Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number.

The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRIzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56°C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121°C for 20 min, boiling at 100°C for 20 min, and heating at 80°C for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) 1ab. For samples treated at 56°C for 30 min, the copy number of the N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80°C for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRIzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRIzol reagent had the least effect on the RNA copy number among the tested methods.

Evaluation of the clinical performance of single-, dual-, and triple-target SARS-CoV-2 RT-qPCR methods.

BACKGROUND: The coronavirus disease 2019 (COVID-19) has become a pandemic. Reverse transcription quantitative PCR (RT-qPCR) has played a vital role in the diagnosis of COVID-19, but the rates of false negatives is not ideal in dealing with this highly infectious virus. It is thus necessary to systematically evaluate the clinical performance of the single-, dual-, triple-target detection kits to guide the clinical diagnosis of this disease. METHODS: A series of reference materials calibrated by droplet digital PCR (ddPCR) and 57 clinical samples were used to evaluate the clinical performance of six single-, dual-, triple-target SARS-CoV-2 nucleic acid detection kits based on RT-qPCR. RESULTS: The dual-target kits, kit B and kit C had the highest and the lowest detection sensitivity, which was 125 copies/mL and 4000 copies/mL, respectively. Among the 57 clinical samples from patients with COVID-19, 47 were tested positive by the kit B, while 35, 29, 28, 30, and 29 were found positive by the kits A, C, D, E, and F, respectively. The number of targets in a detection kit is not a key factor affecting sensitivity, while the amount of sample loading may influence the performance of a detection kit. CONCLUSIONS: This study provides a guide when choosing or developing a nucleic acid detection kit for the diagnosis of COVID-19. Also, the absolute-quantification feature and high-sensitivity performance of ddPCR, suggesting that it can be used to review clinically suspected samples.