Comparative Evaluation of a Dual-target Real-time RT-PCR Assay for COVID-19 Diagnosis and Assessment of Performance in Pooled Saliva and Nasopharyngeal Swab Samples

Overview

Journal Expert Rev Mol Diagn

Specialty Molecular Biology

Date 2021 May 20

PMID 34014785

Citations 4

Authors

Cyril C Y Yip

Kit-Hang Leung

Anthony C K Ng

Kwok-Hung Chan

Kelvin K W To

Jasper F W Chan

Ivan F N Hung

Vincent C C Cheng

Siddharth Sridhar

Affiliations

Soon will be listed here.

Abstract

Objectives: Sensitive molecular diagnostic assays are essential for COVID-19 diagnosis. We evaluated the Hecin Scientific SARS-CoV-2 nucleic acid test kit, a dual-target real-time RT-PCR assay targeting the SARS-CoV-2 N and ORF1ab genes.

Methods: The Hecin test kit's diagnostic performance in detecting SARS-CoV-2 RNA was compared to the LightMix Modular SARS and Wuhan CoV E-gene kit (TIB Molbiol) and an in-house single-tube nested real-time RT-PCR using 296 clinical specimens, 11 proficiency testing samples, and 30 low-positive deep throat saliva and nasopharyngeal swab (NPS) samples pooled into negative samples in ratios of 1:5, 1:10, and 1:30.

Results: The limit-of-detection of the Hecin test kit was around 500 dC/mL for the N and ORF1ab targets. Sensitivity and specificity of the Hecin test kit were 98.1% (95% CI: 93.4-99.8%) and 100% (98.1-100%), respectively, when measured against the reference method. The Hecin test kit showed fair sensitivity (80%) in low-positive NPS samples pooled in ratios of 1:5 and 1:10. Its performance in pooled samples could be dramatically improved by adjusting the assay Ct cutoff.

Conclusion: The Hecin test kit enables sensitive and specific detection of SARS-CoV-2 in clinical samples and pooled samples.

Citing Articles

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Performance evaluation of Biofire Film Array Respiratory Panel 2.1 for SARS-CoV-2 detection in a pediatric hospital setting.

Ramirez M, Zarate M, Rodriguez L, Aquino V PLoS One. 2023; 18(10):e0292314.

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Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis.

Yip C, Sridhar S, Chan W, Ip J, Chu A, Leung K Microbiol Spectr. 2022; 10(6):e0196222.

PMID: 36445095 PMC: 9769742. DOI: 10.1128/spectrum.01962-22.

An improvement of current hypercube pooling PCR tests for SARS-CoV-2 detection.

Wu T, Liao Y, Fuh C, Weng P, Wang J, Chen C Front Public Health. 2022; 10:994712.

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References

Igloi Z, Leven M, Abdel-Karem Abou-Nouar Z, Weller B, Matheeussen V, Coppens J . Comparison of commercial realtime reverse transcription PCR assays for the detection of SARS-CoV-2. J Clin Virol. 2020; 129:104510. PMC: 7833356. DOI: 10.1016/j.jcv.2020.104510. View

Yip C, Sridhar S, Cheng A, Leung K, Choi G, Chen J . Evaluation of the commercially available LightMix® Modular E-gene kit using clinical and proficiency testing specimens for SARS-CoV-2 detection. J Clin Virol. 2020; 129:104476. PMC: 7255195. DOI: 10.1016/j.jcv.2020.104476. View

Yip C, Ho C, Chan J, To K, Chan H, Wong S . Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens. Int J Mol Sci. 2020; 21(7). PMC: 7177594. DOI: 10.3390/ijms21072574. View

Lee J, Kim S, Sung H, Lee S, Lee H, Roh K . Challenges and issues of SARS-CoV-2 pool testing. Lancet Infect Dis. 2020; 20(11):1232-1233. PMC: 7833783. DOI: 10.1016/S1473-3099(20)30516-8. View

Yip C, Sridhar S, Leung K, Ng A, Chan K, Chan J . Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection. Int J Mol Sci. 2020; 21(16). PMC: 7461039. DOI: 10.3390/ijms21165674. View

Yip C, Chan W, Ip J, Seng C, Leung K, Poon R . Nanopore Sequencing Reveals Novel Targets for Detection and Surveillance of Human and Avian Influenza A Viruses. J Clin Microbiol. 2020; 58(5). PMC: 7180252. DOI: 10.1128/JCM.02127-19. View

Chan J, Yip C, To K, Tang T, Wong S, Leung K . Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated and with Clinical Specimens. J Clin Microbiol. 2020; 58(5). PMC: 7180250. DOI: 10.1128/JCM.00310-20. View

Cherif A, Grobe N, Wang X, Kotanko P . Simulation of Pool Testing to Identify Patients With Coronavirus Disease 2019 Under Conditions of Limited Test Availability. JAMA Netw Open. 2020; 3(6):e2013075. PMC: 7315509. DOI: 10.1001/jamanetworkopen.2020.13075. View

Pilcher C, Westreich D, Hudgens M . Group Testing for Severe Acute Respiratory Syndrome- Coronavirus 2 to Enable Rapid Scale-up of Testing and Real-Time Surveillance of Incidence. J Infect Dis. 2020; 222(6):903-909. PMC: 7337777. DOI: 10.1093/infdis/jiaa378. View

10.

Kim S, Lee J, Sung H, Lee H, Han M, Yoo C . Pooling Upper Respiratory Specimens for Rapid Mass Screening of COVID-19 by Real-Time RT-PCR. Emerg Infect Dis. 2020; 26(10):2469-2472. PMC: 7510748. DOI: 10.3201/eid2610.201955. View

11.

Yelin I, Aharony N, Shaer Tamar E, Argoetti A, Messer E, Berenbaum D . Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools. Clin Infect Dis. 2020; 71(16):2073-2078. PMC: 7197588. DOI: 10.1093/cid/ciaa531. View

12.

Chu H, Chan J, Yuen T, Shuai H, Yuan S, Wang Y . Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study. Lancet Microbe. 2020; 1(1):e14-e23. PMC: 7173822. DOI: 10.1016/S2666-5247(20)30004-5. View

13.

Chan J, Yuan S, Kok K, To K, Chu H, Yang J . A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet. 2020; 395(10223):514-523. PMC: 7159286. DOI: 10.1016/S0140-6736(20)30154-9. View

14.

Cheng V, Wong S, Chen J, Yip C, Chuang V, Tsang O . Escalating infection control response to the rapidly evolving epidemiology of the coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 in Hong Kong. Infect Control Hosp Epidemiol. 2020; 41(5):493-498. PMC: 7137535. DOI: 10.1017/ice.2020.58. View

15.

Corman V, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu D . Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25(3). PMC: 6988269. DOI: 10.2807/1560-7917.ES.2020.25.3.2000045. View

16.

Chu A, Chan W, Ip J, Yip C, Chan J, Yuen K . Evaluation of simple nucleic acid extraction methods for the detection of SARS-CoV-2 in nasopharyngeal and saliva specimens during global shortage of extraction kits. J Clin Virol. 2020; 129:104519. PMC: 7309780. DOI: 10.1016/j.jcv.2020.104519. View

17.

Zhu N, Zhang D, Wang W, Li X, Yang B, Song J . A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382(8):727-733. PMC: 7092803. DOI: 10.1056/NEJMoa2001017. View

18.

Chen J, Yip C, Chan J, Poon R, To K, Chan K . Clinical Performance of the Luminex NxTAG CoV Extended Panel for SARS-CoV-2 Detection in Nasopharyngeal Specimens from COVID-19 Patients in Hong Kong. J Clin Microbiol. 2020; 58(8). PMC: 7383549. DOI: 10.1128/JCM.00936-20. View

19.

Aragon-Caqueo D, Fernandez-Salinas J, Laroze D . Optimization of group size in pool testing strategy for SARS-CoV-2: A simple mathematical model. J Med Virol. 2020; 92(10):1988-1994. PMC: 7264525. DOI: 10.1002/jmv.25929. View

20.

Ben-Ami R, Klochendler A, Seidel M, Sido T, Gurel-Gurevich O, Yassour M . Large-scale implementation of pooled RNA extraction and RT-PCR for SARS-CoV-2 detection. Clin Microbiol Infect. 2020; 26(9):1248-1253. PMC: 7308776. DOI: 10.1016/j.cmi.2020.06.009. View