Pooling RT-qPCR testing for SARS-CoV-2 in 1000 individuals of healthy and infection-suspected patients

Abstract

Severe acute respiratory coronavirus 2 (SARS-CoV-2) testing reagents are expected to become scarce worldwide. However, little is known regarding whether pooling of samples accurately detects SARS-CoV-2. To validate the feasibility of pooling samples, serial dilution analysis and spike-in experiments were conducted using synthetic DNA and nucleic acids extracted from SARS-CoV-2-positive and -negative patients. Furthermore, we studied 1000 individuals, 667 of whom were “healthy” individuals (195 healthcare workers and 472 hospitalized patients with disorders other than COVID-19 infection), and 333 infection-suspected patients with cough and fever. Serial dilution analysis showed a limit of detection of around 10–100 viral genome copies according to the protocol of the National Institute of Infectious Diseases, Japan. Spike-in experiments demonstrated that RT-qPCR detected positive signals in pooled samples with SARS-CoV-2-negative and -positive patients at 5-, 10-, 20-fold dilutions. By screening with this pooling strategy, by the end of April 2020 there were 12 SARS-CoV-2-positive patients in 333 infection-suspected patients (3.6%) and zero in 667 “healthy” controls. We obtained these results with a total of 538 runs using the pooling strategy, compared with 1000 standard runs. In a prospective study, we successfully detected SARS-CoV-2 using 10- to 20-fold diluted samples of nasopharyngeal swabs from eighteen COVID-19 patients with wide ranges of viral load. Pooling sample is feasible for conserving test reagents and detecting SARS-CoV-2 in clinical settings. This strategy will help us to research the prevalence infected individuals and provide infected-status information to prevent the spread of the virus and nosocomial transmission.

Modelling population-wide screening of SARS-CoV-2 infection for containing COVID-19 pandemic in Okinawa, Japan

“…Second, a limited testing capacity and weak logistics in procuring personal protective equipment will be demanding. Adopting a pooling strategy [31][32][33] will dramatically dilate Japan's testing capacity and make it possible to repeatedly provide RT-PCR testing. Importing appropriate testing kits for truly recognizing infectiousness based on the previous discussion [34] will be a policy option, although it can be a debatable political choice in terms of health security.…”

Discovery of SARS-CoV-2 strain of P.1 lineage harboring K417T/ E484K / N501Y by whole genome sequencing in the city, Japan

“…Total nucleic acid was isolated from the samples using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) on the KingFisher Duo Prime System (Thermo Fisher Scientific) as we previously described [15, 16]. Briefly, we added 200 µL of VTM, 5 µL of proteinase K, 265 μL of binding solution, 10 μL of total nucleic acid-binding beads, 0.5 mL of wash buffer, and 0.5–1 mL of 80% ethanol to each well of a deep-well 96-well plate.…”

Sample pooling strategies for SARS-CoV-2 detection

“…Hirotsu et al showed that the primer/probe targeting the N1 site succeeded to detect samples with high and moderate viral load, but not those with low viral load when the samples were examined in pools of 20. The N2 site was proven to be more effective target for analyzing the pooled samples, since it was detected even if in cases with low viral load ( Hirotsu et al, 2020 ). Perchetti et al showed that pooling correctly identified SARS-CoV-2 in 94 % of the samples which were classified as samples with median viral load.…”