Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits

Abstract

Background: The severe shortage of nucleic acid extraction kits during the current COVID-19 pandemic represents a key limiting factor in testing capacity.

Objectives: This study compared the results of SARS-CoV-2 RT-PCR using different simple nucleic acid extraction methods on nasopharyngeal and saliva specimens.

Study design: Fifty nasopharyngeal swab and saliva specimens previously tested positive for SARS-CoV-2 were retrieved. Three different methods of nucleic acid extraction were compared. The first method involves incubating the specimen with proteinase K, and then heat treatment at 98 °C for 5 min (PKH); the second method involves heat treatment at 98 °C for 5 min without proteinase K pre-incubation (heat only); the third method involves no pre-processing steps (direct). The products from all 3 methods were tested by SARS-CoV-2 RT-PCR.

Results: PKH had significantly higher positive rate in SARS-CoV-2 RT-PCR (80 %) than those of heat only (58 %; P = 0.001) or direct (56 %; P = 0.002). The median Ct value was significantly earlier for PKH (median Ct: 37.0, IQR 31.7-40) than that of heat only (median Ct: 40, IQR 36.2-41; P < 0.0001) and direct (median Ct, 37.5; IQR 33.9-41.0; P = 0.0049). Subgroup analysis showed that PKH had higher detection rate, lower limit of detection and earlier Ct values than the other two groups for both NPS and saliva specimens.

Conclusions: PKH pre-processing resulted in the highest detection rate of SARS-CoV-2 by RT-PCR, and represents an alternative method for nucleic acid extraction when commercial extraction kits are not available.

Keywords: COVID-19; Detection; Nucleic acid extraction; Proteinase K; RT-PCR; SARS-CoV-2.

Fig. 1

Comparison of RT-PCR results from different nucleic acid extraction methods. The Ct values were compared between the PKH, heat only and direct groups. PKH: specimens pre-incubated with proteinase K for 15 min at 56 °C, followed by 5 min at 98 °C. Heat only: specimens were heated for 5 min at 98 °C. Direct: no pre-processing step before RT-PCR. Original: Total nucleic acid was extracted using NucliSENS easyMAG extraction system. *, <0.05; ****, <0.0001.