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.
OBJECTIVE
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.


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 x 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.


CONCLUSIONS
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.
Ward
P
P
Agriculture and Agri-Food Canada, Food Research and Development Centre, St-Hyacinthe, QC, Canada.
Poitras
E
E
Leblanc
D
D
Letellier
A
A
Brassard
J
J
Plante
D
D
Houde
A
A
D000818:Animals; D017927:Calicivirus, Feline / Q000302:isolation & purification; D002415:Cats; D005243:Feces / Q000821:virology; D016751:Hepatitis E / Q000097:blood / Q000235:genetics / Q000821:virology; D016752:Hepatitis E virus / Q000302:isolation & purification*; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis / Q000097:blood / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology*
D012367:RNA, Viral