Method validation of the Plaque Assay for SARS-CoV-2 Titration
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Abstract
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading around the world. Therefore, efforts have been made to develop a vaccine to stimulate neutralizing antibody responses against the virus. Plaque assay is used for the SARS-CoV-2 titration. Plaque assay is a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. The plaque is developed from a particle of virus which infects and damages cells.
Objectives: The objective of this research was to study method validation of plaque assay for SARS-CoV-2 titration from clinical specimen of a patient infected with SARS-CoV-2 (hCoV-19/Thailand_59/2020 strain).
Methods: The virus was cultured and propagated, the virus-containing supernatant were aliquoted into 700 cyo tubes, then 60 cryo tubes were randomised and taken to study by using the plaque assay. The assay has been confirmed with such parameters as accuracy, precision (repeatability and reproducibility), robustness, and specificity. The study period started from June 2020 to June 2021.
Results: The results showed that the difference between maximum and minimum titre values of the virus in the accuracy study was less than 0.5 logPFU, so the assay was accurate. The geometric mean (GM) of virus titre in the repeatability study was 5.76 logPFU (standard deviation, or SD, 0.05; 95% confidence interval, or 95%CI, 5.65-5.87), and the percent coefficient of variations (%CV) of repeatability was 0.94%. The GM of the virus titre in the reproducibility study was 5.76 logPFU (SD, 0.18; 95%CI, 5.40-6.12), and %CV was 3.12%. The GM of the virus titre in the robustness study was 5.74 logPFU (SD 0.19; 95%CI, 5.35-6.13), and %CV was 3.38. The assay was considered to be repeatable, reproducible, and robust. The Sig (2-tailed) value in the specificity study was 0.038 (being less than 0.05); and the number of plaques in negative serum mixed with SARS-CoV-2 was significantly greater than in positive serum mixed with SARS-CoV-2; thus, the specificity was good.
Conclusions: The plaque assay can be used as a standard method to test for SARS-CoV-2 and to detect neutralizing antibody responses in COVID-19 patients and animals as well as humans after being immunised with any COVID-19 vaccines at biosafety level 3.
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References
2.World Health Organization. Draft landscape of COVID-19 candidate vaccines [Internet]. Geneva: WHO; 2020 [cited 2020 Dec 20]. Available from: https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines
3.Fukuda A, Sengun F, Sarpay HE, Konobe T, Saito S, Umino Y et al. Parameters for plaque formation in the potency assay of Japanese measles vaccines. J Virol Methods 1996;61: 1-6.
4.World Health Organization. Requirements for varicella vaccine (live), technical Report Annex 1 [Internet]. Geneva: WHO; 1994 [cited 2020 Dec 22]. Available from:https://www.who.int/publications/m/item/varicella-vaccine-(live)-annex-1-trs-no-848
5.Husson Van Vliet J. Colinet G, Yane F, Lemoine P. A simplified plaque assay for varicella vaccine. J Virol Methods 1987;18(2-3):113-20.
6.World Health Organization.Recommendation for Japanese encephalitis vaccine (inactivated) for human use (revised 2007). [Internet]. Geneva: WHO; 2020 [cited 2020 Dec 29]. Available from: https://www.who.int/biologicals/vaccines/Annex_1_WHO_TRS_963.pdf?ua=1
7.World Health Organization.Guidelines for the production and control of Japanese Encephalitis vaccine (live) for human use. Technical Report Series No. 910. 2002; Annex 3 [Internet]. Geneva: WHO; 2020 [cited 2020 Dec 29]. Available from: https://www.who.int/biologicals/areas/vaccines/jap_encephalitis/WHO_TRS_910_A3.pdf
8.Forcic D, Kosutic-Gulija T, Santak M, Jug R, Ivancic-Jelecki J, Markusic M, et al. Comparisons of mumps virus potency estimates obtained by 50% cell culture infective dose assay and plaque assay. Vaccine 2010;28(7):1887-92.
9.World Health Organization. Requirements for measles, mumps and rubella vaccines and combined vaccines (live) [Internet]. Geneva: WHO; 1994 [cited 2020 Dec 29]. Available from: http://apps.who.int/iris/bitstream/handle/10665/39048/WHO_TRS_840_(part2).pdf;jsessionid=DEAC76A6984EEDF96AB7E2DB2BA54114?sequence=2
10.World Health Organization. Requirements for poliomyelitis vaccine (Oral) [Internet]. Geneva: WHO; 1990 [cited 2020 Dec 29]. Available from: http://apps.who.int/iris/bitstream/handle/10665/39526/WHO_TRS_800_(part1).pdf?sequence=1
11.World Health Organization. Manual of laboratory methods for testing of vaccines used in the WHO expanded programme on immunization [Internet]. Geneva: WHO; 1997 [cited 2020 Dec 30]. Available from: https://apps.who.int/iris/handle/10665/63576
12.Wang J, Feng H, Zhang S, Ni Z, Ni L, Chen Y, et al. SARS-CoV-2 RNA detection of hospital isolation wards hygiene monitoring during the Coronavirus Disease 2019 outbreak in a Chinese hospital. International Journal of Infectious Diseases 2020;94:103-106.
13.การตรวจสอบความแรงและความคงตัวของวัคซีนป้องกันโรคไข้สมองอักเสบเจอีชนิดเชื้อเป็นลูกผสมโดยเซลล์เพาะเลี้ยง Vero. นนทบุรี: สถาบันชีววัตถุ กรมวิทยาศาสตร์การแพทย์; 2018. หน้า 11.
14.กรมวิทยาศาสตร์การแพทย์, สถาบันชีววัตถุ. การตรวจสอบความถูกต้องทางชีววิธี (Bioassay Validation). นนทบุรี: สถาบันชีววัตถุ กรมวิทยาศาสตร์การแพทย์; 2020. หน้า 1-8.
15.Darling A, Boose J, Spaltro J. Virus Assay Methods: Accuracy and Validation. Biologicals 1998;26(2):105-110.
16.Thomas S, Jarman R, Endy T, Kalayanarooj S, Vaughn D, Nisalak A et al. Dengue Plaque Reduction Neutralization Test (PRNT) in Primary and Secondary Dengue Virus Infections: How Alterations in Assay Conditions Impact Performance.The American Journal of Tropical Medicine and Hygiene 2009;81(5):825-833.
17.Freshey RI. Culture of animal cells: a manual of basic technology (4th ed), Wiley-Liss 1994;290-96.
18.Low IE. Mycoplasma in tissue culture: Overview of detection methods. Health Lab Sci 1976;13:129-36.
19.Chen TR. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell.Res 1997;104:255-62.
20.สุกัลยาณี ไชยมี, สุภาพร ภูมิอมร. การประเมินความถูกต้องของวิธีปฏิกิริยาลูกโซ่โพลีเมอเรสในการตรวจหาการปนเปื้อนเชื้อมัยโคพลาสมาในเซลล์เพาะเลี้ยง. วารสารกรมวิทยาศาสตร์การแพทย์ 2551;50(2):87-102.
21.Phumiamorn S, Kullabutr K, Tepbhuthorn S, Jivapaisarnpong T. Detection of mycoplasma contamination in cell culture: comparison between DNA fluorochom staining and PCR amplification methods. Thai J. Pharm Sci 2006;30:82-92.