Evaluation of extraction-free RT-qPCR methods for SARS-CoV-2 diagnostics

Abstract

Extraction-based real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is currently the “gold standard” in SARS-CoV-2 diagnostics. However, some extraction-free RT-qPCR techniques have recently been developed. In this study, we compared the sensitivity of traditional extraction-based, heated extraction-free, and unheated extraction-free RT-qPCR methods for SARS-CoV-2 detection in nasopharyngeal swabs from symptomatic individuals. The unheated extraction-free method showed perfect agreement with the standard extraction-based RT-qPCR. By contrast, the heat-treated technique was associated with an 8.2% false negativity rate. Unheated extraction-free RT-qPCR for the molecular diagnosis of SARS-CoV-2 is a valuable alternative to the traditional extraction-based methods and may accelerate turnaround times by about two hours.

Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2

“…Another possible shortcoming is that RT-PCR Ct values may differ by brand and protocol; the reported diagnostic accuracy estimates by the Ct category may differ from those observed in other laboratories. For instance, we have previously shown [25] that compared with the extraction-based RT-PCR technique, the extraction-free protocol adopted in the present study is associated with an average increase in Ct values by 2-3 units. Finally, although highly conserved, the N gene may be subject to mutations affecting molecular diagnostics of SARS-CoV-2 infection [29].…”

“…However, a substantial number of samples with Ct > 35 may still produce a viable virus [22][23][24]. Moreover, Ct values across different RT-PCR protocols may vary significantly [25].…”

“…As per the internal SARS-CoV-2 diagnostic protocol adopted by the San Martino Policlinico Hospital, all NP samples were processed by means of a validated [25] unheated RNA extraction-free RT-PCR method. We have previously shown [25] that this technique displays perfect agreement with a traditional extraction-based approach. Briefly, an input volume of 5 µl of specimen was first diluted (1:3) and set up for RT-PCR.…”

“…A total of 5 µL of the extracted RNA in a final volume of 20 µL was used for each reaction. The average time-to-result of the unheated RNA extraction-free RT-PCR method is 156 min [25].…”

Point-of-Care Diagnostic Tools for Surveillance of SARS-CoV-2 Infections

“…The global shortage of RNA extraction kits has had adverse impacts on COVID-19 diagnosis and control worldwide. Though extraction-free RT-qPCR protocols have been considered as alternatives for the standard SARS-CoV-2 RT-PCR method, laboratory optimizations are often required to minimize false-negative rates ( 40 ).…”