Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma.
A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 10(3) copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100 microl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.
Arnauld
C
C
Centre National d'Etudes Vétérinaires et Alimentaires (CNEVA), Unité de Biologie Moléculaire, BP 53, Ploufragan, 22 440, France.
Legeay
O
O
Laurian
Y
Y
Thiery
R
R
Denis
M
M
Blanchard
P
P
Jestin
A
A
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D003124:Colorimetry; D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000821:virology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010322:Parvoviridae Infections / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D017992:Parvovirus / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013552:Swine / Q000821:virology*
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral