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Comparative analysis of different TaqMan real‐time RT‐PCR assays for the detection of swine Hepatitis E virus and integration of Feline calicivirus as internal control
Author(s) -
Ward P.,
Poitras E.,
Leblanc D.,
Letellier A.,
Brassard J.,
Plante D.,
Houde A.
Publication year - 2009
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2008.04104.x
Subject(s) - feline calicivirus , virology , taqman , caliciviridae , biology , calicivirus , hepatitis a virus , real time polymerase chain reaction , virus , hepatitis e virus , viral disease , gene , genotype , genetics
Aims: The aim of this study was to compare the performance of four TaqMan RT‐PCR assays with a commonly used nested RT‐PCR and to include the Feline calicivirus (FCV) as an internal control. Methods and Results: RNA extracted from 87 swine faecal samples and 103 swine blood samples was subjected to different detection systems. Faecal samples naturally contaminated with Hepatitis E virus (HEV) and negative samples were artificially inoculated with 3·2 × 10 3 PFU of FCV. Detection results obtained on faecal and plasma samples were 35·6% and 4·9% with the nested RT‐PCR assay, 8·0% and 0%, 0% and 0%, 13·8% and 0% and 36·8% and 3·9% with TaqMan systems A, B, C and D respectively. The Ct means obtained with the multiplex TaqMan assay were 30·11 and 30·43 for the detection of FCV with HEV contaminated samples and negative samples. Conclusions: The TaqMan system D was more suitable for the detection of swine HEV strains than the three others and FCV was integrated successfully as an internal control. Significance and Impact of the Study: FCV was demonstrated as an efficient control to monitor the RNA extraction process and HEV amplification procedure in a multiplex HEV/FCV TaqMan assay. This control would be helpful in limiting false negative results.