Sanger sequencing-the gatekeeper to exclude false positives in nucleic acid-based diagnostics for infectious diseases

Background False-positive results are a known challenge in polymerase chain reaction (PCR)-based diagnostics for infectious diseases. The widespread public testing during the COVID-19 pandemic brought the issue to unprecedented global attention with immense clinical and societal consequences. Most authors of scientific publications claim contamination due to poor laboratory management as the major cause of false-positive PCR test results. However, the possibility of false positives being generated by the PCR technology itself has not been investigated. Methods The residues of 30 patient nasopharyngeal swab samples, which were certified to be positive for SARS-CoV-2 N gene by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays, were retested by a heminested reverse transcription polymerase chain reaction (RT-PCR), followed by Sanger sequencing to verify the authenticity of the amplified product as the physical evidence for true-positives and to explore the molecular mechanism of generating false positives. In addition, the platelet-rich plasma specimens of 145 people residing in Lyme disease-endemic areas during a Lyme disease season in the United States were used for split-sample nested PCR amplification followed by Sanger sequencing for the detection of Borrelia burgdorferi flaB and 16S rRNA genes and to explore the molecular mechanism of false positives. Results Heminested RT-PCR generated 19 PCR products from 30 SARS-CoV-2 RT-qPCR positive samples 16 of which contained a segment of SARS-CoV-2 N gene verified by Sanger sequencing. Three of the 19 PCR products showed mixtures of nontarget DNA sequences, possibly derived from the chromosomes of human cells, bacteria and fungi in the nasopharynx. Split-sample PCR testing for B. burgdorferi showed that in the absence of the target DNA, the primers designed for Borrelial 16S rRNA gene PCR may amplify segments of the human mitochondrial DNA, causing a false-positive PCR result. Sanger sequencing can eliminate all PCR-induced false positives. This study also showed that when the nested PCR protocol is optimized, the crude DNA extract can be used for initiating a primary PCR without nucleic acid isolation, purification, and quantitation. The nested PCR product can be used directly as the template for Sanger sequencing to facilitate implementation of sequence analysis in diagnostic laboratories.