false-negative results in taqman one-step rt-pcr test: evaluation of endogenous internal control function used in sars-cov-2 detection tests

Background: taqman one-step real-time pcr (rt-pcr) has special importance due to its high sensitivity and specificity in the diagnosis of infectious diseases such as viral infections. in the recent pandemic of sars-cov-2, diagnostic kits based on this method are commonly used for molecular detection. one of the main systematic errors that misinterpret the results is using inaccurate internal control in rt-pcr diagnostic kits. designing primers and probes that span exon-exon junction will avoid genomic dna amplification and lead to obtaining high specific results. objectives: this study aimed to evaluate the endogenous internal control of primers and probe for rnase p rna to reduce falsenegative results in respiratory samples. methods: in this study, 30 samples of patients who were negative for sars-cov-2, influenza a, and influenza b were re-evaluated for sars-cov-2 using newly designed primers and probes for rnase p rna (ultra-specific primers and probe). we also performed bioinformatics analysis on cdc-approved primers and probes of rnase p endogenous internal control. results: in this analysis, we specified the location of these newly designed primers and probe on target mrna and genomic dna. then, the taqman one-step rt-pcr method was performed using both cdc-approved primers and probes along with our ultraspecific primers and probe for rnase p rna. based on bioinformatics analysis, the attachment sites of the cdc-approved primers and probe for endogenous internal control of rnase p are located on the first exon of this gene. in addition to identifying the target gene sequence, these primers and probe also non-specifically detect similar sequences on the genomic dna. conclusions: the present study showed that the use of specific primers and probes introduced bycdcfor sars-cov-2 and influenza virus may cause false results due to non-specific binding to the genomic dna. therefore, choosing the right internal control for rnase p rna can be useful in achieving very accurate results.