key: cord-0719900-mgleo3e6
authors: Kalita, Manash Jyoti; Dutta, Kalpajit; Hazarika, Gautam; Dutta, Ridip; Kalita, Simanta; Das, Partha Pratim; Sarma, Manash P.; Banu, Sofia; Idris, Md. Ghaznavi; Talukdar, Anjan Jyoti; Dutta, Sangitanjan; Sharma, Ajanta; Medhi, Subhash
title: In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity
date: 2021-09-09
journal: Sci Rep
DOI: 10.1038/s41598-021-97502-1
sha: 0c545317d5d74b6e1fbe1470669c8c54166aeba8
doc_id: 719900
cord_uid: mgleo3e6

As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

www.nature.com/scientificreports/ Polymerase Chain Reaction (PCR) is a gold standard technique which offers significant advantages in viral detection and quantification with high reproducibility and sensitivity 9 . Initially, for the detection of COVID-19, a reliable qRT-PCR assay was developed by the Centers for Disease Control and Prevention (CDC), USA. With time, various laboratories across the world developed different primer/probe pairs for specific detection of SARS-CoV-2 with varying sensitivity and accuracy. However, in at least three independent studies it was reported that the primer pairs suggested for N region by US CDC, namely 2019-nCoV_N2 showed significant background cross reactivity 10 , along with non-specific amplification. Another primer 2019-nCoV_N3 has been reported to give false negative result 11 in a study that includes SARS-CoV-2 positive patients besides showing false positive result even in the absence of template 10 . Similarly the most preferable primer RdRp-P2, used in more than 30 European laboratories has been reported to have less sensitivity 12 .

With the surge in disease burden, the demand for rapid screening for SARS-CoV-2 has risen significantly. Undeniably, the present circumstances have driven the need for more specific and sensitive primers for virus detection without false-positive results. We performed literature review of articles via selecting keywords 'RT-PCR detection of COVID-19' , 'primer specificity of SARS-CoV-2 detection assay' , 'sensitivity and specificity of COVID-19 detection test' with probable sensitivity limit or limit of detection (LoD) that are reported based on the analysis. Upon reviewing and analysing the primer pairs that are initially prescribed by different public health agencies, we observed a noticeable difference in detection sensitivity at lower template concentrations besides producing the false-positive results. Such limitations simultaneously drive the need for the availability of more specific primer/probes that can accurately detect COVID-19 cases in robust conditions. Moreover, the establishment of non-hazardous infrastructure encompassing skilled manpower, availability of reagent subsequently increases the per sample cost that appears to be an additional burden for any country with resource constraints.

In the current scenario, rapid and accurate detection of SARS-CoV-2 could significantly influence the management of the pandemic. World Health Organization (WHO) guideline require, at least two different targets on the COVID-19 virus genome for the detection of SARS-CoV-2 through PCR amplification. This study has therefore focused on developing a Fast Multiplex Polymerase Chain Reaction targeting E, S and RdRp gene of SARS-CoV-2, detectable through Agarose gel-based visualization besides HRM coupled multiplex Real-Time PCR based validation using the same primer set. The optimized PCR condition applied for both the traditional method and HRM coupled multiplex Real Time PCR along with the adoption of the sample pooling strategy in the present study established a more sensitive assay protocol with reduced cost.

Sample collection. Archived positive samples of a varied range of C t values ranging from low to high C t value (Tested using Standard Operating Procedure from ICMR-NIV Pune which uses Invitrogen SuperScript III Platinum One-Step Quantitative Kit) were collected from State Level Viral Research and Diagnostics Laboratory (VRDL), Gauhati Medical College and Hospital were used as in the study. A total of 100 known positive and 33 known negative samples were included in the study. Viral RNA extraction. Viral RNA extraction was carried out using AuPreP Viral RNA Extraction Miniprep System (Life technologies, Cat no: RNV-52-906LT) following the manufacturer's protocol. The extracted RNA was then quantified using Nano Drop Spectrophotometer (NanoVue plus, Make: Invitrogen) and the values recorded in ng/µl. The extracted RNA was converted into cDNA immediately.

Reverse transcription. Tetro cDNA Synthesis Kit (Make: Meridian Bioscience, Cat. No: BIO-65043) was used for cDNA preparation using the manufacturer's protocol. 5 µg of the extracted RNA was used for cDNA preparation in a final volume of 20 µl reaction. Each reaction was incubated at 25 °C for 10 min followed by 45 °C for 30 min and finally terminated by incubating at 85 °C for 5 min.

Primer design. The full sequences of SARS-CoV-2 were retrieved from the NCBI Reference Sequence Database. Alignment of the sequences was done in BioEdit software. Primer3 tool (https:// bioin fo. ut. ee/ prime r3-0. 4.0/) was used to design the three primer sets targeting the SARS-CoV-2 specific E gene, S gene and RdRp gene. The selected primer pairs were analyzed using NCBI primer blast for the specificity of the primer set. Blast report showed that all the three primers have specifically amplified the target region of SARS-CoV-2 only. The primers pairs were further analyzed for secondary structure, the amplicons as well as probable self and heterodimer formation tendencies using Idtdna.com (https:// sg. idtdna. com/ pages/ tools/ oligo analy zer). The primer sets were synthesized and delivered by Reprocell Brand: Bioserve (Hyderabad, Telangana. India).

Primer testing and development of fast PCR assay. The accuracy and optimization of each primer set was verified through PCR amplification. Gradient PCR of the test was performed with an annealing temperature profile ranging from 62 to 68 °C based on the melting temperature (T m ) of each of the SARS-CoV-2 specific target primer set. The template concentration was kept up to a maximum of 10 ng of cDNA in a reaction volume of 20 μl along with forward and reverse primer sets at a final concentration of 0.2-0.3 μM each using Emerald Amp GT PCR Master Mix (2x) from Takara Bio Inc. (Cat No. RR310A). The cycling condition includes an initial denaturation step at 95 °C for 5 min. Amplification was carried out for 35 cycles with denaturation at 95 °C for 20 s, followed by a temperature gradient of 62-68 °C for 30 s to obtain the optimum annealing temperatures, and extension at 72 °C for 20 s. A final extension was carried out at 72 °C for 7 min.

Following primer optimization, the cycling condition was modified by reducing the denaturation time to 5 s and eliminating the extension step at 72 °C for 20 s. As such the Fast PCR assay will involve an initial denaturation www.nature.com/scientificreports/ at 95 °C for 5 min. Amplification will be done by denaturation at 95 °C for 5 s, followed by annealing at 66 °C for 15 s and extension at 72 °C for 5 min.

To minimize the amount of sample and reagent usage, preparation time, cost and labor, we developed an alternative protocol by adopting a multiplex PCR protocol for detection of SARS-CoV-2 in which all the primer sets were mixed into one reaction which reduced the total number of reactions to 1 (one) per sample instead of 3 (three) reactions. When mixed, the final concentration of the primer pairs was reduced proportionally to decrease primer-dimer formation. The designed primer pairs targeting the E, S and RdRP genes in the SARS-CoV-2 genome, produced amplicons of different sizes (i.e., 101 bp, 103 bp, and 160 bp, respectively) for easy separation and visualization. The Applied Biosystems Veriti 96-Well Thermal Cycler instrument was used for multiplex PCR. EmeraldAmp GT PCR Master Mix (Cat No: RR310A) was used and each 20 µl reaction mixture contained 10 µl of PCR Master Mix 0.5 µl of each primer and 1 µl of synthesized cDNA. The final concentrations of primers were 0.2 µM (E,S and RdRp gene primers). The thermal cycler was set as per the temperature profile for the Fast PCR assay described earlier.

Sensitivity and specificity of multiplex PCR. The sensitivity of the primer pairs was tested using multiplex PCR with optimized primer pairs for E, S and RdRp gene on archived known SARS-CoV-2 positive and negative samples. Variable C t values of the samples ranging from 19 to 35 makes it possible to analyze the sensitivity of the primer pairs. Apart from this the template was diluted 10 times serially and tested for sensitivity and specificity of the primer pairs using multiplex PCR.

Cross reactivity analysis. To analyse the cross reactivity of the assay procedure with other respiratory viruses, the multiplex PCR was tested against archived Influenza A positive, Influenza B positive and H1N1positive samples. However, as an efficient alternative cross reactivity studies can be performed in-silico where cross reactivity can be defined as 80% or more sequence similarity between a primer and any nucleotide sequence in the target organism 13 . Sequencing and phylogenetic analysis. PCR amplicons of E, S and RdRp gene were sequenced through Sanger sequencing (Bioserve sequencing service from Reprocell USA, inc, Hyderabad, Telangana. India). The obtained sequence was aligned separately for the three different gene targets against the SARS-CoV-2 reference sequence (NC_045512) and other related coronavirus sequences obtained from the NCBI database to analyse the percent identity.

Compliance statement. All the experimental procedures were performed in accordance with the relevant guidelines and regulations provided by Indian Council of Medical Research (ICMR) which is again in accordance with WHO laboratory safety manual related to the novel coronavirus (2019-nCoV) (https:// www. who. int/ docs/ defau lt-source/ coron aviru se/ labor atory-biosa fety-novel-coron avirus-versi on-1-1. pdf? sfvrsn= 912a9 847_2). All the samples were appropriately processed and maintained in a well-ventilated Biosafety cabinet by personnel with demonstrated capability. All bio hazardous waste generated were disinfected using Sodium Hypochlorite (0.1%) for general disinfection and 1% hypochlorite solution was used for any kind of spillage during sample processing. 

Primer optimization. In-silico validation of primer pairs and amplicon sequences showed no secondary structure and the possibility of self or heterodimer formation was not observed. In silico PCR tool nullified the possibility of nonspecific reactions within the same genome as well as genomes from other related species. The optimum annealing temperature of primer pairs was found to be 66 °C. The optimum primer concentration for multiplex PCR was found to be 0.2 μM final concentration of each primer. With this primer concentration, the resulting Agarose gel image showed no primer dimer formation (Fig. 1 ). www.nature.com/scientificreports/ Fast PCR protocol for SARS-CoV-2 detection. Following primer optimization, all three primers were tested separately using a single known positive sample with our optimized PCR cycling condition to observe the amplification efficiency of the protocol. Agarose gel electrophoresis showed distinct band of size 101 bp, 103 bp, and 160 bp for E gene, S gene and RdRp gene respectively without any primer dimer formation (Fig. 2 ).

PCR protocol for SARS-CoV-2 detection and easy implementation in any biological laboratory in the world. Archived positive samples from State Level Viral Research and Diagnostics Laboratory (VRDL) Gauhati Medical College and Hospital was used for cDNA preparations and used as a template for multiplex PCR optimization. The PCR was performed for 35 cycles following the cycling conditions described earlier. The gel electrophoresis showed two sharp bands of variable size i.e. 160 bp for the targeted RdRp gene and another of two overlapping bands of 101 bp and 103 bp for the E gene and S gene of SARS-CoV-2 respectively (Fig. 3 ).

Multiplex PCR protocol for SARS-CoV-2 detection, we further developed a multiplex RT PCR assay with implementation of High-Resolution Melting profile in addition to the thermal cycling profile for obtaining an enhanced separation of melt curve. A very similar result was obtained for HRM coupled multiplex RT PCR assay. The amplification plot showed one combined reaction curve with significantly lower C t value for all the three primers put together apart from the amplification curve for the individual primer put in separate reaction tube (Fig. 4) . The accuracy of the multiplex reaction in real time was confirmed further by analyzing the highresolution melt curve with three distinct peaks (Fig. 5 ) which were further compared with the melt curve peak obtained for the three primers when used separately in distinct tubes (Figs. 6, 7, 8, 9) . Based on these observations we can conclude that the three distinct melt curve peaks were of E, S and RdRp genes. Thus, this result demonstrates that the newly developed HRM coupled multiplex RT PCR protocol can be used for the fast and accurate detection of SARS-CoV-2 without the need of a costly probe based approach of detection. Cross reactivity of the assay. There were no false positive results from any of the primer pairs for the three target genes when the cross reactivity of the assay procedure was analysed with archived Influenza A, Influenza B and H1N1 positive samples. In-silico cross reactivity analysis for all the three primer pairs further showed 100% SARS-CoV-2 specific hit [14] [15] [16] .

Limit of detection (LoD). LoD was determined by using a tenfold serial dilution of the clinical sample SC005G (4.71 × 10 5 RNA copies/ml), which was previously reported to have a low C t value (C t -ORF1b = 19, C t -N gene = 21; Meril COVID-19 detection kit). All the dilutions were replicated 5 times and the average C t value obtained for individual target region for each dilution has been shown in Table 1 All the three targets were detected in 100% www.nature.com/scientificreports/ of the replicates for dilution ranging from 10 −1 (4.71 × 10 4 RNA copies/ml) to 10 -5 (4.71 RNA copies/ml) with a corresponding average C t value of 34.28 for RdRp gene, C t value of 33.77 for E gene, and C t value of 34.30 for S gene (Fig. 10) . At 10 -6 dilution, Target 1 and Target 2 were detected in 40% of the replicates with average C t value of 37.42 for Target 1 (RdRp gene) and 38.21 for Target 2 (E-gene). Target 3 (S-gene) was detected in 60% of replicates with average C t value of 39.53 as shown in Table 1 . However, none of the target was detected in any of the replicates for 10 -7 dilution (Table 1) .

All the 100-specimen showed reportable C t values for all the three targets. Mean C t value for Target 2 was found to be lowest among the tested SARS-CoV-2 positive samples followed by Target 1 and Target 3. However considerable degree of similarity and closeness of C t values among the three targets were observed. When stratified by age, within age Table 1 . Average C t values obtained for three target regions from a series of 7 tenfold serial dilution of a clinical specimen previously detected SARS-CoV-2 positive (SC005G) using Meril covid-19 detection kit. www.nature.com/scientificreports/ group 80-89 years, the C t value for, Target 1 was 4.98 C t lower than the overall mean C t for Target 1, for Target 2 it was 5.06 C t lower and for Target 3 it was found to be 4.37 C t , lower than the overall C t value ( Table 2 ) (Supplement Figure 1 ), which is a representative of high viral load in this age group.

Comparison of sensitivity and specificity of our In house RT-PCR assay.

The sensitivity and specificity of the primer pairs were further analysed by comparing the C t value obtained through our optimized detection protocol with the C t value obtained for the same set of samples (N = 100) using commercially available detection kits used in diagnostic setup (Meril COVID-19 One-Step RT-PCR Kit; Cat no: NCVPCR-02). The average C t value was found to be ≤ 30 for all the three target (Supplement Table 1 ) which is comparable with the Meril COVID-19 RT-PCR kit (C t value ≤ 35, positive) with significantly reduced median C t value obtained with our In house HRM coupled RT-PCR assay (Fig. 11 ).

Upon analysing the Limit of Detection (LoD) for Meril COVID-19 RT-PCR kit using the same serially diluted sample, efficient amplification of the prescribed target Orf 1b and N gene was obtained for the dilution range from 10 -1 to 10 -3 with C t value ≤ 35 for both the target. As such the limit of detection was found to be 471 RNA copies/ml (Fig. 12) . Although amplification was observed for further diluted sample (10 -4 to 10 -6 ) but the C t value obtained followed a gradual increase pattern from the recommended C t value for interpretation of SARS-CoV-2 positive detection (Supplement Table 2 ). No amplification was observed at 10 -7 dilution. However efficient amplification up to 10 -5 dilution (approximately 5 RNA copies per reaction, C t value < 35) using our In house RT-PCR assay is suggestive of enhanced limit of detection of the assay.

Risk of appearing false-negative result in the assay. Our tenfold serial dilution study showed the assay LoD to be 10 -5 dilution (4.71 RNA copies) with corresponding C t value of 34.28 for Target 1, 33.72 for Target 2, and 34.3 for Target 3. At 10 -1 dilution 100% of the specimens were detected for the entire three targets. As such, the risk of false negative result increases from 0% at 10 -1 dilution (approximately 11 C t lower than LoD) to 95% at LoD, which increases further with increase in C t value after LoD. In the studied cohort of SARS-CoV-2 positive samples Mean C t value for Target 1(24.53) and Target 2 (23.96) was 10 C t below the LoD, and for Target 3, mean C t (25.37) value was 9 C t lower than the LoD value. Only 2/100 (2%) positive samples showed a slightly higher C t value only for Target 3 (approx. 35) than the LoD, whereas for Target 1 and 2, the obtained C t values were near the assayed LoD, and as such would be at risk of false negative result. However, the assay efficiently detects 98/100 (98%) samples for Target 1 and Target 2, thereby supporting the enhanced sensitivity for RdRp gene (Target 1) and E gene (Target 2) detection (Fig. 13) .

The time required for the assay. The extraction of RNA from the sample will depend upon the type of kit or protocol used. Most of the commercially available kit requires around 35-45 min for RNA extraction. In the present study, following the RNA extraction, cDNA synthesis required 45 min with the Tetro cDNA synthesis kit (Meridian Bioscience, BIO-65043) followed by PCR amplification of the target genes from SARS-CoV-2 which required 37 min. Subsequently Agarose gel-based visualization needed approximately 30 min. Thus, the Fast Table 2 . Mean C t values obtained for the entire three targets among the positive specimens, stratified by age. Age group 80-89 showed considerably lower C t value for the entire three targets in comparison to mean C t which is representative of high viral load.

No of specimen 

Targeting the nucleic acid evaluation of any living microbial entities is considered the gold standard for the detection of any active infection. The advantage of PCR-based viral detection relies on high reproducibility, sensitivity and ability to quantify vial load with accuracy 9 . In the present study, we developed three sets of primers encompassing the E gene, RdRp gene and S gene of the SARS-CoV-2 genome with enhanced sensitivity and specificity. The primers sets were designed with the additional objective of extended feasibility to further amplify in both gel-based multiplex PCR assay and SYBR green based real-time detection in a single tube. The same sets of primer produced distinct amplification in both conventional as well as HRM coupled multiplex RT-PCR based detection assay. Indeed, with optimized primer concentration and low template concentration, this study seems to provide a cost-effective detection protocol with accuracy, which opens up prospective vistas to develop and commercialize a new detection kit. However, developing a fast-track detection kit from our study protocol needs further research with absolute refinement and necessary modification. Owing to its highly sensitive primer pairs, the assay procedure offers versatility in its application in variable resource settings for efficient and costeffective detection of SARS-CoV-2. Such an innovative approach can additionally set a significant impact on the detection and management of the COVID-19 pandemic and is useful to prepare for the future onset of any acute viral infection of similar nature and magnitude.

The present study has shown promising results in the cross-reactivity assay and Limit of Detection (LoD) analysis. Non-amplification of our targeted genes in Influenza A, Influenza B and H1N1 positive samples further reduces the chance of cross-contamination and diminished the risk of getting false-positive results. Moreover, LoD analysis of the samples up to 10 -5 dilution with 100% efficacy revealed high specificity of the primers even in low template concentration (approximately 5 RNA copies per reaction (C t value < 35) provides added advantages besides exhibiting an obvious comparison with other published assay. Supportive evidence came from the study of Garg et al. 17 which reports that Fosun COVID-19 RT-PCR kit have the lowest LoD (300 Copies/ml, C t < 36) among the seven RT-PCR kit evaluated in the study. However, the efficacy of the reaction reduces considerably in 10 -6 dilution may be due to exhausting template concentration. Further obtaining a C t value ≤ 30 for all the three target in (N = 100) archived positive sample without any false result with our in-house RT-PCR assay is suggestive of its enhanced sensitivity and specificity. Significant variation in the C t values was observed from the original sample C t value derived from COVID-19 positive cases, which indicates the possibility of having varied specificity of the original SARS-CoV-2 detection kit (Meril COVID-19 detection kit, C t ≤ 35, positive)). The penultimate evaluation of the study has been established due to reportable and reproducible C t value that extensively justified our study design. Highest deviation of C t was observed in the segregated age group of 80-89 years from mean C t value of the three target genes by about 4-5 unit, that might be due to pipetting error or sample handling error during experiment setup.

Initially approved SARS-CoV-2 RT-PCR test kit developed by various manufacturers around the world targeted the different number of genes, embedding three and two or even single gene with or without internal control. Accordingly, a diverse range of cost, specificity and efficacy of the kits were observed. For instance, Gene Matrix (USA), Daan Gene Co. Ltd (China), Kogene Biotech (Korea), LabGenomics (South Korea), Primer Design (UK), SansureBiotechInc. (China) used either combination of N, RdRP or E, RdRP or ORF1ab, N probe/primer in the kit 18, 19 . However, all the RT-PCR based kit possess limitation in terms of cost of reagent, reporter dye and synthesis of high-end specificity probe. The cost of the probe-based RT-PCR test is an obligatory limiting factor of COVID-19 management that lay a huge financial burden for any resource-limited country in the world 13 . For most of the presently available RT-PCR assay kits, the estimated cost is around $ 40-50 (Centres for Medicare and Medicaid Services, COVID-19 test pricing. 2020) 14 . Moreover, some detection assays possess constraints both in terms of reagent limitation and low specificity with initial template concentration. Apart from that, whether constant mutation of the SARS-CoV-2 genome is influencing the efficacy of probe-based detection is still not revealed entirely and that needs profound research. However, our detection assay involving the implementation Figure 13 . Distribution of experimentally obtained C t values for the 100 positive specimen for Target 1, Target 2, and Target 3 are plotted. Only 2/100 (2%) positive sample were found with C t values slightly higher than LoD or near the assay LoD and as such the sensitivity of detection may be less than 100%. Remaining 98 specimens showed efficient detection of the entire three targets. www.nature.com/scientificreports/ of high-resolution melting analysis following multiplex PCR amplification using SYBR green master mix further reduces the cost significantly by eliminating the need for probe, and cut down the cost to around $ 13-15. The first recommended detection kit in India was developed and supplied by the National Institute of Virology (NIV), Pune, which used the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (make Invitrogen Catalogue number: 12574026) that requires about 5-6 h for confirmation of the result. While various other commercial kits are available that target major genes of SARS-CoV-2 requires a time of around 1.0 to 3.5 h after sample preparation. In contrast, our protocol observed successful amplification in 37 min using Fast multiplex PCR and 96 min in HRM coupled real-time detection using SYBR green that reduces the time by around 2-threefold. Additionally, the sample pooling strategy can further contribute to a reduction of cost. In addition to that, sample pooling strategy can further reduce the cost per sample in an emergency 20 . However, this approach requires robust standardization as the probability of reducing efficacy due to the exhaustive PCR component seems to be a major limitation associated with the experiment strategy. With the demonstration of absolute reproducible results with our detection assay involving innovative modification of conventional SARS-CoV-2 test protocol, the HRM coupled multiplex RT-PCR assay represents an easy, fast and low-cost option for SARS-CoV-2 detection with resource limitation.

Received: 10 April 2021; Accepted: 13 August 2021

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The author acknowledges the Department of Health Research Government of India for financial support. The authors also thank Viral Research and Diagnostics Laboratory (VRDL), Department of Microbiology, Gauhati Medical College for providing a laboratory facility to perform the study and the Department of Bioengineering & Technology, Gauhati University for overall support. The authors also acknowledges Mr Himangka Kaushik for english editing in the manuscript. 

The authors declare no competing interests.

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