Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2

Abstract

Background and aim: Existing real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2.

Methods: The RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies.

Results: The specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable.

Conclusions: The RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.

Keywords: COVID-19; Coronavirus detection; RT-PSR; SARS-CoV-2; TrueNat test.

Fig. 1

Schematic representation of workflow for the detection of SARS-CoV-2 using RT-PSR assay targeting RdRp gene.

Fig. 2

A representative image showing the visual detection of RT-PSR amplified product followed by agarose gel electrophoresis analysis. Lane 1: Non template control; Lane 2: Positive control (in-vitro synthesized RNA).

Fig. 3

Optimization of temperature, sensitivity, specificity of RT-PSR assay. A. Temperature optimization using visual detection followed by 2 % agarose gel analysis of RT-PSR assay. Lane 1: Non template control; Lane 2: Amplification at 60 °C for 60 min; Lane 3: Amplification at 63 °C for 60 min, Lane 4: Amplification at 65 °C for 60 min. B. Determination of sensitivity of RT-PSR assay using visual detection followed by 2 % agarose gel analysis. Figure showing the amplification using different RNA concentration for RdRp gene (expressed in copy number). Lane 1: Non template control, Lane 2: 2.65 × 10¹¹ RNA copies; Lane 3: 2.65 × 10⁴ RNA copies; Lane 4: 2.65 × 10³ RNA copies; Lane 5: 2.65 × 10² RNA copies; Lane 6: 2.65 × 10 RNA copies. C. Determination of specificity using visual detection followed by 2 % agarose gel analysis. Lane 1: Non template control; Lane 2: In-vitro RNA (positive control); Lane 3: Influenza virus; Lane 4: B. cepacia; Lane 5: M. marinum; Lane 6: A. baumannii and Lane 7: P. aeruginosa.

Fig. 4

Results of internal clinical validation of the optimized RT-PSR assay. A, and B: Visual detection of amplicons followed by agarose gel analysis of clinical samples for first and second phase, respectively. A1: Lane 1: Non template control, Lane 2: Positive control (in-vitro synthesized RNA), Lanes 3–7: Samples showing amplifications. A2: Lane 1: Non template control, Lane 2: Positive control (in-vitro synthesized RNA), Lanes 3–7: Samples showing no amplifications. B1: Lane 1: Non template control, Lane 2: Positive control (in-vitro synthesized RNA), Lanes 3–7: Samples showing amplifications. B2: Lane 1: Non template control, Lane 2: Positive control (in-vitro synthesized RNA), Lanes 3–7: Samples showing no amplifications. C. Results for first phase of sample validation compared to TrueNat confirmed old positive (80.5 %), new positive (95.8 %) and negative (96.6 %). D. Results for second phase of clinical validation compared to TrueNat confirmed positive (93.7 %) and negative (100 %) samples. E. Comparison of results between TrueNat and RT-PSR assay for first phase sample validations. ‘*’ indicates Significant difference between RT-PSR and TrueNat (p = <0.05). ‘ns’ No significant difference between RT-PSR and TrueNat assay (p = >0.05); F. Comparison of results between TrueNat and RT-PSR assay for second phase sample validation. ‘*’ indicates Significant difference between RT-PSR and TrueNat (p = <0.05). ‘ns’ No significant difference between RT-PSR and TrueNat assay (p = >0.05).