Bovine semen samples spiked with bovine herpesvirus 1 (BHV-1) were used to compare dot blot hybridization, polymerase chain reaction (PCR), and virus isolation for detection of BHV-1 in bovine semen. The PCR amplification used primers targeting the BHV-1 thymidine kinase gene and a nucleic acid releasing cocktail (GeneReleaser); the PCR product was used as the DNA probe in dot blot hybridization; virus isolation was done in primary bovine fetal testis (BFT) cell cultures. Semen diluted 1:20 in tissue culture medium had the least cytotoxicity and inhibition of viral cytopathic effects in BFT cells, allowing detection of 1 TCID50/100 microL of BHV-1 suspension by virus isolation. The presence of foreign DNA such as bovine sperm DNA or salmon sperm DNA increased the sensitivity of dot blot hybridization in detecting BHV-1, allowing detection of 20,000 TCID50/100 microL of neat semen. The inhibition of PCR amplification of BHV-1 DNA in bovine semen was eliminated by diluting the samples 1:20 in tissue culture medium. The best PCR amplification was obtained when semen was diluted 1:20 and when a reaction buffer of pH 9.0, with 1.0 mM MgCl2 was used. Under these conditions, the PCR followed by ethidium bromide staining of agarose gels could detect 1 TCID20/100 microL of sample, whereas PCR followed by Southern blot hybridization could detect 0.01 TCID50/100 microL of sample.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
The purpose of this study was to determine the rate of decline and geographical distribution by municipality of clinical and subclinical African swine fever (ASF) in the affected areas of Spain. A second aim was to evaluate the performance of diagnostic tests in the Spanish ASF eradication program. Clinical outbreaks were confirmed using both the direct and indirect immunofluorescence test (and if both were negative, by the hemabsorption test). The serological status of swine was determined by an enzyme-linked immunosorbent assay (ELISA) and suspect serum samples were confirmed by the immunoblot assay.
The number of clinical outbreaks (herds) of ASF for 1989, 1990 and 1991 was 170, 347 and 207, respectively. The numbers of municipalities within each affected province experiencing acute outbreaks for the same time periods were 49, 69 and 48, respectively. Serologically diagnosed animals positive for ASF were 1.1% of animals tested in 1989, 0.5% in 1990 and 0.8% in 1991. The corresponding positive predictive values of the standard ELISA test used were 99.0, 97.9 and 98.8, respectively. Similarly, the number of municipalities within each affected province experiencing serologically positive subclinically infected animals was 269, 178 and 147 for each of the years 1989, 1990 and 1991, respectively. 相似文献