Evaluation of COVID-19 RT-qPCR test in multi-sample pools

Abstract

These authors contributed equally + Correspondence regarding sample collection and medical interpretation should be set to M. H. (m_halberthal@rambam.health.gov.il), Y. G. (y_geffen@rambam.health.gov.il), or M. S-C (M_Szwarcwort@rambam.health.gov.il); correspondence regarding the experimental procedure and data analysis should be sent to R. K. (rkishony@technion.ac.il). AbstractThe recent emergence of SARS-CoV-2 lead to a current pandemic of unprecedented levels. Though diagnostic tests are fundamental to the ability to detect and respond, many health systems are already experiencing shortages of reagents associated with this test. Here, testing a pooling approach for the standard RT-qPCR test, we find that a single positive sample can be detected even in pools of up to 32 samples, with an estimated false negative rate of 10%. Detection of positive samples diluted in even up to 64 samples may also be attainable, though may require additional amplification cycles. As it uses the standard protocols, reagents and equipment, this pooling method can be applied immediately in current clinical testing laboratories. We hope that such implementation of a pool test for COVID-19 would allow expanding current screening capacities thereby enabling the expansion of detection in the community, as well as in close integral groups, such as hospital departments, army units, or factory shifts.

Large-scale implementation of pooled RNA-extraction and RT-PCR for SARS-CoV-2 detection

“…While such approaches for SARS-CoV-2 have been recently suggested (e.g. [16,17]), we have tested direct pooling of lysates of clinical nasopharyngeal samples, with RNA extraction already performed on the pooled samples. First, we tested the pooling of 184 consecutive samples into 23 pools of 8 samples each, and also tested in parallel each sample individually.…”

“…Of the 5 pools which contained one individual sample with an "indeterminate" result (in each pool), one was found to be negative suggesting potential yet negligible loss of sensitivity. 20,21,23), and 4 out of 5 pools containing a single indeterminate sample detected as indeterminate (pools 16,17,18,19,22); Pools containing 1-2 samples with low amount of SARS-CoV-2 are detected at a similar Ct (pools [9][10][11][12][13][14][15][16][17][18], showing clinical sensitivity is retained and the risk of false negatives is minimal. UD= Undetected.…”

“…While several pooling approaches for SARS-CoV-2 detection were recently suggested (e.g. [16][17][18][19]), none demonstrated adherence to current clinical standards, nor shown to work for performing RNA extraction in pools, which is typically the limiting step. Here we describe and demonstrate practical pooling solutions that save time and reagents by performing RNA extraction and RT-PCR on pooled samples.…”

Multi-Stage Group Testing Improves Efficiency of Large-Scale COVID-19 Screening

“…We determined the improvement factor of P3S2, P9S3, P4S2 and P16S4 for prevalence rates up to 30% (Figure 3 A+B) and compared results with previously described schemes, namely the two-stage testing schemes with pool sizes of 10 (P10S2, [6]), or 32 (P32S2, [5]). We also assessed the matrix testing scheme [7] using a matrix of 96 samples (12x8) that groups samples in rows (8 pools comprising 12 samples) and columns (12 pools comprising 8 samples) a method that has also been used for epitope mapping in immunology research application [8].…”

Noisy Pooled PCR for Virus Testing

“…Main idea. Noisy group testing is used to analyze RT-PCR data from mixed or pooled samples, as recently demonstrated for COVID-19 [2]. The goal of group testing is twofold.…”