Phenol-chloroform-based RNA purification for detection of SARS-CoV-2 by RT-qPCR: Comparison with automated systems

Abstract

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic levels. Sufficient testing for SARS-CoV-2 has remained essential for tracking and containing the virus. SARS-CoV-2 testing capabilities are still limited in many countries. Here, we explore the use of conventional RNA purification as an alternative to automated systems for detection of SARS-CoV-2 by RT-qPCR. 87 clinical swab specimens were extracted by conventional phenol-chloroform RNA purification and compared to commercial platforms for RNA extraction and the fully integrated Cobas®6800 diagnostic system. Our results show that the conventional RNA extraction is fully comparable to modern automated systems regarding analytical sensitivity and specificity with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. Moreover, the method is easily scalable and implemented in conventional laboratories as a low cost and suitable alternative to automated systems for the detection of SARS-CoV-2.

Fig 1. Phenol-chloroform extraction of RNA is a viable alternative to automated systems, showing similar levels of sensitivity, specificity and accuracy.

(A) 87 patient specimens with known SARS-CoV-2 status based upon routine testing for SARS-CoV-2 using the Cobas^®6800 platform were compared to results reported for the in-house SARS-CoV-2 RT-qPCR assay after RNA isolation using the Maxwell^® RSC 48 instrument or TRI Reagent^®. Only RNA specimens passing the internal control for the Newcastle Disease Virus vaccine strain (NDV, Ct values <29.5) were used for the analyses (Maxwell^®; 82 samples, TRI Reagent^®; 80 samples). True positive (TP), true negative (TN), false positive (FP), false negative (FN), confidence interval (CI). Sensitivity is defined as the probability a test result is positive for a SARS-CoV-2 positive sample. Specificity is defined as the probability a test result is negative for a SARS-CoV-2 negative sample. Accuracy is defined as the probability a patient sample is correctly evaluated for SARS-CoV-2. (B) Diagram showing a highly significant correlation (r = 0.970, p<0.0001) between obtained Ct values for the SARS-CoV-2 E gene in SARS-CoV-2 positive specimens when assessed by RT-qPCR using the Cobas^®6800 platform and the in-house SARS-CoV-2 RT-qPCR assay after RNA isolation using TRI Reagent^®. (C) Diagram showing a highly significant correlation (r = 0.978, p<0.0001) between obtained Ct values for the SARS-CoV-2 E gene in SARS-CoV-2 positive specimens when assessed by RT-qPCR using the in-house RT-qPCR assay after RNA isolation using the Maxwell^® RSC 48 instrument and TRI Reagent^®. (D) Side-by-side comparison of Ct values obtained for the internal control NDV when assessed by RT-qPCR using identical patient specimens from which RNA was isolated using the Maxwell^® RSC 48 instrument or the AGPC method. Not significant (NS). (E) Pie chart showing the average rate of sample specimens that pass the NDV internal control for RNA extraction and sample quality after isolation of the RNA using the AGPC pipeline (n = 736).