Indian Journal of Public Health

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 66  |  Issue : 3  |  Page : 276--281

A comparative study on the efficiency of commercial reverse transcriptase–Polymerase chain reaction kits for the detection of severe acute respiratory syndrome coronavirus 2 infections


Gopinath Ramalingam1, A Dhanasezhian2, Amudhan Murugesan3, Gowsalya Saminathan3, Lallitha Sivathanu4,  
1 Scientist B, Department of Microbiology Govt Theni Medical College Theni, Tamil Nadu, India
2 Assistant Professor, Department of Microbiology Govt Theni Medical College Theni, Tamil Nadu, India
3 Research Associate, Department of Microbiology Govt Theni Medical College Theni, Tamil Nadu, India
4 Professor and Head, Department of Microbiology, Central Leather Research Institute Adyar, Chennai, Tamil Nadu, India

Correspondence Address:
Lallitha Sivathanu
Department of Microbiology, Central Leather Research Institute Adyar, Chennai, Tamil Nadu
India

Abstract

Background: Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) kits have been reliably employed for the diagnosis of coronavirus disease 2019 (COVID-19) by the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA since the beginning of the disease outbreak. In consideration of reliable diagnosis, apart from RT-PCR, the isothermal nucleic acid amplification-based point-of-care automated kits have also been tagged as a simpler and rapid alternative to the conventional techniques. Currently, the availability of a better diagnostic method for COVID-19 when compared to RT-PCR is nil. The most important step in the detection of SARS-CoV-2 in a RT-PCR diagnostic laboratory is to identify and employ RT-PCR kits with higher sensitivity as well as specificity. Objectives: This study aimed to study commercially available RT-PCR kits for the detection of SARS-CoV-2 infections. Methods: The performance of seven different RT-PCR kits from different manufacturers used for diagnosis of COVID-19 in Govt Theni Medical College and Hospital, Theni, Tamil Nadu were analysed. Nasopharyngeal and oropharyngeal swabs were collected from patients and subjected to RT-PCR using these kits. Results and Conclusion: The sensitivities and batch effects of the assessed kits were slightly different for different targets, for SARS-CoV-2 detection in nasopharyngeal swab specimens. Examination of COVID-19 kits should be done using currently employed kits in routine diagnosis for better efficiency.



How to cite this article:
Ramalingam G, Dhanasezhian A, Murugesan A, Saminathan G, Sivathanu L. A comparative study on the efficiency of commercial reverse transcriptase–Polymerase chain reaction kits for the detection of severe acute respiratory syndrome coronavirus 2 infections.Indian J Public Health 2022;66:276-281


How to cite this URL:
Ramalingam G, Dhanasezhian A, Murugesan A, Saminathan G, Sivathanu L. A comparative study on the efficiency of commercial reverse transcriptase–Polymerase chain reaction kits for the detection of severe acute respiratory syndrome coronavirus 2 infections. Indian J Public Health [serial online] 2022 [cited 2023 Jan 30 ];66:276-281
Available from: https://www.ijph.in/text.asp?2022/66/3/276/356598


Full Text



 Introduction



COVID-19 is viral disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The first reported death occurred on December 31, 2019, in the city of Wuhan, Central China, and then, the disease developed into a pandemic.[1],[2] All countries depend on the early detection of infected individuals for the isolation and mitigation of transmission. Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) diagnosis is the most sensitive and specific assay that is capable of providing crucial etiological evidence for the diagnosis of COVID-19.[3],[4] Recently, the efficiency of RT-PCR for COVID-19 diagnosis has been subjected to questioning.[5] Despite the availability of several COVID-19 RT-PCR diagnostic kits commercially, the detection rates have been considered to be unsatisfactory, and several cases have been detected following the negative detection results that have been obtained from repeated RT-PCR laboratory diagnostic tests.[6] Currently, there is no diagnostic method for COVID-19 that is considered better than the RT-PCR. In this study, we have evaluated the performance of seven commercially available RT-PCR kits for the detection of SARS-CoV-2 that are widely used in Government Theni Medical College and Hospital, Theni, Tamil Nadu. This study aims to compare the sensitivity and specificity among seven commercial COVID-19 RT-PCR diagnostic kits from different manufacturers and suggest comparison methods that may be employed to identify efficient kits for routine diagnostics.

 Materials and Methods



Study design

This was a retrospective study.

Clinical samples

Nasopharyngeal and oropharyngeal swabs were collected from 288 suspected SARS-CoV-2 patients at Government Theni Medical College and Hospital, Theni, Tamil Nadu.

Specimen collection

The specimens were collected in accordance with the guidelines of the Indian Council of Medical Research (ICMR). Nasopharyngeal and oropharyngeal swabs were inserted into sterile vials containing 3 mL of viral transport medium. The specimens were obtained from the patient 2 days after they were diagnosed with COVID-19.

Nucleic acid extraction

The RNA extraction and polymerase chain reaction (PCR) amplification were performed within 2 h after specimen collection. The RNA was extracted from the clinical specimens employing Thermo Scientific™ KingFisher™ Flex Purification System (Cat No. 5400630; Thermo Fisher: Waltham, MA, USA 02451). Approximately 50 μL of total nucleic acid obtained was recovered in nuclease-free tubes and tested immediately.

Selection of kit

Commercially available COVID-19 RT-PCR diagnostic kits [Table 1] from different manufacturers approved by the ICMR were selected for evaluation. Commercial RT-PCR assay kits were selected on the basis of the following criteria such as: (i) if it is a generic RT-PCR, (ii) if it can be performed on standard real-time PCR thermocyclers, and (iii) its availability on the market. All PCRs were run on an ABI 7500 and were then compared to the performance of this kit on the Bio-Rad CFX96 PCR machine.{Table 1}

To verify the sensitivity of SARS-CoV-2 nucleic acid detection kits, seven kits were selected. The kits contained 30 μL (BGI), 25 μL (FTD-SARS-CoV-2), 20 μL (Mylab PathoDetect Kit), 30 μL (SD Biosensor), 20 μL (LabGun COVID-19 ExoFast RT-PCR kit), 25 μL (TaqPath COVID-19 Combo Kit), and 20 μL NIV single-tube kit of the reaction system, including 5–10 μL, of the RNA template.

Analytical performance of seven nucleic acid amplification kits

The analytical sensitivity and specificity along with the positive and negative predictive values (PPV and NPV) in concordance with the accuracy for each of the kits were assessed using clinical diagnostic reports as the reference standard.

Reproducibility of reverse transcriptase–polymerase chain reaction kits

Reproducibility for the RT-PCR kits was evaluated using a positive sample verified by all kits, and the analysis was measured in triplicate by three different operators. The mean value, standard deviations, and the coefficients of variation were also calculated

Statistical analysis

The statistical analyses were performed using the SPSS software version 21.0 (IBM Corp. IBM SPSS Statistics version 21, Armonk, NY, United States of America). The sensitivity, specificity, PPV, and NPV of the SARS-CoV-2 RT-PCR were evaluated using online MedCalc and compared with seven commercial COVID-19 RT-PCR diagnostic kits from different manufacturers (MedCalc Software bvba, Belgium; version 18.9; 2018).

 Results



A total of 288 samples were tested for SARS-CoV-2. A total of 288 samples were tested for SARS CoV-2. Out of 288, 102 samples were positive and the rest negative; we retested all positive and 50 negative samples and finally 50 positive samples and 50 negative samples were selected for the study. Twenty-five samples had high viral loads with a low cutoff threshold (<25) and 25 samples had low viral loads with a high cycle threshold (Ct) value (>25). The specific cutoff values of the seven kits are listed in [Table 2]. The selected 100 samples were tested by RT-PCR for SARS-CoV-2 using seven commercial kits. All seven commercial kits were evaluated with 50 positive and 50 negative samples. Results showed that all kits performed well when strongly positive samples were tested and all seven kits detected all samples with low Ct value (<30). In the case of samples with Ct in the range of 35-38, only 2 kits were able to correctly identify the positive case. [Table 3] shows the detection of targets genes and computed tomography in the 50 positive COVID-19 patients using seven commercial RT-PCR kits. All seven commercial kits were evaluated with 50 positive and 50 negative samples. Results showed that all kits performed well when strongly positive samples were tested and all seven kits detected all samples with low Ct value (<30). [Table 3] shows the detection of target genes and computed tomography in the 50 positive COVID-19 patients using seven commercial RT-PCR kits.{Table 2}{Table 3}

Performance characteristics of seven coronavirus disease 2019 reverse transcriptase–polymerase chain reaction diagnostic kits

From the clinical samples tested (n = 100), which comprised both confirmed (n = 50) and negative (n = 50) cases by clinical manifestation and standard RT-PCR methods, the analytical performances from the seven kits were evaluated [Table 4]. The analytical specificity and PPV of all seven kits were 100% (95% confidence interval [CI]: 92.89%–100.00%). The positive detection numbers of 50 nasopharyngeal and oropharyngeal swab specimens from the confirmed COVID-19 patients using seven commercial RT-PCR kits were 45, 44, 44, 46, 45, 45, and 46, respectively, and the sensitivities were 94.00%, 98.00%, 92.00%, 100.00%, 92.00%, 94.00%, and 100.00%, respectively. Differences observed between the seven kits were statistically significant (P < 0.001). When testing weak positive samples (Ct value > 30), TaqPath Kit and NIV single-tube RT-PCR kit showed 100% (95% CI: 92.89%–100.00%) sensitivity, specificity, PPV, NPV, and accuracy. The analytical sensitivity, NPV, and accuracy of LabGun ExoFast (94.00%, 94.34%, and 97.00%), BGI (98.00%, 98.04%, and 99.00%), SD Biosensor (92.00%, 92.59%, and 96.00%), FTD-SARS-CoV-2 (92.00%, 92.59%, and 96.00%), and Mylab PathoDetect Kit (94.00%, 94.34%, and 97.00%) of weakly positive samples, respectively.{Table 4}

 Discussion



The development of COVID-19 into a global pandemic has made it a worldwide public health concern.[5] Accurate results on time are considered to be important for the decision-making during outbreaks, both in the inpatient and outpatient department settings. For patients admitted to the hospital, the results are critical for medical management and in the implementation of infection control measures. The results also play an important role in the outpatient setting such as in providing community prevalence data and identification of disease hot spots that help the decision-makers to implement social distancing, curfew in infected areas, and in the disease-combating strategy. A short turnaround time of COVID-19 test reports is essential for the judicious use of limited resources, such as the availability of beds, isolation rooms, and real-time cohorting decisions. In addition to all these, results on time are required to ensure the safety of health-care workers and in the minimizing of their exposure as levels of personal protective equipment required by health-care professionals depending on whether the patient is COVID-19 positive or negative.

Here, we provide a direct comparison of key performance characteristics of commercially available RT-PCR kits for the detection of SARS-CoV-2. While most assays showed good sensitivity and specificity for at least one or two of the included targets, others were significantly less sensitive. As all kits contain multiple PCR targets with at least one PCR target approaching similar sensitivity as the reference E or N gene, it can be concluded that the majority of commercial PCR kits can be used for the diagnosis of SARS-CoV-2 infection if single-target detection is considered sufficient. However, confirmation of positive RT-PCR results is advised, especially when a low incidence in the population is expected. Seven kits showed comparable sensitivity with the reference gene assays for multiple PCR targets and would be excellent candidates for further clinical evaluation by laboratories interested in implementing RT-PCR diagnostic capacity. This study results show higher analytical sensitivities, specificity, and accuracy of the TaqPath Kit and NIV single-tube RT-PCR when compared to the other RT-PCR kits.

 Conclusion



The sensitivities and batch effects of the assessed kits were slightly different for different targets, for SARS-CoV-2 detection in nasopharyngeal swab specimens. Therefore, these data suggest that suspected COVID-19 cases with low RNA concentration or at the initial stages of the disease should be examined using different COVID-19 kits to a feasible extent and that comparison of commercial COVID-19 RT-PCR kits should be performed before using new batches of the kits in routine diagnostics. In addition, with the increasing number of commercial COVID-19 kits, it is necessary for researchers to share information such as multicenter kit comparison methods and the detection abilities of various commercial RT-PCR diagnostic kits for SARS-CoV-2 infections.

Acknowledgment

We thank NIV-ICMR and TNMSC for providing the commercial RT-PCR kits.

Financial support and sponsorship

This study was financially supported by the ICMR/TNMSC.

Conflicts of interest

There are no conflicts of interest.

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