| Summary: | 碩士 === 國立中興大學 === 獸醫學系暨研究所 === 101 === Mycobacterium avium subsp. paratuberculosis (MAP) is one of the most widespread and economically important zoonotic pathogens of ruminants and human, which may cause Johne''s disease in ruminants. The cost of Johne''s disease to the cattle industry is staggering with an estimated $1.5 billion loss every year in the USA. In recent years, the prevalence of MAP in the USA and Taiwan had increased remarkably. Accurate diagnostic tests for MAP such as ELISA and PCR are routinely used to identified the disease-free animals, however, the sensitivity and specificity of ELISA and PCR are unsatisfied. The purpose of this study was to detect the presence of MAP in fecal samples of dairy cows by Loop-mediated Isothermal Amplification (LAMP) system and nested PCR-ELISA, which were compared by using bacteriological culture as the gold standard. The samples were collected from two sources, 21 laboratory-confirmed samples and 11 field samples of suspected cases with positive result of ELISA assay. All 32 faecal samples were detected by bacteriological culture, PCR, nested PCR, LAMP and nested PCR-ELISA. The results indicated that PCR can detect 100 pg/μl of MAP, while LAMP and nested PCR can detect 1 pg/μl of MAP. However, nested PCR-ELISA can detect 0.1 pg/μl of MAP. None of the negative controls including E. coli, Staphylococcus sp. and Enterococcus sp. was detected by PCR, nested PCR, LAMP and nested PCR-ELISA. In 16 positive and 9 negative laboratory-confirmed samples, the results from LAMP and nested PCR-ELISA were identical to those from bacteriological culture, which yielded a sensitivity of 100% and specificity of 100%. However, all of the 9 field samples detected by PCR, nested PCR, LAMP and nested PCR-ELISA showed positive results, which were inconsistent with those by bacteriological culture. It is inferred that the storage condition of faecal samples may affect the results of bacteriological culture. In conclusion, the accuracies for detection of MAP in faecal samples by LAMP and nested PCR-ELISA are relatively high as compared to the bacteriological culture.
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