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

Abstract

Introduction: Testing for active SARS-CoV-2 infection is a fundamental tool in the public health measures taken to control the COVID-19 pandemic. Because of the overwhelming use of SARS-CoV-2 reverse transcription (RT)-PCR tests worldwide, the availability of test kits has become a major bottleneck and the need to increase testing throughput is rising. We aim to overcome these challenges by pooling samples together, and performing RNA extraction and RT-PCR in pools.

Methods: We tested the efficiency and sensitivity of pooling strategies for RNA extraction and RT-PCR detection of SARS-CoV-2. We tested 184 samples both individually and in pools to estimate the effects of pooling. We further implemented Dorfman pooling with a pool size of eight samples in large-scale clinical tests.

Results: We demonstrated pooling strategies that increase testing throughput while maintaining high sensitivity. A comparison of 184 samples tested individually and in pools of eight samples showed that test results were not significantly affected. Implementing the eight-sample Dorfman pooling to test 26 576 samples from asymptomatic individuals, we identified 31 (0.12%) SARS-CoV-2 positive samples, achieving a 7.3-fold increase in throughput.

Discussion: Pooling approaches for SARS-CoV-2 testing allow a drastic increase in throughput while maintaining clinical sensitivity. We report the successful large-scale pooled screening of asymptomatic populations.

Keywords: COVID-19; Diagnostics; Group testing; Infectious diseases; RT-PCR.

Fig. 1

Pooling eight lysates retains clinical sensitivity. Shown are results of 23 pooling experiments, with eight lysates in each pool; 15 pools with positive samples indeed come up positive (pools 1–15), three pools without positive samples come up negative (pools 20, 21, 23) and four out of five pools containing a single indeterminate sample detected as indeterminate (pools 16, 17, 18, 19, 22); Pools containing one or two samples with low amount of SARS-CoV-2 are detected at a similar Ct (pools 9–18), showing clinical sensitivity is retained and the risk of false negatives is minimal. Ct, threshold cycle.

Fig. 2

Matrix pooling. (a) Scheme for 5 × 5 matrix pooling. Twenty-five samples sorted in a 5 × 5 matrix and each row and each column is pooled into a total of ten pools, on which RNA extraction, reverse transcription and qPCR are performed. In this illustration row B and column 3 are positive (black stars), hence sample B3 is the only positive sample. If more than one row and one column are positive then all samples in intersections need to be retested, as some may be negative. (b) Three 5 × 5 pool matrices were generated (30 pools from 75 lysates). Each matrix (25 lysates that were previously tested individually) included a single lysate positive for SARS-COV-2. As expected, only six pools (one row and one column per matrix) were positive for SARS-COV-2, while 24 pools had threshold cycle (Ct) > 40 (Undetected). Reverse transcription-PCR Ct values of positive pools were nearly identical in the column pool (green) and the row pool (blue), and similar to the values of the individual test of the positive sample (grey).