METHOD FOR DETECTING GENETIC EVENTS

Abstract

The present invention relates to a method of detecting a genetic event, the method comprising steps of partitioning a sample from a subject into a plurality of partitions, and carrying out a digital polymerase chain reaction (dPCR) assay, to determine occurrence of said genetic event.

Claims

1-15. (canceled)

16. A method of detecting a genetic event, the method comprising the steps of: a) partitioning a sample from a subject into a plurality of partitions, wherein said partitions comprise an amplification mixture comprising: i. a polymerase; ii. at least two pairs of nucleic acid primers, each pair of nucleic acid primers being capable of hybridizing to a respective nucleic acid target of the genetic event; and iii. at least two nucleic acid probes, each nucleic acid probe being capable of hybridizing to a respective nucleic acid target of the genetic event, and being attached or linked to a different detectable fluorescent label, the different detectable fluorescent labels having non-overlapping excitation wavelength ranges, non-overlapping emission wavelength ranges, or a combination thereof; b) performing a digital polymerase chain reaction (dPCR) assay on the plurality of partitions under conditions suitable to amplify each nucleic acid target of the genetic event; c) providing a conclusion regarding the presence of said genetic event based on whether fluorescence in at least one partition of the plurality of partitions is detected or not, wherein fluorescence in a given partition is indicative of amplification of at least one nucleic acid target of the genetic event in said given partition; wherein the at least two nucleic acid targets are located, upon spontaneous fragmentation, artificial fragmentation, or a combination thereof of nucleic acids in the sample, on separate fragments of said nucleic acids, thereby statistically behaving independently from each other during partitioning of the sample into a plurality of partitions, and therefore behaving independently from each other in the dPCR assay, the genetic event being a monosomy, a trisomy or a tetrasomy.

17. The method according to claim 16, further comprising a step of computing, for each nucleic acid target for which corresponding fluorescence is detected, a respective concentration, and wherein step c) comprises concluding that said genetic event is present when fluorescence is detected in at least one partition of the plurality of partitions and when, for each nucleic acid target for which corresponding fluorescence is detected, the respective concentration is outside a corresponding predetermined concentration range.

18. The method according to claim 16, wherein step c) comprises concluding that the genetic event is present when fluorescence is not detected in the plurality of partitions.

19. The method according to claim 16, wherein each nucleic acid target of the genetic event is an RNA target, and the method comprises a step of reverse-transcribing the RNA target, thereby obtaining at least one reverse-transcribed cDNA target.

20. The method according to claim 19, wherein the step of reverse-transcribing is carried out after step a).

21. The method according to claim 16, wherein the spontaneous fragmentation, the artificial fragmentation, or the combination thereof of the nucleic acids occurs in the sample, before step a) of partitioning the sample into a plurality of partitions.

22. The method according to claim 16, comprising, before step a), a step of artificially fragmentating nucleic acids in the sample, in conditions suitable to have the at least two nucleic acid targets located on separate fragments of said nucleic acids, thereby statistically behaving independently from each other during the partitioning of the sample into a plurality of partitions at step a).

23. The method according to claim 22, wherein artificially fragmentating nucleic acids is carried out by contacting the sample with at least one sequence-specific endonuclease.

24. The method according to claim 23, wherein the at least one sequence-specific endonuclease is selected from sequence-specific endonucleases capable of cleaving the nucleic acids between the at least two nucleic acid targets, thereby having the at least two nucleic acid targets located on separate fragments of said nucleic acids upon artificial fragmentation and statistically behaving independently from each other during the partitioning of the sample into a plurality of partitions at step a).

25. The method according to claim 16, wherein the genetic event is at least one of: Down syndrome 21, Edwards syndrome 18, Patau syndrome 13, trisomy 9, tetrasomy 9p, Warkany syndrome 2, cat eye syndrome, trisomy 22, trisomy 16, 1q21.1 deletion syndrome, 1q21.1 duplication syndrome, TAR syndrome, 1p36 deletion syndrome, Wolf-Hirschhorn syndrome, cri du chat syndrome, chromosome 5q deletion syndrome, Williams syndrome, Jacobsen syndrome, Miller-Dieker syndrome, Smith-Magenis syndrome, DiGeorge syndrome, 22q11.2 distal deletion syndrome, 22q13 deletion syndrome, Angelman syndrome, Prader-Willi syndrome, distal 18q-, proximal 18q-, Turner syndrome, Klinefelter syndrome, XXYY syndrome, XXXY syndrome, 49XXXYY syndrome, 49XXXXY syndrome, triple X syndrome, tetrasomy X, 49XXXXX, Jacobs syndrome, 48XYYY, 49XYYYY, 45X/46XY, 46XX/46XY.

26. A non-invasive prenatal testing method, comprising performing the steps of the method according to claim 16, the sample being a blood sample from a pregnant female subject carrying an embryo or a fetus, and wherein step c) is a step of providing a conclusion regarding the presence of said genetic event in the fetus of the embryo based on whether fluorescence in at least one partition of the plurality of partitions is detected or not.

27. A system for performing the method according to claim 16, the system comprising: at least one container suitable for storing a sample from a subject; a subsystem and reactants for performing a digital PCR assay, said reactants including an amplification mixture comprising: i. a polymerase; ii. at least two pairs of nucleic acid primers, each pair of nucleic acid primers being capable of hybridizing to a respective nucleic acid target of the genetic event; and iii. at least two nucleic acid probes, each nucleic acid probe being capable of hybridizing to a respective nucleic acid target of the genetic event, and being attached or linked to a different detectable fluorescent label, the different detectable fluorescent labels having non-overlapping excitation wavelength ranges, non-overlapping emission wavelength ranges, or a combination thereof; a subsystem for detecting a fluorescence signal; the system optionally further comprising at least one of: a subsystem and reactants for extracting nucleic acids; a subsystem and reactants for artificially fragmenting nucleic acids; pipetting means.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0290] FIG. 1 is a set of two graphs showing the sensitivity of the Covid-19 digital PCR detection kit for SARS-CoV-2 detection. Serial dilutions of SARS-CoV-2 synthetic targets were assayed in triplicates in a background of 1 ng of human RNA. A total of 8 .Math.L of positive controls was added to each 25 .Math.L reaction. The vertical bars represent the theoretical 95% Poisson confidence intervals for the pools of 3 replicates. Empty circles represent replicates where SARS-CoV-2 sequences were not detected. Upper graph: copies (cp) of SARS-CoV-2 N gene/.Math.L of positive control; lower graph: copies (cp) of SARS-CoV-2 ORFlab gene/.Math.L of positive control.

[0291] FIG. 2 is a flowchart illustrating the structure of the comparative study of Example 4.

[0292] FIG. 3 is a histogram showing the distribution of Ct values for the E gene and Orf gene, as measured using individual reference RT-PCR with COBAS® 6800, for the 25 positive samples.

[0293] FIG. 4 is a set of two graphs showing the number of positive droplets for the N gene (FIG. 4A) and ORFlab gene (FIG. 4B) per reaction versus the predicted equivalent Ct value of the N gene and the ORF lab gene, respectively.

[0294] FIG. 5 is a workflow illustrating a complete, automatable process for sensitive pooled testing based on the method of the invention.

EXAMPLES

[0295] The present invention is further illustrated by the following examples. The examples provided hereinafter relate to the detection of Sars-CoV-2 in a population of several subjects. However, the person skilled in the art understands that these teachings can be directly applied, without changes, to the detection of a genetic event in a single subject, i.e., without pooling samples from a population of several subjects.

Example 1

Covid-19 Digital PCR Detection Kit

[0296] The kit contains a specific ready-to-use system for detection of Sars-CoV-2 by one-step reverse transcription PCR using a digital PCR platform. A master mix contains mix for the specific amplification of virus RNA. The kit uses primers and probes targeting the SARS-CoV-2 ORFlab and N genes, which are labeled with HEX and FAM fluorescence, respectively, and all other necessary detection reagents to prepare the reactions. After pressurization of the digital PCR system, the reaction solution is dispersed into 25000-30000 droplets in a well of the digital PCR chip. When a droplet contains the RNA of SARS-CoV-2, the corresponding primers and probes will pair with the nucleic acids, and reverse transcription (with a qScript XLT reverse transcriptase) followed by PCR amplification (with an AccuStart II Taq DNA polymerase) can occur and generate a fluorescence signal. In addition, the kit contains a set of primers and probes complementary to an internal human control (IC) and labeled with the CY5 fluorophore to identify possible PCR inhibition.

Assay Procedure

[0297] The naica® System (Stilla Technologies) with a Sapphire Chip was used to partition the samples and carry out amplification, with the program described in Table 3.

TABLE-US-00003 Step Temp (°C) Time Cycle 1 Partition 40° C. 12 min 1 2 cDNA synthesis 50° C. 10 min 1 3 Initial denaturation 95° C. 1 min 1 4 Denaturation 95° C. 10 sec 45 5 Annealing/Extension 55° C. 30 sec 6 Release / 33 min /

Results Analysis

[0298] Using a three-channel digital PCR system (naica® System, Stilla Technologies), the chips were moved after PCR to the naica® Prism3 device and results were analyzed using the “Crystal reader” software.

[0299] Results could be determined using the matrix described in Table 4 below.

TABLE-US-00004 Sample Negative control Result Channel 1 (FAM) Channel 2 (HEX) Channel 3 (CY5) N gene (FAM) ORF1ab gene (HEX) N gene ORF1ab gene Internal Control + + + - or ND Positive + + < 10 - or ND If one target is + and one target is ND + - or ND If one target is + and one target is ND < 10 - or ND Inconclusive ND ND + - Positive ND ND + If only one or if two targets, is ND (no +) Inconclusive If one target is ND and one target is - + If only one or if two targets, is ND (no +) If one target is ND and one target is - + - - + - or ND Negative - or ND - or ND < 10 - or ND Inconclusive If one target is + and one target is - < 10 or + - or ND Negative Any Any Any + Invalid “+” means the number of positive droplets ≥ 3 “ND” means 1 or 2 positive droplets “-” means 0 positive droplets “Positive” means that SARS-Co V-2 was detected in the sample “Negative” means that no SARS-CoV-2 was detected in the sample (because absent or below the detection limit).

Assay Performance

Limit of Detection (LoD) - Analytical Sensitivity

[0300] A seven-fold serial dilution of a positive control containing synthetic RNA sequences targeted by the ORF1ab and N genes was assayed in triplicates. The limit of detection was defined as the lowest number of copies per .Math.L in the 10.5 .Math.L of RNA template of positive control detected in a sample in all replicates.

[0301] The limit of detection at a 95% confidence level was 480 copies/mL in 10.5 .Math.L of input RNA.

Inclusivity (analytical Sensitivity)

[0302] The complete genome sequence corresponding to the SARS-CoV-2 isolate Wuhan-Hu-1 with NCBI Reference Sequence NC_045512.2 was aligned to the Betacoronavirus sequences of the NCBI database using Megablast optimized for highly similar sequences. A total of 96 complete genome sequences of SARS-CoV-2 with > 99% percentage of identity were retrieved. Primers and probes targeting the ORF1ab and N genes were aligned to the complete genome sequences using MAFFT version 7 and displayed 100% homology in 100% of their length.

Cross-Reactivity (Analytical Specificity)

[0303] In silico study: the amplicons generated by primers targeting the ORF1ab and N genes were aligned to the sequence of the SARS coronavirus Frankfurt 1 strain (GenBank: AY291315.1) and the closely related bat SARS-like coronavirus isolate bat-SL-CoVZC45 (GenBank: MG772933.1). The ORF1ab detection model displayed 5 and 6 mismatches with the SARS coronavirus Frankfurt 1 strain and bat-SL-CoVZC45 sequence, respectively. The N detection model displayed 8 and 14 mismatches with the SARS coronavirus Frankfurt 1 strain and bat-SL-CoVZC45 sequence, respectively.

[0304] In vitro study: A total of 15 replicates containing 1 .Math.L of a human total RNA solution at 1 ng/.Math.L were analyzed using the kit. No false positive droplets were measured in all negative control replicates, so no false positives were detected for the ORF1ab or N gene.

[0305] The sequenced strains of the SARS-CoV-2 virus can be detected, and no crossover with human coronaviruses HCoV-229E, HCoV-HKU1, HCoV-OC43 or HCoV-NL63 was observed.

Example 2

qPCR Versus Digital PCR

[0306] Samples from 44 patients tested for Covid19 by qPCR at the institute hospital-universitaire (IHU) “Mediterranee Infection” (Marseille, France) were retrieved and tested by digital PCR using the Covid-19 digital PCR detection kit of Example 1.

[0307] Results initially obtained by qPCR are as shown in Table 5 below.

TABLE-US-00005 Result Number of patients Negative 14 Positive 12 Inconclusive (Ct > 35) 18

[0308] As can be seen, out of 44 patients, the cycle threshold (Ct) was above 35 cycles for 40% of them (18/44), and their results were considered inconclusive.

Digital PCR to Confirm Negative qPCR Results

[0309] The samples from the 14 patients determined to be negative by qPCR were tested by digital PCR using the Covid-19 digital PCR detection kit of Example 1. Results are shown in Table 6 below.

TABLE-US-00006 Sample N gene (FAM) ORF1ab gene (HEX) IC (CY5) C (cp/uL) Nbr Pos C (cp/uL) Nbr Pos C (cp/uL) Nbr Pos #1 0 0 0 0 8.36 101 #2 0 0 0 0 34.2 469 #3 0 0 0 0 21 277 #4 0 0 0 0 5.6 56 #5 0 0 0 0 13.5 129 #6 0 0 0 0 13.3 81 #7 0 0 0.09 1 37 390 #8 0 0 0 0 22.8 214 #9 0 0 0 0 100.4 1024 #10 0.1 1 0 0 37.5 384 #11 0 0 0 0 42.8 486 #12 0 0 0 0 11.6 142 #13 0.86 10 0.6 7 21.2 244 #14 0 0 0.09 1 125.2 1337 C (cp/uL): copy number concentration Nbr Pos: number of positive droplets

[0310] As can be seen, one patient (#13) tested negative by qPCR was clearly tested positive using the Covid-19 digital PCR detection kit and the matrix described in Table 2 of Example 1.

Digital PCR to Confirm Positive qPCR Results

[0311] Similarly, the samples from the 12 patients determined to be positive by qPCR were tested by digital PCR using the Covid-19 digital PCR detection kit of Example 1.

[0312] All 12 patients were confirmed as positive.

Digital PCR to Determine the Status of Inconclusive Results

[0313] Finally, the samples from 18 patients with inconclusive results were tested by digital PCR using the Covid-19 digital PCR detection kit of Example 1. Results are shown in Table 7 below.

TABLE-US-00007 Sample N gene (FAM) ORF1ab gene (HEX) IC (CY5) Results Ct value in qPCR C (cp/uL) Nbr Pos C (cp/uL) Nbr Pos C (cp/uL) Nbr Pos #27 0 0 0 0 11 132 Negative 37.32 #28 0.7 8 0.44 5 165.4 1806 Positive 35.53 #29 0.26 3 0.09 1 35.2 406 Positive 35.15 #30 0.09 1 0.18 2 77.6 849 Negative 34.65 #31 0 0 0 0 73.4 785 Negative 36.92 #32 0.08 1 0 0 6.72 80 Negative 36.56 #33 0.45 5 0.18 2 1047 8723 Positive 36.75 #34 0.08 1 0.62 8 23.4 298 Positive 35.17 #35 0.17 2 0.26 3 742.3 7057 Positive 34.37 #36 0.31 4 0 0 30.2 387 Positive 37.43 #37 0.16 2 0.39 5 11.9 151 Positive 34.95 #38 0.16 2 0.16 2 0.78 10 Negative 38.17 #39 2.47 34 1.96 27 25.8 352 Positive 33.78 #40 0.22 3 0.44 6 46.9 636 Positive 36.35 #41 0 0 0.16 2 55.5 703 Negative 36.66 #42 0.53 7 0.68 9 48.9 637 Positive 34.3 #43 0 0 0 0 160.9 2206 Negative 36.96 #44 0.2 3 0.27 4 126.3 1801 Positive 36.47

[0314] Viral absence was confirmed in 7/18 patients and viral presence was confirmed in 11/18 patients.

[0315] A retrospective analysis of the Ct values obtained by qPCR (last column of Table 7) shows no correlation between the result and the Ct value, confirming the limits of qPCR.

Conclusion

[0316] In conclusion, the test by digital PCR of the present invention offers a substantial gain of sensibility as compared to the commonly used qPCR tests, and allows to investigate Covid 19 serology of qPCR data > 35 Ct. A statistical analysis of these results allowed to determine a gain of sensitivity between about 32 and 64 times as compared to qPCR.

[0317] Moreover, a statistical analysis of the results obtained in this assay confirmed that each droplet in the digital PCR assay contained in average no more than a single nucleic acid target.

Example 3

Pooled Testing

[0318] An experimental model containing synthetic sequences targeted by the Covid-19 digital PCR detection kit of Example 1 was serially diluted and seven dilution points were assessed in triplicate. A total of 1 ng of human RNA was added to each replicate.

[0319] The results shown in FIG. 1 indicate a robust and specific detection of SARS-CoV-2 sequences down to 0.6 copies (cp)/.Math.L of positive control (5 cp/25 .Math.L, reaction) of the N gene and down to 0.9 cp/.Math.L of positive control (7 cp/25 .Math.L reaction) of the ORF1ab gene in all tested samples (dilution factor 16).

[0320] Further dilutions showed an extremely sensitive but stochastic detection down to 0.25 cp/.Math.L of positive control (2 cp/25 .Math.L reaction) for both genes (dilution factor up to 64).

[0321] In parallel, a total of 15 controls containing only human RNA (1 ng/25 .Math.L reaction) were tested as negative controls, and no false positives were observed.

[0322] These results indicate that the Covid-19 digital PCR detection kit of Example 1 is suitable for performing a “large pool” testing approach, by pooling and testing samples from a number of individuals in a single digital PCR assay, with a sensitive detection.

[0323] These results are in line with the results of Example 2, which allowed to determine a gain of sensitivity between about 32 and 64 times as compared to qPCR; hence it is conceivable that the Covid-19 digital PCR detection kit of Example 1 is suitable for testing a pooled sample comprising between 32 to 64 individual samples, in a single digital PCR assay, with at least the same sensitivity as qPCR.

[0324] This sensitivity and the precision can further be improved by using more targets on the viral RNA, since, as confirmed in Example 2, each droplet in the digital PCR assay contains in average no more than a single nucleic acid target. This is due to the fact that viral RNA is spontaneously cleaved during processing of the sample (in between 5 to 10 pieces), and that targets are chosen in such a way that they are sufficiently distant from each other to be on different pieces of the cleaved viral RNA.

[0325] Using more targets on the viral RNA, it is then conceivable, with a three-channel digital PCR system, such as the naica® System (Stilla Technologies), to dedicate the third channel to a viral target instead of an internal control (since a pooled sample of several individual samples will statistically always be positive for the internal control).

[0326] Additionally or alternatively, it is also conceivable to test more than one target in each channel; and/or to use a digital PCR system with more than 3 channels.

Example 4

Pooled Testing

Material and Methods

Specimens Collection, Storage and Pooling

[0327] Nasopharyngeal swabs of 448 patients screened for Covid-19 in a Parisian hospital (France) between May 6.sup.th and May 26.sup.th, 2020 were included. All samples were collected in universal transport medium (UTM) (Virocult® or eSwabTM) and tested, within 15 hours maximum upon collection, for SARS-CoV-2 detection according to the local standards. Briefly, 400 .Math.L of transport medium were tested by real-time Retro-Transcriptase Polymerase Chain Reaction (RT-PCR) (Cobas SARS-CoV-2 test, Roche). All the remaining volume of transport medium of all specimens were kept at +5° C.

[0328] No later than 24 hours after routine screening, all samples with a leftover UTM volume above 600 .Math.L were systematically included in the grouped analysis. Thus, 125 .Math.L of each included specimen were sampled and randomly mixed with seven others to generate a total of 56 pools of 8 specimens with a final volume of 1 mL per pool. Nucleic acids were extracted from each pool prior to viral titration by digital Retro-Transcriptase Polymerase Chain Reaction (RT-dPCR). The remaining volume of transport medium were stored at +5° C.

[0329] According to the current French ethical laws, samples used in the current study were only included after the completion of all analysis required for the patient’s care.

Detection of SARS-CoV-2 by Routine Individual RT-PCR Testing

[0330] All 448 specimens were analyzed individually on a Cobas® 6800 system (Roche, Switzerland) for Covid-19 screening using the Cobas® SARS-CoV-2 Test kit, which targets conserved regions for ORF-1a/b and E-gene, according to the manufacturer’s recommendations. Briefly, for each specimen 400 .Math.L of transport medium were mixed with 400 .Math.L of Cobas® lysis buffer and loaded on the analyzer. During the run, extracts were eluted in 50 .Math.L of which 27 .Math.L were used in the RT-PCR amplification of E and ORF1ab. A sample was considered positive for routine screening of Covid-19 (“COBAS +”) if either target had a Ct value below 40 PCR cycles.

[0331] All samples which had different results for RT-PCR and RT-dPCR were reassessed on the Cobas® 6800 system. In case of low remaining amounts of transport medium, the nasal swabs were vortexed again into the remaining transport medium diluted 1 to 10 with new transport medium.

Nucleic Acid Extraction

[0332] All nucleic acids extractions for droplet PCR assays were performed on a MagNA Pure LC 2.0 (Roche, Switzerland) using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche, Switzerland) following manufacturer’s instructions.

[0333] For all sample pools, the total pool volume of 1 mL was used for the extraction. For individual samples, 200 .Math.L was diluted with 800 .Math.L of buffer before extraction.

[0334] Nucleic acids were eluted from 1 mL to 50 .Math.L of the elution buffer provided with the kit and stored at +5° C. for a maximum of 12 hours before analysis.

Preparation of Pools of 16 and 32 Individuals

[0335] After co-extraction of the 56 pools of 8 specimen (P8 extracts) and prior to viral titration by RT-dPCR, 28 pools of 16 individual samples (P16 pools) were obtained by mixing 15 .Math.L of two P8 extracts and 14 pools of 32 (P32 pools) were obtained by mixing 10 .Math.L of two P16 pools.

Detection of SARS-CoV-2 by Pooled RT-dPCR Testing

[0336] SARS-CoV-2 titration of the pooled samples by RT-dPCR was performed on the naica® system (Stilla Technologies, France) within the next three hours after extraction, using the COVID-19 Multiplex Digital PCR Detection Kit (Stilla Technologies, France/Apexbio, China). This one-step reverse-transcription and triplex PCR kit amplifies one sequence in the N gene, one sequence in the ORF1ab region of SARS-CoV-2 and an endogenous internal control (IC) to assess the quality of the sample and extraction. These sequences are targeted by three TaqMan probes respectively labelled with a FAM, HEX and Cy®5 fluorophore.

[0337] As recommended by the kit manufacturer, the PCR mix for a single reaction contained 12.5 .Math.L of dPCR MasterMix 1, 1 .Math.L of dPCR Mix 2, 1 .Math.L of COVID-19 Assay and 10.5 .Math.L of either, P8, P16, P32, positive control, negative control or individual extract. 25 .Math.L of this PCR mix were loaded in the inlet ports of the Sapphire chips (Stilla Technologies, France) and the chips were placed in the naica® Geode (Stilla Technologies, France) for droplets generation, reverse transcription and PCR amplification following the kit manufacturer’s instructions.

[0338] After amplification, the chips were transferred to the naica® Prism3 (Stilla technologies, France) for fluorescence reading in the three detection channels and data were analyzed with Crystal Miner Software (Stilla Technologies, France) following the kit manufacturer’s instructions.

Individual Confirmatory Testing for SARS-CoV-2 by RT-PCR and RT-dPCR

[0339] When needed, a confirmatory RT-PCR test was performed on individual samples. 200 .Math.L of transport medium from samples diluted into 800 .Math.L of phosphate buffered saline (PBS) were extracted individually on the MagNAPure LC 2.0 instrument following the same protocol as before. Confirmatory RT-PCR testing was perform using the RealStar® SARS-CoV-2 RT-PCR Kit (Altona Diagnostics, Germany) on an ABI 7500 thermocycler (ThermoFisher Scientific, United-States). This kit targets all lineage B-betacoronaviruses by amplifying a sequence of the E gene and a sequence of the S gene specific to SARS-CoV-2 as well as a heterologous internal control. In this study, a sample is considered positive to SARS-CoV-2 if the Ct value of either target is below 40 PCR cycles. Confirmatory testing by RT-dPCR was performed following the same method as described above.

Summary of the Method of the Comparative Study

[0340] Overall, all samples from the cohort of 448 patients are tested for SARS-CoV-2 by [0341] i) individual RT-PCR (Cobas® 6800), [0342] ii) RT-dPCR in 56 pools of 8, [0343] iii) RT-dPCR in 28 pools of 16, and [0344] iv) RT-dPCR in 14 pools of 32; and results are compared between all four test protocols. In case of discordance between the results of individual RT-PCR testing and pooled testing in RT-dPCR, samples were re-analyzed individually by RT-dPCR and RT-PCR (Altona).

[0345] The protocol is illustrated in FIG. 2.

LoB/LoD Evaluation for SARS-CoV-2 Detection Using RT-dPCR

[0346] The Limit of Blank (LoB) and Limit of Dection (LoD) wer evaluated for SARS-CoV-2 detection using the pooling approach used in the study on a cohort of 256 pre-epidemic nasal swab samples (negative control samples) that were collected between Dec. 1, 2019 and Jan. 31, 2020 and for which transport medium was stored at -20° C.

[0347] Specimens were randomly pooled into 32 pools of 8 negative controls which were co-extracted and analyzed by RT-dPCR using the same protocol described above. The results for all 32 pools are given in Table 8.

TABLE-US-00008 Sample ID Number of droplets Positive droplets (N+ORFlab) Positive droplets (N) Positive droplets (ORFlab) IC (cp/.Math.L) Pool 1 19460 0 0 0 31430 Pool 2 20641 0 0 0 10985 Pool 3 21449 0 0 0 4054 Pool 4 20983 0 0 0 15143 Pool 5 19881 1 1 0 7401 Pool 6 20609 0 0 0 8783 Pool 7 19044 0 0 0 6249 Pool 8 20787 0 0 0 8759 Pool 9 21470 0 0 0 10271 Pool 10 18952 0 0 0 17578 Pool 11 23424 0 0 0 8222 Pool 12 22748 0 0 0 9875 Pool 13 23747 0 0 0 10275 Pool 14 24886 0 0 0 4266 Pool 15 24079 0 0 0 10264 Pool 16 24718 0 0 0 13219 Pool 17 24228 0 0 0 10186 Pool 18 23742 0 0 0 8431 Pool 19 23880 0 0 0 7628 Pool 20 23613 0 0 0 3111 Pool 21 25723 0 0 0 8987 Pool 22 25522 0 0 0 7411 Pool 23 24781 0 0 0 6894 Pool 24 7074 0 0 0 5884 Pool 25 27003 0 0 0 8911 Pool 26 27138 0 0 0 5552 Pool 27 24425 0 0 0 7886 Pool 28 27017 0 0 0 7484 Pool 29 26722 0 0 0 9963 Pool 30 26088 0 0 0 2492 Pool 31 26747 0 0 0 5703 Pool 32 26728 0 0 0 5825

[0348] The LoB at 95 % confidence level for the N target and the ORF1ab target is determined to be of 2 and 0 positive droplets respectively. The LoD at 95 % confidence level for each target is of 0.46 copies/.Math.L and 0.22 copies/.Math.L respectively.

[0349] Consequently, a threshold of a least 3 positive droplets in aggregate between both the N target and ORF1ab target is used to classify a sample as positive to SARS-CoV-2 by RT-dPCR in this study.

Results

Cohort Description From Routine RT-PCR Testing

[0350] Using routine RT-PCR testing, 25 samples were identified as positive out of the 448 samples tested, corresponding to an average test positivity rate of 5.5%. The positivity rate decreased during the study, in correlation with the decrease in the disease prevalence observed during the month of May in France. The positivity rate was of 8.5% for the first 224 samples and of 2.5% for the last 224 samples.

[0351] The average Ct value was of 30.0 and 27.3 for the E gene and ORF gene respectively, with minimum values of 16.5 and 16.3 and maximum values of 38.7 and 34.8 (FIG. 3).

Results From Pooled RT-dPCR Testing

[0352] Table 9 presents the overall results for the detection of SARS-CoV-2 by RT-dPCR for the pooled extracts (P8) and the subsequent pools of 16 (P16) and 32 (P32).

TABLE-US-00009 Number of “COBAS +” samples in pool Results forP8 extracts Results for P16 pools Results for P32 pools Total dPCR - dPCR + Total dPCR - dPCR + Total dPCR - dPCR + 0 35 32 3 12 11 1 2 2 0 1 18 1 17 10 2 8 6 0 6 2 2 0 2 4 0 4 3 0 3 3 1 0 1 1 0 1 1 0 1 4 0 0 0 1 0 1 1 0 1 5 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 1 0 1 Total 56 33 23 28 12 16 14 2 12

[0353] Because sample pooling was performed randomly as samples came in the laboratory for routing RT-PCR testing, the pools contain variable numbers of RT-PCR positive samples (“COBAS +” samples).

[0354] Given the positivity rate of RT-PCR at the time of the study, the majority of P8 extracts had no “COBAS +” samples (35 out of 56) and amongst the 23 pools that contained at least one “COBAS +” sample, 18 had one single “COBAS +” sample.

[0355] For the largest pool size of 32 samples, the distribution is opposite. Only two P32 pools have no “COBAS +” samples. Amongst the twelve P32 pools with at least one “COBAS +” sample, only 6 had one single “COBAS +” sample and the maximum number of “COBAS +” samples was 6.

[0356] These observed distributions are in good agreement with theoretical distributions predicted assuming a test positivity rate of 5.5% and random distribution.

Detailed Results for RT-dPCR in Groups of 8

[0357] The results for SARS-CoV-2 detection by RT-dPCR in groups of 8 are in concordance with the reference individual RT-PCR testing for 56 pools (corresponding for 448 samples), out of which 33 are pools with all samples negative by individual RT-PCR testing (“COBAS - pools”) and 23 are pools that contain at least one sample positive by individual RT-PCR testing (“COBAS + pools”).

[0358] 4 pools had discording results: [0359] 3 “COBAS pools” were found positive by RT-dPCR (“COBAS -/dPCR +” discordances): P8_20, P8_28 and P8_39; [0360] 1 “COBAS + pool” was found negative by RT-dPCR (“COBAS +/dPCR -” discordance): P8_02.

[0361] Out of the eight individual samples with Ct > 35 for the E gene, six ended up to be the only positive sample in a P8 extract. These six P8 extracts all tested positive by RT-dPCR, indicating that pooled testing by RT-dPCR is capable of detecting samples with late Ct values and low viral RNA concentrations. The highest detected Ct values for the E gene and ORF1ab is of [38.7; not detected].

[0362] The Ct values for the sample associated with the “COBAS +/dPCR -” discordance were of 34 and 32.3 for the E gene and ORF1ab respectively. This sample is referred to as “Sample_25659” for later discussion. “COBAS -/dPCR +” discordances are discussed further below.

[0363] Table 10 shows a confusion matrix for P8 extracts.

TABLE-US-00010 Expected negatives (RT-PCR) Expected positives (RT-PCR) Total Negatives in digital PCR 32 1 33 Positives in digital PCR 3 20 23 Total 35 21 56

Detailed Results for RT-dPCR in Groups of 16

[0364] The results for SARS-CoV-2 detection by RT-dPCR in pools of 16 are in concordance with individual RT-PCR testing for 25 pools (corresponding for 400 samples), out of which 11 were “COBAS - pools” and 14 were “COBAS + pools”.

[0365] 3 pools had discording results: [0366] 1 “COBAS -/dPCR +” discordance: P16_14; [0367] 2 “COBAS +/dPCR -” discordances: P16_13 and P16_28.

[0368] Out of the 8 individual samples with Ct > 35 for the E gene, 5 ended up to be the only positive sample in a P16 pool two out of these five P16 pools tested negative by RT-dPCR and were responsible for the 2 “COBAS +/dPCR -” discordances.

[0369] The E gene and OFR1ab Ct values for these two samples were of [36.7; not detected] and [36.3; 34.2], while the highest detected Ct values were [38.3; not detected].

[0370] Table 11 shows a confusion matrix for P16 pools.

TABLE-US-00011 Expected negatives (RT-PCR) Expected positives (RT-PCR) Total Negatives in digital PCR 11 2 13 Positives in digital PCR 1 14 15 Total 12 16 28

Detailed Results for RT-dPCR in Groups of 32

[0371] The results for SARS-CoV-2 detection by RT-dPCR in pools of 32 are in concordance with individual RT-PCR testing for all 14 pools (corresponding for 448 samples).

[0372] Out of the eight individual samples with Ct > 35 for the E gene, three ended up to be the only positive sample in a P32 pool. All such three P32 pools tested positive by RT-dPCR. The highest detected Ct values for the E gene and ORF1ab was of [36.7; not detected].

[0373] Surprisingly, the two samples that were associated with the 2 “COBAS +/dPCR -” discordances (with Ct values of [36.7; not detected] and [36.3; 34.2] respectively) were both amongst the three P32 pools discussed above. Consequently, these two samples were successfully detected by RT-dPCR in pools of 32 (P32_07 & P32_14) but not detected in the pools of 16 (P16_13 & P16_28).

[0374] In one case (P32_07), the pool of 32 was a combination of two P16 pools with opposite discordances: P16_13 (COBAS +/dPCR -) and P16_14 (COBAS -/dPCR +). It is likely that the RNA templates from the P16_14 pool were those detected in the P32_07 pool.

[0375] In the case of P32_14, the number of positive droplets observed in RT-dPCR was right above the threshold of three positive droplets (N=2; ORF1ab=1) while for the corresponding pool of 16 (P16_28), the number of positive droplets was just below with 2 droplets (N=2; ORF1ab=1). In this case, statistical variations due to sampling error could explain the observation.

[0376] However, the dataset for P32 pools was not large enough to draw reliable conclusions regarding the sensitivity of pooled testing with pools of 32, in particular because most pools contained more than 1 positive sample.

[0377] Table 12 shows a confusion matrix for P32 pools.

TABLE-US-00012 Expected negatives (RT-PCR) Expected positives (RT-PCR) Total Negatives in digital PCR 2 0 2 Positives in digital PCR 0 12 12 Total 2 12 14

Further Investigation of “COBAS -/dPCR + ” Discordances

[0378] Three P8 extracts (P8_20, P8_28 and P8_39) and one P16 pool (P16_14) were tested positive by digital PCR while containing only samples that tested negatives by individual RT-PCR with the Cobas® 6800 system. P16_14 originates from a combination of P8_27 (COBAS -/dPCR -) and P8_28.

[0379] To further investigate these “COBAS -/dPCR +” discordances, confirmatory testing RT-dPCR was performed on all individual samples from the three P8 extracts. For each pool, one sample tested positive by individual RT-dPCR, with measured concentrations of viral RNA ranging from 128 copies per reaction to 2 copies per reaction for the N gene, and from 106 to 1 copies for the ORF1ab gene.

[0380] The 3 samples identified were retested on the Cobas® 6800 system and by confirmatory individual RT-PCR using the Altona kit. 2 samples tested positive using the Altona kit and only the sample with the highest measured concentration by RT-dPCR (Sample 56075) tested positive on the Cobas® 6800 with a high Ct value of 36.7 for the E gene while the ORF gene was not detected.

[0381] The sample that tested negative with the Altona kit had borderline levels of positive droplets in RT-dPCR (N=2; ORF1ab=1) and is likely to be below the level of detection of the Altona kit and Cobas® 6800 system.

[0382] Based on these results, the “COBAS -/dPCR +” samples are interpreted to be true positive samples that were not correctly assessed by the initial reference RT-PCR test on the Cobas® 6800 system.

[0383] Table 13 shows the results of the individual reassessment by both RT-dPCR and RT-PCR (Altona and Cobas®) for the samples from the 3 “COBAS -/dPCR +” discordant pools. NT: not tested; ND: not detected.

TABLE-US-00013 Sample ID COBAS reference P8 extract number dPCR+/dPCR- N cp/rnx ORF1ab cp/rnx IC cp/rnx Altona E gene Altona S gene COBAS N COBAS OFR 51996 COBAS- 20 dPCR- 1 0 7 502 NT NT NT NT 52019 COBAS- 20 dPCR- 0 0 25 944 NT NT NT NT 52031 COBAS- 20 dPCR- 0 0 68 983 NT NT NT NT 52035 COBAS- 20 dPCR- 0 0 28 215 NT NT NT NT 52042 COBAS- 20 dPCR+ 16 19 89 747 33 31.9 ND ND 52047 COBAS- 20 dPCR- 0 0 122 825 NT NT NT NT 52060 COBAS- 20 dPCR- 0 0 17 237 NT NT NT NT 52062 COBAS- 20 dPCR- 0 0 105 711 NT NT NT NT 56075 COBAS- 28 dPCR+ 128 106 69 268 29 28.4 36.7 ND 56077 COBAS- 28 dPCR- 0 0 34 656 NT NT NT NT 56083 COBAS- 28 dPCR- 0 0 71 410 NT NT NT NT 56191 COBAS- 28 dPCR- 0 0 22 471 NT NT NT NT 56211 COBAS- 28 dPCR- 0 0 21 185 NT NT NT NT 56275 COBAS- 28 dPCR- 0 0 22 771 NT NT NT NT 56303 COBAS- 28 dPCR- 0 0 56 797 NT NT NT NT 56307 COBAS- 28 dPCR- 0 0 59 159 NT NT NT NT 60241 COBAS- 39 dPCR- 0 0 46 988 NT NT NT NT 60281 COBAS- 39 dPCR- 1 0 73 581 NT NT NT NT 60310 COBAS- 39 dPCR- 0 0 11 420 NT NT NT NT 60334 COBAS- 39 dPCR- 0 0 32 663 NT NT NT NT 60345 COBAS- 39 dPCR- 0 0 81 004 NT NT NT NT 60362 COBAS- 39 dPCR- 0 0 36 943 NT NT NT NT 60389 COBAS- 39 dPCR- 0 0 825 NT NT NT NT 60401 COBAS- 39 dPCR+ 2 1 43 651 ND ND ND ND

Further Investigation of the Sample Cobas +/dPCR -

[0384] One pool of 8 (P8_02) tested negative by digital PCR while it contained one sample (Sample _25659) which tested positive by reference RT-PCR with Ct value of 34 and 32.3 for the E gene and ORF1ab respectively. These Ct values are not particularly high and pools containing samples with significantly higher Ct values, up to 38, were reliable detected in this study.

[0385] Sample _25659 was subsequently re-extracted individually and retested by individual digital PCR testing, confirmatory RT-PCR using the Altona kit and Cobas® routine screening method.

[0386] Sample _25659 was found to be borderline negative by individual digital PCR (N=2; ORF1ab=0) but Ct values of 37.3 and 34.9 were found for E and ORF respectively in the second Cobas® assessment.

Correlation Between Digital PCR Measurements and Ct Values

[0387] It is noteworthy that a good correlation is observed between the number of positive droplets observed in RT-dPCR and the log-average Ct value of the individual samples contained in the group, as seen in FIG. 4A (for the N gene) and FIG. 4B (for the ORF1ab gene).

Discussion

[0388] Based on these results, we assessed the sensitivity of group testing combined with digital PCR, for the 3 different group sizes investigated. To do so, we suggest a practical protocol in which the first step of group screening is performed by RT-dPCR as disclosed herein, and in which the second step of individual re-testing of positive groups is performed using RT-dPCR as well (FIG. 5).

[0389] In this sensitivity analysis, the 3 samples associated with the COBAS -/dPCR + discordances are considered to be true positive samples containing the SARS-CoV-2 virus responsible of Covid-19. The sample associated with the COBAS +/dPCR - is also considered a true positive sample. Further investigation by sequencing is underway on these four samples in an effort to rigorously assess the nature of the nucleic acids responsible for the discording results.

[0390] Based on these assumptions, this study suggests that group testing by RT-dPCR has a better sensitivity than individual RT-PCR testing for pools of 8 samples. In the cohort of 448 samples, a total of 23 groups of 8 tested positive and included 27 true positive samples. Only 25 samples were identified as positive using reference individual RT-PCR testing. This corresponds to a +8% improvement in sensitivity.

[0391] In the case of pools of 16, the data indicates that grouped testing by RT-dPCR has similar sensitivity to individual RT-PCR testing. A total of 15 groups of 16 tested positive and included 26 true positive samples. Compared with the 25 true positive samples identified by RT-PCR, this corresponds to +4% improvement in sensitivity.

[0392] Testing in pools of 32 by RT-dPCR has 100% concordance with the reference RT-PCR testing. Re-testing positive groups by RT-dPCR would have likely led to better sensitivity than RT-PCR.

[0393] We also discuss an alternative and more cost-effective group testing protocol in which the re-testing step would be performed using RT-PCR with Cobas or Altona kits. In these protocols, the sensitivity becomes dependent on the RT-PCR kit used. With an Altona kit, our data suggest that testing with pools of 8 and 16 would still have a better or similar sensitivity.

[0394] Overall, our data indicates that Covid-19 pooled testing by digital PCR, followed by re-testing by RT-PCR or digital PCR, has better to similar sensitivity than individual RT-PCR testing, with large group sizes of up to 16, and possibly 32 samples. By comparison, multiple studies have found that Covid-19 group testing by RT-PCR had lower sensitivity than individual testing, the loss of sensitivity increasing with the group size.

[0395] Here, the gain in sensitivity of the method disclosed herein is due to a concentration effect due to performing the pooling prior extraction and performing the extraction step from a large volume of 1 mL of pooled transport medium, and to the intrinsic superior sensitivity of digital PCR compared to RT-PCR.

[0396] The loss of sensitivity is one of the main reasons why group testing has not been widely adopted for Covid-19 testing, whilst research groups have advocated for its implementation as a solution to the worldwide unmet demand for tests and reagent shortage.

[0397] This study suggests that the high-sensitivity of group testing by digital PCR makes the approach viable for large-scale, low-cost patient screening with minimum reagent consumption.