Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Internal Reference

Reverse transcription-polymerase chain reaction(RT-PCR)is an essential method for specific diagnosis of SARS-CoV-2 infection.Unfortunately,false negative test results are often reported.In this study,we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens.Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercialfluorescent RT-PCR kit targeting the ORF1 ab and N regions of SARS-CoV-2 genome.The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30(RPP30)in these specimens was also assessed by RT-PCR.Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed.By using the ROC-AUC analysis,we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity(95.03%–95.26%)and specificity(83.72%–98.55%)for respective combination of specimen type and ampli-fication reaction.Using these Ct cutoff values,false negative results could be reliably identified.Therefore,the presence of cellular materials,likely infected host cells,are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens.RPP30 could serve as an indicator for cellular content,or a surrogate indicator for specimen quality.In addition,our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.