Extraction-free protocol combining Proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

Abstract

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT- qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2.

Natural spring water gargle samples as an alternative to nasopharyngeal swabs for SARS‐CoV‐2 detection using a laboratory‐developed test

“…Another possibility would be the use of proteinase K (PK) in the CHUQ protocol. Indeed, some studies suggested that pretreatment of samples with PK increased the sensitivity of direct rRT‐PCR for SARS‐CoV‐2 detection, 28,29 particularly in low positive samples with high C t values 13 . It has been suggested that PK could degrade or inactivate RNAses, allowing to purify a better quality of RNA 30 .…”

Direct NP- A cost-effective extraction-free RT-qPCR based test for SARS-CoV-2

“…Several methods used combinations of treatments for releasing viral RNA into the reaction for various types of samples. For example, either enzymatic or combination of enzymatic and heat denaturation have been used for Saliva, Sputum, and NP swab samples [ 11 , 12 , 13 , 15 , 16 , 17 , 29 ]. Our data showed that heat denaturation for 2 min alone is sufficient for detecting viral RNA into the sample with no significant loss of PPA when compared to CDC assay ( Table 3 ).…”

“…Several extraction-free methods were proposed for SARS-CoV-2 testing from various specimen types, such as NP swabs, saliva, and sputum [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. These methods use a combination of enzymatic digestion with heat treatment for sample preparation before RT-qPCR.…”

RT-LAMP-CRISPR-Cas13a technology as a promising diagnostic tool for the SARS-CoV-2 virus

“…For sample processing, a proteinase K-heat inactivation protocol (PK-HID) was applied to samples from swabs stored in viral transport medium (Gibco ® ) [37]. Thus, 95 µl aliquots of samples were treated for 15 minutes at 55 o C with 5 µl of proteinase K (10 mg/ml, stock) prepared at 1 mg/ml in 100 µl of final volume and heat-inactivated at 98 o C for 5 minutes.…”