RESUMEN
We established a fluorescent quantitative PCR (qPCR)method for the detection of Chlamydophila abortus (C. abortus),and replaced the method of smear staining which has subjective influence on the detection of C.abortus inactivated vaccine titer.According to the conserved sequence of the large cysteine-rich periplasmic protein(envB)of C.abortus,a specific primer was designed and the EnvB-PMD19T positive plasmid was used as the reference standard,optimization condition,sensi-tivity assay,specificity assay,repeatability assay and the bacteria loads of organs from mouse have been done.The results showed that the standard curve established with positive plasmid had a liner response from 1×102copies/μL to 1×106copies/μL with the correlation coefficient of 97%,sensitive for detecting C.abortus with the detection limit of 10 copies/μL,and re-peatable and stable with the coefficients of variation less than 2%.According to the result,the established method can detect the bacteria loads in organ of mouse,which provide a reliable method for evaluation of inactivated C.abortus vaccine.
RESUMEN
<p><b>BACKGROUND</b>Echinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China. Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections.</p><p><b>METHODS</b>The genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.) granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program, and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method.</p><p><b>RESULTS</b>Fifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4.</p><p><b>CONCLUSION</b>We found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological methods for diagnosis of metacestodosis.</p>