Method of detecting sars-cov-2

Abstract

Provided is method of detecting SARS-CoV-2 in a sample, including amplifying polynucleotides in the sample to form subgenomic amplicons using a forward subgenomic primer and a reverse subgenomic primer, wherein the forward subgenomic primer hybridizes to a forward subgenomic primer target of a subgenomic SARS-CoV-2 N protein transcript or its complement and the reverse subgenomic primer hybridizes to a reverse subgenomic primer target of the subgenomic SARS-CoV-2 transcript or its complement, and detecting an amount of hybridization of a subgenomic SARS-CoV-2 transcript probe to the subgenomic amplicons, wherein the subgenomic SARS-CoV-2 transcript probe hybridizes to a probe target of the subgenomic amplicons, wherein the probe target of the subgenomic amplicons includes at least a portion of a leader sequence, a transcriptional regulatory sequence, and at least a portion of a junction region between the transcriptional regulatory sequence and a coding sequence of the subgenomic SARS-CoV-2 transcript and the probe target is between the forward subgenomic primer target and the reverse subgenomic primer target

Description

METHOD OF DETECTING SARS-CoV-2CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority from U.S. Provisional Patent Application No. 63 / 509,550, filed June 22, 2023, the entire content of which is incorporated herein by reference.GOVERNMENT RIGHTS STATEMENT

[0002] This invention was made with Government support under grant number U50 CK000516 awarded by the Centers for Disease Control and Prevention and grant number U50 CK000486 awarded by the Centers for Disease Control and Prevention. The Government has certain rights in the invention.

[0003] The instant application contains an electronic sequence listing. The contents of the electronic sequence listing 0332093 AWO_Sequence_Listing.xml; Size: 8,541 bytes; and Date of Creation: June 20, 2024, is herein incorporated by reference in its entirety.BACKGROUND

[0004] Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has caused a global pandemic, affected global health and damaged the world economy. The laboratory community responded rapidly, developing sensitive molecular tests that detect the viral genomic RNA with high sensitivity and specificity. However, it was discovered relatively early that viral genomic RNA remains detectable many weeks after complete clinical recovery (1,2), which caused confusion for policy making regarding duration of isolation of positive patients and quarantine of asymptomatic patients. The majority of viral transmission occurs immediately before and after the onset of clinical symptoms (3). However, the precise duration of contagiousness after the onset of clinical symptoms can be unclear, variable, and difficult to ascertain. Although most of the available molecular tests offer a sensitive means of diagnosing COVID-19, they are unable to differentiate between the presence of remnant noninfectious, viral RNA and infectious, replication-competent virus (4,5). While culture isolation of virus may be an indication of contagiousness, routine viral culture for SARS-CoV-2 is impractical and notstandardized, with an ongoing requirement for BSL-3 containment, the labor intensive nature of the procedures, and the fact that the vast majority of diagnostic laboratories do not perform viral culture. As a means of diagnosis, it is impractical due to the time, expertise and resources needed. SARS-CoV-2 however, has been recovered in viral culture from immunocompromised patients several months after their primary infection (6), indicating that some patients are able to actively replicate and transmit the virus beyond the period of their acute illness. Several studies (7-11) have suggested some correlation between cycle threshold (CT) values and positive viral culture. However, using CT values in clinical settings as an indicator of infectivity is still generally advised against by most professional societies, as supported by multiple studies that demonstrate significant variations in the correlations between viral load and CT across different platforms and chemistries, and even across runs within the same method. There is very little correlation between CT values and infectivity. The only acceptable alternative is a validated quantitative molecular assay (12). The present disclosure is directed to overcoming these and other deficiencies in the art.SUMMARY

[0005] In an aspect, provided is method of detecting SARS-CoV-2 in a sample, including amplifying polynucleotides in the sample to form subgenomic amplicons using a forward subgenomic primer and a reverse subgenomic primer, wherein the forward subgenomic primer hybridizes to a forward subgenomic primer target of a subgenomic SARS-CoV-2 N protein transcript or its complement and the reverse subgenomic primer hybridizes to a reverse subgenomic primer target of the subgenomic SARS-CoV-2 transcript or its complement, and detecting an amount of hybridization of a subgenomic SARS-CoV-2 transcript probe to the subgenomic amplicons, wherein the subgenomic SARS-CoV-2 transcript probe hybridizes to a probe target of the subgenomic amplicons, wherein the probe target of the subgenomic amplicons may include at least a portion of a leader sequence, a transcriptional regulatory sequence, and at least a portion of a junction region between the transcriptional regulatory sequence and a coding sequence of the subgenomic SARS-CoV-2 transcript and the probe target is between the forward subgenomic primer target and the reverse subgenomic primer target.

[0006] The forward subgenomic primer target may be in a leader sequence of the subgenomic SARS-CoV-2 transcript and the reverse subgenomic primer target may be in acoding sequence of the subgenomic SARS-CoV-2 transcript. The forward subgenomic primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides. The reverse subgenomic primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides. The forward subgenomic primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides and the reverse subgenomic primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides.

[0007] A sequence of the forward subgenomic primer may include a polynucleotide sequence as set forth in SEQ ID NO: 4. The sequence of the forward subgenomic primer may be the polynucleotide sequence as set forth in SEQ ID NO: 4. A sequence of the reverse subgenomic primer may include a polynucleotide sequence as set forth in SEQ ID NO: 5. The sequence of the reverse subgenomic primer may be the polynucleotide sequence as set forth in SEQ ID NO: 5.

[0008] A sequence of the forward subgenomic primer may include a polynucleotide sequence as set forth in SEQ ID NO: 4 and a sequence of the reverse subgenomic primer may include a polynucleotide sequence as set forth in SEQ ID NO: 5. In still another example, the sequence of the forward subgenomic primer may be the polynucleotide sequence as set forth in SEQ ID NO: 4 and the reverse subgenomic primer may be the polynucleotide sequence as set forth in SEQ ID NO: 5. The subgenomic SARS-CoV-2 transcript probe may include a length having a range of from between about 8 nucleotides to about 50 nucleotides.

[0009] A sequence of the subgenomic SARS-CoV-2 probe may include a polynucleotide sequence as set forth in SEQ ID NO: 6. The sequence of the subgenomic SARS-CoV-2 probe may include a polynucleotide sequence as set forth in SEQ ID NO: 6, wherein nucleotide 3, 11, 15, 16, 20 and 21 comprise locked nucleic acids. The subgenomic SARS-CoV-2 probe may include a subgenomic SARS-CoV-2 probe dye. The subgenomic SARS-CoV-2 probe may include a subgenomic SARS-CoV-2 probe quencher. The subgenomic SARS-CoV-2 probe may include a subgenomic SARS-CoV-2 probe dye and a subgenomic SARS-CoV-2 probe quencher.

[0010] The amplifying further may include forming genomic amplicons using a forward genomic primer and a reverse genomic primer, wherein the forward genomic primer hybridizes to a forward genomic primer target of SARS-CoV-2 genome or its complement and the reverse genomic primer hybridizes to a reverse genomic primer target of the SARS-CoV-2 genome or its complement, and further may include detecting an amount of hybridization of a genomic SARS-CoV-2 probe to the genomic amplicons, and determining a difference between the amount of hybridization of the probe for the subgenomic SARS-CoV-2 transcript to the amount of hybridization of the probe to the SARS-CoV-2 genome.

[0011] The SARS-CoV-2 genome target may include at least a portion of one or both of an ORF la genome region and an ORF lb genome region. At least a portion of one or both of an ORFla genome region and an ORF lb genome region may include one or both of the forward genomic primer target of the SARS-CoV-2 genome and the reverse genomic primer target of the SARS-CoV-2 genome. In yet another example, the forward genomic primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides. The reverse genomic primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides. The forward genomic primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides and the reverse genomic primer may include a length having a range of from 10 nucleotides to 30 nucleotides.

[0012] A sequence of the forward genomic primer may include a polynucleotide sequence as set forth in SEQ ID NO: 1. The sequence of the forward genomic primer may be the polynucleotide sequence as set forth in SEQ ID NO: 1. A sequence of the reverse genomic primer may include a polynucleotide sequence as set forth in SEQ ID NO: 2. The sequence of the reverse genomic primer may be the polynucleotide sequence as set forth in SEQ ID NO: 2. A sequence of the forward genomic primer may include a polynucleotide sequence as set forth in SEQ ID NO: 1 and a sequence of the reverse genomic primer may include a polynucleotide sequence as set forth in SEQ ID NO: 2. The sequence of the forward genomic primer may be the polynucleotide sequence as set forth in SEQ ID NO: 1 and the reverse genomic primer may be the polynucleotide sequence as set forth in SEQ ID NO: 2.

[0013] The genomic SARS-CoV-2 probe may include a length having a range of from between about 8 nucleotides to about 50 nucleotides. A sequence of the genomic SARS-CoV-2 probe may include a polynucleotide sequence as set forth in SEQ ID NO: 3. The genomic SARS- CoV-2 probe may include a genomic SARS-CoV-2 probe dye. The genomic SARS-CoV-2 probe may include a genomic SARS-CoV-2 probe quencher. The genomic SARS-CoV-2 probe may include a genomic SARS-CoV-2 probe dye and a genomic SARS-CoV-2 probe quencher.

[0014] The amplifying further may include forming control amplicons using a forward control primer and a reverse control primer, wherein the forward control primer hybridizes to aforward control primer target of a control transcript or its complement and the reverse control primer hybridizes to a reverse genomic primer target of the control transcript or its complement, and further may include detecting an amount of hybridization of a control transcript probe to the control amplicons. The control transcript may be a housekeeping gene transcript. The control transcript may be human RNase P transcript.

[0015] The forward control primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides. The reverse control primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides. The forward control primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides and the reverse control primer may include a length having a range of from about 10 nucleotides to about 30 nucleotides. A sequence of the forward control primer may include a polynucleotide sequence as set forth in SEQ ID NO: 7. The sequence of the forward control primer may be the polynucleotide sequence as set forth in SEQ ID NO: 7. A sequence of the reverse control primer may include a polynucleotide sequence as set forth in SEQ ID NO: 8. The sequence of the reverse control primer may be the polynucleotide sequence as set forth in SEQ ID NO: 8.

[0016] A sequence of the forward control primer may include a polynucleotide sequence as set forth in SEQ ID NO: 7 and a sequence of the reverse control primer may include a polynucleotide sequence as set forth in SEQ ID NO: 8. The sequence of the forward control primer may be the polynucleotide sequence as set forth in SEQ ID NO: 7 and the reverse control primer may be the polynucleotide sequence as set forth in SEQ ID NO: 8. The control probe may include a length having a range of from between about 8 nucleotides to about 50 nucleotides. A sequence of the control probe may include a polynucleotide sequence as set forth in SEQ ID NO: 9. The control probe may include a control probe dye. The control probe may include a control probe quencher. The control probe may include a control probe dye and a control probe quencher.

[0017] One or more nucleotide of one or more of the subgenomic SARS-CoV-2 probe, the genomic SARS-CoV-2 probe, and the control probe, may include a modified nucleotide. One or more nucleotide of the subgenomic SARS-CoV-2 probe may include a modified nucleotide. One or more modified nucleotide may include a locked nucleic acid.

[0018] Replicating SARS-CoV-2 virus in a subject may include performing the method wherein the sample may include a sample from the subject, the identifying may include detectingan amount of hybridization of the subgenomic SARS-CoV-2 transcript probe to the subgenomic amplicons relative to the amount of hybridization of the genomic SARS-CoV-2 transcript probe to the genomic amplicons, and the relative level may be at or above a predetermined threshold.

[0019] The detecting may include performing quantitative PCR and the threshold may be calculated according to the following Formula I: threshold = 2'ACt, wherein Ct may be cycle threshold, and ACt = Ct (subgenomic SARS-CoV-2 transcript) - Ct (genomic SARS-CoV-2 transcript). The detecting may include performing quantitative PCR and the threshold may be calculated according to the following Formula II: threshold = 2-AACt, wherein Ct may be cycle threshold, and AACt = (Ct (subgenomic SARS-CoV-2 transcript) - Ct (genomic SARS-CoV-2 transcript)) - Ct (control transcript). Threshold may be about 0.5 million, or about 0.75 million, or about 1 million, or 1.25 million or about 1.5 million, or about 1.75 million, or about 2 million.

[0020] The subject may be a human or a non-human primate. The subject may be a nonhuman animal. The subject may be a bat, a cervid, a caniformid, a feliformid, a manid, a mustelid, a horse, a cow, a goat, a sheep, a pig, or a rodent.

[0021] The sample may be selected from a nasal swab, a nasopharyngeal swab, an oropharyngeal swab, a throat swab, a nasal secretion, saliva, mucous, throat secretion, a lower respiratory specimen, whole blood, blood component, tissues, plasma, serum, stool, and urine.

[0022] The subject may be treated when the level may be at or above the threshold, including administering a treatment for SARS-CoV-2 to the subject. The treatment may include an anti-SARS-CoV-2 antibody, an antiviral composition, SARS-CoV-2 convalescent plasma, or any combination of two or more of the foregoing. The treatment may include an anti-SARS- CoV-2 antibody. The treatment may include an antiviral composition.

[0023] The subject may be selected for a transmission prevention. The transmission prevention may be selected from mask-wearing, face shield- wearing, social distancing, quarantining, social isolation, environmental decontamination procedures, or any two or more of any of the foregoing.

[0024] A kit may include one or more of any of the foregoing forward subgenomic primer, the foregoing reverse subgenomic primer, and the foregoing subgenomic SARS-CoV-2 transcript probe. A kit further may include any one or more of the foregoing forward genomic primer, the foregoing reverse genomic primer, and the foregoing genomic SARS-CoV-2 transcript primer. A kit further may include one or more of the foregoing forward control primer,the foregoing reverse control primer, and the foregoing control transcript primer. A kit further may include any one or more of a subgenomic transcript positive control, a subgenomic transcript negative control, a genomic transcript positive control, a genomic transcript negative control, a control transcript positive control, and a control transcript negative control. A kit further may include one or both of a replicating SARS-CoV-2 virus negative control and a replicating SARS-CoV-2 virus positive control. A kit further may include one or more of a subgenomic probe control, a genomic probe control, and a control probe control. A kit further may include one or more additional reagents for use in quantitative polymerase chain reaction.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

[0026] FIG. 1 shows a graphical representation of subgenomic (sg) N gene assay design. The sgN assay incorporates a forward primer, designed to target a region in the leader sequence; a reverse primer in the N gene ORF; and a probe designed to bridge the region covering the 3’ end of the leader, the TRS and N gene specific sequence between the TRS and initiation codon. TRS=transcription regulatory sequence, LBJ=leader-body-junction, ORF = open reading frame.

[0027] FIG. 2 shows a graphical representation of subgenomic amplicon design. RT-PCR was performed on total nucleic acid extracted from SARS-CoV-2 isolate USA-WA1 / 2020 , cultured in VeroE6 cells. Single-stranded cDNA was first created using random hexamers, followed by PCR amplification using a universal leader-specific forward primer (Uni-forward, dark green) (11) and a downstream subgenomic RNA-specific reverse primer (bright green). PCR amplicons were visualized by gel electrophoresis, purified, quantified, and diluted to approximately 106 DNA copies / uL for use as template in the SMART assay. The first 500 bases of each subgenomic RNA transcript are represented, including the leader sequence, TRS, and coding region of each gene (colored rectangles).

[0028] FIGs. 3A and 3B show verification of subgenomic N RNA detection from multiple SARS-CoV-2 lineages. Nucleic acid from SARS-CoV-2 isolates representing multiple pangolin lineages was amplified using the sgN forward and reverse primers, modified with Ml 3 tags. Electrophoresis using 1% agarose gels displayed single bands, of similar size to thesubgenomic N RNA control, observed on all samples. Amplicons were sequenced and analyzed using Geneious Prime 2020.2.5. 3A. SARS-CoV-2 isolates Alpha (B.l.1.7), Epsilon (B.1.429) and Iota (B.1.526) 3B. Additional experiments were repeated using nucleic acid from Delta (B.1.617.2) and Omicron (BA.l) positive respiratory specimens and their corresponding isolates. NTC=No Template Control.

[0029] FIGs. 4A and 4B show genomic and subgenomic RNA production in VeroE6 / TMPRSS2 cells. VeroE6 / TMPRSS2 cells were infected with SARS-CoV-2 at an MOI = 0.01 in 6-well plates. Cells and supernatants were harvested from quadruplicate wells at 1, 3, 6, 9, 12, 24, 48, 72, and 96 hours post-infection, extracted, and tested using the SMART assay. ORF lab and sgN gene RNA copy numbers were quantified against target-specific standard curves. Mean log 10 RNA copy numbers + / - SD are shown for each timepoint. 4A. Intracellular 4B. Extracellular.

[0030] FIG. 5 shows ORF lab and sgN gene assay efficiencies. Quantified in-vitro transcribed RNA transcripts containing target assay amplicon sequences were 10-fold serially diluted from 1E+06 gc / uL to 10 gc / uL and run in duplicate with the SMART assay. Mean CT values were plotted and linear regression analysis was performed on each.

[0031] FIG. 6 shows d distribution of SMART assay CT values from culture-positive and culture-negative SARS-CoV-2-positive clinical respiratory specimens. Residual specimens from 355 SARS-CoV-2 positive patients were inoculated onto VeroE6 or VeroE6 / TMPRSS2 cells for virus isolation. ORF lab, sgN, and human RNase P CT values were also determined from residual specimens at inoculation. Center line in each plot, median; bottom and top lines, first and third quartiles. Red: ORF lab, blue: sgN, green: human RNase P. Samples with ORF lab CT values less than or equal to 23 were automatically considered “SMART positive”. Samples with ORF lab CT values greater than or equal to 35 were automatically considered “SMART negative”. Samples with ORF lab CT values between 23 and 35 were analyzed by determining the SMART score: 24(sgN CT-°RFlab CT)’RP CT].

[0032] FIG. 7 shows amplification of SARS-CoV-2 from respiratory samples with varying viral loads, represented by three ORFlab Ct value ranges. 302 of the 355 respiratory samples inoculated for culture had positive ORFlab values after re-testing the original specimen with the SMART assay. 99% (71 / 72) of samples with an ORFlab CT less than 23 (high viral load) were culture positive, whereas only 17% (4 / 23) of those with an ORFlab CT value greaterthan 35 (low viral load) were culture positive. 207 (58%) of the samples displayed ORFlab Ct values between 23 and 35, with 117 (57%) of those culture positive. ORFlab values in this Ct range were chosen for SMART score calculation.

[0033] FIG. 8 shows distributions of SMART scores from 10 individual sample training sets, each consisting of 30 culture positive and 30 culture negative samples, taken from the 355 sample pool. The SMART scores were applied to a box and whisker plot. Lower 25th percentile scores from the 10 culture negative training sets were averaged to determine the SMART score cutoff for the entire data set. A SMART score greater than 1,276,740 was considered SMART positive, if the ORFlab CT was between 23-35.

[0034] FIG. 9 shows SMART scores calculated from respiratory specimens with ORFlab CT values between 23 and 35. Box and whiskers plot of 207 samples analyzed by culture and SMART assay (N=207), with ORFlab CT values 23-35. Solid line represents median. Upper and lower bars represent minimum and maximum values. Blue; culture positive (N=l 17) , Red; culture negative (N=90) ****P-value=<0.0001; Mann Whitney test.

[0035] FIG. 10 shows an ROC curve of SMART score analyses on samples with ORFlab CT values between 23 and 35. ROC curve (black) shows the ability to detect actively replicating SARS-CoV-2 virus in a respiratory sample, using the SMART score calculation on samples with ORFlab Ct values between 23-35 (N=207). The ability to detect actively replicating virus was determined by viral culture in VeroE6 and VeroE6 / TMPRSS2 cells. Area under the curve (AUC): 0.71. P-value: <0.0001. The red dotted line represents a non -discriminatory test using the SMART score.

[0036] FIG. 11 shows a summary of SARS-CoV-2 lineages s analyzed by SMART and viral culture. Lineage information was available on 93 of the 355 samples used in the analysis. Whole genome sequencing was performed on clinical samples using the Illumina MiSeq ARTIC protocol, as previously described (24), to determine PANGO lineage.

[0037] FIGs. 12A-12B show examples of detection of amplification of ORFlab and sgN RNA transcripts in a digital PCR method using amplification and probe primers having the same sequences as disclosed herein for detection in a qPCR method (SEQ ID NOs: 1-3 and SEQ ID Nos: 4-6, respectively).DETAILED DESCRIPTION

[0038] Understanding the status of active viral reproduction is essential to successfully quarantine and treat patients infected with SARS-CoV-2. RT-PCR studies have demonstrated that viral genomic RNA can be detected for weeks after the onset of symptoms which in most patients, likely does not represent active infection (7,2). However, in some patients, active viral reproduction has been documented for weeks (6). As disclosed herein, in contrast to controversy in the relevant field relating to subgenomic viral RNA’s potential use for detecting active SAR.S- CoV-2 infection (19), detection of subgenomic RNA transcripts expressed by SARS-CoV-2 represents productive infection and a tractable marker for monitoring infectivity (11). Disclosed herein is a subgenomic RNA based RT-PCR test that utilizes a unique assay design and a complex algorithm yielding a prediction of active SARS-CoV-2 replication in a patient. The assay is referred to herein as subgenomic mRNA active replication test (referred to herein as “SMART”). As further described herein, in an example, nasopharyngeal samples from 355 patients w7ere subjected to culture conditions and SMART assay and results showed a strong correlation between the two with a positive predictive value of 74 and negative predictive value of 92. SMART may be used to identify patients warranting and not warranting selection for application of a method for transmission prevention.

[0039] As observed with other coronaviruses, during replication, SARS-CoV-2 produces subgenomic-length transcripts, each containing a common leader sequence. Subgenomic RNAs (sgRNA) are transcribed following host cell infection and are not believed to be commonly packaged into new virions. Thus, detection of sgRNA may signify presence of actively infectious or infecting virus in a sample or actively infected cells, whereas detection of genomic transcripts may fail to distinguish active infection, during which infection may be transmissible, from historic infection or a later stage of infection during which genomic viral material may be present but a person may no longer be infectious to others or pose a relatively low or lower risk of being infectious.

[0040] Thus, as disclosed herein, an assay for detecting sgRNA may detect active infection as distinguished from historical presence of virus in a non-infectious subject. sgRNA may serve as a surrogate marker for replication-competent virus. Provided is a subgenomic RNA based quantitative real-time polymerase chain reaction (RT-PCR or qPCR) test that may differentiate between culture-positive and culture-negative samples with improved sensitivity and specificity compared to conventional assays. The test may use purified RNA fromnasopharyngeal swabs as sample, and detect genomic and subgenomic viral RNA while using a housekeeping RNA transcript as control. This test may distinguish patients who are secreting replication-competent virus from those secreting non-infectious RNA with relatively high sensitivity and specificity, and therefore help identify those who may benefit most from one or more treatment, or for whom implementation of a transmission prevention regimen may effectively reduce the infection of others.

[0041] PCR may be used to amplify copies of sequences of viral nucleic acids present in a sample. A subgenomic transcript may be amplified by performing PCR with a forward and reverse primer bookending an amplifiable subgenomic or genomic transcript of interest, respectively. FIG. 1 depicts a nongenomic sequence of viral N gene. The sequence includes a leader sequence, a transcription regulatory sequence (TRS), a leader-body -junction (LBJ), and an open reading frame for the N gene (N gene ORF). Performing PCR using a forward primer that hybridizes to a portion of the leader sequence (forward subgenomic primer target) and a reverse primer that hybridizes to a portion of the ORF (reverse subgenomic primer target) generates amplicons from a nongenomic transcripts that span the leader, TRS, LBJ, and ORF. Importantly, such primers will not lead to amplification of a genomic transcript during PCR. Thus, increased presence of amplicons generated by such primers during successive cycles of PCR indicates that a subgenomic viral transcript was present in the sample, indicating active infection.

[0042] A target as referred to herein includes to a sequence of polynucleotides present or potentially present in a sample, whose detection may be sought. For example, a target may be a portion of genomic SARS-CoV-2 RNA or a portion of genomic SARS-CoV-2 RNA. In another example, a target may be a portion of RNA transcripts detected as a control for assay sensitivity, such as a RNA transcript of a subject’s genome. For detection, a target may be amplified by PCR to produce amplicons, and amplicons detected with a probe. A target may have a portion to which a forward PCR probe hybridizes (forward primer target) and a portion to which a reverse PCR probe hybridizes (reverse primer target). Thermal cycling of sample in the presence of a forward primer that specifically hybridizes to a forward primer target and a reverse primer that specifically hybridizes to a reverse primer target, in the presence of polymerase, free nucleotides, and other reagents as may be appropriate to a given assay, results in generation of amplicons having a nucleotide sequence corresponding to that of the target as spanned by and including the primer targets.

[0043] Between the primer targets on the target sequence is a probe target. A probe target is a sequence to which a probe oligonucleotide specifically hybridizes. Assays such as qPCR operate by detecting a quantity of probes that hybridize to amplicons of targets during repeated cycles of amplification. As the number of cycles of amplification increases during a thermocycling process, more amplicons corresponding to target sequences may be produced (e.g., if target is present in the sample), leading to increased amounts of probe target being present (probe target being present between forward and reverse primer targets in a target and, therefore, included within amplicons thereof). Hybridizing of a probe to a probe target may be detected and quantified during the course of amplification cycles, such as after rounds of amplification. An increase in a detectable signal indicating probe hybridization to probe target indicates an increase in the amount of target amplicons present in the assay.

[0044] Probe hybridization may be detected by optical detection of fluorescence emission from a dye that specifically hybridizes to an amplicon of a target. For example, typically, in qPCR, a probe may include a dye such as a fluorescent label attached to an end. Stimulation of a dye with electromagnetic radiation of a given excitation wavelength range may induce the probe to emit electromagnetic radiation in a different, emission wavelength range. Thus, detection of electromagnetic radiation within a dye’s emission wavelength range following stimulation with the electromagnetic radiation having the relevant excitation wavelength range indicates presence of the dye, i.e. of the probe to which the dye is attached. One or more filter may be used to narrow the range of detectable emission radiation for a given dye.

[0045] A probe may also include, attached to an opposite end to which a dye is attached, a quencher. A quencher may absorb electromagnetic radiation, such as that emitted by the dye when in proximity thereto. Thus, where a probe includes a dye and a quencher, electromagnetic radiation within a emission wavelength range might not be detected following stimulation with electromagnetic radiation within the excitation wavelength range even through the probe may be present. In particular, proximity of a dye to a quencher may be such that the quencher absorbs all or most of the radiation emitted by a dye. When a probe is not hybridized to a target, conformation of the unhybridized probe may be such that, on average, a quencher and dye may be in sufficient proximity to dampen or block emission of a detectable quantity of radiation within an emission wavelength range. Thus, exposing probes including a dye and a quencher, in the absence of amplicons including the probe target sequence, to electromagnetic radiationwithin an excitation wavelength range for the dye, may lead to no or low levels of detectable emission of electromagnetic radiation within an emission wavelength range of the dye. Such a level of detectable emission may be quantified following optical detection thereof and correspond to background emission, i.e., a level of emission detectable with no or negligible hybridization of probe to probe target.

[0046] When a probe hybridizes to a probe target, however, the hybridization of nucleotides of the probe along a stretch of probe target may result in distancing the end of the probe connected to the dye from the end of the probe connected to the quencher, and thus distancing of the dye from the quencher, more so than may occur on average for an unhybridized probe. When such a probe including a dye and a quencher is hybridized to a probe target, absorbance of the dye’s emitted radiation by the quencher may be decreased or eliminated, such that exposure of such a probe to a probe target to electromagnetic radiation within an excitation wavelength range for the dye may result in optical detection of electromagnetic radiation within an emission wavelength range of a magnitude detectable when such a probe is not hybridized to probe target. The amount of emission radiation detected in such a process may be detected optically and quantified, and positively correlate to an amount of probe hybridizing to probe target.

[0047] qPCR permits detection of target in a sample by performing PCR using a forward and reverse primer in the presence of probe including a dye and quencher. In successive rounds of amplification, the reaction solution may be exposed to electromagnetic radiation within an excitation wavelength range and emission of electromagnetic radiation within an emission wavelength range detected. In the absence of target, radiation detected within the emission range may be low, such as a background level. Background emission signifies absence of hybridization of target to a target probe sequence, or such low levels of such hybridization that radiation emitted by dye of hybridized probes is at a level too low for detection. Where target is present in the sample, successive rounds of PCR may result in an exponential increase in the number of amplicons present in the sample. In that case, after a certain number of cycles of PCR, a sufficiently high number of probes would be able to hybridize to a sufficiently high number of amplicons to result in a level of electromagnetic radiation within an emission wavelength range to be detectable above background. Detection of a level of electromagnetic radiation within an emission wavelength range thereby indicates presence of amplicons in the sample, andcorresponds to detection of the target in the sample.

[0048] In general, an amount of target present in a sample may be inversely proportional to the number of PCR cycles required to generate enough amplicons to be detectable and signify presence of the target n the sample. That is, relatively lower levels of target in a sample may require more cycles of PCR to be performed before enough amplicons including probe target are generated to permit detection thereof, and relatively higher levels of target in a sample may require fewer cycles of PCR to be performed before enough amplicons including probe target are generated to permit detection thereof. In qPCR, this aspect is reflected in the term cycle threshold (CT). Cycle threshold is the number of PCR cycles that needed to be performed on a sample before electromagnetic radiation within an emission wavelength range above background is detected. A relatively lower cycle threshold for a sample indicates presence of a relatively higher concentration of target in the sample, and vice versa.

[0049] In some examples, a multiplex qPCR process may be used. In multiplex qPCR, a sample may be probed for detection of more than one target at the same time. For example, a sample may be run through successive amplification cycles of PCR in the presence of a forward and reverse primer pair for two different targets, each having a different forward primer target, reverse primer target, and probe target. A probe specific for each target may also be included in the reaction. The two probes may be distinguishable from each other in terms of detection parameters employed. For example, a probe specific for a first probe target may have an electromagnetic radiation excitation wavelength range that differs from that of a probe specific for a second probe target. In that case, following rounds of PCR, a solution may be exposed to electromagnetic radiation within the first excitation wavelength range and emission of electromagnetic radiation within the emission wavelength range of the first probe detection, followed by detection of emission of electromagnetic radiation within the emission wavelength range of the second probe during exposure to electromagnetic radiation within the second excitation wavelength range. In another example, the two dyes may emit at different wavelengths, such that detection of electromagnetic radiation of one wavelength emitted by a first dye may be detected and distinguished from electromagnetic radiation of a second wavelength emitted by a second dye. Wavelengths of excitation radiation to which samples are exposed, and wavelengths of emission radiation detectable during exposure, may also be filtered or limited, such as where excitation wavelength ranges, emission wavelength ranges, or both, fortwo or more dyes used during multiplex qPCR may be distinguished from each other.

[0050] Numerous dyes are known to e useful in qPCR, including in multiplex qPCR, for detecting presence of target amplicons. Non-limiting examples include FAM, HEX, Cy3, TEX 615, LC Red 640, Tye 665, Cy5, TAMRA, 4,5-dichloro-dimethoxy-fluorescein (JOE®) and others. Numerous quenchers are also available, such as BLACK HOLE QUENCHER (BHQ) 1, 2, or 3, TAMRA, or others.

[0051] Detection of the presence of infection SARS-CoV-2 in an individual, as distinguished from presence of SARS-CoV-2 genetic material or transcripts in an individual attributable to historical SARS-CoV-2 infection or otherwise non-infections levels of such material present in a subject, may be accomplished in accordance with aspects of the present disclosure. Genomic SARS-CoV-2 genetic material or transcripts may be present in an induvial in whom SARS-CoV-2 is actively replicating in cells and generating levels of sheddable viral particles posing an infectivity risk to those the individual comes in contact with. However, such material or transcripts in an individual may also be present in an individual who poses only a low or no infectivity risk, such as an individual who is recovering from SARS-CoV-2 infection but is not infectious, or is responding immunologically to SARS-CoV-2 infection but is not infectious, or previously was infected but is no longer infected or infectious. Thus, merely detecting presence of genomic SARS-CoV-2 material or transcripts in an individual may not indicate the individual is infectious or poses a risk for being infectious.

[0052] By comparison subgenomic transcripts of SARC-CoV-2 may specifically be generated during “active” infection, such as when an individual’s cells are infected with SARS- CoV-2 and generating relatively high levels of viral particles such that they would be likely to be shed by the individual in a sufficiently high level to pose an elevated or high infectivity risk for those in whom the individual were to come into contact with. Detection of subgenomic SARS- CoV-2 genetic material in an individual may therefore indicate that the individual poses an infectivity risk, being capable of transmitting infectious material. Thus, a disclosed herein, subgenomic SARS-CoV-2 transcripts or genetic material may be detected, as distinguishable from genomic SARS-CoV-2 transcripts or genetic material.

[0053] In some respects, sequences of sub-genomic SARS-CoV-2 transcripts or genetic material sequences share similarities with or may be indistinguishable from sequences of SARS- CoV-2 genomic sequences. Disclosed herein is a process for detecting sub-genomic SARS-CoV-2 distinguishable from genomic SARS-CoV-2 sequences. FIG . 1 shows an example of a subgenomic transcript for SARS-CoV-2 N protein. Sub-genomic SARS-CoV-2 sequences differ from genomic sequences in that the former include a 5' leader sequence, followed by a transcription regulatory sequence (TRS), followed by a leader body junction sequence (LBJ), followed by an open reading frame for the encoded protein, N protein in the example depicted in FIG. 1. Amplicons may be generated using a forward primer that specifically hybridizes to a forward primer target in a leader sequence and a reverse primer that specifically hybridizes to a reverse primer target near the 5' end of the open reading frame. Such amplicons would be generated during PCR when sub-genomic SARS-CoV-2 N protein transcripts were present in the sample but not in the absence thereof. A probe for detection may be used that spans from the 3' end of a leader sequence, to the transcription regulatory sequence, to the 5' end of the leader body junction. Detection of hybridization of a probe to such a probe target signifies presence of amplicons of sub-genomic material or transcripts in the PCR reaction and, thus, in the sample taken from a subject. In turn, this indicates the individual’s SARS-CoV-2 infectivity.

[0054] A forward primer length and a reverse primer length may each be designed as appropriate for specificity to respective targets and parameters employed during a PCR run, as would be understood by skilled persons. A forward or reverse primer may be, in an example, from about 10 nucleotides in length to about 30 nucleotides in length. For example, a forward primer or a reverse primer may be about 10 nucleotides in length, about 11 nucleotides in length, about 12 nucleotides in length, about 13 nucleotides in length, about 14 nucleotides in length, about 15 nucleotides in length, about 16 nucleotides in length, about 17 nucleotides in length, about 18 nucleotides in length, about 19 nucleotides in length, about 20 nucleotides in length, about 21 nucleotides in length, about 22 nucleotides in length, about 23 nucleotides in length, about 24 nucleotides in length, about 25 nucleotides in length, about 26 nucleotides in length, about 27 nucleotides in length, about 28 nucleotides in length, about 29 nucleotides in length, or about 30 nucleotides in length. Where a primer of a primer pair hybridizes to a leader sequence such as is illustrated in FIG. 1, the other primer of the primer pair may specifically hybridize to a 5' region of an open reading frame for a SARS-CoV-2 protein, including N protein as depicted in FIG. 1, or S protein, 3a protein, E protein, M protein, 6 protein, 7a protein, or 8 protein.

[0055] In an example, a forward or reverse primer may include nucleotides that do not hybridize to a forward primer target or reverse primer target, such as 5' to a sequence of a primerthe hybridizes specifically to a forward primer target, or may include one or more nucleotide between a 5' and 3' end of the primer that is not complementary to a nucleotide with which it aligns when the primer hybridizes to its primer target. In some examples, imperfect or incomplete primer hybridization may be modified to include one or more such non-hybridizing nucleotide for the purpose of tuning conditions of hybridization specificity of primer to primer target, and / or to introduce one or more nucleotide not present in a target into amplicons generated during PCR.

[0056] A probe length may be designed as appropriate for specificity relative to a probe target and parameters employed during amplicon detection as would e understood by skilled persons. A probe may be, in an example, between about 8 nucleotides long and about 50 nucleotides long. For example, a probe may be about 8 nucleotides long, about 8 nucleotides long, about 9 nucleotides long, about 10 nucleotides long, about 11 nucleotides long, about 12 nucleotides long, about 13 nucleotides long, about 14 nucleotides long, about 15 nucleotides long, about 16 nucleotides long, about 17 nucleotides long, about 18 nucleotides long, about 19 nucleotides long, about 20 nucleotides long, about 21 nucleotides long, about 22 nucleotides long, about 23 nucleotides long, about 24 nucleotides long, about 25 nucleotides long, about 26 nucleotides long, about 27 nucleotides long, about 28 nucleotides long, about 29 nucleotides long, about 30 nucleotides long, about 31 nucleotides long, about 32 nucleotides long, about 33 nucleotides long, about 34 nucleotides long, about 35 nucleotides long, about 36 nucleotides long, about 37 nucleotides long, about 38 nucleotides long, about 39 nucleotides long, about 40 nucleotides long, about 41 nucleotides long, about 42 nucleotides long, about 43 nucleotides long, about 44 nucleotides long, about 45 nucleotides long, about 46 nucleotides long, about nucleotides long, about 47 nucleotides long, about 48 nucleotides long, about 49 nucleotides long, or about 50 nucleotides long.

[0057] A probe may have a sequence that specifically hybridizes to a SARS-CoV-2 probe target comprising a 3' end of a leader sequence, a transcription regulatory sequence and a 5' end of a leader body junction sequence. A SARS-CoV-2 subgenomic transcript for various proteins, including N protein S protein, 3a protein, E protein, M protein, 6 protein, 7a protein, or 8 protein, may include a probe target sequence to which a probe having a sequence that specifically hybridizes to a SARS-CoV-2 probe target comprising a 3’ end of a leader sequence, a transcription regulatory sequence and a 5' end of a leader body junction sequence mayspecifically hybridize. Thus, in an example, such a probe may be used for detection of amplicons generated when one primer of a primer pair hybridizes to a leader sequence and the other primer of the primer pair specifically hybridizes to a 5' region of an open reading frame for a SARS- CoV-2 protein, including N protein, S protein, 3a protein, E protein, M protein, 6 protein, 7a protein, or 8 protein.

[0058] A probe may include one or more non-naturally occurring nucleotide, referred to herein generally as a modified nucleotide or xeno nucleotide. See, for example, Duffy et al., Modified nucleic acids: replication, evolution, and next-generation therapeutics, BMC Biology (2020) 18: 112, incorporated herein by reference in its entirety. Modified nucleotides or xeno nucleic acids may exhibit modified backbones, sugars, or nucleobases, or novel bases or base pairs. An oligonucleotide probe including one or more modified nucleotide may hybridize with a particular naturally occurring target sequence, wherein each of the one or more modified nucleotide may complement and bind to a corresponding, naturally occurring nucleotide in the target sequence, but do so in such a manner as imparts to the probe a higher or more specific binding than may occur in the absence of one or more of the modified nucleotides.

[0059] Non-limiting examples of modified nucleotides that may be included in a probe as disclosed herein include sugar modification (such as 2'-fluoro, 2'-O-methyl, locked nucleic acid wherein in which a methylene bridge bond links the 2' oxygen and the 4' carbon of the RNA pentose ring, 2'-fluoro arabinose nucleic acid, hexitol nucleic acid, or 2'-O-methoxyethyl), sugar / b ackbone modification (such as mirror DNA, (l'-3')-p-L-ribulo nucleic acid, a-L-threose nucleic acid, 3 '-2' phosphonom ethyl -threosyl nucleic acid, or 2'-deoxyxylonucleic acid), backbone modification (such as phosphorothioate or borano-phosphate), base modification (such as C7-modified deaza-adenine, C7-modified deaza-guanosine, C5-modified cytosine, or C5- modified uridine, wherein the modification includes substitution of a side chain at the indicated position with H, Cl, F, or other halogen, or alkyl phosphonate nucleic acid or peptide nucleic acid) or unnatural base pairs). Any one or more of any of the foregoing may be substituted for any one or more of any nucleotide of a nucleotide of a probe as disclosed herein, independently. Inclusion of an appropriately selected and placed one or more modified nucleotide in a probe as disclosed herein may significantly increase specificity and hybridization of a probe to a probe target and improve detection of replicating SARS-CoV-2. In another example, a probe may be attached to a moiety or group that may increase hybridization, such as a minor groove bindingprotein motif, which stabilizes a target-probe duplex.

[0060] A cycle threshold value may be determined for one or more target in the course of performing qPCR as disclosed herein. For example, a qPCR instrument may include components for thermal cycling and exposing reaction solutions to excitation radiation, optical components for detecting emission radiation, and be connected to one or more microprocessors and memory storage units. The one or more microprocessors and memory storage units may include instructions for and be configured to control operation of the qPCR instrument, and to identify and record levels of emission radiation detected during processing. The one or more microprocessors may be programmed to determine how many cycles of amplification occurred before a level of emission radiation for a given probe-probe target hybridization was detected that exceeds background, as a determination of cycle threshold for the relevant target. Cycle thresholds of multiple targets may be so identified and recorded, for use in further analysis. Where a series of cycles is completed without emitted radiation for a given target having been detected (indicating no or relatively low or relatively negligible amounts of the target in the sample), an arbitrary cycle threshold number may be attributed, such as the total number of cycles of PCR performed, or the total number of cycles performed plus an integer, such as 1.

[0061] As further disclosed herein, a cycle thresholds determined for a sub-genomic SARS-CoV-2 target may be compared to a cycle threshold for a SARS-CoV-2 genomic target. For example, a low level of a sub-genomic target being present in a sample may yield a cycle threshold that is higher that would be seen for a sample containing a higher amount of target.Cycle threshold for a target may indicate relative levels of target between two samples. However, it may be difficult or of relatively lower reliability to extrapolate a cycle threshold per se for a SARS-CoV-2 sub-genomic target in a sample to a determination of probable infectivity of a subject from whom the sample was obtained. As disclosed herein, a highly sensitive and reliable indication of infectivity may be obtained by performing a comparative calculation relating cycle threshold of sub-genomic SARS-CoV-2 transcript and cycle threshold of genomic SARS-CoV-2 transcript. For example, cycle threshold for genomic SARS-CoV-2 and genomic SARS-CoV-2 may be determined, then a relative amount of sub-genomic SARS-CoV-2 transcript relative to SARS-CoV-2 genomic transcript in the sample calculated using cycle threshold values. In an example, a relative amount may be determine according to the following Formula I: relative amount = 2'ACt, whereinCt is cycle threshold, and ACt = Ct (sub-genomic SARS-CoV-2 transcript) - Ct (genomic SARS- CoV-2 transcript).

[0062] As disclosed herein, according to Formula I, a measure of a relative amount of sub-genomic SARS-CoV-2 subgenomic transcript in a sample relative to an amount of genomic SARS-CoV-2 transcript in a sample may be obtained by determining cycle threshold for a subgenomic target, cycle threshold for a genomic target, the difference between the two (ACt), then calculating 2 to the exponent -ACt.

[0063] In another example, a further control measure may be included in a qPCR assay, further improving sensitivity and reliability of a method for detecting replicating SARS-CoV-2 in a subject. In addition to performing qPCR for identification of cycle threshold for a non- genomic SARS-CoV-2 nongenomic target and a SARS-CoV-2 genomic target, a further target may be amplified an applied as a control as to the SARS-CoV-2 targets. For example, a control transcript encoded by the genome of the subject a sample was taken from may provide a target. Detecting presence of such a control transcript of a subject in a sample may serve as a control for variables such as sample quality, sample processing prior to qPCR assay performance, sample degradation, etc. Variability between samples as to detected amounts of SARS-CoV-2 genomic transcript and detected amounts of SARS-CoV-2 non-genomic transcript, and sample-to-sample variability between measures of levels of one relative to the other, attributable to methodological factors independent of different levels of replicating SARS-CoV-2 virus in subjects from whom samples were taken, may be minimized by controlling for such factors.

[0064] For example, a control target may include what is referred to as a housekeeping transcript or gene. A housekeeping genes may be a generally constitutively expressed gene, such as may be required for the maintenance of basal cellular functions, necessary for cell existence or survival, etc. A housekeeping gene may be expressed in many or all cells or cell types of a subject, irrespective of presence or absence of SARS-CoV-2 virus, replicating or otherwise, in a subject, and may be present in irrespective of tissue type, developmental stage, cell cycle state, or cellular signal, or health of an individual. A number of such genes have been identified and measurement of their expression in a sample may be used to control for incidental variables in factors unrelated to SARS-CoV-2 viral presence in a subject. Some non-limiting examples of a control or housekeeping genes include: RNAse P; 18S ribosomal RNA; Actin, beta; Glyceraldehyde-3 -phosphate dehydrogenase; Phosphoglycerate kinase 1; Peptidylprolylisomerase A; Ribosomal protein LI 3a; Ribosomal protein, large, PO; Acidic ribosomal phosphoprotein PO; Tyrosine 3 -monooxygenase / tryptophan5 -monooxygenase activation protein, zeta polypeptide; Succinate dehydrogenase complex, subunit A, flavoprotein (Fp); Transferrin receptor; Glucuronidase, beta; Hydroxymethylbilane synthase; Hypoxanthine phosphoribosyltransferase 1; and TATA box binding protein. Other transcripts, of other housekeeping or other genes, may be used as a control. In an example, more than one control transcript may be detected as a control in a multiplex qPCR in accordance with the present disclosure.

[0065] In a qPCR process, an assay may include a forward primer and reverse primer, specific for a forward primer target and a reverse primer target, respectively, for a control transcript such as a housekeeping transcript. A sequence for a control target probe may be present in the control target between the forward and reverse primer targets, such that generation of amplicons from the control transcript using the forward and reverse control transcript primers during PCR results in amplicons including control probe target sequences. A control target probe may be included in a qPCR process for detection of control target amplicons, much as described above in connection with performing qPCR for detection of SARS-CoV-2 transcripts (e.g., including a dye and a quencher). And as with SARS-CoV-2 subgenomic and genomic targets, a cycle threshold for a control target may be determined. In an example, a multiplex qPCR process may be performed, wherein primer pairs and a probe for two or more of a sub-genomic SARS- CoV-2 transcript, a genomic SARS-CoV-2 transcript, and a control transcript, may be used, with dyes and quenchers and as may be necessary filtration of excitation and / or emission spectra so as to permit differentiation between signal indicating presence of each of the foregoing from each of the others.

[0066] In an example, qPCR for SARS-CoV-2 subgenomic and genomic and control transcript may all be performed, for determination of cycle threshold of each type of transcript. A SARS-CoV-2 genomic probe may be attached to a genomic probe dye and a genomic probe quencher, a SARS-CoV-2 sub-genomic probe may be attached to a sub-genomic probe dye and a sub-genomic probe quencher, and a control probe may be attached to a control dye and a control quencher.

[0067] A cycle threshold for a control transcript may be included in determining presence of sub-genomic SARS-CoV-2 in a sample. For example, a relative amount of sub-genomicSARS-CoV-2 transcript relative to genomic SARS-CoV-2 transcript in a sample , based on cycle thresholds of each of the foregoing and for a control transcript, may be determined according to the following Formula II: relative amount = 2'AACt, whereinCt is cycle threshold, and AACt = (Ct (subgenomic SARS-CoV-2 transcript) - Ct (genomic SARS-CoV-2 transcript)) - Ct (control transcript).

[0068] As disclosed herein, according to Formula II, a measure of a relative amount of sub-genomic SARS-CoV-2 subgenomic transcript in a sample relative to an amount of genomic SARS-CoV-2 transcript in a sample may be obtained by determining cycle threshold for a subgenomic target, cycle threshold for a genomic target, and the difference between the two (a first difference), determining the cycle threshold for a control transcript and the difference between it and the first difference (second difference), then calculating 2 to the exponent -AACt.

[0069] As disclosed herein, a predetermined threshold value of relative amount may be used to determine whether a relative amount calculated for a sample indicates presence of replicating SARS-CoV-2 in the subject from whom the sample was taken. A relative amount calculated according to Formula II may be compared to a predetermined threshold. If the relative amount equals or exceeds the predetermined threshold, the subject from whom the sample was taken may be determined to be infected with replicating SARS-CoV-2. A relative amount calculated according to Formula II may be compared to a predetermined threshold. If the relative amount is less than the predetermined threshold, the subject from whom the sample was taken may be determined not to be infected with replicating SARS-CoV-2.

[0070] As disclosed herein, a predetermined threshold of a relative amount according to Formula II may be about 0.5 million, or about 0.75 million, or about 1.0 million, or about 1.25 million, or about 1.5 million, or about 1.75 million, or about 2.0 million. A predetermined threshold of a relative amount according to Formula II may be in a range from about 0.3 million to about 5 million. A predetermined threshold of a relative amount according to Formula II may be about 0.3 million, or about 0.4 million, or about 0.5 million, or about 0.6 million, or about 0.7 million, or about 0.8 million, or about 0.9 million, or about 1.0 million, or about 1.1 million, or about 1.2 million, or about 1.3 million, or about 1.4 million, or about 1.5 million, or about 1.6 million, or about 1.7 million, or about 1.8 million, or about 1.9 million, or about 2.0 million, or about 2.1 million, or about 2.2 million, or about 2.3 million, or about 2.4 million, or about 2.5million, or about 2.6 million, or about 2.7 million, or about 2.8 million, or about 2.9 million, or about 3.0 million, or about 3.1 million, or about 3.2 million, or about 3.3 million, or about 3.4 million, or about 3.5 million, or about 3.6 million, or about 3.7 million, or about 3.8 million, or about 3.9 million, or about 4.0 million, or about 4.1 million, or about 4.2 million, or about 4.3 million, or about 4.4 million, or about 4.5 million, or about 4.6 million, or about 4.7 million, or about 4.8 million, or about 4.9 million, or about 5.0 million. A predetermined threshold of a relative amount according to Formula II may be about 0.05 million between any of the foregoing that differ from each other by about 0.1 million. A predetermined threshold of a relative amount according to Formula II may be about 0.025 million between any of the foregoing that differ from each other by about 0.05 million.

[0071] In another example, viral load of replicating SARS-CoV-2 in a subject may be determined. For example, a qPCR process may include calculation of a standard curve of viral load, by preparing control samples with a predetermined amount of viral load and determining the Ct for each and using the Ct to generate a standard curve. Cycle threshold of a sample may then be calculated and compared to the standard curve and the standard curve viral load corresponding to the sample’s cycle threshold taken as an indication of viral load of the sample. A predetermined threshold may be chosen whereby a cycle threshold below the predetermined threshold taken as an indication of infection of the individual with a replicating SARS-CoV-2 viral load indicative of the subject being infective or in an actively infected state.

[0072] In another example, rather than qPCR, viral load may be determined according to digital PCR, a sensitive method that can detect rare events like single-nucleotide mutations and trace amounts of DNA. Using digital PCR, replicating SARS-CoV-2 viral load for a subject may be determined, using statistical methods to quantify nucleic acids by counting positive amplification signals. If viral load is at or above a predetermined threshold, the subject may be infective or in an actively infected state. In digital PCR, a sequencing reaction is partitioned into thousands of separated reactions of submicroliter volume each (for example, by a droplet generator for droplet digital PCR, or using a digital PCR nanoplate), each acting as a distinct chamber for a PCR reaction. Target polynucleotides are separated into the droplets such that the number of droplets including target polynucleotides is directly proportional to the number of target polynucleotides present in the sample. Amplification by PCR occurs only in droplets containing target sequences, and the number of droplets in which a target is amplified can bedetected and quantified. Identifying the number of droplets in which PCR reaction product becomes detectable (such as by using a flow cytometer for droplet digital PCR or other digital PCR platform apparatus), such as after a certain number of cycles of PCR, therefore gives an estimation of the total number of target polynucleotides in a sample. The number of positive and negative droplets is counted to determine the concentration of the target sequence, and Poisson statistics are used to calculate the original DNA concentration. See Wagner et al., 2023, A multiplexed, paired-pooled droplet digital PCR assay for detection of SARS-CoV-2 in saliva, Sci Rep 13 ( 1 ): 3075 and Abasiyanik et al., 2021, Sensitive detection and quantification of SARS- CoV-2 in saliva, Sci Rep, 11(1): 12425, incorporated herein by reference in their entireties.

[0073]

[0074] As disclosed herein, a method according to any of the foregoing examples may be performed to detect presence of replicating SARS-CoV-2 virus in a subject. The subject may be a human or a non-human primate. The subject may be a non-human animal. The subject may be, without limitation, a bat, a cervid, a caniformid, a feliformid, a manid, a mustelid, a horse, a cow, a goat, a sheep, a pig, or a rodent.

[0075] As disclosed herein, a sample may be any sample taken from a subject that may be expected or predicted to include SARS-CoV-2 virus in the event the subject were infected with SARS-CoV-2 virus, including replicating virus. A sample may include, without limitation, a nasal swab, a nasopharyngeal swab, an oropharyngeal swab, a throat swab, a nasal secretion, saliva, mucous, throat secretion, a lower respiratory specimen, whole blood, blood component, tissues, plasma, serum, stool, or urine.

[0076] Detection of replicating SARS-CoV-2 virus in an individual according to any of the foregoing examples may indicate that the subject may benefit from one or more treatment intended to treat infection, improve symptoms, reduce viral replication in the subject, or otherwise reduce or prevent any other deleterious health effect for the individual. A subject in whom replicating SARS-CoV-2 virus may be detected according to any of the foregoing methods, such as where a predetermined threshold relative level is met or exceeded according to for example Formula I or Formula II, may be suitable for receipt of administration of a treatment for SARS-CoV-2 or symptoms negative health effects of SARS-CoV-2 viral infection. Nonlimiting examples of such a treatment may include convalescent plasma, one or more anti-SARS- CoV-2 therapeutic antibody (such as but not limited to bamlanivimab plus etesevimab,casirivimab plus imdevimab, sotrovimab, or bebtelovimab), one or more antiviral composition (such as, but not limited to, paxlovid, remdesivir, or molnupiravir), a steroid hormone (such but not limited to as a corticosteroid hormone receptor agonist such as prednisone), or any combination of two or more of the foregoing.

[0077] In some examples, presence of replicating SARS-CoV-2 in a subject may render them particularly susceptible to one or more of the foregoing treatments. Detection of replicating SARS-CoV-2 virus in a subject in accordance with the present disclosure may therefore present a particularly useful method for identifying individuals for who such one or more treatment is particularly advisable. Similarly, lack of detection of replicating SARS-CoV-2 virus in an individual following assaying a sample from the subject in accordance with a method as disclosed herein may indicate that the subject may not benefit one or more such treatment. A subject in whom replicating SARS-CoV-2 virus may not be detected according to any of the foregoing methods, such as where a predetermined threshold relative level is not met according to for example Formula I or Formula II, may not be suitable for receipt of administration of one or more said treatment. For example, avoidance of one or more negative side effect may be a countervailing consideration in a treatment decision for an individual suspected of being infected with replicating SARS-CoV-2. An ability to determine that said individual is not infected with replicating SARS-CoV-2 may help in making a treatment determination to refrain from administering one of more such treatment to said subject and thereby avoid possible negative side effects.

[0078] An ability to determine that said individual is not infected with replicating SARS- CoV-2 may help in making a treatment determination to refrain from administering one of more such treatment to said subject and thereby conserve supplies of such treatment for administration to another subject in whom replicating SARS-CoV-2 is detected, such as when such other subject may be more likely to respond to such treatment by nature of being infected with replicating SARS-CoV-2.

[0079] Detection of replicating SARS-CoV-2 virus in an individual according to any of the foregoing examples may indicate that the subject poses an infection risk for individuals with whom the subject may come in contact or proximity. A method as disclosed herein may be used to identify an individual for whom a transmission prevention is advisable or recommended as a measure of public health. A transmission prevention may be any step or process taken orimplemented to reduce probability of an individual infected with replicating SARS-CoV-2 from transmitting infection to another one or more individual. A subject in whom replicating SARS- CoV-2 virus may be detected according to any of the foregoing methods, such as where a predetermined threshold relative level is met or exceeded according to for example Formula I or Formula II, may be suitable for a transmission prevention. Non-limiting examples of such a transmission prevention may include wearing of a mask, wearing of a face shield, quarantine or isolation from others, decontamination of an environment occupied by such subject, etc.

[0080] Insofar as a transmission prevention may have undesirable consequences, such as negative mental, emotional, or psychological effects of social isolation, difficulty in caring for others such as family members who rely on the subject’s care, missing school or work or other events, etc., it may be beneficial to identify, according a method in accordance with the present disclosure, a subject who is not infected with replicating SARS-CoV-2. Lack of detection of replicating SARS-CoV-2 virus in an individual following assaying a sample from the subject in accordance with a method as disclosed herein, such as where a predetermined threshold relative level is not met according to for example Formula I or Formula II, may indicate that the subject is not infectious because the subject is not infected with replicating SARS-CoV-2 or not at an infective level.

[0081] In an example, disclosed herein is a kit. A kit may include one or more of a forward subgenomic primer, a reverse subgenomic primer, and a subgenomic SARS-CoV-2 transcript probe as disclosed herein. The kit may further include one or more of the forward genomic primer, the reverse genomic primer, and the genomic SARS-CoV-2 transcript primer as disclosed herein. The kit may further include one or more of the forward control primer, the reverse control primer, and the control transcript primer. The kit may further include one or more of a subgenomic transcript positive control, a subgenomic transcript negative control, a genomic transcript positive control, a genomic transcript negative control, a control transcript positive control, and a control transcript negative control. The kit may further include one or both of a replicating SARS-CoV-2 virus negative control and a replicating SARS-CoV-2 virus positive control. The kit may further include one or more of a subgenomic probe control, a genomic probe control, and a control probe control. The kit may further include one or more additional reagents for use in quantitative polymerase chain reaction. The kit may include any two or moreof any of the foregoing components. Such a kit may include one or more components for use in a method for detection of replicating SARS-CoV-2 in a sample, such as a sample from a subject.

[0082] Although some non-limiting examples have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the present disclosure and these are therefore considered to be within the scope of the present disclosure as defined in the claims that follow.

[0083] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail herein (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein.EXAMPLES

[0084] The following examples are intended to illustrate particular embodiments of the present disclosure, but are by no means intended to limit the scope thereof.

[0085] EXAMPLE 1 : MATERIALS AND METHODS

[0086] Cells and media

[0087] VeroE6 cells (owned and maintained by the Wadsworth Center, New York State Department of Health, hereinafter “Wadsworth” or “Wadsworth Center”), and VeroE6 / TMPRSS2, a modified VeroE6 cell line expressing the transmembrane serine protease, TMPRSS2, were used for virus isolation from clinical specimens and in-vitro growth kinetics studies. VeroE6 / TMPRSS2 cells were purchased from the Japanese Collection of Research Bioresources (JCRB Cell Bank), cell number JCRB1819, Agreement # A2000230 (References Pubmed id 32165541 and 31013314).

[0088] VeroE6 and VeroE6 / TMPRSS2 cells were seeded in T25 flasks at 1.5 x 105cells / mL, with appropriate growth media, three days prior to infection. VeroE6 cells were maintained in Eagle’s minimum essential medium (EMEM), containing sodium bicarbonate, sodium pyruvate, non-essential amino acids, 100 units / ml penicillin, 100 pg / ml streptomycin (pen / strep) and 10% fetal bovine serum (FBS; all MilliporeSigma, St. Louis, MO).VeroE6 / TMPRSS2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with sodium bicarbonate and 10% FBS (all MilliporeSigma), as well as Img / lmL geneticin G418 (Gibco).

[0089] Clinical Samples

[0090] A total of 355 upper respiratory swabs were selected for SMART assay analysis and viral culture. Primary samples included SARS-CoV-2 positive upper respiratory swabs with a range of CT values, collected in New York State from March 2021 to October 2021. These samples were originally submitted to the Wadsworth Center for routine diagnostic SARS-CoV-2 testing, or as part of an ongoing enhanced genomic surveillance program in New York State to perform whole genome sequencing to determine prevalence of circulating lineages and monitor for emerging variants. A complete SARS-CoV-2 genome and PANGO lineage was available for 93 of the 355 samples. Diagnostic testing was performed using one of several molecular FDA EUA tests, including the New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel, the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, NEUMODX™ SARS-CoV-2 Assay, or the Cepheid XPERT® Xpress SARS- CoV-2 assay. Nucleic acid extraction for SMART assay analysis was performed in parallel with cell culture inoculation from thawed samples, retrieved from -80°C archives for comparative testing. Patient demographics are shown in Table E Patients were deidentified and no additional clinical information was available.

[0091] Table 1 : Demographics of SARS-CoV-2 positive patients included in the SMART study. (N=355)

[0092] Virus isolation from clinical specimens

[0093] Before inoculation of cultures, 100 to 500uL of each clinical respiratory swab specimen in viral or universal transport media (VTM / UTM), was processed, by adding 100 to 500uL pen / strep (lOunits / mL of each) and 50 to 250uL of nystatin (Img / mL; both MilliporeSigma). T25-flasks containing 85-95% confluent VeroE6 or VeroE6 / TMPRSS2 cell monolayers were inoculated with processed specimens, adsorbed l-2hr at 37°C with 5% CO2, and fed with 5mL viral growth media as described above, with the following exceptions: FBS reduced to 2%, and EMEM was supplemented with 40mM final concentration HEPES.

[0094] Following inoculation, all incubation and procedures were performed in a biosafety level 3 (BSL-3) laboratory and infected monolayers were checked daily for cytopathic effect (CPE). Flasks were harvested when CPE was observed in at least 50% of the monolayer, or at day seven post-infection; whichever came first. Regardless of CPE, all flasks were harvested and tested by real-time RT-PCR assay to confirm viral growth in culture. Harvested cultures with real-time CT values more than 3 cycles lower than the CT value of the corresponding primary sample (indicating a 10-fold or higher increase in viral load), were considered positive for viral growth.

[0095] Intracellular and extracellular SARS-CoV-2 growth kinetics

[0096] Six-well plates containing confluent monolayers of VeroE6 / TMPRSS2 cells were inoculated with SARS-CoV-2 (Pango lineage B.l), originally isolated in VeroE6 cells from an upper respiratory clinical specimen, at a MOI of 0.01 PFU / cell. Quadruplicate wells were inoculated and analyzed on separate plates for each of the following nine timepoints: 1, 3, 6, 9, 12, 24, 48, 72, and 96 hrs post-infection.

[0097] Briefly, growth media was removed from each well, inoculated with lOOuL of virus, and allowed to adsorb for 1 hr @ 37°C and 5% CO2. Inoculum was then removed from wells, rinsed once with maintenance medium, and replaced with 3mL maintenance medium. Inoculated plates were incubated at 37°C and 5% CO2 and sampled at each time point. Sample collections consisted of the following: lOOuL supernatant was removed from each well and placed in 2mL NUCLISENS® lysis buffer (bioMerieux). An additional lOOuL of supernatant was removed and added to 900uL EMEM + 20% FBS for frozen storage. The remaining supernatant was discarded, cells rinsed once with 2mL maintenance medium, and an additional ImL of fresh medium containing 20% FBS was added. The infected monolayer was harvested by gentle scraping and lOOuL of the harvested cell suspension was lysed in 2mL NUCLISENS® lysis buffer. The remaining cells were archived in frozen storage.

[0098] The SMART assay, described in detail below, was performed on the supernatants and cell harvests from each well at each time point. Growth curves were plotted using the quadruplicate mean genomic and subgenomic N RNA concentration and standard deviation (logioRNA copies / mL) calculated from the real-time assay results at each time point.

[0099] Viral nucleic acid extraction

[0100] Total nucleic acid extraction was performed with the bioMerieux NUCLISENS® EASYMAG® platform (bioMerieux Inc, Durham, NC). Unless otherwise stated, lOOuL of primary sample or viral harvest was added to 2mL NUCLISENS® lysis buffer (bioMerieux) and extracted into lOOuL of eluate.

[0101] Real-time RT-PCR

[0102] When necessary, clinical specimens were re-extracted and re-tested by real-time RT-PCR to recheck the CT value before virus culture. Real-time RT-PCR was performed using the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, according to the Instructions for Use (IFU).

[0103] Cross-reactivity studies to other SARS-CoV-2 subgenomic RNAs were also performed using real-time RT-PCR assays designed for the detection of the E gene (Corman et al, Euro Surveillance, 2020), and subgenomic E gene (Wolfel et al, Nature 2020).

[0104] Control virus and viral RNA quantification

[0105] SARS-CoV-2 isolate, USA-WA1 / 2020 (Pango lineage A), was obtained from BEI Resources (NIH / ATCC, Manassas, VA) and amplified in VeroE6 cells. Cultured viral RNA was extracted and quantified by real-time RT-PCR against standard curves generated with synthesized ORF lab and sgN RNA transcripts.

[0106] SMART assay

[0107] The SMART assay is a multiplexed real-time RT-PCR consisting of three assays: one targeting the genomic ORF lab gene of SARS-CoV-2 (22), a second targeting the SARS- CoV-2 subgenomic N (sgN) RNA transcript, and a third targeting a human RNase P housekeeping gene, which is used as an internal control, sample quality, and normalization assay.

[0108] SMART assay amplification was performed on extracted nucleic acid using the LUNA® Universal One-Step RT-qPCR kit (New England BioLabs, Ipswich, MA). Each 20pL reaction consisted of 5 pL of RNA template, Luna 4X Buffer, 500nM primer, 50 to lOOnM of probe for each target, and water.

[0109] Thermocycling parameters were 30 seconds at 25°C, 10 minutes at 55°C, one minute at 95°C, then 45 cycles of 10 seconds at 95°C and 30 seconds at 58°C.

[0110] sgN transcript real-time RT-PCR assay design

[0111] Assay design was performed with Geneious Prime 2020.2.5, using reference genome SARS-CoV-2 isolate Wuhan-Hu-1, GenBank accession number MN908947.3. In order to obtain the greatest sensitivity and specificity for the sgN transcript target, the sgN real-time RT-PCR assay, designed for the SMART multiplex, incorporates a forward primer targeting a region in a leader sequence, a reverse primer in an open reading frame of a gene, and a probe that bridges a 3’ end of a leader sequence, a transcriptional regulatory sequence (TRS), and a junction between a TRS and a transcription initiation codon (FIG. 1). Increased specificity for the sgN may result from the probe spanning regions described above. In an example, oligonucleotide’s may include one or more modified nucleotide such as locked nucleic acid (LNA) at one or more various position as disclosed herein.

[0112] Primers and Probes

[0113] Primers and probes for the SMART assay, having sequences designed by inventors, were obtained from Sigma-Aldrich (St. Louis, MO). Real-time subgenomic N primers, modified with M13 sequence tags, were obtained from Invitrogen (Waltham, MA). The CDC SARS-CoV-2 EUA primers and probe, leader-specific forward primer (Wolfel et al 2020), E gene real-time primers and probe (Sarbeco), and the subgenomic-specific reverse primers were all purchased from Integrated DNA Technologies (Coralville, IA). Sequences are presented in Table 2 below:

[0114] Table 2: Oligonucleotide Sequences

[0115] In working examples disclosed herein, some nucleotides in SEQ ID NO: 6 were locked nucleic acids (LNA), in which a methylene bridge bond links the 2' oxygen and the 4' carbon of the RNA pentose ring. LNAs in SEQ ID NO: 6 are indicated by being underlined in Table 3. In the qPCR working examples disclosed herein, fluorescent dyes were attached to the 5' end of probes (FAM for ORF lab-Probe, HEX for sgN-Probe, and Cy5 for RP -Probe) andquenchers were attached to the 3' end of probes (BHQ1 for ORF1 ab-Probe and sgN-Probe and BHQ2 for RP-Probe).

[0116] Table 3: Analytical sensitivity of individual SMART assay components

[0117] aTotal nucleic acid purified from SARS-CoV-2 isolate (Pango lineage A)

[0118] bSynthetic RNA transcript containing the sequence of the SARS-CoV-2 ORF lab real-time RT-PCR amplicon manufactured by Aanotech LLC, Rensselaer, NY

[0119] cSynthetic RNA transcript containing the sequence of the SARS-CoV-2 subgenomic N real-time RT-PCR amplicon

[0120] dSynthetic full-length SARS-CoV-2 DNA genome, manufactured by CODEXDNA, Inc. Used as a genomic-specific control.

[0121] SMART assay validation

[0122] Assay controls

[0123] Multiple quantified RNA and DNA controls were used for assay development, optimization and assessment of assay performance. Quantified RNA transcripts containing expected amplicon sequences of N1 and N2 assays of the CDC SARS-CoV-2 EUA, as well as the expected amplicon sequence for the sgN gene assay, were obtained from bio-Synthesis, Inc (Lewisville, TX). For an ORF lab control, PCR product was generated from a synthetic DNA representing nucleotide region 11342-1 1460 of NOB I accession no. MT106054.1 . The PCR product was then cloned into a PCR4 Topo vector, the sense strand RNA synthesized with a T7 RNA polymerase and purified RNA quantified using a Qubit fluorometer (ThermoFisher Scientific, Waltham, MA).

[0124] A full-length, quantified synthetic SARS-CoV-2 DNA reference genome (GenBank Accession# MN908947.3, with spike protein variant D614G), was also obtained fromTelesis Bio (formally Codex DNA, San Diego, CA), and used as a genomic-only control to assess cross-reactivity of the sgN gene assay against SARS-CoV-2 genomic sequence.

[0125] Amplification Efficiency

[0126] Multiple 10-fold serial dilutions of quantified RNA transcripts containing ORFlab and sgN target assay amplicon sequences, ranging from 1.0E+06 gc / uL to 10 gc / uL, were tested, in duplicate, with the SMART assay. CT values were plotted against logio RNA copy number using Microsoft Excel, and linear regression analysis was performed using the trendline created with the data.

[0127] Analytical Sensitivity

[0128] Multiple 10-fold serial dilutions of quantified RNA transcripts containing ORFlab and sgN target assay amplicon sequences, quantified synthetic full-length genomic SARS-CoV-2 DNA, and quantified SARS-CoV-2 isolate USA-WA1 / 2020 RNA, were tested, in duplicate, to individually determine the analytical sensitivity of the ORFlab and sgN assays in the SMART assay. Concentrations ranged from 1.00E+06 gene copies / uL to 1 gene copy / uL.

[0129] Analytical Specificity

[0130] General assay specificity was assessed by real-time RT-PCR using DNA and RNA preparations from 46 different viral and bacterial human respiratory pathogens (Table 4). Frozen stocks of purified nucleic acid, each at approximately 1.00E+06 gene copies / pL, were used for testing.

[0131] Table 4: Specificity of SMART assay components against multiple viral and bacterial respiratory pathogens

[0132] To verify the absence of cross reactivity of the sgN gene assay on other subgenomic transcripts, DNA amplicons, representing portions of sgS, sg3a, sgE, sgM, sg7a, and sg8 viral transcripts, were created in-house to use as templates for the SMART real-time RT- PCR. Due to difficulty in amplifying and purifying the sg6 RNA transcript, a synthetic doublestranded DNA fragment was later obtained from bio-Synthesis, Inc (Lewisville, TX). The genomic maps of the relevant gene portions are depicted in Figure 2. Briefly, cDNA synthesis from SARS-CoV-2 isolate USA-WA1 / 2020 RNA was performed with the SuperScript™ VILO cDNA Synthesis kit (Invitrogen, Waltham, MA), followed by amplification of the cDNA with Thermo Scientific™ DreamTaq™ Hot Start Green PCR Master Mix (Waltham, MA). PCR was performed using a leader-specific forward primer paired with a subgenomic target-specific reverse primer designed to bind approximately 350-500 nucleotides downstream of the leader sequence. Amplicons were visualized by gel electrophoresis, purified, quantified, and sequenced. Each amplicon was diluted to approximately 1.00E+06 copies / uL and used as template for cross-reactivity studies.

[0133] The subgenomic DNA amplicons were also used to assess and compare the specificity of a previously published assay for the detection of subgenomic E gene (sgE) viral transcripts (Wolfel et al). This sgE assay is used in combination with a genomic E gene assay to determine active replication of SARS-CoV-2 in respiratory samples. sgS, sg3a, sgE, sgM, sg7a, sg8, and sgN amplicons were used as template in both the SARS-CoV-2 genomic E gene realtime RT-PCR assay (Sarbeco), and the sgE real-time RT-PCR assay used for detection of subgenomic RNA.

[0134] Inclusivity

[0135] To verify our sgN assay design, as well as the sequence of the subgenomic N RNA transcript from multiple SARS-CoV-2 lineages, dideoxy DNA sequencing was performed on amplicons created with our sgN real-time RT-PCR forward and reverse primers. Isolates of five different SARS-CoV-2 variants were selected: Alpha (B. l.1.7), Epsilon (B.1.429), Iota (B.1.526), Delta (B.1.617.2) and Omicron (BA.l). Purified RNA was amplified with the QSCRIPT® XLT One-Step RT-PCR Kit (Quanta Biosciences, Beverly, MA) using our established sgN primers modified with M13 tails. Amplicons were analyzed by gel electrophoresis and sequenced using M13 primers. Sequences were aligned to predicted sgN RNA transcript sequence, originally determined using sequence from SARS-CoV-2 isolateWuhan-Hu-1 (GenBank accession number MN908947.3).

[0136] Data Analysis and SMART score calculation

[0137] Data analysis was performed using a modified version of a delta delta (AA) cycle threshold (CT) method (AACT), also known as a comparative CT method (Livak K.J., and Schmittgen T.D. 2001, Methods 24 (4): 402-8), in which 2'AACTis used to determine the fold change in expression between multiple genes in different samples. CT is a measure used in quantitative real-time PCR (also referred to as RT-PCT or qPCR) in which amplification of a target amplicon from a source such as polynucleotides from a biological sample proceeds in repeated cycles while the number of amplicons synthesized, which may increase exponentially in successive cycles, is detected, typically by fluorescence, which in turn may also increase with successive cycles. Once a level of fluorescence indicative of presence of a target amplicon above a threshold level such as background fluorescence is detected, the number of cycles of PCR that took place until such threshold fluorescence was detected is referred to as the cycle threshold or CT. Generally, the more abundance a target sequence is in a sample, the lower the CT will be for amplicons of that target, because the number of amplicons required to permit detection of the threshold level of fluorescence will require fewer amplification cycles to be produced.

[0138] Typically, CT values of individual genes are first normalized to a housekeeping gene to yield a ACT value for individual genes in a given sample. Conventionally, a ratio is then generated between treated vs untreated samples to give a AACT. This takes into consideration the different amplification efficiencies of the individual qPCR reactions. As disclosed herein, a different measure was devised, as the amplification efficiencies observed in both the ORF lab and sgN real-time RT-PCR reactions were found to be very similar (>95%, see Figure 5). We therefore first normalized the sgN CT value to the ORF lab CT (to generate the ACT) and then normalize this to a housekeeping gene (e.g., the RNase P) CT, to generate the AACT. In an example, the following formula was applied to calculate the fold difference between sgN and ORF lab, normalized to RNase P (for each sample) which we term the “SMART Score”:2 -AACT= 2- [(sgNCt- ORFlab ^-RNasePC?] (FormulaJ)

[0139] SMART analysis (Establishment of the SMART score threshold)

[0140] To determine the SMART score value, a series of ten training sets were generated, each consisting of calculated SMART scores from 30 culture positive and 30 culture negative samples, randomly selected from the sample dataset. A box and whiskers plot wasconstructed for each training set, and the lower 25thpercentile value from each culture negative training plot was averaged to determine the SMART score cutoff value.

[0141] EXAMPLE 2: SMART assay validation

[0142] Amplification efficiency

[0143] Multiple 10-fold serial dilutions of the quantified ORFlab and sgN RNA transcript controls, ranging each from 1.0E+06 gc / uL to 10 gc / uL, were tested with the SMART assay to determine and compare amplification efficiencies. Linear regression analysis indicated R2values of 0.99 for both ORFlab and sgN, with efficiency values of 95.0% and 99.5% for the ORFlab and sgN assays, respectively. (Figure 5).

[0144] Sensitivity

[0145] As shown in Table 3, when the component sgN and ORFlab assays were tested against cultured virus RNA, synthetic ORFlab, sgN RNA transcript, and full length synthetic genomic DNA, sensitivities ranged from 1-15 gc / uL.

[0146] Specificity

[0147] No cross-reactivity was observed with the SMART assay when challenged with multiple viral and bacterial respiratory pathogens (Table 4). When analyzed using amplicons representing other SARS-CoV-2 subgenomic RNA transcripts, minor cross-reactivity was seen with the sgN assay on sgE (CT 37.24 / 39.60) and sg8 (CT 41.31 / neg) amplicons (Table 4).Results were as expected after challenge with the full-length synthetic SARS-CoV-2 genomic DNA, constructed to represent a genomic-only SARS-CoV-2 template.

[0148] ORFlab Ct values were 19.68 / 19.79, and no cross-reactivity was observed with the sgN assay. Specificity testing with the genomic and subgenomic E gene real-time RT-PCR assays against the subgenomic amplicons revealed more cross-reactivity than that of the SMART assay. Positive results were observed in both genomic and subgenomic assays using sg3a, sgM, sg7a, and sg8 amplicon templates, with Ct values as low as 23 using the sgM amplicon as template (Table 4).

[0149] Inclusivity

[0150] Nucleic acid from Alpha (B.1.1.7), Epsilon (B.1.429), Iota (B.1.526), Delta(B.1.617.2), and Omicron (BA.l) variants was amplified and sequenced using the sgN assay to verify amplification and detection across multiple SARS-CoV-2 lineages. Gel visualization in Figure 3 revealed single bands with the same amplicon size as the sgN transcript control for allSARS-CoV-2 variants tested. Sequence analyses of all amplicons confirmed the presence of the expected leader sequence, six-nucleotide TRS, and junction sequence.

[0151] Isolation of virus from clinical specimens

[0152] A total of 355 upper respiratory specimens were both inoculated into culture and analyzed by the SMART assay. Original CT values of the clinical specimens ranged from 11.00 to 38.15, using one of the initial SARS-CoV-2 real-time RT-PCR screening assays described above in methods. SARS-CoV-2 was successfully isolated from 192 (54%) of the 355 specimens analyzed.

[0153] Intracellular and extracellular SARS-CoV-2 growth kinetics

[0154] When the production of genomic and subgenomic RNA was measured in inoculated TMPRSS2 cultures over time, by quantifying the RNA species with specific SMART component assays, differences were observed in the RNA ratios in intracellular harvests compared to extracellular culture fluid. Genomic and subgenomic RNA remained at very similar levels in the intracellular fluid throughout the 96 hour culture period. However, in the extracellular fluid, genomic RNA increased more rapidly to more than a log higher than subgenomic RNA within the first 12-24 hours and remained at that higher level for the duration of the culture period (Figure 4).

[0155] SMART analysis-clinical specimens

[0156] Upon extraction and re-testing of inoculated primary samples with the SMART assay, 266 of the 355 (75%) were positive for both ORFlab and sgN targets, and 48 (14%) samples were negative for both ORFlab and sgN targets. Five samples (1%) were negative for ORFlab and positive for sgN, and 36 (10%) were positive for ORFlab and negative for sgN. Samples negative for both ORFlab and sgN targets were excluded from the dataset used to determine the SMART score threshold. For SMART score calculations, samples with negative ORFlab and sgN real-time RT-PCR values were assigned a Ct value of 45.

[0157] Figure 6 displays the distribution of Ct values obtained with the SMART assay on all 355 clinical specimens analyzed, separated by target and culture result. As expected, Ct values of both ORFlab and sgN targets were lower in those samples that were culture positive.

[0158] Additionally, in our dataset of 355 samples, 99% of samples with an ORFlab CT less than 23 grew in culture (71 / 72) whereas only 17% (4 / 23) with ORFlab CT values between 35-44 were culture-positive. (Figure 7). We therefore applied the algorithm such that anysample with an ORF lab CT value less than 23 automatically received a positive “SMART result”, and any sample with an ORF lab CT value greater than 35 automatically received a negative “SMART result” (Table 6). Whereas for the 134 specimens analyzed with ORFlab CT values between 23-35, there was a mixture of samples that grew and those that did not grow in culture. We applied the SMART score calculation, followed by the threshold value of 523,823, to determine the “SMART result” for this last set of samples. The tabulated SMART results from the entire sample set, containing positive ORFlab CT values, were then compared to the observed culture results (Table 7). Findings demonstrated the SMART result to have a sensitivity and specificity of 95% and 60% respectively, and a positive and negative predictive value of 74% and 92% respectively.

[0159] Table 6: SMART Algorithm

[0160] Table 7: Comparison of SMART algorithm with culture on clinical specimens

[0161]

[0162] Establishment of the SMART score threshold

[0163] In order to determine the efficacy of using the ratio of subgenomic and genomic RNA in predicting culture positivity, we generated a series of ten trainer sets from eachconsisting of calculated SMART scores from 30 culture positive and 30 culture negative samples, randomly selected from the 355 sample dataset. A box and whiskers plot was constructed for each trainer set (Figure 8). The lower 25thpercentile value from each culture negative trainer set plot was averaged to determine the SMART score cutoff value. A SMART score of 1,219,173 was determined as the assay threshold. This value was used such that any sample with a higher SMART score was considered positive.

[0164] Discussion

[0165] High negative and positive predictive values (NPV and PPV) for infection with replication SARS-CoV-2 are desirable and are obtained by a method as disclosed herein, with the results. Disclosed herein is an assay, termed SMART, that combines specific molecular assays for subgenomic SARS-CoV-2 transcript, genomic RNA and a cellular control gene, together with calculations to generate a score that assesses the likelihood that the patient is still generating replicative virus.

[0166] When applied to an initial set of 355 patient samples, the assay identified those that were infectious, as determined by culture positivity, with a high level of sensitivity and NPV, although specificity and PPV were lower. Given the current dilemma with identifying these patients, this is a reasonable level of accuracy.

[0167] Table 5: Specificity of SMART genomic (ORF lab) and subgenomic (sgN) real- time RT-PCR assays against multiple DNA amplicons representing SARS-CoV-2 RNA targets.SMART specificity was also compared to that of published real-time RT-PCR assays targeting the SARS-CoV-2 E-gene (Sarbeco) and subgenomic E gene (sgE) RNA.aReal-time RT-PCR detection assay for SARS-CoV-2 E gene (Corman et al, 2020)bsgE real-time RT-PCR assay using a leader-specific forward primer (Wolfel et al, 2020), as well as a probe and reverse primer identical to those published in Corman et al 2020, positioned downstream of the E gene start codon.

[0168] EXAMPLE 3 : Digital PCR

[0169] Digital PCR was performed used the same assay disclosed herein for detection by real-time quantitative PCR.. The PCR amplification primers and probes had the sequences of those used for amplification and detection of ORF lab and sgN RNA (SEQ ID Nos: 1-6). The positive controls were copy -number-validated, single strand RNA. These were generated by PCR from positive patient samples, using the same primers as used in the assay. The PCR product was then cloned into a PCR4 Topo vector, the sense strand RNA synthesized with a T7 RNA polymerase. Results of curves showing detected copies as a measure of expected for ORF lab and sgN RNA transcripts are shown in FIGs. 12A and 12B, respectively. Linear curves reflect the sensitivity and accuracy of measurement of genomic and subgenomic copy number in patient samples for use in digital PCR, for detection of active, replicating virus in patient samples as distinguishable from presence of polynucleotides from non-replicating virus.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting SARS-CoV-2 in a sample, comprising: amplifying polynucleotides in the sample to form subgenomic amplicons using a forward subgenomic primer and a reverse subgenomic primer, wherein the forward subgenomic primer hybridizes to a forward subgenomic primer target of a subgenomic SARS-CoV-2 N protein transcript or its complement and the reverse subgenomic primer hybridizes to a reverse subgenomic primer target of the subgenomic SARS-CoV-2 transcript or its complement, and detecting an amount of hybridization of a subgenomic SARS-CoV-2 transcript probe to the subgenomic amplicons, wherein the subgenomic SARS-CoV-2 transcript probe hybridizes to a probe target of the subgenomic amplicons, wherein the probe target of the subgenomic amplicons comprises at least a portion of a leader sequence, a transcriptional regulatory sequence, and at least a portion of a junction region between the transcriptional regulatory sequence and a coding sequence of the subgenomic SARS-CoV-2 transcript and the probe target is between the forward subgenomic primer target and the reverse subgenomic primer target.

2. The method of claim 1, wherein the forward subgenomic primer target is in a leader sequence of the subgenomic SARS-CoV-2 transcript and the reverse subgenomic primer target is in a coding sequence of the subgenomic SARS-CoV-2 transcript.

3. The method of claim 1 or claim 2, wherein the forward subgenomic primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides.

4. The method of any one of claims 1 through 3, wherein the reverse subgenomic primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides.

5. The method of any one of claims 1 through 4, wherein the forward subgenomic primer comprises a length having a range of from about 10 nucleotides to about 30nucleotides and the reverse subgenomic primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides.

6. The method of any one of claims 1 through 5, wherein a sequence of the forward subgenomic primer comprises a polynucleotide sequence as set forth in 4.

7. The method of claim 6, wherein the sequence of the forward subgenomic primer consists of the polynucleotide sequence as set forth in 4.

8. The method of any one of claims 1 through 7, wherein a sequence of the reverse subgenomic primer comprises a polynucleotide sequence as set forth in 5.

9. The method of claim 8, wherein the sequence of the reverse subgenomic primer consists of the polynucleotide sequence as set forth in 5.

10. The method of any one of claims 1 through 9, wherein a sequence of the forward subgenomic primer comprises a polynucleotide sequence as set forth in 4 and a sequence of the reverse subgenomic primer comprises a polynucleotide sequence as set forth in 5.

11. The method of claim 10, wherein the sequence of the forward subgenomic primer consists of the polynucleotide sequence as set forth in 4 and the reverse subgenomic primer consists of the polynucleotide sequence as set forth in 5.

12. The method of any one of claims 1 through 11, wherein the subgenomic SARS-CoV-2 transcript probe comprises a length having a range of from between about 8 nucleotides to about 50 nucleotides.

13. The method of any one of claims 1 through 12, wherein a sequence of the subgenomic SARS-CoV-2 probe comprises a polynucleotide sequence as set forth in 6.

14. The method of any one of claims 1 through 13, wherein the sequence of the subgenomic SARS-CoV-2 probe comprises a polynucleotide sequence as set forth in 6, wherein nucleotides 3, 11, 15, 16, 20, and 21 comprise locked nucleic acids.

15. The method of any one of claims 1 through 14, where the subgenomic SARS-CoV-2 probe comprises a subgenomic SARS-CoV-2 probe dye.

16. The method of any one of claims 1 through 15, where the subgenomic SARS-CoV-2 probe comprises a subgenomic SARS-CoV-2 probe quencher.

17. The method of any one of claims 1 through 16, where the subgenomic SARS-CoV-2 probe comprises a subgenomic SARS-CoV-2 probe dye and a subgenomic SARS- CoV-2 probe quencher.

18. The method of any one of claims 1 through 17, wherein the amplifying further comprises forming genomic amplicons using a forward genomic primer and a reverse genomic primer, wherein the forward genomic primer hybridizes to a forward genomic primer target of SARS-CoV-2 genome or its complement and the reverse genomic primer hybridizes to a reverse genomic primer target of the SARS-CoV-2 genome or its complement, and further comprising detecting an amount of hybridization of a genomic SARS-CoV-2 probe to the genomic amplicons, and determining a difference between the amount of hybridization of the probe for the subgenomic SARS-CoV-2 transcript to the amount of hybridization of the probe to the SARS- CoV-2 genome.

19. The method of 18, wherein the SARS-CoV-2 genome target comprises at least a portion of one or both of an ORF la genome region and an ORF lb genome region.

20. The method of claim 18 or 19, wherein at least a portion of one or both of an ORF la genome region and an ORF lb genome region comprises one or both of the forwardgenomic primer target of the SARS-CoV-2 genome and the reverse genomic primer target of the SARS-CoV-2 genome.

21. The method of any one of claims 18 through 20, wherein the forward genomic primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides.

22. The method of any one of claims 19 through 22, wherein the reverse genomic primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides.

23. The method of any one of claims 18 through 22, wherein the forward genomic primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides and the reverse genomic primer comprises a length having a range of from 10 nucleotides to 30 nucleotides.

24. The method of any one of claims 18 through 23, wherein a sequence of the forward genomic primer comprises a polynucleotide sequence as set forth in 1.

25. The method of claim 24, wherein the sequence of the forward genomic primer consists of the polynucleotide sequence as set forth in 1.

26. The method of any one of claims 18 through 25, wherein a sequence of the reverse genomic primer comprises a polynucleotide sequence as set forth in 2.

27. The method of claim 26, wherein the sequence of the reverse genomic primer consists of the polynucleotide sequence as set forth in 2.

28. The method of any one of claims 18 through 27, wherein a sequence of the forward genomic primer comprises a polynucleotide sequence as set forth in 1 and a sequence of the reverse genomic primer comprises a polynucleotide sequence as set forth in 2.

29. The method of claim 28, wherein the sequence of the forward genomic primer consists of the polynucleotide sequence as set forth in 1 and the reverse genomic primer consists of the polynucleotide sequence as set forth in 2.

30. The method of any one of claims 18 through 29, wherein the genomic SARS-CoV-2 probe comprises a length having a range of from between about 8 nucleotides to about 50 nucleotides.

31. The method of any one of claims 19 through 30, wherein a sequence of the genomic SARS-CoV-2 probe comprises a polynucleotide sequence as set forth in 3.

32. The method of any one of claims 18 through 31, where the genomic SARS-CoV-2 probe comprises a genomic SARS-CoV-2 probe dye.

33. The method of any one of claims 18 through 32, where the genomic SARS-CoV-2 probe comprises a genomic SARS-CoV-2 probe quencher.

34. The method of any one of claims 18 through 33, where the genomic SARS-CoV-2 probe comprises a genomic SARS-CoV-2 probe dye and a genomic SARS-CoV-2 probe quencher.

35. The method of any one of claims 1 through 34, wherein the amplifying further comprises forming control amplicons using a forward control primer and a reverse control primer, wherein the forward control primer hybridizes to a forward control primer target of a control transcript or its complement and the reverse control primer hybridizes to a reverse genomic primer target of the control transcript or its complement, and further comprising detecting an amount of hybridization of a control transcript probe to the control amplicons.

36. The method of claim 35, wherein the control transcript is a housekeeping gene transcript.

37. The method of claim 35, wherein the control transcript is human RNAse P transcript.

38. The method of any one of claims 35 through 37, wherein the forward control primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides.

39. The method of any one of claims 35 through 38 wherein the reverse control primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides.

40. The method of any one of claims 35 through 39, wherein the forward control primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides and the reverse control primer comprises a length having a range of from about 10 nucleotides to about 30 nucleotides.

41. The method of any one of claims 35 through 40, wherein a sequence of the forward control primer comprises a polynucleotide sequence as set forth in 742. The method of claim 41, wherein the sequence of the forward control primer consists of the polynucleotide sequence as set forth in 7.

43. The method of any one of claims 35 through 42, wherein a sequence of the reverse control primer comprises a polynucleotide sequence as set forth in 8.

44. The method of claim 43, wherein the sequence of the reverse control primer consists of the polynucleotide sequence as set forth in 8.

45. The method of any one of claims 35 through 44, wherein a sequence of the forward control primer comprises a polynucleotide sequence as set forth in7 and a sequence of the reverse control primer comprises a polynucleotide sequence as set forth in 8.

46. The method of claim 45, wherein the sequence of the forward control primer consists of the polynucleotide sequence as set forth in 7 and the reverse control primer consists of the polynucleotide sequence as set forth in 8.

47. The method of any one of claims 35 through 46, wherein the control probe comprises a length having a range of from between about 8 nucleotides to about 50 nucleotides.

48. The method of any one of claims 35 through 47, wherein a sequence of the control probe comprises a polynucleotide sequence as set forth in 9.

49. The method of any one of claims 35 through 48, where the control probe comprises a control probe dye.

50. The method of any one of claims 35 through 49, where the control probe comprises a control probe quencher.

51. The method of any one of claims 35 through 50, where the control probe comprises a control probe dye and a control probe quencher.

52. The method of any one of claims 1 through 51, wherein one or more nucleotide of one or more of the subgenomic SARS-CoV-2 probe, the genomic SARS-CoV-2 probe, and the control probe, comprises a modified nucleotide.

53. The method of any one of claims 1 through 52, wherein one or more nucleotide of the subgenomic SARS-CoV-2 probe comprises a modified nucleotide.

54. The method of claim 53, wherein one or more modified nucleotide comprises a locked nucleic acid.

55. A method of identifying replicating SARS-CoV-2 virus in a subject, comprising performing the method of any one of claims 18 through 54, wherein the sample comprises a sample from the subject, the identifying comprises detecting a relative amount of hybridization of the subgenomic SARS-CoV-2 transcript probe to the subgenomic amplicons relative to the amount of hybridization of the genomic SARS-CoV-2 transcript probe to the genomic amplicons, and the relative amount is at or above a predetermined threshold.

56. The method of claim 55, wherein the detecting comprises performing quantitative PCR and the threshold is calculated according to the following Formula I: relative amount = 2’ACt, whereinCt is cycle threshold, and ACt = Ct (subgenomic SARS-CoV-2 transcript) - Ct (genomic SARS-CoV-2 transcript).

57. The method of claim 56, wherein the detecting comprises performing quantitative PCR and the threshold is calculated according to the following Formula II: relative amount = 2’AACt, whereinCt is cycle threshold, and AACt = (Ct (subgenomic SARS-CoV-2 transcript) - Ct (genomic SARS-CoV-2 transcript)) - Ct (control transcript).

58. The method of any one of claims 55 through 57, wherein the threshold is about 0.5 million, or about 0.75 million, or about 1 million, or about 1.25 million, or about 1.5, or about 1.75 million, or about 2 million.

59. The method of any one of claims 55 through 58, wherein the subject is a human or a non-human primate.

60. The method of any one of claims 55 through 58, wherein the subject is non-human animal.

61. The method of claim 60, wherein the subject is a bat, a cervid, a caniformid, a feliformid, a manid, a mustelid, a horse, a cow, a goat, a sheep, a pig, or a rodent.

62. The method of any one of claims 1 through 61, wherein the sample is selected from a nasal swab, a nasopharyngeal swab, an oropharyngeal swab, a throat swab, a nasal secretion, saliva, mucous, throat secretion, a lower respiratory specimen, whole blood, blood component, tissues, plasma, serum, stool, and urine.

63. A method of treating the subject of any one of claims 55 through 62 when the level is at or above the threshold, comprising administering a treatment for SARS-CoV-2 to the subject.

64. The method of claim 63, wherein the treatment comprises an anti-SARS- CoV-2 antibody, an antiviral composition, SARS-CoV-2 convalescent plasma, or any combination of two or more of the foregoing.

65. The method of claim 64, wherein the treatment comprises an anti-SARS- CoV-2 antibody.

66. The method of claim 65, wherein the treatment comprises an antiviral composition.

67. The method of any one of claims 55 through 66, further comprising selecting the subject for a transmission prevention.

68. The method of claim 67, wherein the transmission prevention is selected from mask-wearing, face shield-wearing, social distancing, quarantining, social isolation, environmental decontamination procedures, or any two or more of any of the foregoing69. A kit comprising one or more of the forward subgenomic primer, the reverse subgenomic primer, and the subgenomic SARS-CoV-2 transcript probe of any one of claims 1 through 54.

70. The kit of claim 69, further comprising one or more of the forward genomic primer, the reverse genomic primer, and the genomic SARS-CoV-2 transcript primer of any one of claims 18 through 54.

71. The kit of claim 69 or 70, further comprising one or more of the forward control primer, the reverse control primer, and the control transcript primer of any one of claim 35 through 54.

72. The kit of any one of claims 69 through 71, further comprising one or more of a subgenomic transcript positive control, a subgenomic transcript negative control, a genomic transcript positive control, a genomic transcript negative control, a control transcript positive control, and a control transcript negative control.

73. The kit of any one of claims 69 through 72, further comprising one or both of a replicating SARS-CoV-2 virus negative control and a replicating SARS-CoV-2 virus positive control.

74. The kit of any one of claims 69 through 73, further comprising one or more of a subgenomic probe control, a genomic probe control, and a control probe control.

75. The kit of any one of claims 69 through 74, further comprising one or more additional reagents for use in quantitative polymerase chain reaction.

Images