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Quantification of Norwalk virus inocula: Comparison of endpoint titration and real‐time reverse transcription‐PCR methods
Author(s) -
Liu Pengbo,
Hsiao HuiMien,
Jaykus LeeAnn,
Moe Christine
Publication year - 2010
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.21851
Subject(s) - virology , biology , real time polymerase chain reaction , reverse transcriptase , norwalk virus , reverse transcription polymerase chain reaction , virus , microbiology and biotechnology , genome , rna , norovirus , gene , messenger rna , genetics
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 × 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. J. Med. Virol. 82:1612–1616, 2010. © 2010 Wiley‐Liss, Inc.