key: cord-0799004-72gmj59l
authors: Petrovan, Vlad; Vrajmasu, Virgil; Dimon, Paula; Zaulet, Mihaela
title: Evaluation of commercial qPCR kits for detection of SARS-CoV-2 in pooled samples
date: 2020-05-28
journal: bioRxiv
DOI: 10.1101/2020.05.28.120667
sha: d30b71b5c91ed4a18a6627cf93e524287b6b96d1
doc_id: 799004
cord_uid: 72gmj59l

Due to the current pandemic, global shortage of reagents has drawn interest in developing alternatives to increase the number coronavirus tests. One such alternative is sample pooling. Here we compared commercial kits that are used in COVID-19 diagnostics, in terms of sensitivity and feasibility for use in pooling. We showed that pooling of up to 60 samples did not affect the efficiency of the kits. Also, the RNA dependent RNA polymerase (RdRp) is a more suitable target in pooled samples than the Envelope (E) protein. This approach could provide an easy method of screening large number of samples and help adjust different government regulations.

The recent emergence of the novel severe acute respiratory syndrome coronavirus 2 34 (SARS-CoV-2) in December 2019 from Wuhan, China, has caused more than 5 million cases 35 with an estimate of more than 300 000 deaths associated with coronavirus disease (COVID-19) 36 (1). Clinical manifestation of COVID-19 infection is variable ranging from asymptomatic to 37 severe disease, with symptoms including respiratory distress, fever, cough, dyspnea, and viral 38 pneumonia (2) . Since there is currently no targeted therapeutics against SARS-CoV-2 and CoV-2 and other related betacoronaviruses, such as the closely related SARS-CoV (3, 5) . The 48 majority of qPCR tests are using different sample matrixes, represented by either swabs or 49 sputum, since they contain relatively high titer virus, due to the initial viral replication in the 50 upper respiratory tract (6) . However, the global need for a new surveillance approach reflects the 51 requirement to adapt to the increased demand of number of molecular tests to adjust the 52 lockdown policies. 53 Diagnostic pooling has been already shown to be effective both in veterinary medicine, 54 detecting various diseases induced by swine influenza, African swine fever virus or foot-and-55 mouth disease virus (7; 8; 9) and in human medicine for human immunodeficiency virus (HIV) 56 and other transfusion-transmittable diseases (10; 11) . Recently, the same approach showed 57 encouraging results for SARS-CoV-2 when a pool of up to 7 samples was used before the 58 extraction and up to 60 samples could be pooled after (12) . 59 Therefore, our main goal was to evaluate and compare some commercial kits currently 60 used for COVID-19 diagnostics, using the sample pooling approach. We also showed that the 61 high sensitivity of RNA dependent RNA polymerase (RdRp) compared to other targets, for 62 detection of SARS-CoV-2 in pooled samples. Step Mix (Nippon Genetics Europe Gmbh, Duren, Germany). The high specificity of the 90 commercial assays is based on the unique sequence of the primers specific for the SARS-CoV-2 91 genomic sequence along with optimal PCR conditions used for amplification. This assay does not (Table 2) . Seven patients 144 were tested negative by PCR for the initial screening, but were positive when the PCR was 145 repeated after 2 weeks interval. Mild or asymptomatic patients did not have any other co-146 morbidities, and clinical signs were limited to either fever, cough and/or shortness of breath, as 147 shown in Table 2 . Therefore massive scaling up COVID-19 testing is 168 the temporary solution until immunity levels are achieved. One of the approaches that can be 169 easily applied in order to increase the number of tests represents sample pooling. We showed that 170 using a range of negative sample matrixes with one representative sample, ranging from 5 to 60 171 pools only leads to only an incremental increase in the Ct values, for the RdRp target. This is 172 consistent with the initial report for SARS-CoV-2 pooling, however the number of samples used 173 for pooling before RNA extraction, was limited to 7 (12) . This approach would be feasible for 174 laboratories that are performing large-volume testing and considering screening with the 175 commercial kits evaluated in this study. Moreover, laboratories may consider testing as many as 176 60 samples using different sample matrixes, using the standard protocols, as an option for cost 177 savings without compromising the capacity to detect SARS-CoV-2. However, there are several 178 limitations that might arise using the pooled sample approach.

One limitation of this approach is that it seems only the RdRp gene is suitable for 180 detection of SARS-CoV-2 in pools larger than 30 samples, which might arise in false negative 181 results due to equipment variation and sample handling. However, this could be easily circumvented by integrating additional SARS-CoV-2 specific targets. Moreover, the complexity 183 of the disease can influence the sensitivity and specificity of the assay (15) . Our results are 184 showing that RdRp would be the ideal target for sample pooling, rather than E gene. This agrees 185 with the initial development of molecular tests for SARS-CoV-2 detection, when RdRp gene 186 assays 3.6 copies per reaction for the RdRp assay (4; 13) . 187 In this research, we showed that sample pooling for SARS-CoV-2 diagnostic is a feasible 188 measure using commercial kits widely available. 

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