WO2022115450A2

WO2022115450A2 - Rapid processing of biological samples for sars-cov-2-rna detection

Info

Publication number: WO2022115450A2
Application number: PCT/US2021/060557
Authority: WO
Inventors: Piotr Chomczynski
Original assignee: Piotr Chomczynski
Priority date: 2020-11-24
Filing date: 2021-11-23
Publication date: 2022-06-02
Also published as: WO2022115450A3

Abstract

Viral detection in biological samples; composition for processing biological specimens; processes for viral detection; specifically, SARS-CoV-2 RNA detection; and a kit for use in such viral detection processes.

Description

RAPID PROCESSING OF BIOLOGICAL SAMPLES FOR SARS-COV-2-RNA DETECTION

Technical Field

[0001] Virus detection in biological samples.

Background

[0002] The COVID-19 pandemic has created a great demand for tests to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19). Currently, the most prevalent test for SARS-CoV- 2 is based on detection of its RNA genome by RT-PCR.

[0003] A viral detection test starts with a specimen collection (CDC 2019 Diagnostic Panel). The current SARS-CoV-2 diagnostic tests recommended by the Centers for Disease Control and Prevention (CDC) in the US and by the World health Organization (WHO) comprise specimen collection from the upper respiratory tract using a swab, or from the lower respiratory tract by sputum (saliva) collection. Swab specimens collected from the nose or throat are immersed in a viral transport media (VTM) or universal transport medium (UTM) to form a viral suspension. The viral suspensions are used immediately for testing or are stored at 2 - 8 C for up to 72 h, or at -20 for longer storage. A typical VTM or UTM is composed of physiological saline solution or a cell culture medium such as Hanks’ or Eagle’s medium plus antibiotics.

[0004] Wyllie et al (Wyllie, 2020) disclosed that saliva is the preferred source for SARS- CoV-2 specimen collection for testing. Collected specimens of saliva are stored at 4C for up to 2 days. An aliquot of viral suspension is taken for a diagnostic test. A comprehensive review of storage and testing of samples containing SARS-CoV-2 was presented by Esbin et al (Esbin, 2020). [0005] Rogers et al, (Rogers, 2020) reported no change in detection of SARS-CoV-2 after storage of saliva samples at room temperature for one week. However, it was noted that viral titers may be impacted by clinical course at time of sample acquisition and the quality of the sample specimen collection. The saliva samples used for this testing were processed in 1xPBS.

[0006] Viral transfer media do not inactivate viruses and create the possibility of infection by people handling viral samples (Dewar, 2020). Heating of saliva infected with SARS-CoV-2 was used as a simple method to inactivate the virus. Heating of saliva for 30 min at 57 C or 15 min at 60 C or at 65 C for 30 min was used to inactivate the virus (Wang, 2020, Li 2020).

[0007] The most frequently used viral RNA detection tests comprise two steps: a viral RNA purification step followed by a viral RNA detection step employing RT-PCR and SARS-CoV-2 RNA-specific probe kit. Effective target sequences for the specific RT-PCR detection comprise sequences located in the N gene of SARS-CoV-2, and the most effective sequence is N1 sequence in the N gene (CDC 2019 Diagnostic Panel).

[0008] For the SARS-CoV-2 RNA test, the detection sensitivity of RT-PCR performed with purified RNA is about Ct36 for 10 copies of viral RNA per reaction using primers for the N gene, N1 detection probe (Chu, 2020). In another disclosure, automated detection of SARS-CoV-2 employing cartridges for RNA isolation and detection had a lower limit of detection (LOD) of 14.8 copies per reaction and 365 copies per ml of viral specimen using primers for the S gene (Li, 2020). [0009] Alternatively, RT and a Loop Mediated Isothermal Amplification (LAMP) can be used to detect covid RNA. A SARS-CoV-2 RT-LAMP test performed using purified RNA had a detection sensitivity of 100 copies of viral RNA per reaction (Park, 2020).

[0010] With the aim to streamline diagnostic tests for detecting viral RNA, tests were developed to detect viral RNA directly in biological samples, without RNA purification. A direct RT-PCR detection test for SARS-CoV-2 comprising heating of a viral suspension in VTM followed by RT-PCR was recently described by Bruce et al (Bruce, 2020). For a low viral load (Ct>30) this test was not satisfactory and required additional testing. Sensitivity of this test is below sensitivity of the test performed with purified SARS-CoV-2 RNA. The N1 probe set was shown to be optimal for direct detection tests by RT-PCR. A similar decrease in detection sensitivity of the direct RT-PCR test for SARS-CoV-2 using purified SARS-CoV-2 RNA was described by Arumugam, 2020.

[0011] In another disclosure of the direct detection test, Li (2019) described detection of a single-stranded RNA Zika virus using direct RT-PCR performed with an inhibitor- tolerant DNA polymerase. For Zika testing, the limit of detection was 95 copies of Zika RNA per reaction.

[0012] A direct RT-PCR performed with saliva specimens treated with non-ionic detergents, such as Tween® 20, Triton® X-100, NP 40®, were reported (Smyrlaki, 2020 and Ranoa, 2020). Smyrlaki et al. reported that non-ionic detergents decrease SARS- CoV-2 RNA detection sensitivity in RT-qPCR by about 1 Ct to 2 Ct but they allow detection of SARS-CoV-2 RNA by direct RT-qPCR. Ranoa, et al. reported a positive effect of non ionic detergents on RT-qPCR with saliva specimens diluted with a buffer and subsequently heated at 65 c or 95 C. [0013] Previously, we disclosed direct DNA detection using alkaline-glycol lysis of biological samples (US 7,727,718, Chomczynski 2006). These disclosures described the use of glycols for DNA analysis in biological samples. It was found that concentrated glycols in alkaline aqueous solution act as chaotropes, increasing the alkalinity of a solution and significantly contributing to lysis of biological samples. These properties of glycols were beneficial for making a sample of DNA available for direct PCR analysis without purification of DNA. Compositions and methods described in these disclosures were not recommended for RNA detection because RNA was degraded, precipitated and not available for RNA analysis. Contrary to this, the current application discloses that a modified glycol-alkaline lysis can be successfully used for the detection of RNA in biological specimens, and specifically for detection of SARS-CoV-2 RNA in biological fluids.

[0014] In view of the world-wide pandemic of SARS-CoV-2 infections, the development of fast, sensitive, reliable, and economical diagnostic test is critical for its application to millions of subjects.

[0015] The SARS-CoV-2 detection test of this Invention, based on Alkaline Glycol (AG) Processing, allows rapid, reliable and sensitive detection of viral RNA that can be automated or performed in manual settings. It allows the processing of saliva or swab specimens to detect SARS-CoV-2 RNA by RT-PCR in less than 1 h. The use of AG Processing eliminates the need for an RNA purification step, and heating of specimens is not necessary. SARS-CoV-2 containing specimens are processed by incubation in the AG Processing Compositions at room temperature. The processing is based on the glycol-alkaline treatment of biological specimens, preferentially at pH 12.2 to pH 12.8 During this process, viral RNA is made available for the RT reaction, and inhibitors of RT- PCR are sufficiently inactivated that the sensitivity for detecting the target RNA is on par with tests using purified RNA. A simple incubation in the Direct Processing Composition makes unnecessary the use of purification and processing cartridges commonly used in current clinical tests for the detection of SARS-CoV-2.

Detailed Description

DEFINITIONS OF TERMS used in the description.

[0016] Terms defined in the text of the application are specifically defined for the purpose of presenting the currently described Invention:

[0017] Biological specimen or biological sample, a specimen or sample containing biological matter derived from tissues or body fluids of human, animal or plant origin.

[0018] Dalton, symbol Da, unit of molar mass, 1 Da = 1 g/mol.

[0019] RT, a reaction catalyzed by Reverse Transcriptase generating complementary DNA from an RNA template.

[0020] RT-LAMP, loop mediated isothermal amplification RT-LAMP used to synthesize (cDNA) from RNA sequences. Amplification of this cDNA occurs by strand- displacing using DNA polymerase and four primers recognizing complementary regions within the target DNA sequence.

[0021] RT-PCR, reverse transcription polymerase chain reaction that combines the reverse transcription of an RNA template to produce complementary DNA and amplification of DNA by polymerase chain reaction (PCR) using sequence-specific primers complementary to a target DNA region. [0022] RT-qPCR, quantitative RT-PCR, a sub-group of RT-PCR, it is used to quantitate (quantify) the amount of RNA molecules, including viral RNA and messenger RNA.

[0023] Direct RT-qPCR, RT-qPCR for detection of RNA wherein a biological sample is directly subjected to RT-qPCR for quantitation (quantification) of sample’s RNA, without the RNA purification step. Direct RT-qPCR may involve simple treatments of sample such as heating and/or incubation with compound(s) to improving RNA detection.

[0024] Ct, critical threshold (CT) in quantitative PCR and RT-qPCR, the amplification cycle where the fluorescence curve exhibits the greatest curvature and exceeds the background fluorescent threshold. Ct is used as a quantitate measure of product accumulation in PCR.

[0025] AG Processing, alkaline glycol processing employing AG Processing compositions, a method of this Invention, for processing of biological samples containing RNA, including viral RNA in saliva, to make RNA present in these samples available for detection in direct RT-qPCR, RT-PCR and other RNA detection tests using reverse transcription.

[0026] AG Processing compositions, compositions comprising glycols and alkaline compounds of this Invention for use in AG Processing of biological specimens.

[0027] Effective compound, effective amount, a compound or amount of a compound(s) that is sufficient for a desirable change in at least one symptom of a disorder, and/or a desirable change in a biological process. The change due to the effective amount or effective compound should be at least about 15% of the value without thereof.

[0028] LLOD, lowest limit of detection [0029] PEG 200, polyethylene glycol (CAS 25322-68-3) with average molecular weight 200.

[0030] Alkaline glycol solution, alkaline PEG 200 solution, a solution containing: 65 g of PEG 200, 0.252 g KOH and 34.75 g ml water, wherein PEG 200 is 65% and KOH is 45 mM.

[0031] %, refers to the percent weight of a compound to the total weight of a solution containing said compound. For example, 20% glycol in aqueous solution contains 20g glycol, and 80 g water.

[0032] % (v/v) refers to the volume percent of a compound or mix of compounds in a solution containing said compound(s).

[0033] Singular forms, also indicated by “a”, “an” and “the”, include plural referents unless the context clearly indicates otherwise. In some sentences in the text plural is indicated by (s) added at the end of a word.

[0034] VTM, viral transfer medium, a fluid used for storage and transfer of biological specimens containing viruses, sometimes specified as universal transfer medium (UTM). Typically used with swabs containing specimens from upper respiratory tract or with buccal swabs.

[0035] VPC, virus preservation composition comprising compounds of this Invention in a solution or as mix of dried compounds for preservation of RNA, including viral RNA, in biological specimens for use in RNA detection tests, comprising direct RT-qPCR.

[0036] The current Invention discloses compositions and a process for detection of SARS-CoV-2 comprising collection, processing and detecting SARS-CoV-2 RNA in biological specimens containing SARS-CoV-2. The Invention uses alkaline glycol processing (AG Processing) of virus containing biological specimens for use in viral RNA detection tests, including RT-PCR. Contrary to the previous disclosures (‘718, Chomczynski 2006) the current Invention discloses that AG Processing employing alkaline glycol compositions with limited concentration of a glycol, <25% (v/v) at pH >11 .5, can be used to detect viral RNA in biological specimens. AG Processing of the Invention results in sufficient lysis of viruses and inactivation of inhibitors of RT-PCR present in biological specimens to allow effective detection of viral RNA by direct RT-qPCR, performed without a viral RNA purification step. By eliminating the viral RNA purification step, the compositions and methods of the current Invention simplify and streamline tests for detecting viral RNA. At the same time, the sensitivity of viral RNA detection of the Invention is on par with detection methods that comprise purification of viral RNA.

[0037] The AG Processing compositions of the Invention comprise glycols and polyalkylene glycols such as: polyethylene glycols, propylene glycol, polypropylene glycols. The preferred glycols are polyethylene glycols. Polyethylene glycols or PEGs used in the present Invention are commercially available with molecular weight ranging from 200 to 100,000 Da. For AG Processing, the preferred molecular weight range is from 200 Da to 10,000 Da, and the most preferred molecular weight is 200 Da. The concentration of glycols in the compositions for AG Processing ranges from about 10% to below 25%, and the preferred concentration is between 20% and 23%.

[0038] AG Processing compositions further comprise virus-infected biological specimens including saliva specimens and biological specimens in viral transfer medium (VTM). The most preferred AG Processing compositions are made to comprise two volumes of saliva or VTM and one volume of alkaline PEG 200 solution. The alkaline PEG 200 solution is an aqueous solution comprising 65 % PEG 200, and 45 mM KOH. Thus, the most preferred AG Processing composition comprises, by volume: 67% (v/v) of saliva and 21 .7% (v/v) PEG 200, and by weight: 65% of saliva and 22.8% PEG 200.

[0039] Since the amounts of glycols in the AG Processing composition are from about 10% to below 25% and volume of an alkali is about 0.3%, the volume of biological specimen could be up to about 90% of the volume of AG Processing composition. The lowest volume of a biological specimen in the AG Processing composition could be as low a volume of few animal cells. In the case of small volume specimens, AG Processing compositions comprise the alkaline glycol solution and additional amount of water to reach final glycol concentration and pH in the range specified in this Invention. This is exemplified in the Example 8 wherein 100 mg of tissue is processed with 1.9 ml of water and 1 ml of the alkaline glycol solution.

[0040] During AG Processing, viral RNA is liberated from the viral envelope and is available for enzymatic reactions including reverse transcription and RT-PCR. Biological specimens such as saliva contain carbohydrates and proteins, including ribonucleases, that inhibit RT-PCR. AG Processing compositions with glycols and high alkaline pH reduce the actions of these inhibitors and allow effective detection of viral RNA by direct RT-PCR, without RNA purification. The alkaline pH of AG Processing compositions ranges from pH 11.5 to pH 14.0, and the preferred pH range is from pH 11.8 to pH 13.0, and the most preferred range is between pH 12.2 and pH 12.8. The alkaline pH of AG Processing compositions is maintained by both a strong mineral base such as KOH and/or NaOH, and glycols.

[0041] For evaluating SARS-CoV-2-RNA detection in this invention, AG Processing compositions were spiked with known quantities of inactivated SARS-CoV-2, such as gamma irradiated SARS-CoV-2, heat denatured SARS-CoV-2, or synthetic SARS-CoV-2 RNA. AG Processing of the virus-containing AG Processing composition is carried out by incubating the compositions for 5 min to 30 min at room temperature. If necessary, the incubation time in AG processing composition can be extended to 1 h. After incubation, an aliquot of the AG Processed composition is added to an RT-qPCR reaction mix designed for SARS-CoV-2 RNA detection. After mixing the AG Processed composition with the RT- qPCR reaction mix, the pH of the resulting composition substantially decreases and it is in the range of 8.0 to 8.5, as required for optimal RT-qPCR performance. Thus, no pH adjustment step for RT-qPCR is necessary to reduce the high alkalinity of the AG processing composition.

[0042] In the current Invention, the SARS-CoV-2 RNA detection test comprises a probe for detecting the N1 region of the nucleocapsid gene of the SARS-CoV-2 genome. In addition, a probe detecting the N2 region of the nucleocapsid gene or other probes specific to SARS-Cov-2 RNA was also used (CDC 2019 Diagnostic Panel).

[0043] The effectiveness of AG Processing for detection of SARS-CoV-2 RNA by direct RT-qPCR (no RNA purification), is evidenced in Example 1. Results in Table E1 indicate that AG Processing substantially decreases the number of amplification cycles necessary to detect SARS-CoV-2 RNA. AG Processing of saliva allows detection of SARS-CoV-2 RNA with 6 to 7 fewer amplification cycles than in RT-qPCR reactions performed with no- AG processing of saliva samples. This corresponds to a 64- to 128-fold increase in sensitivity of RT-qPCR detection.

[0044] AG Processing works with several commercially available RT-PCR kits. Results in Example 2 show that AG processing effectively improves detection of SARS-CoV-2 RNA performed with several commercial kits. The increase in sensitivity, measured by a decrease in the number of amplification cycles necessary for viral detection, was at least

70-fold. [0045] Direct RT-PCR performed with AG Processing has a wide detection range when used with saliva or with a viral transfer medium containing viral suspensions. The results in Example 3 show detection of SARS-CoV-2 RNA with AG Processing of saliva, and detection of SARS-CoV-2 with AG Processing of viral suspension in VTM and Hanks medium. For all three specimens, the detection ranged from 400,000 copies to 5 copies of SARS-CoV-2 RNA per reaction.

[0046] In Example 4, processing was performed with two RT-PCR kits and saliva specimens containing a low copy number of SARS-CoV-2 RNA. The LLOD for TaqPath kits was 2 copies per reaction. Similar results were obtained with GoTag and KiCgStart kits. Because the RT-qPCR reaction mix contains 3.3 pi of saliva, this corresponds to 0.6 copies of SARS-CoV-2 per pi of saliva, and 600 copies per ml of saliva. In additional experiments performed in accord with the FDA 20/19 rule, the same 2 copy LLOD was observed with TaqPath, Kick Start and GO Taq kits, wherein 20 assays were performed for each copy number. Wyllie et al (Wyllie, 2020) reported that human saliva contains from 10¹⁰ to 10⁴ SARS-CoV-2 viruses per ml of saliva in subjects infected with the virus. Thus, AG Processing of this Invention is well within the minimal detection limit for clinical applications.

[0047] In the Invention, biological samples are processed at pH> 11 .5, and the most recommended pH is pH>12.2. At this pH, some degradation of RNA to smaller fragments occurs. Unexpectedly, fragmentation of RNA during AG Processing does not significantly affect detection of viral RNA by RT-qPCR. Results in Example 5 show that extending AG Processing of the virus-containing saliva to 45 min does not significantly change the detection of SARS-CoV-2 RNA. These results indicate that during preparation of the assay for RT-PCR, the AG Processing composition containing SARS-CoV-2 can be maintained at room temperature for 45 min. [0048] Heating can be incorporated into the AG Processing protocol. As disclosed in the Introduction, heating of saliva is known to improve SARS-CoV-2 RNA detection. In addition, heating of SARS-CoV-2 at a temperature >57 C can inactivate the virus. When adding a heating step to the treatment in this Invention, saliva specimens should be heated before AG Processing. Ranoa et al (Ranoa, 2020) described the direct RT-qPCR using heated saliva. In their protocol, saliva is processed for the heating step by mixing with one volume of a buffer. After heating, diluted saliva is mixed with a detergent. In the AG Processing protocol of this Invention, heating is performed on unprocessed saliva specimens. It can be done with saliva specimens as collected from donors, in the original collection tubes. Inactivation of viruses in the saliva specimen, before any saliva processing, substantially increases the safety of handling saliva specimens.

[0049] Example 6 shows variable effects of heating on detection of SARS-COV-2 RNA in saliva. Human saliva contains variable amounts of mucous and other constituents, which can affect the RT-PCR assay. For low mucus saliva, heating at 65 C does not significantly affect detection of SARS-CoV-2 RNA, but heating at 95 C has a negative effect on detection of viral RNA, as two additional amplification cycles are necessary to detect viral RNA. In contrast, heating of high mucus saliva at 65 C and 95 C substantially improves detection of viral RNA, as evidenced by a 5 Ct decrease in amplification cycles necessary to detect SARS-CoV-2 RNA. Saliva-dependent effects of heating at 95 C on detection of SARS-Cov-2 by direct RT-PCR in swab-derived specimens were previously reported (Bruce, 2020). In the current Invention, negative effects of heating saliva at 95 C in detection of SARS-CoV-2 RNA were also observed using commercially available pooled saliva specimens.

[0050] Non-ionic detergents are known to support PCR and RT-PCR. Concentrations up to 5 % for Triton® X100 and up to 10% for Tween® 20, were used in the RT-PCR assay to improve detection of SARS-CoV-2 RNA (Smyrlaki, 2020). Subsequently, the use of non-anionic detergents for SARS-CoV-2 RNA detection was combined with heating of saliva diluted in a buffer. Addition of Tween 20, after the heating step, to the diluted saliva improved detection of SARS-Cov-2 RNA by about 2 Ct (Ranoa, 2020).

[0051] Non-ionic detergents, comprising Tween® 20, Triton® X-100, NP®-40 and Brij®35, are also beneficial for detecting SARS-CoV-2 RNA in the current Invention. Results presented in Example 6 evidence the beneficial effects of Tween 20 addition to the RT- PCR assay for detection of SARS-CoV-2 RNA in AG processed saliva. Both heated and non-heated saliva samples were used in these tests, and the detergent was added into the reaction mix. As summarized in Table 6B, Tween 20 and heating, when applied separately, have similar beneficial effects on detection of SARS-CoV-2 RNA. The combined effects of the two treatments are not additive. When used in combination with Tween 20, heating does not have a significant effect on detection of SARS-CoV-2 RNA. An exception is the inhibition of the SARS-CoV-2 RNA detection in the presence of Tween 20 in the low mucus saliva heated at 95 C.

[0052] In this invention, a non-ionic detergent(s) is added directly into the RT-qPCR reaction mix and not into saliva specimens, as it was reported in previous disclosures. In this invention, addition of non-ionic detergent to saliva specimens causes time-dependent degradation of SARS-CoV-2 RNA. In RT-qPCR tests, exposure of saliva to 1% Tween 20, at room temperature, causes substantial decrease of detectable SARS-CoV-2 RNA. For example, in short, up 1 h, exposure of saliva to 1% Tween 20 results in 1 Ct to 2 Ct increase in number of amplification cycles necessary to detect SARS-CoV-2 RNA. When saliva specimens are incubated for 20 h at room temperature with 1% Tween 20 it takes 10 amplification cycles more to detect SARS-CoV-2 RNA, an increase from 26 Ct to 36

Ct. [0053] In the current Invention, the recommended composition of a kit for the RT-qPCR reaction mix comprises non-ionic detergent, including Tween 20, Triton X-100, NP 40 and Brij 35, in concentration from about 0.1% to about 5%.

[0054] In the presence of Tween 20, heating is not necessary to achieve optimal performance of the SARS-CoV-2 detection test. However, by inactivation of SARS-CoV- 2, heating at a temperature >57 C provides additional safety for handling saliva specimens.

[0055] AG Processing can be also used to detect in saliva RNA molecules other than a viral RNA. Example 7 evidences detection of the RPP30 endogenous mRNA transcript in human saliva with the use of AG Processing and RT-qPCR. Specific primers were used to detect only RPP30 transcripts and not genomic DNA sequences. No amplification was observed in non-AG Processed saliva.

[0056] AG Processing is also able to process biological materials other than saliva. Example 7 evidences detection of APOB (apolipoprotein B) mRNA in rat liver and spleen using AG processing and RT-PCR. Rat liver has a high level of APOB gene expression and spleen has only a residual APOB expression. This is reflected in a Ct of 17.64 in the reaction with homogenate obtained from 4.5 μg of liver, and a Ct of 34.27 in the reaction with 5.7 μg of homogenate obtained from spleen. These results demonstrate that, in addition to saliva, AG Processing can be used for fast detection of mRNA derived from other biological sources, comprising tissues homogenates and cell lysates.

[0057] Collection of a virus-containing specimen in the current Invention comprises collection of specimens from the lower respiratory tract comprising saliva (sputum), collection of buccal swabs, and swabs collected from the upper respiratory tract. In this invention, after collection of buccal swabs or swabs from a nose and/or throat, the swabs can be immersed in a transport medium, and preferentially in the viral preservation composition (VPC) of the current Invention. The resulting SARS-CoV-2 suspensions are used immediately for testing or can be stored at 4 C or -70 C for later use.

[0058] The preferred specimen of this Invention is saliva (sputum). Typically, 1 ml to 3 ml and preferably 1 ml of saliva is collected, but other volumes smaller or larger can also be collected. The collected saliva may be used immediately, stored at room temperature for 5 h, or at 4C for 2 days, or at - 20 for longer times.

[0059] Saliva, a biological fluid that support live viruses in vivo is also capable of maintaining viruses under in vitro conditions. As in the case of SARS-CoV-2, the single- stranded RNA genome is protected from degradation by a capsid comprised of protein matrix combining nucleoprotein, membrane protein and spike glycoprotein (Mousavizadeh, 2020). However, after saliva is collected, growth of diverse bacteria and other microorganisms, either opportunistic contamination or commensals, may cause degradation of viruses. In mass testing, specimen collection may not always provide hospital-level hygienic conditions. Studying this subject, we observed that several saliva specimens in our collection developed a foul smell after 48 h at room temperature storage, indicating bacterial growth.

[0060] Currently, this problem is addressed by adding antibiotics and antimicrobial compositions to VTM.

[0061] The antimicrobial compositions used in the current invention comprise saliva and VTM supplemented with thymol (CAS 89-83-8), 2-phenylphenol (CAS 9043-7) and Tween 20 (CAS 9005-64-5). After supplementation of saliva or VTM, the final concentration is in the range: for thymol from 50 pg/ml to 1 mg/ml, for 2-phenylphenol from 0.2 pg/ml to 30 mg/ml, and for Tween 20 from 0.2 mg/ml to 50 mg/ml. Thymol and 2-phenylphenol are known antimicrobial compounds. Both have a wide spectrum of antimicrobial action. Thymol has been recently added by the US EPA to the list of effective compounds for use against SARS-CoV-2 (EPA reg. no. 84683 - 3 and 87742-1). Thymol can bind to proteins and inhibit a variety of enzymatic reactions (Meeran 2017). 2-phenylphenol and its sodium and potassium salts are used as active ingredients in broad-spectrum fungicides and surface biocides (SCCS 2015). Antiviral activity of 2-phenylphenol has been was reported (Sattar, 1993). Tween 20, as a surfactant, can target a weak spot in the SARS coronavirus capsids by rupturing the capsid’s lipid bilayer. Tween 20 and Triton X100 were found effective in supporting direct RT-qPCR test for SARS-CoV-2 RNA performed with detergent-inactivated saliva samples (Smyrlaki, 2020).

[0062] The antimicrobial composition of this invention is prepared by dissolving thymol, 2-phenylphenol and Tween 20 in ethylene glycol. The resulting solution, Viral Preservation Composition (VPC) comprises thymol at 25 mg/ml, 2-phenylphenol at 20 pg/ml, and Tween 20 at 50 mg/ml. At these concentrations, VPC is a 50x stock solution, i.e., 20 pi of VPC is added per ml of saliva or VTM, to obtain a final VPC concentration of 0.5 mg/ml of thymol, 0.4 pg/ml of 2 phenylphenol, 10 mg/ml of Tween 20. Solutions with other final concentrations, in the range specified for these three compounds in the Invention, and compositions containing only thymol and 2 phenylphenol can be also prepared and used in the Invention. For use in RT-qPCR in this invention, in combination with Tween 20, maximum non-inhibitory concentration of 2 phenylphenol is 0.5 pg/ml of saliva.

[0063] In this invention, the use of non-inhibitory compounds in AG Compositions and RT-qPCR is critical for the effectiveness of SARS-CoV-2 detection. It is disclosed in the current Invention that, unexpectedly, the selected phenolic compounds, thymol and 2 phenylphenol, do not inhibit RT-PCR. Experiment 9 documents the lack of an inhibitory effect of thymol and 2-phenylphenol on detection of SARS-CoV-2 RNA by direct RT-qPCR performed with AG Processed saliva.

[0064] Example 10 provides evidence of the efficacy of the present invention process and compositions.

[0065] Example 11 describes kit that can be used to perform saliva (sputum) collection and AG Processing of the Invention. The 2-part kit contains a tube with a screw and a sputum collection funnel. In the simplified, but less safe, approach, the collection kit is only a tube with a screw cup. The tube, made of clear plastic, and about 15 mm wide, with volume markings, for example at 2 ml, 3 ml and 5 ml capacity. The collection tube may contain VPC added as solution in ethylene glycol. VPC can also be also added to the collection tube in the powderized form, or as ethanol solution that after ethanol evaporation leaves thin layer of VPC at the bottom of tube.

[0066] The current Invention integrates handling and processing of SARS-CoV-2 specimens to make them ready for RT-PCR detection. Collected saliva or swab suspension samples are preserved for testing by supplementation with the antimicrobial VPS composition of this invention and are able to be stored at room temperature before testing. For persons skilled in the art, it is apparent that other biological samples, including samples of human, animal, plant, yeast, fungi and bacteria can be stored and processed for RNA detection using the compositions of this Invention. For example, for RNA detection by RT-PCR in saliva include monitoring RNA transcripts that serve as chemical signature of particular gene expression such as oral cell carcinomas and testing for HIV positivity (Tiwari, 2011 ).

[0067] The compositions and methods disclosed in this Invention can be used with a variety of RNA detection procedures, and especially in procedures comprising an RT step. In addition to RT-PCR, AG Processing Composition can be used in RT-LAMP. However, in this Invention, the use of AG Processing Composition with saliva for direct RT-LAMP reduced the sensitivity for detection of SARS-CoV-2 RNA. The LLOD was 100 copies of SARS-CoV-2 RNA per reaction. This is in agreement with a previous observation by Park at al. (Park, 2020).

[0068] The compositions and methods disclosed in this Application are not meant to be limiting as many variations are possible as may be made by those skilled in the art. The present Invention will be understood more readily by reference to the examples provided herein. The examples are provided as an illustration and are not intended to be limiting of the Invention.

EXAMPLES

MATERIALS and METHODS USED IN THE EXAMPLES

[0069] The RT-qPCR tests were performed using commercially available one-step RT- qPCR kits. The following kits were used:

[0070] TaqPathTM 1-Step RT-qPCR Master Mix, CG (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. A15300). qScriptTM XLT 1 -Step RT-qPCR ToughMixTM ROXTM (Quantabio, Beverly, MA, USA; cat. no. 95133-500). GoTaq® Probe 1-Step RT- qPCR System (Promega Corporation, Madison Wl, USA; cat. no A6121). KiCqStart® Probe qPCR ReadyMixTM, ROXTM (MilliporeSigma, St. Louis, MO, USA; cat. no. KCQS06). The RT-qPCR assays were set-up as per each manufacturer’s protocols.

[0071] The reference materials used as substitutes for SARS-CoV-2 live virus were deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA- WA1/2020, Heat inactivated, NR-52286; (1.16 x 10⁹ genome equivalents per ml), and Gamma-Irradiated, NR-52287 (1 .7 x 10⁹ genome equivalents per ml).

[0072] In this report irradiated heat inactivated SARS-CoV-2 is referred as hi-Co19, and gamma-irradiated SARS-CoV-2 is referred as ir-Co19.

[0073] Additional reference materials were:

[0074] synthetic SARS-CoV-2 RNA (syn-Co19 RNA), 99.9% genome coverage, Control 1 (MT007544.1), and Control 2 (MN 908947.3) from Twist Bioscience, San Francisco, CA, USA.

[0075] Extracted SARS-CoV-2 RNA (ex-Co19 RNA). SARS-CoV-2 RNA was extracted from heat inactivated SARS-CoV-2 using QIAamp® Viral RNA Mini Kit (Qiagen GmbH, Hilden, Germany). After extraction, SARS-CoV-2 RNA was quantified using a standard curve referenced to synthetic SARS-CoV-2 RNA

[0076] IDT 2019-nCoV N Positive Control plasmid, containing SARS-CoV-2 nucleocapsid gene cDNA at 2 x 10⁵ copies/pL; cat. no. 10006625 from Integrated DNA Technologies, Coralville, IA, USA

[0077] RPP30 primer set: "PrimeTime qPCR Probe Assay”, catalog no. Hs.PT.58.19785851 for the human RPP30 gene (exon 1 -2) assay. Dyes used were 5' 6- FAM/ZEN/3’ IBFQ. The Probe assay and Dyes from Integrated DNA Technologies, Coralville, IA, USA.

[0078] The qPCR primers and detection probes were sourced from Integrated DNA Technologies, Coralville, IA, USA (cat. no. 10006770). They are manufactured using the CDC sequences and QC qualified under a CDC E.U.A. [0079] Human saliva samples were obtained as pooled donor saliva (Lee BioSolutions, Maryland Heights, Ml, USA) and from single donors collected by MRC. Human saliva samples used in experiments had pH ranging from 8.3 to 5.8. The collection of human saliva from single donors was performed according to MRC C19 protocol, Collection of saliva for detection of viral ribonucleic acid (RNA), reviewed and approved by IntegReview IRB, Austin, TX.

[0080] Determination of mucus content in saliva. Aliquots of 1 g of saliva specimens were sedimented at 10,000 g for 10 minutes at room temperature. After sedimentation, the supernatant fraction was removed and wet weights of the mucus pellets were recorded. In the group of ten individual donors, saliva from nine donors had wet weight of mucus content ranging from 3.9% to 4.3% of the saliva weight, which was assigned as a high mucus content. Saliva from of one donor had a wet mucus content of 1 .6%. The pooled donor saliva was sedimented by the supplier and upon the second sedimentation at time of analysis, the polled saliva had the pelleted mucus content of 0.3%. A mucus content ranging from 1 .6 % to 0.3% was assigned as low mucus content.

[0081] The alkaline-glycol processing (AG Processing) of saliva containing SARS- CoV-2 reference materials.

[0082] AG Processing of saliva was performed by mixing of one volume of the aqueous solution containing 65% (v/v) polyethylene glycol 200 (PEG200) (CAS 25322-68-3) and 45 mM KOH with two volumes of human saliva. The resulting AG Processing composition comprised: 21 .7% (v/v) PEG200, 67 % (v/v) saliva and 15 mM KOH, with pH ranging from 12.2 to 12.8, depending on pH of a saliva specimen. The AG Processing composition was spiked with known quantities of a SARS-CoV-2 reference material and mixed for few seconds using a benchtop vortex until the solution appeared uniform. For AG Processing, the virus-containing AG Processing composition was incubated for 5 min to 30 min at room temperature.

[0083] Direct RT-qPCR detection of SARS-CoV-2 RNA.

[0084] An aliquot the virus-containing AG Processed composition was used for RT- qPCR performed with commercially available kits. RT-qPCR reaction mixes were prepared following manufacturers’ protocols using master mix, qPCR primer-probe mix, and Tris-HCI, pH 8 or 8.5, in accord with optimal requirements of kits. An aliquot of 5 μL of the AG Processed composition was added to 15 μL of RT-qPCR reaction mix. RT- qPCR was performed according to the method specified by each respective kit manufacturer protocol for 45 reaction cycles. In effect, each RT-qPCR contained 3.3 μl of processed saliva. Each reaction was performed in triplicate. Results are presented as a mean of Ct of the reaction triplicates. Positive controls consisting of synthetic SARS-CoV- 2 RNA or SARS-CoV2-cDNA plasmid were included on each reaction plate. Negative RT- qPCR controls consisting of AG Processing composition without reference material, and of water without reference material were included on each reaction plate. Results were considered valid if 100% of controls provided positive or negative results, respectively.

[0085] Reaction mixes for RT-PCR in this invention can also be supplemented by PCR enhancers including: betaine, DMSO, bovine serum albumin, and tetraalkylammonium derivatives.

EXAMPLE 1. Detection of SARS-CoV-2 RNA by direct RT-qPCR in AG processed human saliva specimens.

Table E1. Detection of SARS-CoV-2 RNA by direct RT-qPCR in AG processed human saliva specimens.

Composition RNA copies (cps) Ct Ct Sd Dev D Ct AG saliva 10,000 of ir-Co19 22.27 0.06

Water saliva 10,000 of ir-Co19 28.71 0.21 6.4

AG saliva 10 of ir-Co19 32.59 0.14

Water saliva 10 of ir-Co19 39.02 0.20 6.4

AG saliva 10,000 cps hi-Co19 23.88 0.02 Water saliva 10,000 cps hi-Co19 29.05 0.12 5.2 AG saliva 10 cps hi-C19 34.25 0.54

Water saliva 10 cps hi-C19 0 6.0

AG saliva 10,000 cps syn-Co19 RNA 25.79 0.05 Water saliva 10,000 cps syn-Co19 RNA 35.36 0.62 9.6

[0086] AG saliva- AG Processed saliva specimens, water saliva- water processed saliva.

[0087] AG processing and direct RT-qPCR were performed as described in Methods. The AG Processing compositions were spiked with gamma-irradiated SARS-CoV-2 (ir- Co19) or heat-inactivated SARS-CoV-2 (hi-Co19) or synthetic SARS-CoV-2 RNA (syn- Co19 RNA) to contain 10,000 copies or 10 copies of viral genome per 5 pi, as indicated in Table E1. After 15 min processing at room temperature, a 5 pi aliquot of the AG Processing composition was added to 15 mI of the 1 step KiCqStart RT-PCR reaction mix with 1 .0 mI of 1 M tris HCI pH 8, and with primer and probe set for SARS-CoV-2 N1 target detection.

[0088] Results in Table E1 indicate that in saliva specimens spiked with 10,000 viral copies, detection of SARS-CoV-2 RNA in the AG Processed saliva is observed at about Ct 23 (amplification cycle 23); while in water-processed saliva, virus RNA detection is observed at Ct 29 (amplification cycle 29). In reactions with 10 copies of SARS-CoV2,

AG Processing decreases the number of amplification cycles needed to detect viral RNA from undetectable at 40 cycles to a threshold detection of Ct 33. Thus, AG Processing allows for detection of COVID-19 RNA 6 to 7 amplification cycles earlier than in reactions performed without AG processing. An even higher increase in sensitivity of detection of viral

RNA was observed in reactions performed with synthetic COVID RNA. The AG Processing allowed for detection of synthetic viral RNA after the 25^th amplification cycle (Ct 25); whereas, in reactions without AG processing, synthetic viral RNA was detected after the

35^th amplification cycle (Ct 35).

EXAMPLE 2

Table E2. Detection of SARS-CoV-2 in human saliva using AG Processing and various RT-qPCR kits.

Template Processing GoTaq TaqPath qScript source composition Ct Ct Ct hi-Co19 AG saliva 30.82 24,91 25.80 hi-Co19 Water saliva 35.71 32.11 31.82 ex-Co19 RNA water 25.93 25.52 26.35 [0089] AG saliva- AG Processed saliva specimens, water saliva- water processed saliva.

[0090] AG processing and direct RT-qPCR were performed as described in Methods. The AG Processing compositions were spiked with heat-inactivated SARS-CoV-2 (i-Co19) or extracted SARS-CoV-2 RNA to obtain concentration of 3,000 copies of viral genome per pi. After 15 min of AG processing at room temperature, 5 pi aliquot of the processed compositions, was added to 15 mI of the reaction mix containing 1 .5 mI of 1 M tris HCI pH 8, and a 1 step RT-PCR kit with primer and probe set for SARS-CoV-2 N1 target detection. The following RT-PCR kits were used: GoTaq, Promega; TaqPath, Thermo Fisher; qScript, Quantabio.

[0091] Results in Table E2 indicate that AG Processing effectively improves detection of C19 RNA with all three kits. The increase in sensitivity, evident as a decrease in the number of amplification cycles needed to detect viral RNA was at least 70-fold (4.89 Ct decrease using GoTaq) and up to 300-fold (7.4 Ct decrease with TaqPath). Positive control in this Example included 3,000 copies per reaction of the extracted SARS-CoV-2 RNA.

EXAMPLE 3. RT-qPCR detection range of SARS-CoV-2 RNA in AG Processed saliva and in AG Processed swab suspensions in VTM or in Hanks’ medium.

[0092] Figure E3-A. RT-qPCR detection range for SARS-CoV-2 RNA with AG

Processed saliva specimens.

[0093] Figure E3-B. RT-qPCR detection range for SARS-CoV-2 RNA with AG

Processed swab specimens in VTM or Hanks’ storage medium.

[0094] AG processing and direct RT-qPCR were performed as described in

Methods. Swabs (Copan Diagnostic Inc, Murrieta, CA) were dipped in saliva, VTM, or Hank’s Buffered Salt Solution (HBSS). AG Processing compositions were prepared by mixing two volumes of swab containing saliva or UTM or HBSS with one volume of alkaline glycol solution. The AG Processing compositions were spiked with variable amount of gamma-irradiated SARS-CoV-2, from 400K copies to 10 copies per 5 pi, as indicated in Figure E3. A 5 mI aliquots of the AG Processed compositions were added to 15 mI KiCqStart RT-PCR reaction containing primer and probe set for SARS-CoV-2 N1 target detection.

[0095] The results in Figure E3 show detection of SARS-CoV-2 RNA in all three specimens, with the detection range from 400,000 copies to 5 copies of SARS-CoV-2 RNA per reaction. EXAMPLE 4. Detection of low copy number of SARS-CoV-2 in AG Processed saliva. [0096] Table E4. Detection of low copy number of SARS-CoV-2 in AG Processed saliva.

SARS-CoV-2 TaqPath kit per reaction Ct Ct Ct

4 copies 35.00 34.49 35.58 3 copies 35.24 35.02 35.26 2 copies 34.31 37.87 34.99

1 copy 37.84 und 36.18

[0097] AG processing and direct RT-qPCR were performed as described in Methods. AG Processing compositions were prepared as described in Example 1 and spiked with a variable amount of gamma-irradiated SARS-CoV-2, from 4 copies to 1 copy per 5 pi of AG Processing composition, as indicated in Table E4. A 5 mI aliquot of the AG Processing composition was added to 15 mI of the reaction mix containing TaqPath RT-qPCR kit with primer and probe sets for SARS-CoV-2 N1 target detection. Ct numbers represent the mean of triplicate determinations for each copy number.

[0098] The results of triplicate determinations shown in Table E4 indicate a LLOD of 2 copies per reaction. EXAMPLE 5. SARS-CoV-2 RNA detection in the AG Processing composition stored at room temperature for up to 24 h.

[0099] Table E5. SARS-CoV-2 RNA detection in AG Processing composition stored at room temperature for up to 24 h.

[00100] AG processing and direct RT-qPCR were performed as described in Methods. The AG Processing compositions were spiked with: 10,000 copies of gamma-irradiated SARS-CoV-2 (ir-Co19), or 10,000 copies of synthetic SARS-CoV-2 RNA (syn-Co19 RNA), per 5 pi. The spiked compositions were stored for up to 24 h at room temperature. At the times indicated in Table E5, an aliquot of 5 mI was taken for RT-qPCR performed with KiCqStart kit and qScript kit. The positive control was extracted SARS-CoV-2 RNA.

[00101] Table E5 shows unchanged detection of gamma irradiated SARS-CoV-2 and synthetic SARS-CoV-2 RNA in AG Processing compositions stored at room temperature for at least 1 h. Stability of the detection was shown with both. This was shown with RT-qPCR performed with the KiCqStart kit and the qScript RT-PCR kit.

EXAMPLE 6. Effects of heating and Tween 20 on detection of SARS-Cov-2 RNA by

RT-PCR in AG Processed saliva.

[00102] Table E6A. Effects of heating and Tween 20 on detection of SARS-Cov-2 RNA by RT-PCR in AG Processed saliva.

[00103] Determination of mucus content in saliva, AG processing and direct RT-qPCR are described in Methods. Low mucus and high mucus saliva specimens contained 1 .6% of mucus and 4% of mucus, respectively. Saliva specimens were spiked with 10,000 copies of gamma irradiated SARS-CoV-2 per 5mI of saliva and incubated for 30 min either at room temperature, at 65 C or at 95 C. After incubation, AG Processing compositions were prepared by mixing one volume of the alkaline PEG 200 solution with two volumes of saliva specimens as described in Example 1. After 15 min processing at room temperature, a 5 mI aliquot of the AG Processing composition was added to 15 mI of the RT-PCR reaction mix with 1 .0 mI of 1 M tris HCI pH 8, and a 1 step KiCqStart RT-PCR kit with primer and probe set for SARS-CoV-2 N1 target detection. As indicated in Table E6A, some RT-qPCR reactions were supplemented with Tween® 20 (CAS 9005-64-5) to reach 0.2% concentration in the reaction mix.

[00104] Table E6B. Effect of Tween 20 and heating on detection of SARS-CoV-2.

[00105] Results presented in Table E6A were analyzed to evaluate the effects of heating and Tween 20 on detection of SARS-CoV-2 RNA by direct RT-qPCR performed with AG processing of saliva specimens. The effects are presented as D Ct values. D Ct values were calculated by subtracting Ct values of RT-qPCR reactions performed without heating and/or without Tween 20 addition from Ct values of RT-qPCR reactions performed with heating and/or Tween addition.

[00106] Heating effects: In RT-qPCR with low mucus saliva, heating at 65 C has no significant effect on detection of SARS-CoV-2 RNA, while heating at 95 C increases by 2 the number of amplification cycles necessary to detect the viral RNA. In RT-qPCR with high mucus saliva, heating at 65 C and 95 C improves detection of the viral RNA by decreasing the number of amplification cycles (by about 6) necessary to detect the viral RNA.

[00107] Tween 20 effects: In low mucus saliva, Tween 20 improves detection of SARS- CoV-2 RNA in saliva incubated at room temperature and at 65 C (by about 1 Ct), and has no effect on detection of viral RNA in saliva incubated at 95 C. In high mucus saliva, Tween 20 improves SARS-CoV-2 RNA detection by 7 Ct, and this effect diminishes in heated saliva, with 2.3 Ct improvement at 65 C and no significant effect at 95C.

EXAMPLE 7. Detection of endogenous RPP30 gene transcript in the AG Processed saliva using direct RT-qPCR.

[00108] Table E7. Detection of endogenous RPP30 gene transcript in the AG Processed saliva using direct RT-qPCR.

Template

Source Transcript Ct

AG saliva endogenous RPP30 36.20

AG saliva, no RT endogenous RPP30 not detected

Water saliva endogenous RPP30 not detected

[00109] AG Processing was performed as in Example 1 . A 5 pi aliquot of AG Processed composition was added to 15 mI of the RT-qPCR reaction mix containing 1.0 mI of 1 M tris HCI pH 8, and Go Taq kit components with primer and probe sets for the RPP30 RNA detection. An RPP30 probe used in this assay spanned the splicing junction between exon one and exon two of the human RPP30 gene transcript. This allowed specific detection of cDNA derived from the mRNA RPP30 transcript and eliminated the possibility of detecting cDNA fragments derived from genomic DNA present in saliva. As additional control, RT-qPCR amplification with the GoTaq kit was performed in the absence of reverse transcriptase. The results shown in Table E7 confirm that no amplification is seen when the RT enzyme is excluded from the reaction. PCR was performed for 45 amplification cycles. Negative controls, performed with water instead of RNA template, were all negative. All reactions were performed in triplicate. No amplification of RPP30 by direct RT-PCR was observed in non-AG Processed, wherein water substituted AG Processing.

EXAMPLE 8. Detection of endogenous APOB gene transcripts in the AG Processed rat liver homogenate using direct RT-qPCR.

[00110] Table E8. Detection of endogenous APOB gene transcripts in the AG

Processed rat liver homogenate using direct RT-qPCR.

[00111] Rat liver and rat spleen were homogenized in AG Processing compositions. 100 mg of tissue were homogenized in a solution comprising: 1.9 ml water and 1 ml of aqueous solution containing 65% PEG200 and 45 mM KOH. The resulting homogenate contained PEG 200 at concentration of 22% and its pH was 12.87 for liver and pH 12.80 for spleen. The homogenates were centrifuged at 10,000 g for 10 min. at room temperature. The resulting supernatant was collected and used for detection of the APOB transcript by RT-qPCR. Aliquots of the homogenate corresponding (after dilution) to various amounts of the tissue were added to 15 pi of the Go Taq reaction mix supplemented with 1 .0 mI of 1 M tris HCI pH 8, and with primer and probe sets for APOB transcript. The probe spanned the junction of two exons of the APOB gene. This allowed specific detection of cDNA derived from mRNA APOB transcript and eliminated possible detection of cDNA derived from genomic DNA. Negative controls performed with water instead of RNA template were all negative. All reactions were performed in duplicate.

[00112] Results in Table E8 show a very high level of APOB transcript in rat liver, with Ct 19 with 1.1 ng of tissue, and a very low level of APOB transcript in rat spleen, Ct 34 with 5.7 μg of tissue, reflecting the typical expression pattern of this gene.

EXAMPLE 9. Detection of SARS-CoV-2 RNA by RT-qPCR in AG Processed saliva supplemented with Viral Preservation Solution (VPS) containing thymol and 2 phenylphenol.

[00113] Table E9. Detection of SARS-CoV-2 RNA by RT-qPCR in AG Processed saliva supplemented with Viral Preservation Solution (VPS) containing thymol, 2 phenylphenol and Tween 20.

[00114] VPC components: ethylene glycol as a solvent with: thymol-25 mg/ml, 2 phenylphenol-20 μg/ml and Tween 20-10 mg/ml. 20 μl of VPS was added per ml o saliva containing 10,000 copies of gamma irradiated SARS-CoV-2 per 3.3 μI. After VPS addition, saliva contained: thymol at 0.5 mg/ml, 2 phenylphenol at 2μg/ml and Tween 20 at 10 mg/ml. Two volumes of saliva-VPS or saliva alone was mixed with one volume of the alkaline glycol solution and AG Processing and RT-qPCR detection of SARS- CoV-2 RNA was carried out as described in Methods. 5 μI of the processed saliva composition was added to 15 mI of the RT-PCR reaction mix with 1 .0 μI of 1 M tris HCI pH 8, and a 1 step KiCqStart RT-PCR kit with primer and probe set for SARS-CoV-2 N1 target detection.

[00115] EXAMPLE 10.

[00116] .Detection of SARS-Cov-2 RNA in human saliva using AG Processing and RT- qPCR

[00117] Saliva donor: 31 year female, donor GR, with mild symptoms of a slight headache on 10/30 and loss of smell on 11/3

[00118] On 10/4, about 1 ml of saliva was self-collected in a 1 .5 ml tube by the donor, and within about 30 minutes it was delivered to an MRC technician at the MRC laboratory. The technician used PPE and there was no contact with the donor and the technician.

[00119] At MRC, the sample tube was immediately incubated at 95 C for 30 min to inactivate any SARS-CoV-2.

[00120] At MRC, the inactivated saliva specimen was AG processed and tested by for SARS-CoV-2 by RT-qPCR.

[00121] On 11/4/2020, in accord with a standard protocol: 100 mI of saliva was mixed with 50 mI of the alkaline glycol solution for the AG processing, and a 5 mI aliquot of the processed solution was used for RT-qPCR, performed in triplicates, with TaqPath 1 step RT-PCR kit with N1 primer set and 0.2 % Tween 20. The positive control was an 800 bp synthetic SARS-CoV-2 RNA fragment for the N1 target sequence (50,000 copies per reaction). The 11/4/2020 test resulted in detection of SARS-CoV-2 RNA with a Ct of 32.91 (Std Dev 0.06). In the same reaction plate, tests from 3 additional donors were negative. On 11/9/2020, saliva from the same donor was tested under the same conditions as on 11/4/2020 and RT-qPCR detected SARS-Cov-2 RNA at a Ct of 34.43 (Std Dev 0.74). The results are tabulated below:

[00122] Results document detection of SARS-CoV-2 RNA in saliva from donor GR. After 5 days, there was increase in number of amplification cycles to detect the viral RNA of 2.8 Ct. in detection of SARS-CoV-2 RNA. This translates to about a tenfold decrease of the viral count in the GR donor saliva. After 9 days there was no detectable SARS-CoV-2 in saliva of donor GR. This was correlated with donor’s health improvement and no headaches. However, loss of smell and taste was still persistent.

[00123] In accordance with the MRC IRB protocol, immediately after detecting SARS- CoV-2 RNA, MRC informed the GR donor of positive test results. The donor contacted Hamilton County CARES Covid-19 Testing and was tested on 11/4. She received confirmation from the Hamilton County testing site of positive test results for Covid 19 on 11/6. The Hamilton County test was the RT-PCR detection of SARS-CoV-2 RNA performed on a nasopharangeal swab sample.

EXAMPLE 11. A kit for collection and processing of saliva specimens.

[00124] A commercial kit, such as: Saliva Collectors, manufactured by Biocomma LTD, ShenZhen, China.

[00125] Comprised with: collection tube, saliva collecting funnel, and screw cap to cap the collection tube. The collection tube should have a 2 ml and 3 ml markings for collecting 2 ml of saliva and for 1 ml of the alkaline glycol solution of this Invention. The alkaline glycol solution is added just before the RT-qPCR test. The collection tube may contain a of viral preservation composition (VPC). For example, 40 pi of VPSC 50 times concentrate, containing 20 mg of Tween 20, 1 .0 mg of thymol and 0.8 pg of 2- phenylphenol. The solvent for VPC solution is ethylene glycol.

[00126] The size of saliva collector and the amount of collected saliva may vary, depending on a diagnostic requirement.

[00127] While the present invention has been illustrated by the description of one or more embodiments thereof, and while the embodiments have been described in considerable detail, they are not intended to restrict or in any way limit the scope of the appended claims to such detail. The various features shown and described herein may be used alone or in any combination. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative apparatus and methods and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the scope or spirit of Applicants’ general inventive concept.

Claims

WHAT IS CLAIMED IS:

Claim 1 . Alkaline glycol processing compositions for processing of biological specimens for detection of target RNA, comprising: a biological specimen, glycol(s) at concentration ranging from about 10% to >25%, and alkali capable of maintaining pH >11 .5 in said processing compositions.

Claim 2. Processing compositions of claim 1 wherein pH is >12

Claim 3. Compositions of claim 1 wherein glycol(s) comprise polyalkylene glycol or mix of glycols including propylene glycol, polyethylene glycols, polypropylene glycols and polyglycol.

Claim 4. Wherein glycols of claim 3 comprise polyethylene glycol including polyethylene (PEG) glycol 200

Claim 5. Compositions of claim 1 wherein a biological specimen comprises 0.02% to 90% of the total volume of said compositions.

Claim 6. Compositions of claim 1 wherein a biological specimen comprises a biological fluid containing RNA viruses, and it comprises: saliva, blood, serum, or a virus-containing storage medium.

Claim 7. Compositions of claim 1 wherein a biological specimen comprises a solid biological tissue, wherein a solid biological tissue is in a liquified form such as lysate or homogenate.

Claim 8. Compositions of claims 1 , 6 and 7 wherein a biological specimen contains SARS-CoV-2.

Claim 9. Compositions of claims 1 , 6 and 7 wherein a biological specimen, either fluid or in in liquid medium, comprises an antimicrobial composition that inhibit degradation of viruses and sufficiently preserve them for RNA-based detection tests, when the specimen is stored at room temperature before mixing with other compounds of claim 1 .

Claim 10. Compositions of claim 9 wherein antimicrobial compositions comprise: thymol, 2 phenylphenol and Tween 20, or mix of these compounds.

Claim 11 . Compositions of claims 9 and 10 wherein antimicrobial compositions comprise per ml of said compositions: thymol at about 50 μg to about 1 mg, 2 phenylphenol at about 0.1 μg to about 3 mg and Tween 20 at about 0.2 mg to about 10 mg.

Claim 12. Compositions of claim 1 wherein alkali compounds are selected form strong bases, including sodium hydroxide and potassium hydroxide, capable of maintaining pH >11 .5 of the compositions.

Claim 13. Compositions of claim 1 further comprising RT-PCR enhancers comprising detergent(s), antioxidant(s), and salt(s).

Claim 14. Compositions of claim 13 wherein detergent additive comprise non anionic detergent(s) selected from a group comprising: Tween 20, Triton X100, NP- 40 and Brij 35.

Claim 15. A process for detection of target RNA in biological samples, including detection of SARS-CoV-2 RNA, using processing compositions of claim 1 wherein: a) a biological specimen of claim 1 is processed by mixing with other components of claim 1 , to form Alkaline Glycol Processing Composition comprising polyalkylene glycol(s) and sufficient amount of alkali to maintain pH > 11 .5 in the Processing Composition. b) a biological specimen of claim 1 is processed by mixing with other components of claim 1 to from Alkaline Glycol Processing Composition comprising polyalkylene glycol(s) and sufficient amount of alkali to maintain pH > 12.2 in the Processing Composition. c) a biological specimen of claim 1 is stored at room temperature in compositions of claims 9, 10 and 11 , before it is mixed with compounds of claiml to form Alkaline Glycol Processing Composition. d) Alkaline Glycol Processing Compositions described in a), b) and c.) process biological specimens at room temperature to make viral RNA present in the specimens available for the reverse transcription, and sufficiently inactivate inhibitors of reverse transcription and inhibitors of subsequent enzymatic reactions to be used in the viral RNA direct detection tests. e) Following processing, aliquots of the Alkaline Glycol Processing Compositions of claim 15d are taken for a viral RNA detection test, including RT-qPCR tests.

16. A kit for detection of RNA target sequence, including viral RNA, comprising: a) a specimen collection and storage tube without or with the antimicrobial compositions of claims 9 to 11. b) a specimen collection tube of claim 16a with sufficient volume to mix collected specimen, comprising saliva or swab containing viral transfer medium, with the alkaline glycol solution to form Alkaline Glycol Processing Composition of claim 1 and, c) a direct RT-qPCR kit optimized for detection of an RNA target sequence(s) in the presence of Alkaline Glycol Processing Composition.