Method validation of the Plaque Assay for SARS-CoV-2 Titration

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Kornnika Kullabutr
Asmah Yusho
Sompong Sapsutthipas
Supaporn Chumpol
Kanokphon Ritthitham
Supaporn Phumiamorn


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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Kullabutr K, Yusho A, Sapsutthipas S, Chumpol S, Ritthitham K, Phumiamorn S. Method validation of the Plaque Assay for SARS-CoV-2 Titration. TFDJ [Internet]. 2021 Oct. 14 [cited 2024 Jun. 14];28(3):10-21. Available from:
Research Article


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