Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 Detection

SARS-CoV-2 is a newly emerged coronavirus that was isolated from human infections in recent months. Since drugs and vaccines of Covid-19 are still being developed, accurate pathogen detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been reliably used for the detection and confirmation of SARS-CoV-2 since the beginning of outbreak, whereas isothermal nucleic acid amplification based point of care automated assays has also been considered as a simpler and rapid alternative. However, since these assays have only been developed and applied for clinical use within a short timeframe, their analytical performance has not been adequately compared to-date. We describe a comparative study between a newly developed cross primer isothermal amplification (CPA) assay (Kit A) and five RT-PCR assays (Kits B to F), using clinical diagnosis as the reference standard to evaluate their sensitivity, specificity, predictive values and accuracy analysis. Clinical samples used (n=52) included throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprised of positive (n=26) and cleared cases (n=26) by clinical diagnosis. For the CPA assay (Kit A), the sensitivity, specificity, positive and negative predictive values and accuracy were 100%. Among the five RT-PCR kits, Kits B, C and F had good agreement with clinical diagnosis (Kappa≥0.75), Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all assays were statistically significant (P<0.001). The reproducibility of RT-PCR assays was determined using a positive sample that was verified by all assays, with standard deviations (SD) between 0.35 and 0.87, and coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. To further evaluate the CPA assay (Kit A) compared to Kits B and F, throat swabs from close contacts of cases (n=200) were analyzed. All three kits identified the same positive samples and showed total agreement. This is the first comparative study to evaluate a CPA assay and RT-PCR assays for SARS-CoV-2 detection, which could serve as a reference for clinical laboratories and inform testing protocols amid the rapidly evolving COVID-19 pandemic.