Pooling of SARS-CoV-2 samples to increase molecular testing throughput

J Clin Virol. 2020 Oct;131:104570. doi: 10.1016/j.jcv.2020.104570. Epub 2020 Aug 2.


Background: SARS-CoV-2 testing demand has outpaced its supply. Pooling samples for lower risk populations has the potential to accommodate increased demand for SARS-CoV-2 molecular testing.

Objective: To evaluate the sensitivity, specificity, and reproducibility of 4-way pooling of SARS-CoV-2 specimens for high-throughput RT-PCR.

Study design: Individual samples were pooled 1:4 through automated liquid handling, extracted, and assayed by our emergency use authorized CDC-based RT-PCR laboratory developed test. Positive samples were serially diluted and theoretical and empirical PCR cycle thresholds were evaluated. Thirty-two distinct positive samples were pooled into negative specimens and individual CTs were compared to pooled CTs. Low positive samples were repeated for reproducibility and 32 four-way pools of negative specimens were assayed to determine specificity.

Results: Four-way pooling was associated with a loss of sensitivity of 1.7 and 2.0 CTs for our N1 and N2 targets, respectively. Pooling correctly identified SARS-CoV-2 in 94 % (n = 30/32) of samples tested. The two low positive specimens (neat CT > 35) not detected by pooling were individually repeated and detected 75 % (n=6/8) and 37.5 % (n = 3/8) of the time, respectively. All specimens individually determined negative were also negative by pooling.

Conclusion: We report that 1:4 pooling of samples is specific and associated with an expected 2 CT loss in analytical sensitivity. Instead of running each sample individually, pooling of four samples will allow for a greater throughput and conserve scarce reagents.

Keywords: COVID-19; Diagnostics; Disease surveillance; Pooling; RT-PCR; SARS-CoV-2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Betacoronavirus / isolation & purification*
  • COVID-19
  • COVID-19 Testing
  • Clinical Laboratory Techniques / methods*
  • Coronavirus Infections / diagnosis*
  • Epidemiological Monitoring
  • High-Throughput Screening Assays
  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Polymerase Chain Reaction
  • RNA, Viral / isolation & purification
  • Reproducibility of Results
  • SARS-CoV-2
  • Sensitivity and Specificity
  • Specimen Handling / methods*


  • RNA, Viral