Evaluation of False Negative Among SARS-COV-2 Patients with Negative Real-time PCR Result Using Nested-RT PCR

Background: Fast and precise detection of SARS-CoV-2 RNA in clinical samples and subsequent quarantine are two critical factors in preventing virus transmission and distribution through the community. The false-negative result is a major problem in the SARS-CoV-2 detection because of the kind of sample (swab sample), sampling error, and sensitivity of PCR test, which can be reduced by a much more sensitive test such as nested PCR. Objectives: This study aimed to evaluate the false-negative rate among samples that were negative by a real-time PCR test using RT-nested PCR. Methods: One hundred eighty-four negative samples were included in the study, and nucleic acid was extracted using a commercial kit based on a silica filter column and then subjected to RT-nested PCR using three sets of primers targeting Orf1ab, N, and RdRp regions. Results: Among 184 negative swab samples for SARS-CoV-2, 27 (14.6%) cases were positive for the Orf1ab gene using RT-nested PCR. The samples were tested using N and RdRp primer sets. Also, seven (3.8%) cases were positive for the N gene, and four (2.1%) cases were positive for the RdRp gene. Conclusions: The results indicated that RT-nested PCR could be more sensitive than real-time PCR and reduce the false-negative rate.