Comparison of polymerase chain reaction and dot hybridization with enzyme-linked immunoassay, virological examination and polyacrylamide gel electrophoresis for the detection of porcine rotavirus in faecal specimens.
The purpose of this study was the evaluation of RT-PCR (reverse transcription-polymerase chain reaction) technique and dot blot hybridization with PCR-generated probe for the detection of group A rotavirus in faecal samples derived from diarrhoeal piglets. They were compared with virological examination (isolation of the virus), polyacrylamide gel electrophoresis (PAGE) and ELISA. The specificity and sensitivity of each assay was assessed against a 'gold standard' which was created on the basis of the virological examination and PAGE results. One hundred and seventeen faecal samples taken from piglets with the signs of diarrhoea were used as research material. cDNA probe labelled with digoxigenin, complementary to the region between 1 and 650 nucleotides of the segment 9 of porcine OSU and Gottfried reference strains was used. The probe detected in the dot blot hybridization 1 ng of dsRNA of the reference porcine strains. Using that test it was shown that 23 positive samples out of 117 samples of diarrhoeic piglets were detected. The RT-PCR technique appeared to be the most specific and sensitive diagnostic method. From 117 amplified faecal samples, the products of RT-PCR reactions were obtained from 24 samples. By means of this method it was possible to find 104 virions per 1 g of sample. Overall comparison of the results showed respective sensitivities and specificities of 100% and 98.9% for RT-PCR, 95.7 and 98.3% for hybridization, 100 and 94.7% for ELISA, 78.3 and 100% for virological examination, 91.3 and 100% for PAGE.
Winiarczyk
S
S
Department of Epizootiology, Faculty of Veterinary Medicine, Agricultural Academy, Lublin, Poland.
Gradzki
Z
Z
D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005243:Feces / Q000821:virology*; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012401:Rotavirus / Q000276:immunology / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral