Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: a reliable, faster and economical method.

Background: Corona virus disease 2019 (COVID-19) which initially started as a cluster of pneumonia cases in the Wuhan city of China has now become a full blown pandemic. Timely diagnosis of COVID-19 is the key in containing the pandemic and breaking the chain of In low and middle income countries availability of testing kits has become the major bottle neck in testing. Novel methods like pooling of samples are the need of the hour. Method: Extracted RNA samples were randomly placed in pools of 8 on a 96 well plate. Both individual RNA (ID) and pooled RNA RT-qPCR for the screening E gene were done in the same plate and the positivity for the E gene was seen. Results: The present study demonstrated that pool testing with 8 RNA samples can easily detect even up to a single positive sample with Ct value as high as 38. The present study also showed that the results of pool testing is not affected by number of positive samples in a pool. Conclusion: Pooling of 8 RNA samples can reduce the time and expense by one eighth, and can help expand diagnostic capabilities, especially during constrained supply of reagents and PCR kits for the diagnosis of SARS-CoV-2 infection.


INTRODUCTION
Combined nasopharyngeal and oropharyngeal swabs were collected by healthcare workers  80 The 5 µl of the extracted RNA elute/sample was subjected to RT-qPCR for the qualitative 81 detection of SARS-CoV-2 RNA utilizing with AgPath-IDTM One-Step RT-PCR Reagents All rights reserved. No reuse allowed without permission.

PERFORMANCE OF RT-qPCR IN THE LABORATORY
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.25.20079095 doi: medRxiv preprint (Thermo Fisher Scientific) using a Applied biosystem (ABI) 7500 Real Time PCR system 83 (Thermo Fisher Scientific) and LightMix® SarbecoV E-gene (TIB MOLBIOL). Reactions 84 were heated to 55 o C for 5 minutes for reverse transcription, denatured in 95 o C for 5 85 minutes and then 45 cycles of amplification were carried out for 95ºC for 5 seconds and 60 o C 86 for 15 seconds using FAM parameter for E gene. This assay targets the detection of E gene 87 for SARS as well as nCoV-2. The primer details are given in Table 1. All samples that were 88 screened positive for E gene were confirmed by performance of RT-qPCR for the detection 89 of specific RdRP gene of SARS-CoV-2 using LightMix® Modular SARS-CoV-2 RdRP (TIB 90 MOLBIOL) using similar PCR conditions as described above.

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RNA samples that were obtained after extraction were randomly pooled initially in pools of 93 2, 4, 6, 8, 16 RNA elutes on a 96 well plate. The best results matching with individual RNA All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. . 96 well plate were used as shown in figure 1.Both ID and pooled RNA RT-qPCR for the 96 screening E gene was done in the same plate (figure 1).5 µl of the RNA sample was taken for 97 the PCR and pooling of 5 µl (8x5= 40 µl) was done for the pooled test. After thorough 98 mixing, 5 ul of the pooled RNA was taken for the pooled PCR. The volume of RNA sample 99 was kept similar in both the ID as well as pooled PCR so that assay sensitivity is not affected. 100 ID PCR and pooled PCR was performed in the same run , keeping the entire conditions 101 uniform.
102 Results were seen on the ABI software and each reaction was read for E gene after 103 confirmation of the performance of EAC as well as positive control and negative control 104 results. Ct value for each positive test was recorded and as per the WHO criteria, sample with 105 Ct value ≤ 40 were considered as positive. All initial E gene positive were confirmed as All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

108
The data was compared for the ability of detection of E gene by ID test and pooled test using 109 one sample paired t-test (Table 1). Data of Ct value is shown as mean ± standard deviations.

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The agreement between Ct values obtained for positive sample in one positive sample pool 111 with ID test was done applying intra class correlation ICC followed by Bland and Altman 112 graph. A P value of 0.05 or less was considered statistically significant.

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The study was approved by the Institutional ethics committee and patient consent form was 115 waived off because of the use of deidentified discarded RNA samples.

RESULTS
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Out of 280 samples that were tested, 40 were positive and 240 negative for SARS CoV-2 E 119 gene with a positivity rate of 16.7% (95 CI; 12.2%-22.0%).All 40 were also positive for 120 RdRP gene. Results were communicated to the patients as per the ID test results obtained.

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There were 13 pools where all the ID tests were negative and pool results were also negative. All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.25.20079095 doi: medRxiv preprint In 22 pools one or more positive sample was detected in the ID test (Table 2) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The reliability coefficient was found to be 74.5% . All tests were within 2 SD limit except 1.

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The limit of biasness were -8.4 to 2.3. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.25.20079095 doi: medRxiv preprint the need of the hour. Pooled testing can solve this problem. In another study on pool testing All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted April 29, 2020.