a.  Selection of RT-PCR kits

This observational study was conducted at the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India and the laboratory has been recognised as a COVID-19 RT-PCR testing centre by ICMR, India. Till date, the laboratory has used more than 15 RT-PCR kits for the testing and those were supplied through government agencies i.e., National Institute of Virology (NIV), ICMR, Pune and Madhya Pradesh Public Health Services Cooperation Limited (MPPHSCL), Bhopal, Madhya Pradesh, India. For the present study, seven different COVID-19 RT-PCR kits were selected on the basis of multiple SARS-CoV-2 specific gene targets in a single tube with simultaneous detection of each target on different detection channel. Present study was carried in a period of a month time (October 2020) and none of the manufacturers were involved in the assessment and interpretation of the study results.

The following seven kits were included for the study [Table/Fig-1]: (1) DiagSure nCOV-19 Detection assay (GCC Biotech Pvt., Ltd., India); (2) Meril COVID-19 One-step RT-PCR Kit (Meril Diagnostics Pvt., Ltd., India); (3) TruPCR SARS-CoV-2 RT-qPCR Kit (Kilpest India Ltd., India); (4) Q-line Molecular Coronavirus (COVID-19) RT-PCR kit (POCT Services Pvt., Ltd., India); (5) VIRALDTECT II Multiplex real time RT-PCR for COVID-19 (Genes2me Pvt., Ltd., India); (6) Allplex 2019-nCoV assay (Seegene, South Korea); and (7) TaqPath COVID-19 Combo Kit (Applied Bio-Systems, USA).

b.  Sources of specimen

This study included left over RNA (RNA samples were stored at -80^o C in deep freezer) from the clinical specimens, which were tested earlier in the laboratory as part of routine diagnostics using (NIV Pune supplied) two step PCR kit; (i) screening test for E gene andRNP gene; and (ii) confirmatory test for RdRp gene and ORF-1ab gene.

Sample size for this study was selected by analysing previous related research work done by other scientific groups [8-10]. In this study, 30 different cut-off range SARS-CoV-2 positive RNA samples and 20 negative RNA samples were included. RNA samples with inconclusive test results were excluded in this study. A total of 50 RNA samples (left over RNA) included: (i) Group A: 10 positive RNA samples with high viral loads with a low cut-off threshold (<20); (ii) Group B: 10 positive RNA samples with a medium cut-off threshold (21-30); (iii) Group C: 10 positive RNA samples with low viral loads with a high Ct value (>30); and (iv) Group D: 20 negative RNA samples were included. These selected groups were tested for SARS CoV-2 by using seven different RT-PCR kits.

c.  Real Time-PCR Assay

Earlier the RNA was extracted using HiPurA Viral RNA purification kit (HiMedia, Mumbai, India). All RT-PCR tests were performed on QuantStudio 7 Flex (Applied Biosystems, CA, USA) or CFX96 Touch (BIO-RAD, CA, USA) and thermocycling settings and result interpretation was performed as per manufacturer’s instructions. Positive and negative controls of each kit were included for PCR run validation. RT-PCR instrument set up of each kit is summarised in [Table/Fig-2].

Ct values of all the genes of the RT-PCR kits were recorded. Mean and Standard Deviation (SD) of the tested genes were analysed using Microsoft Excel 2010.

All seven RT-PCR kits were evaluated with four different groups of known RNA samples. Obtained cut-off threshold values of each group were recorded. Mean and SD of the Ct value of the individual tested genes were calculated for the analysis of the results [Table/Fig-3]. All RT-PCR kits performed satisfactorily regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Ct value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values.

PCR run time and other kit parameters were determined for each RT-PCR kits [Table/Fig-2]. As compared to previously used two step PCR kit (screening and confirmatory tests), all these single tube multiplex kits were found to be user friendly, time and resource saving. Among all the RT-PCR kits evaluated, TaqPath COVID-19 combo kit was able to complete the PCR run in the least time (~67 minute) and this is the only kit targeting the S gene of SARS-CoV-2 besides ORF-1ab and N gene. Most of the genes included in the kits are labelled with basic flurophores that can be detected in the most of the RT-PCR platforms, while Allplex 2019-nCoV assay kit and TaqPath COVID-19 combo kit need higher end RT-PCR instruments having wider fluorescence detection range. Those kits contain human housekeeping gene as internal control having advantage over other kits contains normal internal control.

The present SARS-CoV-2 pandemic resulted in the quick setup of laboratories for the COVID-19 molecular diagnosis. The correct diagnosis is more important to identify, control and break the chain of SARS-CoV-2 transmission. Poor diagnosis may lead to false negative test which increases the spread of infection and false positive result may lead to the unnecessary treatment and mental trauma to the patients and their families [11].

RT-PCR is a gold standard test for COVID-19 diagnosis and the result outcome depends upon the performance of the RT-PCR kit being used. Till date, ICMR, New Delhi has evaluated the performance of 321 COVID-19 RT PCR kits and 147 kits performance were found satisfactory [12]. In-house performance assessment of RT-PCR kits in COVID-19 testing laboratory is still limited. However, some studies evaluated performance of COVID-19 RT-PCR kits in single and pooled clinical specimens [8-10,13].

Outcome of this study indicate detection of SARS-CoV-2 was comparable by all RT-PCR kits and kits performed satisfactorily regarding the reproducibility. Authors found that all kits performed similarly in all low, medium and high Ct group (100% sensitivity and 100% specificity). Authors have not included inconclusive RNA samples as our key purpose of this study was to evaluate kits performance among each other and our analysis indicates that all RT-PCR tests look good to diagnose and differentiate COVID-19 positive and negative samples.

Four of the selected kits (DiagSure, Meril, TruPCR and Q line) contain two sets of SARS-CoV-2 specific gene primers and probes while VIRALDTECT, Allplex and Taqpath kits are containing three sets of SARS-CoV-2 specific gene primers and probes including internal control gene. Kits having three SARS-CoV-2 specific gene targets are superior than two gene targets as some studies indicate mutation of SARS-CoV-2 genome [14,15] as RNA viruses have high mutation rates [16] which leads to false-negative results and that can be reduced by targeting more sequences within the viral genome [17]. Most recently, a new SARS-CoV-2 variant (SARS-CoV-2 VOC 202012/01) has been identified in United Kingdom which is believed to be more infectious [18]. However, at present only sequencing method is available for the detection of the same [19-21].

The final outcome of the RT-PCR results also depends upon the proper sample collection of nasopharyngeal and throat swab. Inappropriate sample collection and improper sample transportation temperature will eventually influence the entire process and leads to false results [22-24]. These false results can be avoided by using human housekeeping gene as an internal control in RT-PCR kits. Meril, Q line, VIRALDTECT II and TruPCR kits contains primers and probes for endogenous internal control and have an additional advantage to find out proper sample collection and RNA extraction process.

During this outbreak situation, thousands of samples are collected and being sent to the laboratories for the diagnosis of COVID-19 and most labs do not have sufficient space to store thousands of samples at a time and improper storage leads to sample degradation which increases the chance of false results. Hence, prompt processing of this sample is recommended. Turnaround time is purely depending upon the sample processing and the RT-PCR run time. RT-PCR kits having shorter run time will reduce the turnaround time. In this study, authors have observed that RT-PCR run time of kits ranged between approx one to two hours. TaqPath COVID-19 combo kit able to complete the PCR run in the least time compared with other kits.

Quant Studio 7 Pro and BIO-RAD CFX96 Touch Real-Time PCR instruments were used for this study and both were calibrated with the most of the fluorescent dyes generally used in PCR application like FAM, HEX/VIC, ROX, Cy5, Texas Red etc., [25,26]. However, normally many labs do not have such high-end calibrated PCR instruments and they further need to compensate the same with additional cost burden. This study can help to the laboratories engaged in COVID-19 testing for selecting appropriate kits which best suit the machine available in their laboratory.

This study has some limitations. First, authors have received kits free of cost for SARS-CoV-2 diagnosis from NIV, Pune via NIREH, Bhopal and later on from MPPHSCL, Bhopal, Madhya Pradesh consequently were not able to compare the cost effectiveness of RT-PCR kits on the basis of cost per test. Second, this evaluation used small sample size and total 50 RNA sample (30 SARS-CoV-2 positive RNA sample with three different Ct range and 20 negative RNA sample) were chosen in this study and aimed to check whether all kits were able to detect positive and negative sample equally and correctly.

It was concluded that all commercially available RT-PCR kits included in this study can be used for the routine molecular diagnosis of COVID-19. Considering high sample load per day, it might be advisable to use those kits having less RT-PCR run time for timely diagnosis of symptomatic COVID-19 patients.