A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2

Abstract

SARS-CoV-2 causes substantial morbidity and mortality in elderly and immunocompromised individuals, particularly in retirement homes, where transmission from asymptomatic staff and visitors may introduce the infection. Here we present a cheap and fast screening method based on direct RT-qPCR to detect SARS-CoV-2 in single or pooled gargle lavages ("mouthwashes"). This method detects individuals with large viral loads (Ct≤29) and we use it to test all staff at a nursing home daily over a period of three weeks in order to reduce the risk that the infection penetrates the facility. This or similar approaches can be implemented to protect hospitals, nursing homes and other institutions in this and future viral epidemics.

Fig 1. RT-qPCR of 10 previously SARS-CoV-2 positive (P) and 10 previously SARS-CoV-2 negative (N) mouthwashes.

RNA was either extracted from 200 μl of mouthwashes (black bars) or 1 μl of the mouthwashes was added directly to the RT-qPCR reaction mix without prior extraction (colored bars). Dashes at N/A indicate no amplification of the viral target.

Fig 2. Roche RNA extraction/RT-qPCR and NEB Luna RT-qPCR of seven reference samples.

Dots at N/A show no amplification of the viral target.

Fig 3

(A) Roche RNA extraction/RT-qPCR and NEB Luna RT-qPCR for SARS-CoV-2 detection in 62 mouthwash samples from hospital patients. The NEB method detects the virus when Ct of the Roche method is less than 29 (blue box). Dots at N/A show no amplification of the viral target. (B) Correlation of Ct values between direct NEB assays and RNA extraction followed by Roche assays for mouthwash samples positive with both methods.

Fig 4. Daily testing of personnel of a nursing home with or without pooling of mouthwashes.

Red color indicate a SARS-CoV-2 positive individual/sample.

Fig 5. Tests of seven pools of 26 mouthwash samples, which in six cases contain one positive sample.

Dashes at N/A show no amplification of the viral target.