Evaluation of three extraction-free SARS-CoV-2 RT-PCR assays: A feasible alternative approach with low technical requirements

Abstract

The worldwide demand for SARS-CoV-2 RT-PCR testing resulted in a shortage of diagnostic kits. RNA extraction step constitutes a major bottleneck to perform diagnostic. The aim of this study was to assess performances of different extraction-free SARS-CoV-2 RT-PCR assays compared to a reference RT-PCR assay. The panel of evaluation consisted of 94 samples: 69 positive and 25 negative for SARS-CoV-2 by reference RT-PCR. Three extraction-free RT-PCR assays were assessed: (i) PrimeDirect® Probe RT-qPCR Mix (Takara), (ii) PrimeScript®RT-PCR (Takara), and (iii) SARS-CoV-2 SANSURE®BIOTECH Novel Coronavirus (Sansure). The overall sensitivity of PrimeDirect, PrimeScript and Sansure assays was 55.1%, 69.6% and 69.6%, respectively. The sensitivity increased among samples with Ct < 30: 91.9% (n = 34/37), 89.2% (n = 33/37) and 94.6% (n = 35/37) for PrimeDirect, PrimeScript and Sansure assays, respectively. The specificity was 88%, 100% and 100% for PrimeDirect, PrimeScript and Sansure assays, respectively. In the present study, we showed a good sensitivity of extraction-free PCR assays, especially for high viral loads (Ct < 30), except PrimeDirect that displayed imperfect sensitivity and specificity. Despite a lower sensitivity for low viral loads, extraction-free reagents can provide a valuable option, cheaper, easier and less reagent consuming for SARS-CoV-2 diagnostic, especially in laboratory with lower experience and equipment for molecular assays.

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…For RT-PCR-based extraction-free test kits, currently, the relevant health authorities like FDA, WHO, or CDC have approved at least ten types of such kits and examples of these kits include Xpert®Xpress [166] , SalivaDirect [167] , SwabExpress [168] , PrimeDirect® qRT-PCR Mix [169] , Prime® ScriptRT-PCR [169] and few more [16] , [68] . These test assays or kits could be used to detect various genetic targets of SARS-CoV-2 such as N, E, S, RdRp, and ORF genes and the test sensitivity and specificity would vary greatly from one type of assay to another [166] , [167] , [169] , [170] , [171] , [172] .…”

“…For RT-PCR-based extraction-free test kits, currently, the relevant health authorities like FDA, WHO, or CDC have approved at least ten types of such kits and examples of these kits include Xpert®Xpress [166] , SalivaDirect [167] , SwabExpress [168] , PrimeDirect® qRT-PCR Mix [169] , Prime® ScriptRT-PCR [169] and few more [16] , [68] . These test assays or kits could be used to detect various genetic targets of SARS-CoV-2 such as N, E, S, RdRp, and ORF genes and the test sensitivity and specificity would vary greatly from one type of assay to another [166] , [167] , [169] , [170] , [171] , [172] . In general, the test kits that are made to detect the N gene of the virus are found to have a better sensitivity (>80%) [168] , [170] , [173] , [174] and this is higher compared to test kits that are specifically designed to detect other molecular targets of SARS-CoV-2 like RdRp and E (<70%) [169] .…”

“…These test assays or kits could be used to detect various genetic targets of SARS-CoV-2 such as N, E, S, RdRp, and ORF genes and the test sensitivity and specificity would vary greatly from one type of assay to another [166] , [167] , [169] , [170] , [171] , [172] . In general, the test kits that are made to detect the N gene of the virus are found to have a better sensitivity (>80%) [168] , [170] , [173] , [174] and this is higher compared to test kits that are specifically designed to detect other molecular targets of SARS-CoV-2 like RdRp and E (<70%) [169] . This suggested that the N target could be a better and more reliable target to be used for rapid detection of SARS-CoV-2 as the rapid diagnostic kits are more sensitive to detect this target.…”

“…This suggested that the N target could be a better and more reliable target to be used for rapid detection of SARS-CoV-2 as the rapid diagnostic kits are more sensitive to detect this target. In terms of the test specificity, most of the introduced SARS-CoV-2 RT-PCR extraction-free test kits were reported to have high test specificity (>80%) [166] , [167] , [168] , [169] , [170] , [171] , except that the test specificity of some kits was unclear like VereRT TM ZeroPrep TM COVID-19 PCR kit [172] and qScript ® XLT qRT-PCR test kit [173] . However, both these test kits have generally low LOD (<12 RNA copies in 1 μL) and high test sensitivity (>85) [172] , [173] .…”

Temporal stability and detection sensitivity of the dry swab-based diagnosis of SARS-CoV-2

“…Along with this, there are multiple challenges such as shortage of Ribonucleic acid (RNA) isolation reagents, samples transport facilities, equipment breakdown, and limited human/financial resources (Fomsgaard & Rosenstierne, 2020). In an attempt to overcome these hurdles, multiple studies attempted to present the advantages of RNA extraction-free direct Reverse Transcription-Polymerase Chain Reaction (RT-PCR), without compromising the sensitivity of detection (Bruce et al, 2020; Kiran et al, 2020; Padgett et al, 2020; Parikh et al, 2021; Smyrlaki et al, 2020; Visseaux et al, 2021) which is being effectively implemented in few countries (ICMR, 2021; Srivatsan, Heidl, et al, 2020).…”