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. 2022 Jan 25;39(1):48-54.
doi: 10.5152/balkanmedj.2021.21135. Epub 2021 Dec 20.

Detection of SARS-CoV-2 RNA in Upper Respiratory Swap Samples by Pooling Method

Yesim Tuyji Tok  1 , Mert Ahmet Kuşkucu  1 , Hazal Erdem  2 , Devrim Sarıbal  3 , Seda Salman Yılmaz  4 , Okan Kadir Nohut  5 , Rıdvan Karaali  2 , İlker İnanç Balkan  2 , Bilgül Mete  2 , Ömer Fehmi Tabak  2 , Gökhan Aygün  1 , Kenan Midilli  1
Affiliations

Detection of SARS-CoV-2 RNA in Upper Respiratory Swap Samples by Pooling Method

Yesim Tuyji Tok et al. Balkan Med J. .

Abstract

Background: Widespread and effective use of molecular diagnostic tests is indispensable for protecting public health and containing the severe respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. More than 1 year into the pandemic, as resources have reached a point of depletion, grouping samples in pools of certain sizes appears to be a reasonable method to reduce both the costs and the processing time without necessitating additional training, equipment, or materials.

Aims: To assess whether the pooling strategy that was used in past outbreaks and is used in blood tests prior to transfusion for screening large populations can also be used in SARS CoV-2 tests.

Study design: Diagnostic accuracy study.

Methods: This prospective study was conducted with 2815 samples, sent to the coronavirus disease 2019 (COVID-19) Laboratory of our hospital between February 12 and 21, 2021, to be tested for the presence of SARS-CoV-2. The samples were examined individually and in pools of five 100 μl taken from each sequential sample, using 3 different SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) kits, the Allplex™ 2019-nCoV Assay kit (Seegene, Republic of Korea), the GeneMAP™ 2019-nCoV detection V.3 kit (GenMark, Türkiye), and the Bio-Speedy™ SARS-CoV-2 Double Gene™ RT-qPCR kit (Bioeksen, Türkiye) on the BioRAD CFX96™ Touch (Bio-Rad Laboratories Inc., Hercules, CA, USA) platform available in our laboratory.

Results: Following the extraction of serial dilutions prepared from the SARS-CoV-2 RNA positive (cycle of threshold: 20) sample, the standard curves of RT-PCR were analyzed. By evaluating the efficiency (E) values, all 3 kits showed high sensitivity and similar results; while the highest level was detected with the Allplex™ 2019-nCoV Assay kit in the nucleocapsid (N) gene (E: 124%), the lowest was detected with the Double Gene™ RT-qPCR kit in the N and ORF 1ab genes (E: 90%). Of the samples included in the study, only 1 positive sample with low viral load was found to be negative when studied by pooling. The total number of kits to be used in pooled tests and then to individually retest the 5 samples in positive pools was calculated as 827 and the savings rate as 69.91% (1968/2815).

Conclusion: The pooling strategy is an effective approach to extend the impact of limited testing resources and reagents available in certain periods of the COVID-19 pandemic. Testing by pooling samples requires improvement of RNA extraction methods and careful monitoring of RT-PCR test sensitivity to avoid missing low-positive entities. Therefore, based on the prevalence of COVID-19 in their regions, laboratories should conduct their own validation of pooling studies for RNA extraction and amplification methods they use.

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Conflict of interest statement

Conflict of Interest: The authors have no conflict of interest to declare.

Funding: This study was carried out with the contribution of the İstanbul University-Cerrahpaşa Scientific Research and Project Fund.

Figures

Figure 1
Figure 1
Comparison of the efficacy of Allplex™ 2019-nCoV Assay, GeneMAP™ 2019-nCoV detection V.3, and Double Gene™ RT-qPCR kits for the E, N, RdRP, and ORF1ab gene regions.
Figure 2
Figure 2
Analytical sensitivities of Allplex™ 2019-nCoV Assay, GeneMAP™ 2019-nCoV detection V.3, and Double Gene™ RT-qPCR kits for E, N, RdRP, and ORF1ab gene regions.
Figure 3
Figure 3
Number of samples, positivity rate, and savings rates by day in the 5-pooling study.
See this image and copyright information in PMC

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