AccuPower® RV1 Real-Time RT-PCR 키트와 AccuPower® RV1 Multiplex 키트의 SARS-CoV-2 및 인플루엔자 바이러스 검출에 대한 평가
Evaluation of the AccuPower® RV1 Real-Time RT-PCR Kit and the AccuPower® RV1 Multiplex Kit for SARS-CoV-2 and Influenza Virus Detection
서울대학교병원 진단검사의학과1, 서울대학교병원 임상유전체의학과2, 서울대학교병원운영 서울특별시보라매병원 진단검사의학과3, 녹십자의료재단4
Departments of Laboratory Medicine1 and Genomic Medicine2, Seoul National University Hospital, Seoul; Department of Laboratory Medicine3, Seoul Metropolitan Government-Seoul National University Boramae Medical Center, Seoul; Department of Laboratory Medicine4, Green Cross Laboratories, Yongin, KoreaCorrespondence to:
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lab Med Online 2021; 11(4): 290-296
Published October 1, 2021
Copyright © The Korean Society for Laboratory Medicine.
방법: 우리는 1,098개의 임상검체를 이용하여 AccuPower® RV1 Real-Time RT-PCR 키트와 AccuPower® RV1 Multiplex 키트를 평가하였다. SARS-CoV-2의 존재는 Allplex™ 2019-nCoV Assay (Seegene, Korea)와 Standard M nCoV Real-Time Detection Kit (SD Biosensor, Korea)를 이용하여 재확인하였다. 인플루엔자 바이러스는 Allplex™ Respiratory Panel 1 (Seegene)을 이용하여 재확인하였다.
결과: AccuPower® RV1 Real-Time RT-PCR 키트의 양성 및 음성일치도는 100%였다. AccuPower® RV1 Multiplex 키트의 양성일치도는 SARS-CoV-2에 대해서는 100%였고 인플루엔자 A와 B에 대해서는 98.77%였다. SARS-CoV-2와 인플루엔자 A, B에 대한 카파 값은 0.99를 넘었다. SARS-CoV-2는 객담과 비인두 및 구강인두 스왑 검체를 이용하여 평가하였다. 검체 종류에 따른 검출률의 차이는 없었다.
결론: AccuPower® RV1 Real-Time RT-PCR 키트와 AccuPower® RV1 Multiplex 키트는 임상적으로 사용되는 데 적합한 성능을 보여주었다.
Methods: We clinically evaluated the AccuPower® RV1 Real-Time RT-PCR Kit and AccuPower® RV1 Multiplex Kit by comparing their results for 1,098 clinical samples. The presence of SARS-CoV-2 was confirmed using the Allplex™ 2019-nCoV Assay (Seegene, Korea) and Standard M nCoV Real-Time Detection Kit (SD Biosensor, Korea). Influenza viruses were detected using the Allplex™ Respiratory Panel 1 (Seegene).
Results: The comparative positive and negative agreement values of the AccuPower® RV1 Real-Time RT-PCR Kit for SARS-CoV-2 and influenza A and B were 100%. The positive agreement of the AccuPower® RV1 Multiplex Kit was 100% for SARS-CoV-2 and 98.77% for influenza A and B. The kappa values for SARS-CoV-2 and influenza A and B were >0.99. SARS-CoV-2 was evaluated using both sputum and nasopharyngeal or oropharyngeal swabs. There was no difference in the detection rates for each type.
Conclusions: The findings confirm the clinically comparable performances of the AccuPower® RV1 Real-Time RT-PCR Kit and the AccuPower® RV1 Multiplex Kit.
Coronavirus disease 2019 (COVID-19) was first detected in Wuhan, Hubei Province, China. The disease is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [1, 2]. To date, there have been over 100 million cases in the ongoing SARS-CoV-2 pandemic, with over 2 million deaths globally, according to the COVID-19 dashboard, Center for Systems Science and Engineering, Johns Hopkins University. We cannot accurately diagnose patients with COVID-19 based on the observable symptoms alone because these are nonspecific, such as fever, cough, sputum production, and shortness of breath, and are shared by other respiratory infections . Infiuenza virus is a common cause of respiratory infections. Influenza and SARS-CoV-2 infections present similar symptoms and modes of transmission, including transmission through contact, droplets, and fomites .
Alveolar type II cells (AT2 pneumocytes), the primary site of infiuenza replication, especially infiuenza A, have been implicated as targets for SARS-CoV-2 infection . There is scant evidence regarding the interaction between COVID-19 and infiuenza. One case report described that symptoms worsened when patients were co-infected with SARS-CoV-2 and infiuenza A . Another report described rapid respiratory deterioration in four patients infected with SARS-CoV-2 and infiuenza A or B . Moreover, simultaneous infection with SARS-CoV-2 and other respiratory pathogens may infiuence the morbidity and prognosis in patients . It is important to accurately detect SARS-CoV-2 and infiuenza viruses, including possible coinfections in the community .
Reverse-transcription PCR (RT-PCR) is highly sensitive and specific and has become the standard test for detecting respiratory viruses, including SARS-CoV-2 . This study aimed to evaluate a multiplex PCR to detect SARS-CoV-2 and infiuenza A and B viruses using the ExiStation™ system (Bioneer, Daejeon, Korea).
MATERIALS AND METHODS
1. Clinical specimens
This study was approved by the Institutional Review Board of the Seoul National University Hospital. The study included 1,098 clinical respiratory specimens obtained from the Seoul National University Hospital, Seoul National University Boramae Medical Center, and Green Cross Laboratory. A total of 378 sputum samples and 720 nasopharyngeal/oropharyngeal swabs were collected from patients infected with SARS-CoV-2 or infiuenza. All the sputum samples and 382 nasopharyngeal or oropharyngeal swabs were analyzed for the presence of SARS-CoV-2. The remaining 338 nasopharyngeal or oropharyngeal swabs were tested for infiuenza A and B.
Clinical testing was performed using retrospective clinical samples. We analyzed the samples using the Allplex™ 2019-nCoV Assay (Seegene, Seoul, Korea) and STANDARD M nCoV Real-Time Detection kit (SD Biosensor, Suwon, Korea) for SARS-CoV-2, and Allplex™ Respiratory Panel 1 (Seegene) for infiuenza viruses. All specimens were stored at -70°C until testing with the AccuPower® RV1 Real-Time RT-PCR Kit (Bioneer) and the AccuPower® RV1 Multiplex Kit (Bioneer). The samples were not frozen or thawed more than once.
2. AccuPower® RV1 Real-Time RT-PCR Kit and AccuPower® RV1 Multiplex Kit
The AccuPower® RV1 Kits can detect SARS-CoV-2, infiuenza A (Infiuenza A-H1, Infiuenza A-H3N2, and Infiuenza A-pdm09), and infiuenza B with different Ct values. The test was performed using an
BIONEER’s nucleic acid extraction eluted 400 μL of the clinical sample in a final volume of 100 μL using an automated extraction protocol on ExiStation™ and ExiPrep™ 48 Viral DNA/RNA Kit (Bioneer). The
We used 50 μL aliquots for the AccuPower® RV1 Real-Time RT-PCR Kit analysis and 10 μL aliquots for the AccuPower® RV1 Multiplex Kit analysis. The AccuPower® RV1 Real-Time RT-PCR Kit was optimized for
The samples that exhibited discrepancies in their results were confirmed by sequencing. We extracted the total RNA from the samples according to the manufacturer’s instructions using the ExiStation™ and ExiPrep™ 48 Viral DNA/RNA Kit. We referred to the WHO protocol for the PCR conditions and primer sequences used for amplification ．Primer sequences for infiuenza were as follows: infiuenza type A M30F2/08: 5′-ATG AGY CTT YTA ACC GAG GTC GAA ACG-3′ and infiuenza type A M264R3/08: 5′-TGG ACA AAN CGT CTA CGC TGC AG-3′ for the
4. Analytical sensitivity and specificity
We determined the analytical sensitivity of SARS-CoV-2 using 3-fold serially diluted virus samples. For sputum, 900, 300, 100, 33.3, 11.1, and 3.7 copies/reaction, and for swabs, 300, 100, 33.3, 11.1, 3.7, and 1.23 copies/reaction, were tested in 72 replicates for six concentrations. Similarly, influenza A and B were diluted 3-fold from 600 copies/reaction to 0.27 copies/reaction and tested in 72 replicates for six concentrations.
SARS-CoV-2 was obtained from SeraCare Life Sciences (Milford, MA, USA) and infiuenza viruses from Zeptometrix (Buffalo, NY, USA). Protocols for analytical sensitivity followed the Clinical and Laboratory Standards Institute guideline EP17-A2 . Analytical sensitivity was estimated via probit regression analysis using R Studio (version 3.6.1).
The analytical specificities of the AccuPower® kits were tested using 40 different pathogens. DNA or RNA was extracted from each reference panel and assayed using the same protocols used for clinical samples.
5. Statistical analysis
We assessed significant differences between the two methods using 2×2 contingency tables and the VassarStats website (http://vassarstats.net/) and performed agreement statistics (kappa calculation) to compare the detection sensitivity of the AccuPower® kits and the comparative methods. Clinical sensitivity, specificity, and kappa statistics were reported with 95% confidence intervals (CIs).
1. Comparison of the AccuPower® Kits and the confirmation results
Out of the 760 samples analyzed for SARS-CoV-2, 384 (190 sputum and 194 swab samples) were found to be positive, and 376 (188 sputum and 188 swab samples) were negative. Out of the samples tested for infiuenza, 81 were positive, and 95 were negative (Table 1).
For SARS-CoV-2, the evaluation was conducted at three institutions. The Green Cross Laboratory results were confirmed using the Allplex™ 2019-nCoV Assay and Seoul National University Hospital and Boramae Medical Center results using the STANDARD M nCoV Real-Time Detection Kit. The SARS-CoV-2 results analyzed with AccuPower® RV1 Kits were compared with the confirmed results from each institution (Table 2). Both the positive and negative agreement between the results from AccuPower® RV1 kits and the confirmation results for SARS-CoV-2 was 100%. The kappa values of the two methods were 1.0. Both the positive and negative agreement values of infiuenza A and infiuenza B between the results from AccuPower® RV1 Real-Time RT-PCR Kit and Allplex™ Respiratory Panel 1 were 100%. However, the positive and negative agreement between infiuenza A and B between the results from AccuPower® RV1 Multiplex Kit and Allplex™ Respiratory Panel 1 was 98.77%. The kappa value was 0.99.
The sequencing results for the discordant samples was positive. However, upon re-testing the samples with the comparative reagent (Allplex™ Respiratory Panel 1) and the AccuPower® RV1 Real-Time RT-PCR Kit, the Ct values were detected near the cut-off in both kits. Therefore, the samples were low titer samples, and the discrepancy was attributed to the difference in the minimum nucleic acid concentration needed for use in the two tests.
2. Analytical sensitivity and specificity of the AccuPower® RV1 Kits
The analytical sensitivities of the AccuPower® RV1 Real-Time RT-PCR Kit were 64.57 copies/reaction (95% CI: 42.66–97.72), 56.23 copies/reaction (95% CI: 37.15–87.10), 38.90 copies/reaction (95% CI: 26.92–56.23), 37.15 copies/reaction (95% CI: 26.92–50.12), and 27.54 copies/reaction (95% CI: 19.05–39.81) for infiuenza A-H1N1 seasonal, H1N1 pdm, H3N2, infiuenza B-Yamagata, and infiuenza B-Victoria, respectively. The analytical sensitivities for the
The analytical sensitivities of the AccuPower® RV1 Multiplex Kits were 47.86 copies/reaction (95% CI: 34.67–66.07), 37.15 copies/reaction (95% CI: 26.30–52.48), 33.11 copies/reaction (95% CI: 22.91–46.77), 33.88 copies/reaction (95% CI: 25.12–45.71), and 26.92 copies/reaction (95% CI: 21.38–33.11) for infiuenza A-H1N1 seasonal, H1N1 pdm, H3N2, infiuenza B-Yamagata, and infiuenza B-Victoria, respectively. The analytical sensitivities for the
We tested 40 different viruses and bacterial reference strains using the protocols used for the clinical samples to assess the cross-reactivity results and the detection specificity of the AccuPower® kits (Table 3). The test results were negative (34 species), except for the specific target (related SARS-CoV-2, Infiuenza A, and Infiuenza B). No non-specific positive reactions were observed.
Data on SARS-CoV-2 and infiuenza virus co-infection are limited. Infiuenza infection was classified as one of the characteristics of SARS-CoV-2 positive patients, and a small number of positive cases were studied. However, many studies have reported a correlation between the likelihood of co-infection and the severity or mortality rate due to co-infection [11-13]. Therefore, co-infection of SARS-CoV-2 and infiuenza viruses can pose serious risks. We can prepare for potential pandemics due to such co-infections by routinely testing for respiratory viruses other than SARS-CoV-2.
The AccuPower® RV1 Real-Time RT-PCR Kit contains dried primary materials required for PCR in one tube optimized for the
We evaluated the cross-reactivity with 40 respiratory pathogens, including SARS-CoV-2, infiuenza A, and infiuenza B, and confirmed that there was no cross-reactivity to pathogens with similar symptoms.
We evaluated the limit of detection (LoD) in the sputum and nasopharyngeal/oropharyngeal swabs. The LoD in sputum was approximately 100 copies/test, and that in the swabs was approximately 40 copies/test in both kits. The rate of positivity is higher in sputum samples than that in the swabs and is less affected by the onset of symptoms or the time of collection . However, sputum samples are usually viscous and contain many PCR and extraction inhibitors; therefore, we recommend performing a pre-treatment process for nucleic acid extraction for real-time PCR. Homogenization of sputum with proteinase K, dithiothreitol, or N-acetyl-L-cysteine (NALC) can increase the sensitivity of the test, and hence, the rate of positivity [15, 16].
We confirmed the clinical sensitivity and specificity by comparing the confirmation results of the AccuPower® RV1 Real-Time RT-PCR Kit and the AccuPower® RV1 Multiplex Kit using residual clinical samples collected from three institutions. The sensitivity and specificity were 100%, which is consistent with the confirmation results for both positive and negative samples of SARS-CoV-2. One infiuenza A sample and some infiuenza B samples displayed a discrepant result in the AccuPower® RV1 Multiplex Kit. Sensitivity and specificity were 98.77%. The kappa values for all targets were ≥0.99 and were considered clinically valid when compared with the results of the confirmation tests.
A limitation of our study is that we did not include asymptomatic, pre-symptomatic, and co-infection cases, as only residual samples were used. Further studies, including these cases, need to be conducted.
In conclusion, the AccuPower® RV1 Kits produced results comparable to the Allplex™ and Standard M Kit. The AccuPower® RV1 Kits showed a higher concordance with the results of the comparative methods. Thus, the AccuPower® RV1 Kits are a valuable tool to detect infiuenza A, B, SARS-CoV-2, and their co-infections in clinical laboratories.
This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C2038). Some bioresources from the National Biobank of Korea and the Korea Disease Control and Prevention Agency (grant number: 2020-045) were used.
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