WO2021186317A1 - Screening method for the detection nucleic acids - Google Patents

Screening method for the detection nucleic acids Download PDF

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Publication number
WO2021186317A1
WO2021186317A1 PCT/IB2021/052120 IB2021052120W WO2021186317A1 WO 2021186317 A1 WO2021186317 A1 WO 2021186317A1 IB 2021052120 W IB2021052120 W IB 2021052120W WO 2021186317 A1 WO2021186317 A1 WO 2021186317A1
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interest
nucleic acids
reagents
detection
pcr
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PCT/IB2021/052120
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French (fr)
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Huu Tho HO
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Vietnam Military Medical University
Mien Dong Saigon Clinics Co., Ltd.
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Publication of WO2021186317A1 publication Critical patent/WO2021186317A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features

Definitions

  • the present invention relates to a method capable of screening for one or more nucleic acids of interest on a large scale, comprising the steps of providing a sample aliquot from each of a plurality of samples, performing amplification reactions for gene regions containing the nucleic acids of interest for each sample aliquot, pooling the amplified sample aliquots to form a pooled template, testing the pooled template for detecting of the presence of the nucleic acids of interest, whereby identification of the presence of the nucleic acids in the pooled amplicon indicates the presence of the nucleic acids of interest in at least one of the sample aliquots.
  • the nucleic acids of interest in each sample is enriched in the first amplification step and then pooled to make the template for the next amplification.
  • the assay sensitivity is not affected, and applying this method allows for the screening of dozens to thousands of samples per day with a limited number of devices.
  • the present invention relates to a kit for the screening of nucleic acids of interest presented in an aliquot of a specimen in a pool of aliquots of different specimens.
  • pandemics such as SARS, MERS and Influenza A occurred and became global concerns about the spread of the diseases.
  • COVID-19 pandemic caused by SARS-CoV-2 emerged in December 2019 in Wuhan, Hubei, China.
  • COVID-19 presented in 84 countries and territories, with more than 94.000 infected case and about 3000 deaths (updated in March 2020).
  • SARS and MERS pandemics passed the SARS and MERS pandemics in 2003 and 2012.
  • the biggest challenge in preventing and stopping the pandemic is the high transmission rate of the disease, up to 2-4, and the virus can be transmitted from people to people, even from asymptomatic cases.
  • vaccine and treatment of the new disease need time for research, development and trial testing, the best solution for stopping the disease is massive screening for all suspected cases to timely detect infected cases and to take preventive measure to prevent the spread of disease.
  • the invention is proposed to solve above issues.
  • the invention relates to a massive screening method for the detection of one or more nucleic acids of interest.
  • the invention relates to the following embodiments:
  • a massive screening method for the detection of one or more nucleic acids of interest is a testing method for the detection of nucleic acids of interest in an aliquot of a specimen, comprising the steps of:
  • step (c) pooling the amplified sample aliquots of step (b) to form a pooled amplicon
  • the nucleic acid of interest is of DNA nature.
  • the nucleic acid of interest is of RNA nature.
  • primers for amplification of gene regions containing the nucleic acids of interest can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the target nucleic acid, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
  • step (9) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) is performed by nested-PCR or semi-nested PCR which amplifies secondary targets within the product amplified in step (b).
  • step (10) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR.
  • step (d) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by conventional PCR combined with the electrophoresis on agarose gel.
  • step (d) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicons in step (d) can be performed by real-time PCR using specific fluorescent probes.
  • step (d) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR combined with intercalating dye and detecting of the nucleic acids of interest based on specific melting peaks of amplicons.
  • the screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by sequencing.
  • the screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification.
  • step (d) The screening method according to embodiment (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification and detecting the nucleic acids of interest based on changes that can be observed by naked eyes.
  • step (d) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification and detecting the nucleic acids of interest based on a dipstick.
  • step (d) The screening method according to embodiment (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification combined with an intercalating dye (for instance SYBR Green, Evagreen) and specific melting peaks of amplicons.
  • an intercalating dye for instance SYBR Green, Evagreen
  • a kit for the detection of the presence of the nucleic acids of interest in a sample aliquot among plurality of sample aliquots of different clinical samples comprising of:
  • reagents (a) include reagents for PCR (Polymerase Chain Reaction) or reverse transcription PCR (RT-PCR).
  • the reagents (a) include primers for amplification of gene regions containing the nucleic acids of interest, wherein the primers can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the nucleic acid sequences of interest, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
  • reagents (b) include primers for performing nested-PCR or semi-nested PCR that amplifies secondary targets within the products amplified by reagent (a).
  • reagents (b) include reagents for PCR with intercalating dye and combined with artificial intelligence models analyzing data of melting peaks of amplified products.
  • reagents (b) include reagents for isothermal gene amplification and chemicals for the detection of amplified products by naked eyes.
  • reagents (b) include reagents for isothermal gene amplification and dipstick(s) for the detection of the nucleic acid of interest.
  • kits according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification with intercalating dye.
  • the testing method according to the invention is different in that, the pooling is performed after target enrichment step, therefore, the copy number of nucleic acid of interest increases many times and is sufficient for working as template for the subsequent amplification.
  • the number of template is not reduced significantly. A large number of samples can be simultaneously screened with out compromising the sensitivity of the test. Therefore, the testing cost will be reduced as the amount of reagent required is hundred times lower than individual testing.
  • Fig. 1 shows the principle of the multiplex, semi-nested PCR for the detection of SARS- CoV-2 according to the invention.
  • a massive screening method for the detection of one or more nucleic acids of interest is a testing method for the detection of one or more nucleic acids of interest in a sample aliquot, comprising the steps of:
  • step (c) pooling the amplified sample aliquots of step (b) to form a pooled amplicon; (d) testing the pooled amplicon for the detection of the presence of the nucleic acids of interest, whereby identification of the presence of the nucleic acids of interest in the pooled amplicon indicates the presence of the nucleic acids of interest in at least one of the sample aliquots.
  • the nucleic acid of interest is of DNA nature.
  • the nucleic acid of interest is of RNA nature.
  • primers for amplification of gene regions containing the nucleic acids of interest can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the target nucleic acid, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
  • step (9) The screening method according to embodiment (1), wherein detection of the nucleic acids of interest in the pooled amplicon in step (d) is performed by nested-PCR or semi-nested PCR which amplifies secondary targets within the products amplified in step (b).
  • step (10) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR.
  • the screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by conventional PCR method combined with electrophoresis on agarose gel.
  • the screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicons in step (d) can be performed by real-time PCR using specific fluorescent probes.
  • step (d) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR combined with intercalating dye and detecting of the nucleic acids of interest based on specific melting peaks of amplicons.
  • step (d) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by sequencing.
  • step (d) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification.
  • a kit for the detection of the presence of the nucleic acid sequences of interest in a sample aliquot among plurality of sample aliquots of different clinical samples comprising of: (a) Reagents for the amplification of gene regions containing the nucleic acids of interest in each separate sample aliquot;
  • reagents (a) include reagents for PCR (Polymerase Chain Reaction) or reverse transcription PCR (RT-PCR).
  • the reagents (a) include primers for amplification of gene regions containing the nucleic acids of interest, wherein the primers can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the nucleic acid sequences of interest, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
  • reagents (b) include primers for performing nested-PCR or semi-nested PCR that amplifies secondary targets within the products amplified by reagent (a).
  • reagents (b) include reagents for PCR with intercalating dye.
  • reagents (b) include reagents for PCR with intercalating dye and combined with artificial intelligence models analyzing data of melting peaks of amplified products.
  • kits (36) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification and chemicals for the detection of amplified products by naked eyes.
  • kits according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification with intercalating dye.
  • Sample pooling is the method used to reduce the cost of massive screening for suspected cases in populations with low disease frequency.
  • the advantage of the pooled testing is that it reduces the number of tests required, thereby reducing the biological cost needed for the screening of pathogens on a large scale.
  • main disadvantages of pooled testing are problems of assay sensitivity and the more complex composition of the pooled sample, as the amount of inhibitor is higher, that can affect the results.
  • the strategy of sample pooling after target enrichment was developed to maintain assay sensitivity.
  • the target enrichment step can be performed by different amplification methods such as Polymerase Chain Reaction (PCR), reverse transcription-PCR (RT-PCR), Loop mediated polymerase amplification (LAMP), reverse transcription-LAMP (RT-LAMP), or methods of isothermal gene amplification.
  • PCR Polymerase Chain Reaction
  • RT-PCR reverse transcription-PCR
  • LAMP Loop mediated polymerase amplification
  • RT-LAMP reverse transcription-LAMP
  • the copy number of target gene after target enrichment step is
  • the concentration of target gene after target enrichment step is a mixture of target gene after target enrichment step.
  • V a n volume of V ai product is used for sample pooling (V a n ⁇ V ai ). If products of target enrichment step of n samples are pooled with an equal volume, the concentration of DNA containing target gene reduces n times. As a result, the concentration of target gene after target enrichment step (increases M times) and sample pooling (reduces n times) will be
  • the concentration Ci- p0oi is higher than Co many times. Therefore, the sensitivity of the test will not be affected, even if the number of samples n in the pool is hundreds or thousands.
  • a portion of pooled sample is used as the template for the next amplification.
  • specific primers or specific probe(s) are used for detecting of the nucleic acids of interest. Any amplification method can be used, preferably nested-PCR or semi-nested PCR.
  • Primers for amplification can be designed by BLAST-Primer (NCBI). Sequences of genome/nucleic acids of interest can be retrieved from GenBank (NCBI) database and/or other database, e.g. virus database GISAID. The primers are designed for targeting different regions of genome, and the primer with the best limit of detection will be chosen.
  • the products of the target enrichment step of each sample will be sub-pooled with a lower number of samples and tested with the second amplification so that the number of pooling times and the number of amplification reactions are lowest.
  • These sub-pools are used as template for the amplification with the specific primers or specific probes for the detection of the nucleic acids of interest.
  • the positive sub-pool is continued to be sub-pooled with a smaller number of samples for the specific amplification for the detection of nucleic acids of interest, wherein the product of the first amplification are pooled and then used as template for the specific amplificiation; or the product of the first amplification of each sample aliquot is used as template for the specific amplification for the detection of nucleic acids of interest so that the number of pooling times and the number of amplification reactions are lowest.
  • 2 set of primers were designed on 8 distinct regions of 3 genes of SARS-CoV-2 including N, E and ORFlab gene; wherein 8 outer primer pairs and 8 inner primer pairs were included for semi- nested PCR. Sequences of primers are shown in Table 1.
  • Example 2 Reverse transcription and enrichment of multi targets using multiplex RT-PCR
  • the presumptive samples were used before testing clinical samples collected from COVID-19 suspected cases for evaluation of the testing method in the present invention.
  • the presumptive samples were created by spiking respiratory specimens of healthy individuals with the in-vitro transcribed SARS-CoV-2 RNA at different concentrations (presumptive positive samples) or with purified water (presumptive negative sample).
  • RNA extraction After RNA extraction, 94 presumptive samples were performed reverse transcription and target enrichment step, of which 1 sample was mixed with SARS-CoV-2 RNA before performing RNA extraction. A positive control was included to control the RNA extraction step and the amplification of 2 PCR steps. A negative control was also included to control the cross contamination of the manipulation. All primers Fo and Ro were included in 1 reaction to perform the multiplex amplification. The concentration and volume of the PCR components as well as the thermal cycling program are shown in Table 2 and Table 3.
  • Table 2 Components of a reverse transcription and target enrichment reaction using RT-PCR
  • Table 3 Thermal cycling program of the reverse transcription and target enrichment step
  • Example 3 Screening for SARS-CoV-2 using real-time PCR
  • Results of real-time PCR was collected by Rotor-Gene Q system and analyzed by Rotor- Gene Q Software (Qiagen). Each sample was run in duplicate. A negative control and a positive control were included in each PCR run. The average values of Ct and Tm were used for the detection of the pathogen.
  • Example 4 Screening for SARS-CoV-2 RNA in the pooled sample.
  • Ct value and Tm value illustrated the presence of SARS-CoV-2 RNA in the pooled sample
  • sub-pool samples were prepared by pooling of a lower number of samples. The number of samples in a sub-pool depends on the desired number of individual tests for pathogen detection, such as 50 or 10 samples.
  • the amplification was performed in the Rotor-Gene Q system with 20m1 reaction, wherein 5m1 of target enrichment product was used as template.
  • the real-time PCR for positive control and negative control were included. Concentrations and volumes of PCR components as well as thermal cycling program are shown in Table 4 and Table 5.
  • Example 6 Evaluation of the testing method for screening of SARS-CoV-2 in clinical samples
  • RNA samples were collected for evaluation of the testing method in the present invention, including 490 samples (including upper-, lower-respiratory and stool specimens) collected from COVID-19 suspected cases, and 1,030 anonymized upper- respiratory specimens collected from non-exposed volunteers.
  • Detection of SARS-CoV-2 RNA in 490 clinical samples of suspected cases was performed as routine clinical testing based on the Charite real-time RT-PCR assay recommended by WHO.
  • Total RNAs were extracted using QIAmp Viral RNA kit (Qiagen, Hilden, Germany), or MagNA Pure DNA and Viral NA Small Volume kit (Roche, Darmstadt, Germany) according to the manufacturer’s instruction. All RNA samples were stored at -80°C until used.
  • samples were randomly ordered within each batch and identified by barcodes so that the technicians remained blinded to the identities of the samples.
  • a positive and a negative control were included in each batch to monitor the execution of the procedure and ensure the validity of results.
  • RNA samples were thawed just before the amplification, and then performed the reverse transcription to generate cDNA and enriched target regions. Products of the target enrichment step were used as the template for the second amplification for the screening of SARS-CoV-2.
  • Data were collected by Rotor-Gene Q system and analyzed by Rotor-Gene Q software (QIAGEN). Melting spectra of amplified products were analyzed by an artificial intelligence model, that is available at https://covidl9ai.topdatascience.com/.
  • Example 7 Evaluation of the testing method using clinical samples spiked with in-vitro transcribed SARS-CoV-2 RNA at different concentrations
  • the target enrichment products of 93 negative clinical samples were pooled with the target enrichment product of a positive sample (a negative clinical sample spiked with in- vitro SARS-CoV-2 RNA), or with a negative control (negative clinical sample without in-vitro SARS-CoV-2 RNA).
  • the positive samples were negative samples mixed with in-vitro transcribed SARS-CoV-2 target RNA at different concentrations (1, 2, 5, 10, 20, 50, 100 and 500 copies/reaction).
  • each pooled sample includes 93 negative samples and 1 positive sample.
  • Each pooled sample was tested in 10 replications.
  • Results showed that 100% of pooled sample wherein the positive sample contains from 10 copies to 500 copies/reaction were positive. Regarding pools with lower SARS-CoV-2 concentration, 90% of pools that include 1 positive sample with 2 or 5 copies/reaction were positive, while only 50% of pools that include 1 positive sample with 1 copy/reaction were positive. All negative pools showed negative for SARS-CoV-2. Results of the evaluation were summarized in Table 7. Melting spectra of amplified products were illustrated in Figure 2.
  • Table 7 Evaluation of the testing method using clinical samples spiked with in-vitro transcribed SARS-CoV-2 RNA at different concentrations
  • Example 8 Evaluation of pooled testing using clinical samples
  • the pooled testing in the present invention uses a novel pooling strategy toward the screening of nucleic acid sequences of interest presented at low frequency and the pooling after target enrichment step does not affect the sensitivity of the test.
  • One out of 94 samples in the pool containing the nucleic acid of interest can be detected by this method.
  • the use of the pooled testing method in the present invention can save large numbers of biological products required for massive screening for nucleic acids of interest in suspected samples.
  • the testing method in the present invention can be applied for the screening of pathogen in low positive rate population, e.g. severe acute respiratory caused by coronavirus or cancer.
  • the screening cost based on this method will be reduced significantly compared with the individual testing method.
  • positive groups will be promptly quarantined and isolated to prevent disease transmission in the population.

Abstract

The present invention relates to a method for detecting nucleic acids of interest suitable for mass screening, comprising the steps of providing an aliquot of a sample from a plurality of samples, individually performing amplification reactions for gene regions containing nucleic acids of interest, combining aliquots of the amplified sample into a combined amplicon that serves as a template for the next amplification step, detection of nucleic acids of interest in the combined amplicon, whereby the presence of nucleic acids of interest in the combined amplicon indicates the presence of nucleic acids of interest in at least one of the sample aliquots. This method allows simultaneous screening of thousands of samples without compromising the sensitivity of the assay. In addition, the present invention provides a kit for detecting nucleic acids of interest in an aliquot of a sample among a plurality of aliquots of samples from various clinical samples.

Description

SCREENING METHOD FOR THE DETECTION NUCLEIC ACIDS
Technical field
The present invention relates to a method capable of screening for one or more nucleic acids of interest on a large scale, comprising the steps of providing a sample aliquot from each of a plurality of samples, performing amplification reactions for gene regions containing the nucleic acids of interest for each sample aliquot, pooling the amplified sample aliquots to form a pooled template, testing the pooled template for detecting of the presence of the nucleic acids of interest, whereby identification of the presence of the nucleic acids in the pooled amplicon indicates the presence of the nucleic acids of interest in at least one of the sample aliquots. In this method, the nucleic acids of interest in each sample is enriched in the first amplification step and then pooled to make the template for the next amplification. Thereby, the assay sensitivity is not affected, and applying this method allows for the screening of dozens to thousands of samples per day with a limited number of devices. Besides, the present invention relates to a kit for the screening of nucleic acids of interest presented in an aliquot of a specimen in a pool of aliquots of different specimens.
Background Art
In the last few decades, many pandemics such as SARS, MERS and Influenza A occurred and became global concerns about the spread of the diseases. Most recently, the COVID-19 pandemic caused by SARS-CoV-2 emerged in December 2019 in Wuhan, Hubei, China. After 3 months, COVID-19 presented in 84 countries and territories, with more than 94.000 infected case and about 3000 deaths (updated in March 2020). These numbers passed the SARS and MERS pandemics in 2003 and 2012. The biggest challenge in preventing and stopping the pandemic is the high transmission rate of the disease, up to 2-4, and the virus can be transmitted from people to people, even from asymptomatic cases. As vaccine and treatment of the new disease need time for research, development and trial testing, the best solution for stopping the disease is massive screening for all suspected cases to timely detect infected cases and to take preventive measure to prevent the spread of disease.
Currently, methods for detecting of pathogen are mainly based on real-time RT-PCR technique using specific fluorescent probes. However, one real-time PCR system allows for testing of only 240 samples/day at maximum capacity. In term of economy, using of fluorescent probe and applying of at least 1 real-time PCR reaction for every single case raise up the testing cost. Besides, the number of real-time PCR system is limited, even in centralized laboratories. In the COVID-19 pandemic, there has been no available test for dozens of thousands of at risk cases, especially in USA, China, UK, and Italy. As the pandemic is widespread globally, we are facing the serious problem of biological shortage for diagnostic testing. In technical term, current assays for detection of SARS-CoV-2 have maximum 3 target regions, e.g. the assay of USA CDC uses only 3 target regions in N gene. However, the respiratory vims such as SARS- CoV-2 is RNA virus that has high mutation rates. If the mutations simultaneously occur in the same gene, the sensitivity of the test will be affected significantly.
The invention is proposed to solve above issues.
Summary of the invention
The invention relates to a massive screening method for the detection of one or more nucleic acids of interest. In particular, the invention relates to the following embodiments:
(1) A massive screening method for the detection of one or more nucleic acids of interest, in particular, is a testing method for the detection of nucleic acids of interest in an aliquot of a specimen, comprising the steps of:
(a) providing a sample aliquot from each of a plurality of samples, thereby providing a plurality of sample aliquots;
(b) performing amplification reactions of gene regions containing the nucleic acids of interest for each sample aliquot;
(c) pooling the amplified sample aliquots of step (b) to form a pooled amplicon;
(d) testing the pooled amplicon for the detection of the presence of the nucleic acids of interest, whereby identification of the presence of the nucleic acids of interest in the pooled amplicon indicates the presence of the nucleic acids of interest in at least one of the sample aliquots.
(2) The screening method according to embodiment (1), wherein some of sample aliquots can be mixed together to form a pooled sample before performing the amplification of gene regions containing the nucleic acids of interest.
(3) The screening method according to embodiment (1), wherein the amplification is simultaneously performed at multiple gene regions containing the nucleic acids of interest of interest in a reaction.
(4) The screening method according to embodiment (1), the nucleic acid of interest is of DNA nature. (5) The screening method according to embodiment (1), the nucleic acid of interest is of RNA nature.
(6) The screening method according to embodiment (1), wherein the amplification of gene regions containing the nucleic acids of interest can be performed by isothermal gene amplification.
(7) The screening method according to embodiment (1), wherein the amplification of gene regions containing the nucleic acids of interest can be performed by either PCR (Polymerase Chain Reaction) or reverse transcription PCR (RT-PCR).
(8) The screening method according to claim 1-7, wherein, primers for amplification of gene regions containing the nucleic acids of interest can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the target nucleic acid, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
(9) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) is performed by nested-PCR or semi-nested PCR which amplifies secondary targets within the product amplified in step (b).
(10) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR.
(11) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by conventional PCR combined with the electrophoresis on agarose gel.
(12) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicons in step (d) can be performed by real-time PCR using specific fluorescent probes.
(13) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR combined with intercalating dye and detecting of the nucleic acids of interest based on specific melting peaks of amplicons.
(14) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by sequencing. (15) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification.
(16) The screening method according to embodiment (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification combined with specific fluorescent probes.
(17) The screening method according to embodiment (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification and detecting the nucleic acids of interest based on changes that can be observed by naked eyes.
(18) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification and detecting the nucleic acids of interest based on a dipstick.
(19) The screening method according to embodiment (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification combined with an intercalating dye (for instance SYBR Green, Evagreen) and specific melting peaks of amplicons.
(20) The screening method according to embodiment (13) or (19), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) is based on specific melting peaks of amplicons in the support of an artificial intelligence model.
(21) A kit for the detection of the presence of the nucleic acids of interest in a sample aliquot among plurality of sample aliquots of different clinical samples, comprising of:
(a) Reagents for the amplification of gene regions containing the nucleic acids of interest in each sample aliquot;
(b) Reagents for the detection of the nucleic acids of interest in pooled samples after amplification of gene regions containing the nucleic acid sequences of interest.
(22) The kit according to embodiment (21), wherein the reagents (a) is for simultaneous amplification of multiple gene regions containing the nucleic acids of interest in a reaction tube.
(23) The kit according to embodiment (21), wherein the reagents (a) contain enzyme DNA-dependent DNA polymerase. (24) The kit according to embodiment (21), wherein the reagents (a) contain enzyme RNA-dependent DNA polymerase as well.
(25) The kit according to embodiment (21), wherein the reagents (a) include reagents for the isothermal gene amplification.
(26) The kit according to any one of embodiments (21) - (24), wherein the reagents (a) include reagents for PCR (Polymerase Chain Reaction) or reverse transcription PCR (RT-PCR).
(27) The kit according to any one of embodiments (21) - (24) and (26), wherein the reagents (a) include primers for amplification of gene regions containing the nucleic acids of interest, wherein the primers can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the nucleic acid sequences of interest, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
(28) The kit according to embodiment (21), wherein the reagents (b) include primers for performing nested-PCR or semi-nested PCR that amplifies secondary targets within the products amplified by reagent (a).
(29) The kit according to embodiment (21), wherein the reagents (b) include reagents for PCR.
(30) The kit according to embodiment (21), wherein the reagents (b) include reagents for conventional PCR.
(31) The kit according to embodiment (21), wherein the reagents (b) include reagents for real-time PCR with specific fluorescent probes.
(32) The kit according to embodiment (21), wherein the reagents (b) include reagents for PCR with intercalating dye.
(33) The kit according to embodiment (21), wherein the reagents (b) include reagents for PCR with intercalating dye and combined with artificial intelligence models analyzing data of melting peaks of amplified products.
(34) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification.
(35) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification combined with specific fluorescent probes.
(36) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification and chemicals for the detection of amplified products by naked eyes. (37) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification and dipstick(s) for the detection of the nucleic acid of interest.
(38) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification with intercalating dye.
(39) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification with intercalating dye, and combined with artificial intelligence models for analysis of melting peaks of amplified products.
The testing method according to the invention is different in that, the pooling is performed after target enrichment step, therefore, the copy number of nucleic acid of interest increases many times and is sufficient for working as template for the subsequent amplification. When multiple samples are pooled with the same volume, 50 or 100 samples or more, the number of template is not reduced significantly. A large number of samples can be simultaneously screened with out compromising the sensitivity of the test. Therefore, the testing cost will be reduced as the amount of reagent required is hundred times lower than individual testing.
Brief description of the drawing
Fig. 1 shows the principle of the multiplex, semi-nested PCR for the detection of SARS- CoV-2 according to the invention.
Detailed description of the present invention
Screening for infected cases in the community at an early stage is critical to take preventive measures, to control disease transmission and timely start the treatment. Thu, after the successful establishment of the testing method, the inventors have applied it for the screening of SARS-CoV-2.
The invention relates to the following embodiments:
(1) A massive screening method for the detection of one or more nucleic acids of interest, in particular, is a testing method for the detection of one or more nucleic acids of interest in a sample aliquot, comprising the steps of:
(a) providing a sample aliquot from each of a plurality of samples, thereby providing a plurality of sample aliquots;
(b) performing amplification reactions for gene regions containing the nucleic acids of interest for each sample aliquot;
(c) pooling the amplified sample aliquots of step (b) to form a pooled amplicon; (d) testing the pooled amplicon for the detection of the presence of the nucleic acids of interest, whereby identification of the presence of the nucleic acids of interest in the pooled amplicon indicates the presence of the nucleic acids of interest in at least one of the sample aliquots.
(2) The screening method according to embodiment (1), wherein some of sample aliquots can be mixed together to form a pooled sample before performing the amplification of gene regions containing the nucleic acids of interest.
(3) The screening method according to embodiment (1), wherein the amplification is simultaneously performed at multiple gene regions containing the nucleic acids of interest in a reaction.
(4) The screening method according to embodiment (1), the nucleic acid of interest is of DNA nature.
(5) The screening method according to embodiment (1), the nucleic acid of interest is of RNA nature.
(6) The screening method according to embodiment (1), wherein the amplification of gene regions containing the nucleic acids of interest can be performed by isothermal gene amplification.
(7) The screening method according to embodiment (1), wherein the amplification of gene regions containing the nucleic acids of interest can be performed by either PCR (Polymerase Chain Reaction) or reverse transcription PCR (RT-PCR).
(8) The screening method according to claim 1-7, wherein, primers for amplification of gene regions containing the nucleic acids of interest can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the target nucleic acid, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
(9) The screening method according to embodiment (1), wherein detection of the nucleic acids of interest in the pooled amplicon in step (d) is performed by nested-PCR or semi-nested PCR which amplifies secondary targets within the products amplified in step (b).
(10) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR.
(11) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by conventional PCR method combined with electrophoresis on agarose gel. (12) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicons in step (d) can be performed by real-time PCR using specific fluorescent probes.
(13) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR combined with intercalating dye and detecting of the nucleic acids of interest based on specific melting peaks of amplicons.
(14) The screening method according to embodiment (10), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by sequencing.
(15) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification.
(16) The screening method according to embodiment (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in the step (d) can be performed by isothermal gene amplification combined with specific fluorescent probes.
(17) The screening method according to embodiment (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in the step (d) can be performed by isothermal gene amplification and detecting the nucleic acids of interest based on changes that can be observed by naked eyes.
(18) The screening method according to embodiment (1), wherein the detection of the nucleic acids of interest in the pooled amplicon in the step (d) can be performed by isothermal gene amplification and detecting the nucleic acids of interest based on a dipstick.
(19) The screening method according to embodiment (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in the step (d) can be performed by isothermal gene amplification combined with an intercalating dye (for instance SYBR Green, Evagreen) and specific melting peaks of amplicons.
(20) The screening method according to embodiment (13) or (19), wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) is based on specific melting peaks of amplicons in the support of an artificial intelligence model.
(21) A kit for the detection of the presence of the nucleic acid sequences of interest in a sample aliquot among plurality of sample aliquots of different clinical samples, comprising of: (a) Reagents for the amplification of gene regions containing the nucleic acids of interest in each separate sample aliquot;
(b) Reagents for the detection of the nucleic acids of interest in pooled samples after amplification of the gene regions containing the nucleic acids of interest.
(22) The kit according to embodiment (21), wherein the reagents (a) is for simultaneous amplification of multiple gene regions containing the nucleic acids of interest in a reaction tube.
(23) The kit according to embodiment (21), wherein the reagents (a) contain enzyme DNA-dependent DNA polymerase.
(24) The kit according to embodiment (21), wherein the reagents (a) contain enzyme RNA-dependent DNA polymerase as well.
(25) The kit according to embodiment (21), wherein the reagents (a) include reagents for isothermal gene amplification.
(26) The kit according to any one of embodiments (21) - (24), wherein the reagents (a) include reagents for PCR (Polymerase Chain Reaction) or reverse transcription PCR (RT-PCR).
(27) The kit according to any one of embodiments (21) - (24) and (26), wherein the reagents (a) include primers for amplification of gene regions containing the nucleic acids of interest, wherein the primers can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the nucleic acid sequences of interest, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
(28) The kit according to embodiment (21), wherein the reagents (b) include primers for performing nested-PCR or semi-nested PCR that amplifies secondary targets within the products amplified by reagent (a).
(29) The kit according to embodiment (21), wherein the reagents (b) include reagents for PCR.
(30) The kit according to embodiment (21), wherein the reagents (b) include reagents for conventional PCR.
(31) The kit according to embodiment (21), wherein the reagents (b) include reagents for real-time PCR with specific fluorescent probes.
(32) The kit according to embodiment (21), wherein the reagents (b) include reagents for PCR with intercalating dye. (33) The kit according to embodiment (21), wherein the reagents (b) include reagents for PCR with intercalating dye and combined with artificial intelligence models analyzing data of melting peaks of amplified products.
(34) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification.
(35) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification combined with specific fluorescent probes.
(36) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification and chemicals for the detection of amplified products by naked eyes.
(37) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification and dipsticks for the detection of the nucleic acids of interest.
(38) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification with intercalating dye.
(39) The kit according to embodiment (21), wherein the reagents (b) include reagents for isothermal gene amplification with intercalating dye, and combined with artificial intelligence models for analysis of melting peaks of amplified products.
Sample pooling is the method used to reduce the cost of massive screening for suspected cases in populations with low disease frequency. The advantage of the pooled testing is that it reduces the number of tests required, thereby reducing the biological cost needed for the screening of pathogens on a large scale. However, main disadvantages of pooled testing are problems of assay sensitivity and the more complex composition of the pooled sample, as the amount of inhibitor is higher, that can affect the results. In this invention, the strategy of sample pooling after target enrichment was developed to maintain assay sensitivity.
The target enrichment step can be performed by different amplification methods such as Polymerase Chain Reaction (PCR), reverse transcription-PCR (RT-PCR), Loop mediated polymerase amplification (LAMP), reverse transcription-LAMP (RT-LAMP), or methods of isothermal gene amplification. With No copies of target gene G in volume Vo pi and at concentration Co copies/pl (No = Vo*Co) used as template for the target enrichment step at reaction volume Vai:
Concentration of target gene G before target enrichment step reduces Vai/Vo time, therefore it is: CIT = Co*Vo/Vai copies/pl. After target enrichment step, the number and concentration of target gene is increased many times. The number of times that the target gene G amplified compared with the initial concentration is symbolized as M (times), therefore:
The copy number of target gene after target enrichment step is
Nis = M*No = M*Vo*Co (copies)
The concentration of target gene after target enrichment step is
Cis = M*CIT = Co*M* Vo/Vai (copies/pl)
After target enrichment step, a portion (volume Van) of Vai product is used for sample pooling (Van < Vai). If products of target enrichment step of n samples are pooled with an equal volume, the concentration of DNA containing target gene reduces n times. As a result, the concentration of target gene after target enrichment step (increases M times) and sample pooling (reduces n times) will be
Ci -pool = M*CIT = Co*M* Vo/(n*Vai) (copies/pl)
If the target enrichment step is performed with a sufficient number of amplification, the concentration Ci-p0oi is higher than Co many times. Therefore, the sensitivity of the test will not be affected, even if the number of samples n in the pool is hundreds or thousands.
Once products of the target enrichment step were pooled, a portion of pooled sample is used as the template for the next amplification. In this step, specific primers or specific probe(s) are used for detecting of the nucleic acids of interest. Any amplification method can be used, preferably nested-PCR or semi-nested PCR.
Primers for amplification can be designed by BLAST-Primer (NCBI). Sequences of genome/nucleic acids of interest can be retrieved from GenBank (NCBI) database and/or other database, e.g. virus database GISAID. The primers are designed for targeting different regions of genome, and the primer with the best limit of detection will be chosen.
When a pooled sample is reported as positive, the products of the target enrichment step of each sample will be sub-pooled with a lower number of samples and tested with the second amplification so that the number of pooling times and the number of amplification reactions are lowest. These sub-pools are used as template for the amplification with the specific primers or specific probes for the detection of the nucleic acids of interest.
When a sub-pool shows positive, all samples in the remaining sub-pools are reported as negative. The positive sub-pool is continued to be sub-pooled with a smaller number of samples for the specific amplification for the detection of nucleic acids of interest, wherein the product of the first amplification are pooled and then used as template for the specific amplificiation; or the product of the first amplification of each sample aliquot is used as template for the specific amplification for the detection of nucleic acids of interest so that the number of pooling times and the number of amplification reactions are lowest.
Examples
The invention will be described in more detail through the following examples, which detect the acid nucleic sequence specific to SARS-CoV-2. However, the invention is not limited to these particular examples.
Example 1 : Design of primer
To screening for COVID-19 using the testing method in the present invention, 2 set of primers were designed on 8 distinct regions of 3 genes of SARS-CoV-2 including N, E and ORFlab gene; wherein 8 outer primer pairs and 8 inner primer pairs were included for semi- nested PCR. Sequences of primers are shown in Table 1.
Table 1: Primer design for the screening of COVID-19
Figure imgf000014_0001
Figure imgf000015_0001
Example 2: Reverse transcription and enrichment of multi targets using multiplex RT-PCR
As the virus SARS CoV 2 is contagious and can cause dangerous disease, and the number of real samples is limited, the presumptive samples were used before testing clinical samples collected from COVID-19 suspected cases for evaluation of the testing method in the present invention. The presumptive samples were created by spiking respiratory specimens of healthy individuals with the in-vitro transcribed SARS-CoV-2 RNA at different concentrations (presumptive positive samples) or with purified water (presumptive negative sample).
After RNA extraction, 94 presumptive samples were performed reverse transcription and target enrichment step, of which 1 sample was mixed with SARS-CoV-2 RNA before performing RNA extraction. A positive control was included to control the RNA extraction step and the amplification of 2 PCR steps. A negative control was also included to control the cross contamination of the manipulation. All primers Fo and Ro were included in 1 reaction to perform the multiplex amplification. The concentration and volume of the PCR components as well as the thermal cycling program are shown in Table 2 and Table 3.
Table 2: Components of a reverse transcription and target enrichment reaction using RT-PCR
Figure imgf000015_0002
Table 3: Thermal cycling program of the reverse transcription and target enrichment step
Figure imgf000016_0001
Example 3: Screening for SARS-CoV-2 using real-time PCR
After target enrichment, 2pl of each product of 94 samples were pooled together (the number of pooled samples depends on the maximum number of PCR wells on a PCR system). 2m1 of the pooled sample then served as the template for the next amplification for the detection of SARS-CoV-2. Two multiplex PCR were performed in this step, one PCR is specific for SARS-CoV-2 and one PCR is for the detection of betacoronavims. Two reactions used the universal reverse primer Ru and the forward primers Fi, of which the specific amplification reaction for SARS-CoV-2 used Ru and the forward primers Fi-ORFl and Fi-Nl; the amplification reaction for the detection of Betacoronavims used Ru and the forward primers Fi- ORF2, Fi-N2, Fi-N3, Fi-N4 and Fi-E. Principle of the multiplex semi-nested PCR for the specific detection of SAR-CoV-2 is shown in Figure 1 (illustrated only for 2 regions in N gene), wherein 1 is RNA (gene N), 2 is reverse-transcription (RT), 3 is target enrichment, 4 is specific detection and 5 is specific Tm values of amplified products on real-time PCR system.
The amplification was performed in Rotor-Gene Q system with 20m1 reaction, of which 5m1 of pooled sample serves as template. An internal control and a negative control were included. Concentrations, volumes of the reaction components as well as the thermal cycling program are shown in Table 4 and Table 5. Table 4: Components of a real-time PCR for specific detection of SARS-CoV-2
Figure imgf000017_0001
Table 5: Thermal cycling program of the real-time PCR for specific detection of SARS-CoV-2
Figure imgf000017_0002
Results of real-time PCR was collected by Rotor-Gene Q system and analyzed by Rotor- Gene Q Software (Qiagen). Each sample was run in duplicate. A negative control and a positive control were included in each PCR run. The average values of Ct and Tm were used for the detection of the pathogen.
Example 4: Screening for SARS-CoV-2 RNA in the pooled sample. When the Ct value and Tm value illustrated the presence of SARS-CoV-2 RNA in the pooled sample, sub-pool samples were prepared by pooling of a lower number of samples. The number of samples in a sub-pool depends on the desired number of individual tests for pathogen detection, such as 50 or 10 samples.
2pl of target enrichment products of each 10 samples were pooled together. Subsequently, 5pl of pooled template was amplified by the next amplification for pathogen detection. The PCR was performed in the Rotor-Gene Q system with 20m1 reaction. A positive control and a negative control were also included. The concentrations and volumes of PCR components as well as thermal cycling program are shown in Table 4 and Table 5.
Data of real-time PCR were collected by Rotor-Gene Q system and then analyzed by Rotor-Gene Q Software (QIAGEN). Each sample was run in duplicate. A negative control is included for each run. The average values of Ct and Tm of the two replicate were used for data analysis for the detection of pathogen.
Example 5: Procedure for the detection of SARS-CoV-2
When the Ct value and Tm value illustrated the presence of SARS-CoV-2 RNA in a sub pool sample, individual testing was performed for all samples in the sub-pool to identify which sample is positive. All samples in the negative sub-pools were considered as negative for SARS-CoV-2. Two microlit of target enrichment product of each 10 sample in the positive sub pool was used as template for the specific amplification reaction for SARS-CoV-2 detection. In total, 10 PCRs were performed.
The amplification was performed in the Rotor-Gene Q system with 20m1 reaction, wherein 5m1 of target enrichment product was used as template. The real-time PCR for positive control and negative control were included. Concentrations and volumes of PCR components as well as thermal cycling program are shown in Table 4 and Table 5.
Data of real-time PCR were collected by Rotor-Gene Q system and then analyzed by Rotor-Gene Q Software (QIAGEN). Each sample was run in duplicate. The average values of Ct and Tm of the two replications were used for data analysis for pathogen detection.
Example 6: Evaluation of the testing method for screening of SARS-CoV-2 in clinical samples
A total of 1520 clinical specimens were collected for evaluation of the testing method in the present invention, including 490 samples (including upper-, lower-respiratory and stool specimens) collected from COVID-19 suspected cases, and 1,030 anonymized upper- respiratory specimens collected from non-exposed volunteers. Detection of SARS-CoV-2 RNA in 490 clinical samples of suspected cases was performed as routine clinical testing based on the Charite real-time RT-PCR assay recommended by WHO. Total RNAs were extracted using QIAmp Viral RNA kit (Qiagen, Hilden, Germany), or MagNA Pure DNA and Viral NA Small Volume kit (Roche, Darmstadt, Germany) according to the manufacturer’s instruction. All RNA samples were stored at -80°C until used.
To reduce experimental bias, samples were randomly ordered within each batch and identified by barcodes so that the technicians remained blinded to the identities of the samples. A positive and a negative control were included in each batch to monitor the execution of the procedure and ensure the validity of results.
The frozen RNA samples were thawed just before the amplification, and then performed the reverse transcription to generate cDNA and enriched target regions. Products of the target enrichment step were used as the template for the second amplification for the screening of SARS-CoV-2. Data were collected by Rotor-Gene Q system and analyzed by Rotor-Gene Q software (QIAGEN). Melting spectra of amplified products were analyzed by an artificial intelligence model, that is available at https://covidl9ai.topdatascience.com/.
Out of 490 samples collected from suspected cases, 10 samples had been reported as inconclusive after analysis with the Charite assay. By contrast, only one sample was inconclusive, and the other nine out of 10 inconclusive samples were reported negative with the novel testing method in the present invention.
Further investigation confirmed that nine out of 10 samples, reported as inconclusive by the Charite assay, were negative, and thus, concordant with the output of the testing method in the present invention. Among these, four samples collected from suspected cases, who were followed up for 21 days and confirmed as negative for COVID-19 with six testing points; two samples were derived from stool specimens collected from COVID-19 patients at day 8 and day 9 after diagnosis, that were likely supposed to be negative because the number of positives detected in stool specimens of COVID-19 cases was low; one sample was collected from COVID-19 patients, who had recovered, at day 35 after diagnosis; one sample was collected at day 10, wherein both of samples collected at day 7 and day 17 from the same patient were confirmed as negative; and one sample was collected at day 6, wherein another sample collected at day 2 from the same patient was inconclusive.
Detection of SARS-CoV-2 by the testing method in the present invention and Charite assays in 480 remaining specimens is shown in Table 6. The test successfully identified 451 negative samples and 26 positive samples, corresponding to a sensitivity of 100% (95% Cl: 86.8-100) and a specificity of 99.3% (95% Cl: 98.1-99.9). The specificity evaluated on the 1030 samples collected from non-exposed individual was 100%. The Cohen’s Kappa coefficient of 0.942 (with 95% Cl: 0.877-1.00) reveals the almost perfect agreement between two assays in detecting of SARS-CoV-2 RNA. McNemar chi-square test resulted in a two-tailed P = 0.248 inferring that there was no significant difference between 2 methods in the detection of SARS- CoV-2 RNA.
Table 6: Comparison between the testing method in the present invention and Charite assays in the detection of SARS-CoV-2 in suspected cases
Figure imgf000020_0001
Three samples with discordant results, which were reported negative by the WHO test, and positive by the new test were further analyzed by Sanger sequencing of PCR amplicons and investigated clinical symptoms. The sequencing data of target sequences on gene E and Rdrp region of ORFlab gene were successfully generated for 2 out of 3 samples which showed 100% identical to SARS-CoV-2 (compared with the first published sequence in GenBank, Accession number MN908947). These two samples were derived from 2 suspected cases, who had clinical symptoms of coughing, having temperature in one case; and one case had mild symptoms with a lung nodule on the CT scan, and a history of working as a nurse voluntarily working in a quarantined area. The remaining case had been quarantined in another province, and there was no information available.
Example 7: Evaluation of the testing method using clinical samples spiked with in-vitro transcribed SARS-CoV-2 RNA at different concentrations
To evaluate the testing method in detecting SARS-CoV-2 RNA at different concentrations, the target enrichment products of 93 negative clinical samples were pooled with the target enrichment product of a positive sample (a negative clinical sample spiked with in- vitro SARS-CoV-2 RNA), or with a negative control (negative clinical sample without in-vitro SARS-CoV-2 RNA). The positive samples were negative samples mixed with in-vitro transcribed SARS-CoV-2 target RNA at different concentrations (1, 2, 5, 10, 20, 50, 100 and 500 copies/reaction). Thus, each pooled sample includes 93 negative samples and 1 positive sample. Each pooled sample was tested in 10 replications. A portion of each pooled sample was screened for SARS-CoV-2 by 1 PCR tube, and results was then analyzed by melting spectra of amplified products. Results were analyzed and interpreted by the artificial model available at https://covidl9ai.topdatascience.com/.
Results showed that 100% of pooled sample wherein the positive sample contains from 10 copies to 500 copies/reaction were positive. Regarding pools with lower SARS-CoV-2 concentration, 90% of pools that include 1 positive sample with 2 or 5 copies/reaction were positive, while only 50% of pools that include 1 positive sample with 1 copy/reaction were positive. All negative pools showed negative for SARS-CoV-2. Results of the evaluation were summarized in Table 7. Melting spectra of amplified products were illustrated in Figure 2.
Table 7: Evaluation of the testing method using clinical samples spiked with in-vitro transcribed SARS-CoV-2 RNA at different concentrations
Figure imgf000021_0001
Example 8: Evaluation of pooled testing using clinical samples
28 positive clinical samples were tested with the pooling method for the screening of SARS-CoV-2, wherein each pool sample includes target enrichment products of 93 random negative samples and 1 positive sample. Each pool was run in duplicate. Results showed that, all pools showed positive for SARS-CoV-2.
Effects of the invention
The pooled testing in the present invention uses a novel pooling strategy toward the screening of nucleic acid sequences of interest presented at low frequency and the pooling after target enrichment step does not affect the sensitivity of the test. One out of 94 samples in the pool containing the nucleic acid of interest can be detected by this method. Thus, the use of the pooled testing method in the present invention can save large numbers of biological products required for massive screening for nucleic acids of interest in suspected samples.
The testing method in the present invention can be applied for the screening of pathogen in low positive rate population, e.g. severe acute respiratory caused by coronavirus or cancer. The screening cost based on this method will be reduced significantly compared with the individual testing method. For infectious diseases, positive groups will be promptly quarantined and isolated to prevent disease transmission in the population.

Claims

Claims
1. A massive screening method for the detection of the presence of one or more nucleic acids of interest, in particular, is a testing method for the detection of one or more nucleic acids of interest in a sample aliquot, comprising the steps of
(a) providing a sample aliquot from each of a plurality of samples, thereby providing a plurality of sample aliquots;
(b) performing amplification reactions for gene regions containing the nucleic acids of interest for each sample aliquot;
(c) pooling a portion of the amplified aliquots of step (b) to form a pooled amplicon;
(d) testing the pooled amplicon for the detection of the presence of the nucleic acids of interest, whereby the presence of the nucleic acids of interest in the pooled amplicon indicates the presence of the nucleic acids of interest in at least one of the sample aliquots.
2. The screening method according to claim 1, wherein some of sample aliquots can be mixed together to form a pooled sample before performing the amplification of the gene region containing the nucleic acids of interest.
3. The screening method according to claim 1, wherein the amplification is simultaneously performed at multiple gene regions containing the nucleic acids of interest in a reaction.
4. The screening method according to claim 1, wherein the nucleic acid of interest is of DNA nature.
5. The screening method according to claim 1, wherein the nucleic acid of interest is of RNA nature.
6. The screening method according to claim 1, wherein the amplification of gene regions containing the nucleic acids of interest can be performed by isothermal gene amplification.
7. The screening method according to claim 1, wherein the amplification of gene regions containing the nucleic acids of interest can be performed by either PCR (Polymerase Chain Reaction) or reverse transcription PCR (RT-PCR).
8. The screening method according to any one of claims 1-7, wherein primers for amplification of gene regions containing the nucleic acids of interest can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the target nucleic acid, and the second part at the 5’ -end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
9. The screening method according to claim 1, wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) is performed by nested-PCR or semi-nested PCR which amplifies secondary targets within the product amplified in step (b).
10. The screening method according to claim 1, wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR.
11. The screening method according to claim 10, wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by conventional PCR combined with electrophoresis on agarose gel.
12. The screening method according to claim 10, wherein the detection of the nucleic acids of interest in the pooled amplicons in step (d) can be performed by real-time PCR using specific fluorescent probes.
13. The screening method according to claim 10, wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by PCR combined with intercalating dye and detecting of the nucleic acids of interest based on specific melting peaks of amplicons.
14. The screening method according to claim 10, wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) is performed by sequencing.
15. The screening method according to claim 1, wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification.
16. The screening method according to claim 15, wherein the detection of the nucleic acids of interest in the pooled amplicon in the step (d) can be performed by isothermal gene amplification combined with specific fluorescent probes.
17. The screening method according to claim 15, wherein the detection of the nucleic acids of interest in the pooled amplicon in the step (d) can be performed by isothermal gene amplification and detecting the nucleic acids of interest based on changes that can be observed by naked eyes.
18. The screening method according to claim 1, wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) can be performed by isothermal gene amplification and detecting the nucleic acids of interest based on a dipstick.
19. The screening method according to claim (15), wherein the detection of the nucleic acids of interest in the pooled amplicon in the step (d) can be performed by isothermal gene amplification combined with intercalating dye and specific melting peaks of amplicons.
20. The screening method according to claim 13 or 19, wherein the detection of the nucleic acids of interest in the pooled amplicon in step (d) is based on specific melting peaks of amplicons in the support of artificial intelligence models.
21. A kit for the detection of the presence of the nucleic acids of interest in a sample aliquot among plurality of sample aliquots of different clinical samples, comprising of (a) reagents for the amplification of the gene regions containing the nucleic acids of interest in each separate sample aliquot;
(b) reagents for the detection of the nucleic acids of interest in pooled samples after amplification of the gene regions containing the nucleic acids of interest.
22. The kit according to claim 21, wherein the reagents (a) are for simultaneous amplification of multiple gene regions containing the nucleic acids of interest in a reaction tube.
23. The kit according to claim 21, wherein the reagents (a) contain enzyme DNA-dependent DNA polymerase.
24. The kit according to claim 21, wherein the reagents (a) contain enzyme RNA-dependent DNA polymerase as well.
25. The kit according to claim 21, wherein the reagents (a) include reagents for isothermal gene amplification.
26. The kit according to any one of claims 21-24, wherein the reagents (a) include reagents for PCR or reverse transcription PCR (RT-PCR).
27. The kit according to any one of claims 21-24 or 26, wherein the reagents (a) include primers for amplification of gene regions containing the nucleic acids of interest, wherein the primers can be sequences comprising of 2 parts, wherein the first part at the 3’ -end is perfectly matched with the nucleic acid sequences of interest, and the second part at the 5’- end is an unrelated sequence that is not complementary to any nucleic acid sequence of the target genome.
28. The kit according to claim 21, wherein the reagents (b) include primers for performing nested-PCR or semi-nested PCR that amplifies secondary targets within the products amplified by reagent (a).
29. The kit according to claim 21, wherein the reagents (b) include reagents for PCR.
30. The kit according to claim 21, wherein the reagents (b) include reagents for conventional PCR.
31. The kit according to claim 21, wherein the reagents (b) include reagents for real-time PCR with specific fluorescent probes.
32. The kit according to claim 21, wherein the reagents (b) include reagents for PCR with intercalating dye.
33. The kit according to claim 21, wherein the reagents (b) include reagents for PCR with intercalating dye and combined with artificial intelligence models analyzing data of melting peaks of amplified products.
34. The kit according to claim 21, wherein the reagents (b) include reagents for isothermal gene amplification.
35. The kit according to claim 21, wherein the reagents (b) include reagents for isothermal gene amplification combined with specific fluorescent probes.
36. The kit according to claim 21, wherein the reagents (b) include reagents for isothermal gene amplification and chemicals for the detection of amplified products by naked eyes.
37. The kit according to claim 21, wherein the reagents (b) include reagents for isothermal gene amplification and dipsticks for the detection of the nucleic acids of interest.
38. The kit according to claim 21, wherein the reagents (b) include reagents for isothermal gene amplification with intercalating dye.
39. The kit according to claim 21, wherein the reagents (b) include reagents for isothermal gene amplification with intercalating dye, and combined with artificial intelligence models for analysis of melting peaks of amplified products.
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