Introduction

The outbreak of COVID-19 has become a public health emergency. Viral nucleic acid detection by reverse transcription PCR (RT-PCR) is the gold standard method for diagnosis of COVID-19. Droplet digital PCR (ddPCR) is a highly sensitive PCR technology based on the generation of 20,000 nanodrops per tube. This technology is rarely used in clinical laboratories, due to its higher cost when compared with PCR. Cost could be reduced by using pooled testing. A negative test result indicates that all individuals in the pool are negative while a positive result indicates that at least one individual within the pool is positive. Pooled testing may be particularly useful to communities with low prevalence of COVID-19.

Objective

We proposed to validate the use of ddPCR to detect SARS-CoV-2 by pooling.

Materials and methods

Test were performed from April to July 2021, throat swab samples of 1000 patients were collected and soaked in 2mL saline. RNA extraction was done using automatic magnetic extraction, columns extraction kits, and heat (95C for 10 min). ddPCR was performed with a Bio Rad QX200 AUTO DG following manufacturer instructions.

Firstly, positive and negative samples were identified with RT-PCR, pools of different sizes were designed and ddPCR was performed. Data was analyzed with Quanta Soft analysis software v.1.7.4.0917 (Bio-Rad).

This study was granted exception from bioethics committee approval as deidentified remnants samples were used.

Results

We determined the specificity (we measured 100 negatives pools), the limit of detection (three independent octuplicates of the greatest dilution that it is positive) and the robustness of the method (the ability to withstand small but deliberate variations in method parameters by performing 20 repetitions changing the order of pooling and purification; and by measuring RNAs obtained using different extraction method). In the present work, we validated the use of pooled testing by combining up to 34 samples per pool where a single positive sample can be detected with an estimate false negative rate of 5%.

Discussion

This pooling method can be applied immediately in current clinical testing laboratories that count with appropriate equipment. We hope that such implementation of a pool test for SARS-CoV-19 would allow expanding current screening capacities, thereby enabling the expansion of detection in the community, as well as in close organic groups.