considered potentially infectious and handled accordingly.

• Do not eat, drink, smoke, apply cosmetics or handle contact lenses in areas where reagents and human specimens are handled.

• Handle all specimens as if infectious using safe laboratory procedures. Refer to Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with CO VID- 19 https://www.cdc.gov/coronavirus/COVID-19/lab-biosafety-guidelines.html. • Specimen processing should be performed in accordance with national biological safety regulations.

• Specimens should be collected with appropriate infection control precautions.

• Perform all manipulations of samples within a Class II (or higher) biological safety cabinet

• Use personal protective equipment such as (but not limited to) gloves, eye protection, and lab coats when handling kit reagents while performing this assay and handling materials including samples, reagents, pipettes, and other equipment and reagents.

• The procedures in this handbook must be followed as described. Any deviations may result in assay failure or cause erroneous results.

• Good laboratory practice is required to ensure the performance of the kit, with care required to prevent contamination of the kit components. Components should be monitored for contamination and any components thought to have become contaminated should be discarded as standard laboratory waste in a sealed pouch or zip-lock plastic bag.

• As with any molecular test, new mutations within the target regions of the GG SARS-CoV-2 Variant Real Time RT-PCR Kit-2 assay could affect primer and/or probe binding resulting in failure to detect the presence of virus

• The present assay can identify presence or absence of mutation of codon 452, codon 478, codon 484, codon 501 and codon 417 of SARS-CoV-2, but not mutation of the other codon of S.

• The present assay can detect WHO Alpha-type, Beta-type, Gamma-type, Delta-type variant of concern and Kappa-type of variant of interest and can aid to screen other variants of interest such as WHO- Delta Plus, Lambda, Epsilon, Theta, Zeta, Eat and Iota type, but can’t detect other type of variant.

• The present assay can detect WHO alpha-type, beta-type, gamma-type, delta-type VOCs and even other VOIs, but can’t provide exact sequence variation, for which additional sequencing assay is necessary.

• The present assay can’t discriminate beta-type variant and gamma-type variant. Additional sequencing assay is necessary to confirm.

• A negative result for any PCR test does not conclusively rule out the possibility of infection.

• Always include the specified controls for nucleic acid extraction and Real Time RT-PCR with every batch of patient samples. Patient results must only be interpreted if the expected control results are obtained.

• The result of this test must be interpreted in combination with that of clinical study.

• If the amount of SARS-CoV-2 in sample or sample RNA (after extraction) is lower than the limitation of detection of this test, it can be missed and may result in false negative result.

• This test can theoretically make cross reactivity with bat or panglion beta coronavirus although such cross reactivity by contamination of human respiratory tract by those viruses are not likely to happen in real life.

I. Software for Interpretation: GG Variant software vs. 1 (See Fig. 13.)

J. Supplemental Data: Clinical Validation of RT-PCR in comparison with Sequencing.

In this study, DNA-sequencing (ABI 3730, Thermo Fisher, USA) was used to identify the mutation of interest in comparison with our GG CO VID-19 Variant RT-PCR Standard B kit (Biorad_CFX96 machine used for Real Time RT-PCR). Pairwise comparisons of mutation hot spots and in the presence of CO VID-19 were performed. 100 Remnant clinical nasopharyngeal swab specimens from the United States (RDX biosciences, Kenilworth, New Jersey, USA, https://rdxbioscience.com/) previously confirmed for COVID-19 with Applied Biosystems QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher, United States) and EURORealTime SARS-CoV-2 reagent (EUROIMMUN, Mountain Lakes, NJ, United States/ MP 2606-0100) were sent to South Korea. The RNA extraction and PCR was done at delivery by Good Gene, Inc (Seoul, South Korea) and 56 samples were found to be sufficient. The DNA sequencing was carried out at Bionics InC (Seoul, South Korea, http://www.bionicsro.co.kr/) and RT-PCR was carried out at Good Gene, InC. The results were compared and only 45 out of 56 samples were found to be sufficient. The result of the comparison study is attached below.

Table 11.

Example 5 - Assaying Gene Region for the Design LNA Chip for Variant SARS-Cov-2

The gene region to be assayed was selected primarily from Spike protein lesions of SARS-Cov-2, or the middle of them, and the space intergenic region, which are most widely used for identification of the phylogeny of mutation hot spots. As a standard for PCR amplification region, the common base sequence in all mutation hot spots and mutation hot spots of the same genus was placed at 5' end, and the unique base sequence in species (genus) of the target mutation hot spot was placed at the other end. If there is no suitable base sequence, the unique gene and unique base sequences of the target mutation hot spot are selected in other regions for assay. (See Fig. 5A and Fig. 5B (e.g., italicized entries which are exhibit different luminescent properties than the non-italicized entries).) The Design LNA Chip can also be modified to make determinations as exhibited in Fig. 5C and Fig. 5D, where “X” denotes points of modification in comparison to Fig. 5A.

Example 6 - Securement of control mutation hot spot and sample and clone thereof

The strains of the 14 CO VID-19 Variants of standard positive control group and DNAs of CO VID-19 Variants genes were purchased from Korean CDC, BEI, Twist biosciences and a sample including COVID-19 Variants genes were obtained. RNA was isolated therefrom, and then the target gene region to be assayed was amplified by PCR for each mutation hot spot and identified through cloning and sequencing. Plasmid clone was secured for each of them. The cloning experiment was performed by the publicly known method. The method for PCR is the same in following examples, so the description thereof is skipped here.

The PCR products of the genes of the 14 CO VID-19 Variants and the PCR product of human beta-globin gene were isolated on the agarose gel using a Reverse transcriptase kit, and the concentrations were measured using a spectrophotometer or by densitometry of the agarose gel.

Example 7 - Gathering of clinical sample

A suitable method was established for gathering from human body various samples such as nasopharyngeal swab, oropharyngeal swab, nasal swabs, sputum, saliva, and so forth.

The gathered sample should be carried within 48 hours to the laboratory if possible, and should be kept at refrigerating temperature during transportation. If RNA extraction is not carried out right away, this sample was stored at -70°C and later was subjected to RNA isolation. Example 8 - Isolation of RNA and reverse transcription

RNA was isolated and purified using a commercialized kit and various human samples of Example 7. High purity viral RNA was extracted from upper/lower respiratory tract samples carried in viral transport media according to the manufacturer's recommended procedure using a commercially available viral RNA extraction kit such as QIAamp Viral RNA Mini kit and QIAamp DSP Viral RNA Mini Kit (Qiagen) Exgene Viral DNA and RNA Extraction Kit (GeneAll, Cat No.128-150, 16-748; verified the purity and quantity of the extracted RNA by using fluorometer before RT-PCR testing; extracted RNA, which should be used for experiment right away and stored short term at -20°C and stored long term at -70°C.

Reverse transcription was carried with High purity DNA, which was reverse transcribed from above RNA according to the manufacturer's recommended procedure using a commercially available viral DNA reverse transcription kit such as AnyScript™ Reverse Transcriptase Kit (Biotechrabbit, Hennigsdorf, Germany) or AMV Reverse Transcriptase (Promega, Madison, WI, USA).

Example 9 - PCR for establishing conditions of single PCR

Artificial samples were made by adding plasmid clones of target genes, which were obtained in Example 6 for testing on each mutation hot spot in multiple copies of 10, 100, 1,000 and 10,000, to sterilized triply distilled water, a sample storage solution of Example 3 and negative nasopharyngeal swab specimens. Then, single PCR on target genes for each mutation hot spot was preformed repeatedly, thereby establishing conditions for the single PCR. When performing PCR, PCR of human beta-globin gene, which was an internal reference gene, was performed together. Moreover, in consideration of multiplex PCR afterwards, it was designed such that the size of each PCR product was distinctively different from each other, but that the annealing temperature had no big difference.

When the primer designed in the systems and methods herein was used, detection was always possible as long as 10 to 100 copies of plasmid clones are included in 1 mL of sample solution. After performing PCR, the product was identified by subjecting to electrophoresis on 3% agarose gel, and the results are shown in Chip Images 1-17 and the corresponding electrophoresis results. Typically the wells appeared as highly resolved green dots or red dots, wherein the green dots corresponded to the lighter dots in Chip Images 1-17 and the darker dots in Chip Images 1-17. However, there are instances of white dots or partially or poorly resolved green dots or red dots. The electrophoresis results form a profiled that corresponded to a diagnosis.

The composition and conditions for PCR are summarized in table below.

Composition and conditions for single PCR of 14 CO VID- 19 Variants

Example 10 - Single PCR on clinical sample

Various nasopharyngeal swab samples were collected via the method of Example 7. Then, DNA was isolated according to the method of Example 8, and single PCR was performed according to Example 9.

Example 11 - Sequencing of PCR products of clinical sample The PCR products of Example 10 were subjected to sequencing reaction using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit version 1.1 (Perkin Elmer Biosystems, USA), and then subjected to base sequencing using ABI 3130x1 automated sequencer (Perkin Elmer, USA). These procedures were carried out in the following order.

(1) In order to use the PCR products obtained from each sample as main substance in the sequencing reaction, the most suitable concentration was adjusted. For example, if the product is 100 to 200 bp long, a concentration of 1 to 3 ng/pL is needed, and if it is 200 to 500 bp long, about 3-10 ng/pL is needed.

(2) 1 pL of each PCR product, 2 pmol of primer and 8 pL of dye terminator ready reaction mix were fed into a thin wall microcentrifuge tube, and sterilized distilled water was added thereto so that the final volume was 10 pL. Then, the mixture was mixed well by light agitation.

(3) The mixture of (2) was subjected to cycle sequencing reaction using GeneAmp 2700 (PE Biosystems, USA) with a total of 25 cycles, in which each cycle was 10 seconds at 96°C, 5 seconds at 50°C and 4 minutes at 60°C.

(4) The obtained PCR product was added to 62 pL of PPT solution (absolute EtOH 250 mL, 3 M NaOAc 10 mL, DW 50 mL) in a 1.5 mL microtube. After mixing well by vortexing, followed by keeping at -20°C for 15 minutes, centrifuge was performed at 14°C and at 13,000 rpm for 5 minutes to precipitate only the fluorescence-labeled DNA. After carefully removing the supernatant and adding 170 pL of cleaning solution (70% EtOH), centrifuge was performed again. After removing the supernatant and the salt, the product was dried on a 60°C heat block for about 3 minutes.

(5) 10.1 pL of Hi-Di was added to the DNA obtained in (4) and mixed by vortexing for 30 to 60 seconds. 10 pL in the middle was put in a new 0.2 mL strip PCR tube and prepared by reacting at 95°C for 2 minutes and at 4°C for about 3 minutes.

(6) The denatured DNA of (5) was charged to each well of a plate that had been cast, subjected to electrophoresis for 2 to 4 hours, and then base sequence was analyzed using ABI 3130x1 sequencer. From the sequencing assay, the appropriateness of the single PCR for detecting the 14 COVID-19 Variants and the COVID-19 Variants genes was confirmed, and as a result, the database on molecular epidemiology and genotyping of the 14 CO VID-19 Variants was established for Koreans. The sample identified as infected by CO VID-19 through PCR and sequencing was then used for multiplex PCR and for evaluation of the DNA chip.

Example 12 - Establishment of conditions for multiplex PCR Artificial samples were made by adding plasmid clones of specific genes comprising one to four types, which were obtained in Example 6 for testing on each mutation hot spot in multiple copies of 10, 100, 1,000 and 10,000, to sterilized triply distilled water, a sample storage solution of Example 7 and nasopharyngeal swab from healthy person. Then, multiplex PCR was preformed simultaneously by adding the primers of target genes for the 14 CO VID-19 Variants and CO VID-19 Variants genes in one tube.

PCR conditions

Example 13 - PCR on clinical sample

The reverse transcripted DNA of a human nasopharyngeal swab which had been already identified to be infected by CO VID-19 by the single PCR and sequencing in Example 11 was subjected to PCR according to the method established in Example 12.

Example 14 - Probe designing for hybridization assay

In order to prepare a DNA chip for genotyping the hybridization 14 CO VID-19 Variants and the PCR product of the control gene at once on a single chip, first, a combination of oligonucleotide probes having appropriate base sequences was designed. This was a fundamental step of developing the DNA chip of the present invention, that was the process of designing and preparing oligonucleotide probes which will be integrated on the DNA chip. The GeneBank database of the National Center for Biotechnology Information (NCBI) and the database of the genes of the 14 CO VID- 19 Variants obtained in Example 11 and human beta-globin gene found in Koreans were analyzed, and base sequence of each genotype determined. The obtained DNA sequence was subjected to pairwise alignment and multiple sequence alignment by means of ClustalW using the computer program MegAlign™ 5 (DNASTAR, Inc.), and then the phylogenetic tree was completed and the type-specific base sequence of each group was selected. Next, a type-specific probe was designed using the computer program Primer Premier 5 (PREMIER Biosoft International Co.). For the probes, oligonucleotides 20 ± 2 bp and 18 ± 2 bp long were used. A total of 10 genotype-specific probes were designed. The probe for human beta-globin gene is for use as a comer marker of the chip of the systems and methods herein. It may be detected using Cy5.

Example 15 - Preparation of DNA chip

The oligonucleotide probes designed in Example 14 were mixed with appropriate reagents and then integrated on a glass slide for a microscope using an arrayer to prepare an oligonucleotide microarray or an oligo DNA chip for detection and genotyping of CO VID- 19 Variants. Furthermore, a modified chip having 8 grids on one chip wherein 8 different samples may be integrated thereon for simultaneous assay was also prepared (see Fig. 5E).

Integration of probes for CO VID-19 Variants and CO VID-19 Variants genes on DNA chip: Grids were formed in groups such that, after hybridization on a chip, specific mutation hot spot can be easily detected by the fluorescence signal corresponding to the genotype of the COVID-19-causing mutation hot spot. The order of probes and the grid arrangement are schematically shown in Fig. 5E. Fig. 5E showed a photograph of the DNA chip of the systems and methods herein. DNA probes were spotted on a slide at 8 different wells, so that different samples can be detected at the same time and schematically showed the order and location of the DNA probes for genotyping the genomic genes or plasmid genes of the 14 COVID-19 Variants and for genotyping the genes related with COVID-19 Variants. Each oligonucleotide probe was spotted using an arrayer. At this time, the same probes were integrated in duplicate so that each genotype of the mutation hot spot appears at least twice and at most 4 times. One of the most important modifications of the DNA chip of the present intention was to equally divide grids into 8 wells on one chip using a well cover. With this, 8 different samples can be detected on one chip, which is very useful in reducing time, labor and cost.

Preparation of solution for spotting oligonucleotide probes onto chip and division onto master plate involved the oligonucleotide probes designed according to Example 14; synthesized by attaching amine onto C6 position were purified by high performance liquid chromatography (HPLC); and then dissolved in sterilized triply distilled water to a final concentration of 200 pM. Thus, the prepared probes were mixed with microspotting solution Plus (Telechem, TC-MSP, USA) in a proportion of 4.3 times to a final concentration of 38 pM. For example, to 7.6 pL of probes at 200 pM concentration, 32.4 pL of the spotting solution was mixed to make 40 pL. Thus, the prepared mixture was divided into a 96-wel master plate.

Fixation of oligonucleotide probes used an arrayer. The spotting solution containing the probes was transferred from the master plate to a specially coated glass slide and integrated thereto by double hit. A volume of about 0.005 pL on average was integrated in one spot. As for the glass slide, Nuricell aldehyde glass slide (Nuricell, Korea), 7.5 x 2.5 cm in size and coated with super aldehyde, or a product comparable thereto was preferred. For the arrayer, Q arrayer2 (Genetixs, UK), MGII (Biorobotics Inc, MA01801, USA) or an equipment comparable thereto was preferred.

The DNA chip prepared by integrating the probes onto the glass slide was reacted at room temperature for 15 minutes inside a glass jar maintained at a humidity of 80%.

After completion of the reaction, the fixated slide was baked in a drying oven for 1.5 hours at 120°C. Then, the slide was washed in 0.2% sodium dodecylsulfate (SDS) solution twice for 2 minutes, and then transferred to triply distilled water and washed twice for 2 minutes. Thereafter, the slide was dipped in triply distilled water heated to 95°C for 3 minutes, whereby the oligonucleotide probes attached on the slide were denatured, and washed in triply distilled water for 1 minute. After washing, the slide was reduced for 15 minutes in blocking solution (1 g of NaBH₄, 300 mL of PBS and 100 mL of ethanol), washed in 0.2% SDS solution twice for 2 minutes, and then transferred to triply distilled water and washed twice for 2 minutes. Water on the slide was removed by centrifuging at 800 rpm for 1 minute and 30 seconds, and then the slide was put in a slide box and stored in a desiccator at room temperature.

The conditions and qualities of thus prepared chip were observed and controlled as described in Example 16.

Example 16 - Hybridization assay on DNA chip and result analysis

The artificial samples made by mixing the plasmid clones of each COVID-19-causing mutation hot spot and the plasmid clones of human beta-globin gene in various combinations and concentrations in Example 12 were subjected to multiplex PCR. The products were integrated on the COVID-19 DNA chip prepared in Example 15 and subjected to hybridization reaction for multiple times. Then, the chip was analyzed using a fluorescence scanner to establish the optimized conditions. The method thereof is as follows and the results are shown below.

The PCR was performed in accordance with Examples 12 and 13.

For the hybridization reaction, on the slide chip to which the oligonucleotide probes were spotted, each 10 pL of the PCR product of each gene was mixed, with the sample DNA as main substance, to a final volume of 50 pL. After denaturation at 95°C for 5 minutes, the mixture was immediately placed on ice and left to stand for 3 minutes. Thereafter 50 pL of hybridization reaction solution was added thereto to adjust the final volume to 100 pL and then the mixture was reacted for 30 minutes at 45°C with the probes fixated on the slide. The hybridization reaction solution was prepared by mixing 2 mL of 20x SSC, 1.7 mL of 90% glycerol and 6.3 mL of 50 mM phosphate buffer solution to make the final volume 10 mL.

After completion of the hybridization reaction, the well cover was removed from the DNA chip, and the chip was dipped in 3x SSPE solution [NaCl (26.295 g), NafLPOplILO (4.14 g), Na2EDTA (1.11 g) dissolved in 1 L of distilled water, with pH adjusted to 7.4 using 10 N NaOH] and washed for 2 minutes at room temperature for washing. The chip was further washed with lx SSPE solution [NaCl (8.765 g), NaftPChT^O (1.38 g), Na2EDTA (0.37 g) dissolved in 1 L of distilled water, with pH adjusted to 7.4 using 10 N NaOH], washed for 2 minutes at room temperature, and dried by centrifuging at 800 rpm, at room temperature, for 1 minute and 30 seconds.

Scanning analysis was done after removal of nonspecific signals through washing. The dried slide was subjected to analysis of fluorescence signals and images using a fluorescence scanner. As for the scanner, GenePix 4000B Scanner (Axon, USA), ScanArray Lite (Packard Bioscience, USA) or an equipment comparable thereto are preferred. A ct value of 2.5 was used as cutoff.

Example 17 - SEQUENCE TABLES OF SYSTEMS AND METHODS HEREIN

The nucleotide sequences, amino acid mutations, PCR conditions, how to interpret the results, and DNA Chip Primer Sequences from the systems and methods systems herein are listed in the Tables below. The locked LNA areas are differentiated as capital letters in the table below.

Real Time RT-PCR kit for variant test

Table 1. LNA probe sequence

*478, 484, 490, 501 share the same primers

Table 2. Spike protein Primer sequence

*Tm: melting temperature

Table 3. N primers and probes for GSAID

Table 4. RdRp primers and probes for GSAID

Table 5. Nucleotide mutation summary for VOC

Table 6. Nucleotide mutation summary for VOI

Table 7. summary of mutations used in snapshot, RT-PCR, and DNA chip

Table 8. SNAPSHOT combination for mutants used for interpretation

Table 9-1. PCR mixture for 1 tube setting (Basic and Delta kit)

Table 9-2. PCR mixture for 2 tube setting (Standard and Extended kit)

Table 9-3. PCR thermocycling condition

Table 10-1. Basic Kit interpretation

Table 10-2. Delta Kit Interpretation

Table 10-3A Standard-A

Table 10-3B Standard B

Table 10-4. Extended- Korea

Table 10-5. Extended- USA/Korea

Table 10-6. Extended-USA (For cases already diagnosed with COVID-19 (SARS-CoV-2))

Table 11. DNA chip primer sequence

Example 18 - Precision Study Summary

A precision study was conducted and summarized below.

Precision Study

Twist Bioscience Synthetic RNA control

1. Delta 104533 Control 23 (B.1.617.2) LOD=143cp/ul

EPI ISL 1544014 India/MH-NCCS-Pl 162000182735/2021

2. Omicron 105204 Control 48 (B.1.1.529/BA.1) LOD=609 cp/uL EPI ISL 6841980 Hong Kong/HKU-211129-001/2021

Pos: positive result (sigmoidal amplification with Ct

< 40), Neg: negative result (No amplification)

* This may encompass BA.4. and BA.5. believed to be a combination of BA.l. and BA.2..

**This has not been validated on clinical deltacron specimens. This may likely encompass lineage XD/XF BA.2.12.1, believed to be a combination of BA. l. and Delta, (“Deltacron”) but may also be a result of contamination.

***This has not been validated on clinical specimens. This may encompass lineage XE, believed to be a combination of BA.l. and BA.2.

, but may also be a result of contamination.

** Sequencing assay may be indicated if same results persist.

This is a screening test for variant testing and results may be confirmed by sequencing.

Positive

*May include Omicron XE= BA. 1 + BE. 2

* *May include Omicron XD/XS/XF : hybrids of Delta and BA.1

Claims

CLAIMS What is claimed is:

1. A composition comprising a set of locked nucleic acid (LNA)-modified probes and a set of primers, each of the probes comprising two or more nucleotides, wherein the primers comprises the two or more nucleotides targeted for the diagnosis, thereby screening for corona virus disease-2019.

2. The composition of claim 1, wherein the probes and primers are configured for the diagnosis and screening of “variants” of corona virus disease-2019.

3. The composition of claims 1 or 2, wherein the variants comprise Alpha, Beta, Gamma, Delta, Eta, Iota, Kappa, Lambda, Alpha+484, Delta Plus, MU (B.1.621), Epsilon, Zeta, and Theta by WHO classification.

4. The composition of claims 1 - 3, wherein the said LNA probes comprises:

(i) nucleotides around 12-15 base pairs (bp);

(ii) melting temperature (Tm) close to 65°C; and

(iii) discriminating position in LNA probe is at position 2 rather than 1 or 3.

5. The composition of claims 1 - 4, wherein LNA probe comprise combinations of L5F,L18F, T19R, T20N, P26S, Q52R, A67V, D69-70, G75V, T76I, D80A,T95I, D138Y, G142D, D144, E154K, D 157-158, R190S, D215G, D242-244, R246I, D246-252, D253G, K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, A570D, D614G, H655Y, Q677P, Q677H, P681H, P681R, A701V, T716I, T859N, F888L, D950N, S982A, T1027I, Q1071H, D1118H, V1176F mutation hot spots of SARS-CoV-2.

6. The composition of claims 1 - 5, wherein LNA probe is used in real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) test for detection of the variants.

7. The composition of claims 1 - 6, wherein the primers target a portion of the SARS-Cov-2 spike protein in combination as we as an internal control.

8. The composition of claims 1 - 7, wherein the said primers are used together with said LNA probes with a positive, negative, and/or extraction control.

9. The composition of claims 1 - 8, wherein the said modified LNA probes are targeting SARS-Cov-2 S (Spike) protein against the wild type of L5F,L18F, T19R, T20N, P26S, Q52R, A67V, D69-70, G75V, T76I, D80A,T95I, D138Y, G142D, D144, E154K, D157-158, R190S, D215G, D242-244, R246I, D246-

77 252, D253G, K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, A570D, D614G, H655Y, Q677P, Q677H, P681H, P681R, A701V, T716I, T859N, F888L, D950N, S982A, T1027I, Q1071H, D1118H, V1176F mutation hot spots of SARS-CoV-2 for highly selective binding to mutation hot spots of the SARS-CoV-2.

10. The composition of claims 1 - 9, wherein the said modified LNA probes are especially targeting a combination of K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, P681H, P681R in S (Spike) protein.

11. The composition of claims 1 - 10, wherein the said modified LNA probes are targeting, but not limited to; SARS-Cov-2 S (Spike) protein against the wild type of K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, P681H, P681R in S (Spike) proteinfor highly selective binding to mutation hot spots of the SARS-CoV-2 such that resulting binding combinations with said modified LNA probes are deemed most useful in differentiating the variants.

12. The composition of claims 1 - 11, wherein LNA probe is used multiplex real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) test comprising a one-step reaction, two-step reaction, or more than two-step reaction in tube for detection of SARS-Cov-2 variants.

13. The composition of claims 1 - 12, wherein the set of probes in the same tube are labeled with markers different from each other.

14. The composition of claims 1 - 13, wherein the said modified LNA probes including markers, wherein the markers comprise fluorescent dye.

15 The composition of claims 1 - 14, wherein the said modified LNA hydrolysis probes including markers comprising a polynucleotide, a reporter, and a quencher label.

16. A method of determining the genotype at a locus of interest in a sample obtained from a subject, the method comprising a) contacting the sample comprising genetic material with the composition of any of claims 1 to 15; and b) detecting the binding of a set of probes to the wild type of the target material, thereby determining the mutation status at the locus.

17. The method of claims 1 to 16, wherein the mutation locus is a single nucleotide.

78

18. The method of claims 16 to 17, wherein the method comprises a) performing an amplifying step comprising contacting the sample with a set of primers to produce an amplification product including the locus of interest; b) performing a hybridizing step comprising contacting the amplification product of step a) with the composition of any of claims 1 to 17; and c) detecting the hybridizing of a set of probes to the genetic material, thereby determining the genotype at the locus.

19. The method of claim 1 to 18, wherein the locus of interest differentiates variants of SARS-CoV-2 and the amplification product is the S protein of SARS-CoV-2.

20. The method of claim 1 to 19, further comprising:

(i) measuring a presence or an absence of fluorescence in a sample;

(ii) detecting the fluorescence in real-time;

(iii) employing a polymerase enzyme having 5' to 3' exonuclease activity as an amplification; and/or

(iv) employing a reverse transcriptase step;

(v) wherein the sample is a biological sample, preferably a sample selected from the group consisting of a respiratory sample.

21. The method of claim 1 to 20, wherein the reporter marker is selected from the group consisting of fluorescein, LC-Yellow 555, FAM, VIC, HEX, Rhodamine B, Rhodamine 6G, LC-Red 610, LC-Red 640, LC-Red 670, LC-Red 705, Cy3, Cy 3.5, Cy5, Cy5.5; texas red, HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7- dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC( fluorescein isothiocyanate), Oregon green, alexa fluor, JOE (6-Carboxy-4',5’ Dichloro-2',7'- Dimethoxyfluorescein), ROX(6-Carboxyl-X- Rhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(l- Naphthyl) ethylenediamine, Cyanine dye or thiadicarbocyanine).

22. The method of claim 1 to 21, wherein the quencher marker is selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluore scent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), Blackberry Quencher, Iowa black.

23. The method of claim 1 to 22, wherein the library of sets of probes, wherein the sets of probes are spatially separated from each other.

79

24. The method of claim 1 to 23, wherein a presence of amplification product using the LNA modified probes and primers from the real-time RT-PCR prove that a particular codon is of wild type, and an absence of amplification product using the probes and primers described from the real-time RT-PCR prove that the particular codon is of variant, mutant type.

25. The method of claim 1 to 24, wherein the real-time RT-PCR of the S protein of SARS-Cov-2 uses both modified LNA wild-type and mutant-type probes for the codons of hot spot mutations above mentioned against the wild type of K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, F490S, N501Y, P681H, P681R in S (Spike) protein using sequences mentioned in Table 1.

26. The method of claim 1 to 25, wherein the real-time RT-PCR of the S protein of SARS-Cov-2 uses forward and reverse primers for codons of hot spot mutations via the sequences mentioned in Table 1.

27. The method of claim 1 to 26, wherein the real-time RT-PCR of the S protein of SARS-Cov-2 uses forward and reverse primers for the codons of hot spot mutations via the sequences in Table 1, in combination with internal control and N primer and probes.

28. The method of claim 1 to 27, wherein the real-time RT-PCR of the S protein of SARS-Cov-2 uses forward and reverse primers for the codons of hot spot mutations using the sequences in Table 1 in combination with the internal control and the N primer/probes comprises: a presence of amplification product using the LNA modified probes and primers from the realtime RT-PCR prove that a particular codon is of wild type, and an absence of amplification product using the probes and primers described from the real-time RT-PCR prove that the particular codon is of variant, mutant type.

29. The method of claim 1 to 28, wherein the variant, mutant type that World Health Organization, WHO or Communicable disease center, CDC or other organizations define as variants of concern, interest, or Alerts for Further Monitoring.

30. The method of claim 1 to 29, wherein the real-time RT-PCR of the S protein of SARS-Cov-2 uses respiratory comprising nasopharyngeal, oropharyungeal swab, nasal swab, throat swab, sputum, tracheal/bronchial secretion, bronchoalveolar lavage, bronchial lavage, pleural fluid, saliva, blood or blood derivatives, urine, feces

31 The method of claim 1 to 30, further comprising: standard material and negative control, plus minus extraction control run parallel for correct diagnosis, wherein the standard material comprising:

80 (i) In vitro transcript of receptor binding protein (RBD) of Spike protein of S ARS-Cov-2 RNA (genomic RNA) retrieved from multiple RNA controls; those with wild type mutations in all hot spots as proven by sequencing and alpha mutant S ARS-Cov-2 RNA, beta mutant SARS- Cov-2 RNA, gamma mutant SARS-Cov-2 RNA, delta mutant SARS-Cov-2 RNA, kappa mutant SARS-Cov-2 RNA from BEI, TWIST, or Korean CDC;

(ii) RBD IVT RNA with mutant type nucleotide codons for: K417N, K417T, L452R, L452Q, T478K, E484K, E484Q, N501Y, F490S, P681H, P681R for diagnosis of SARS-COv-2 presence (NP, N protein) and RNA dependent RNA polymerase (RdRp) IVT RNA;

(iii) Human beta-actin IVT RNA prepared for internal control QC.

(iv) Negative and positive extraction controls.

32. The method of claim 1 to 31, wherein the real-time RT-PCR of the S protein of SARS-Cov-2 result is interpreted by filling out the form per table by a software.

33. The method of claim 1 to 32, wherein the real-time RT-PCR of the S protein of SARS-Cov-2 result is used for in vitro diagnostic use of SARS-CoV-2 variants, and to determine what kind of a variant the result is categorized as.

34. The method of claim 1 to 32, wherein the real-time RT-PCR is used for the diagnosis and surveillance of COVID-19 mutants in community and hospital settings as a point of care or diagnostic test.

35. A DNA chip for detecting and genotyping COVID-19 Variants and for analyzing their CO VID-19 Variants, comprising oligonucleotide probes having base sequences.

36. The DNA chip for detecting and genotyping CO VID-19 Variants according to claim 35, wherein the oligonucleotide probe comprises a base sequence which binds complementarity to a human beta-globin gene.

37. The DNA chip for detecting and genotyping CO VID-19 Variants according to claim 35, wherein the oligonucleotide probe having a base sequence which binds complementarity to an oligonucleotide having a base sequence with the 5' end labeled with Cy5.

38. The DNA chip for detecting and genotyping CO VID-19 Variants according to claims 35, wherein an area of the DNA chip on which the probe is spotted is partitioned into 8 wells.

39. A kit for detecting and genotyping CO VID-19 Variants and analyzing COVID-19 Variants, comprising the DNA chip according to any one of claims 35 to 38, a primer set for amplifying DNAs of COVID-19 Variants, and a label for detecting the

81 amplified DNAs binding complementarity to the DNA chip.

40. The kit for detecting and genotyping CO VID-19 Variants and analyzing COVID- 19 Variants according to claim 39, wherein the primer set is a primer set for amplifying nucleic acids of COVID- 19.

41. The kit for detecting and genotyping CO VID-19 Variants and analyzing COVID-

19 Variants according to claim 39, wherein the label is selected from a group consisting of Cy5, Cy3, biotinylated material, EDANS (5-(2 - aminoethyl) amino- 1 -naphthalenesulfonic acid), tetramethylrhodamine (TMR), tetramethylrhodamine isothiocyanate (TMRITC), x- rhodamine and Texas Red.

42. The kit for detecting and genotyping CO VID-19 Variants and analyzing COVID- 19 Variants according to claim 41, wherein the label is Cy5 and labeled dCTP and unlabeled dCTP are reacted at a molar ratio of 1: 12.5.

43. A method for detecting and genotyping CO VID-19 Variants and analyzing COVID-19 Variants, comprising: amplifying DNAs of COVID-i9 Variants by single or multiplex PCR using a primer for amplifying nucleic acids of the COVID-19 Variants; and hybridizing the amplified DNAs on the DNA chip according to any one of claims 1 to 4; and detecting the hybridized product.

44. The method for detecting and genotyping COVID-19 Variants and analyzing CO VID-19 Variants according to claim 43, wherein the amplification by single or multiplex PCR is carried out using one or more primer set(s) selected from a group consisting of a primer set for amplifying nucleic acids of COVID- 19 or other respiratory pathogens.

45. The method for detecting and genotyping COVID-19 Variants and analyzing CO VID-19 Variants according to claim 44, wherein the amplification by single or multiplex PCR comprises: mixing the primer set with template DNA, Taq DNA polymerase, dNTP, distilled water and PCR buffer to form a resulting mixture; predenaturing the resulting mixture at 95°C for 10 minutes to form a resulting product; subjecting the resulting product to 40 cycles of denaturation at 94°C for 30 seconds, primer annealing at 58°C for 30 seconds and extension at 72°C for 30 seconds; and subjecting the resulting product to final extension at 72°C for 5

82 minutes.

46. The method for detecting and genotyping COVID-19 Variants and analyzing CO VID-19 Variants according to claim 43, wherein the amplification by multiplex PCR is carried out using a primer set having a combination of base sequences at a molar ratio of 1:1:1:1: 1: 1: 1: 1.

47. The method for detecting and genotyping COVID-19 Variants and analyzing CO VID-19 Variants according to claim 43, wherein the amplification by multiplex PCR is carried out using a primer set having combination of base sequences at a molar ratio of 1:1:1:1: 1.

48. The method for detecting and genotyping COVID-19 Variants and analyzing CO VID-19 Variants according to claim 43, wherein the amplification by multiplex PCR is carried out using a primer set having combination of base sequences at a molar ratio of 1:1:1:1: 1.

49. The method for detecting and genotyping COVID-19 Variants and analyzing CO VID-19 Variants according to claim 46, wherein a PCR product by the primer set having combination of base sequences of has a size as desired by an operator.

50. The method for detecting and genotyping COVID-19 Variants and analyzing CO VID-19 Variants according to claim 47, wherein a PCR product by the primer set having combination of base sequences has a size as desired by an operator.

83