We have reported the simple detection system of Mycobacterium avium subsp. paratuberculosis (MAP) based on LAMP method, which was sensitive and specifically identify MAP DNA by means of white turbidity (J. Clin. Microbiol. 41:4359-65. 2003). Here we present the improved LAMP system with modified primer sets and additional denaturing step of DNA, which allows for a rapid detection of MAP in cattle feces. The new primer sets were designed from first one third region of IS900 sequence. As the additional step, the extracted DNA was denatured by heat-treatment and added to a reaction mixture.

Using the new system, 10 copies of cloned IS900 DNA or 0.005pg of genomic DNA from cultured MAP were detected within 30 minutes. It specifically distinguished MAP from M. avium or M. scroflaceum. Non-specific amplification was not observed within 120 minutes in the LAMP assay with 10 fecal samples which were obtained from Johne's disease free herd. Thus, the improved LAMP system showed high sensitivity and specificity for detecting MAP in fecal samples.

In order to compare with the culture method using Herrold's egg yolk medium with mycobactin, LAMP system was evaluated using 40 cattle fecal samples obtained from MAP infected cattle herds. The samples were collected when the cattle were slaughtered, and were stored at 4°C for less than 3 days before conducting the culture test. Nineteen samples, including 9 culture-positive samples, were positive with LAMP system. On the other hand, twenty one LAMP negative samples were all culture-negative. The LAMP's higher sensitivity would allow for the detection of tiny amount of DNA of dead or dormant MAP cells potentially present in the culture-negative but LAMP positive samples. Additionally, real-time monitoring data of LAMP indicates that there is some correlation between a number of colonies on culture media and amplification time to reach the threshold turbidity. These results suggest that LAMP system is robust and useful method for the detection of MAP DNA, and will contribute to rapid and reliable diagnosis of MAP infected cattle.