The Detection Of Fluorescent Antibody And Establishment Of PCR Method For Goat Pox

Goat pox is a highly contagious disease of goats and sheep characterized by pyrexia, generalized skin and internal pox lesions, and lymphadenopathy. The disease is caused by goat poxvirus(GPV), which is enveloped, double-stranded DNA virus, classified in the genus capripoxvirus of the family poxviridae. Goat pox is ancient disease that is currently endimic in the Middle East, the Indian subcontinent, Bangladesh, and Central and Northern Africa, The desease is defined as a heavy disease of animal by OIE, based on further economic loss in the endemic countries.In recent years, the disease is endemic in Hei longjiang, Sichuan and Yunnan provinces of China. In October 2002, the disease outbroke firstly and spreaded to eight areas of Guizhou province, which was incidence of 66.38% and mortality of 42.06%. In August 2007, the disease outbroke secondly in the district. To diagnose of the borderline cases in an early stage, the methods of serology and molecular biology were carried out, and the achievements is gained as following:1. Establishment of fluorescent antibody test for GPV: The fluorescent antibody was prepared with IgG purified from rabbit anti-goatpox virus(GPV) serum and used to detect the antigen of GPV in BHK-21 infected cells. The results showed, all the cell slides and smears of the infected cells were positive, but the control cells were negative, and the optimal working concentration of the fluorescent antibody was 1:32, and the fluorescence could be inhibited specifically by the standard positive serum of goat pox.2. Establishment of PCR method and sequence analysis for P32 gene: According to the sequences of P32 gene in GenBank, a pair of specific primers were designed, and a PCR method was established successfully. The specificity is approved by digested test of PCR products and the minimum content of PCR detection is 19.06×10~3 pg. And the positive rate of this PCR method for ten pock samples is 100%, which these samples were collected from infected goats in different areas of Guizhou province.The P32 gene of the PCR products were purified by Gel Extract Kit and cloned into the pMD18-T vector, and then transformed into DH5a competent cells. After identification of PCR and restriction enzymes, the positive vectors were sequenced by TaKaRa company. The results showed, the homology of nucleotide sequence between 4 strains and referenced strains of were 97.9%～100%, and that of deduced amino acid sequence were 96.2%～100%.3. Establishment of PCR method and sequence analysis for ITR gene fragments: According to ITR gene sequence in GenBank, a pair of specific primers were designed, and a PCR method was established successfully. The specificity is approved by digested test of PCR products and the minimum content of PCR detection is 24.40 pg. And the positive rate of this PCR method for ten pock samples is also 100%, which these samples were collected from infected goats in different areas of Guizhou province.The ITR gene fragment of the PCR products were purified by Gel Extract Kit and cloned into the pMD18-T vector, and then transformed into DH5a competent cells. After identification of PCR and restriction enzymes, the positive vectors were sequenced by TaKaRa company. The results showed, the homology of nucleotide sequence between 4 strains and referenced strains of were 98.2%～100%, and that of deduced amino acid sequence were 96.5%～100%.4. By comparing the specificity, sensitivity of PCR and the homology of gene sequence about P32 and ITR gene fragment. The PCR method based on ITR gene was superior to that of P32 gene in sensitivity and conservatism. So, a more sensitive and conservative PCR method was provided in detecting capripoxvirus clinically.