Quantification of Norwalk virus inocula: Comparison of endpoint titration and real-time reverse transcription-PCR methods

Journal of Medical Virology
Pengbo LiuChristine Moe

Abstract

Human noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis. In order to fully characterize features such as persistence and infectious dose, precise quantification of virus concentration is necessary. The purpose of this study was to compare two methods [endpoint titration RT-PCR and quantitative RT-PCR (RT-qPCR)] with respect to quantification of Norwalk virus (NV) in inocula made from purified stock suspensions of human fecal specimens. A full-length NV RNA transcript was developed to facilitate quantification using RT-qPCR and provided log linear detection in the range of 49-4.9 x 10(4) genome equivalent copies (GEC) per reaction. Endpoint titration RT-PCR was used to estimate PCR detection units, and RT-qPCR was used to estimate genome copies in two NV inocula (8fIIa and 8fIIb) used in previous human challenge studies. Overall, RT-qPCR was 1.1-1.6 log(10) more sensitive (lower detection limit) than endpoint titration RT-PCR when the same RNA release method, PCR primers and thermocycle program were used. These findings have important implications for many experimental interpretations, not the least of which is estimating the median infectious dose in human challenge studies.

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Citations

Nov 24, 2011·Journal of Microbiological Methods·Pengbo LiuChristine L Moe
Oct 15, 2013·Applied and Environmental Microbiology·Pengbo LiuChristine L Moe
Aug 23, 2011·Applied and Environmental Microbiology·Scot R SeitzChristine L Moe
Nov 29, 2011·Environmental Microbiology·Unai Pérez-SautuAlbert Bosch
Jan 30, 2016·Clinical and Experimental Immunology·K L NewmanJ S Leon
Jul 19, 2014·The Pediatric Infectious Disease Journal·Pengbo LiuChristine Moe
Oct 20, 2019·The Journal of Infectious Diseases·Roberto MateoDavid N Taylor

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