title: Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.
abstract: Nested PCR and qPCR (quantitative PCR) tests based on glycoprotein B (gB) gene were designed for detecting Bovine herpesvirus 6 (BoHV6) in bovine whole blood samples and wild ruminant blood clots (deer and roe-deer). This virus, commonly known as BLHV (bovine lymphotropic herpesvirus) belongs to the Herpesviridae family, subfamily Gammaherpesvirinae and Macavirus genus. DNA isolated from 92 dairy cow blood samples and 69 wild ruminant clots were examined for the presence of BoHV6 using nested PCR and qPCR tests. Viral DNA was detected by using nested PCR in 59 out of 92 bovine blood samples (64.1%), and by qPCR in 68 out of 92 bovine blood samples (73.9%), but none out of 69 DNA samples isolated from wild ruminant blood clots, was positive in both assays. The specificity of nested PCR and qPCR was confirmed by using BoHV1, BoHV4, BoHV6, BFV, BIV, and BLV DNA. The sensitivity of nested PCR and qPCR was determined using a serially 10-fold diluted vector pCR2.1HgB (2 × 10(0)-2 × 10(6)copies/reaction). In this testing, qPCR was more sensitive than the nested PCR, detecting two copies of BoHV6 whilst the limit of detection for nested PCR was 20 copies. In all qPCR assays, the coefficients of determination (R(2)) ranged between 0.990 and 0.999, and the calculated amplification efficiencies (Eff%) within the range of 89.7-106.9. The intra- and inter-assay CV (coefficient of variation) values did not exceed 4%.
authors: National Veterinary Research Institute, Department of Biochemistry, Partyzantów 57, 24-100 Puławy, Poland. Electronic address: kubip@piwet.pulawy.pl.
authors: Piotr
authors: P
authors: Kubiś
authors: Magdalena
authors: M
authors: Materniak
authors: Jacek
authors: J
authors: Kuźmak
mesh_terms: D000818:Animals; D001769:Blood / Q000821:virology*; D002417:Cattle; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D003670:Deer; D018142:Gammaherpesvirinae / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000662:veterinary* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods*; D019562:Viral Load / Q000379:methods*
chemical_list: D004279:DNA, Viral
keywords: BoHV6; Nested PCR; gB; qPCR