Sensitive and specific detection of Yersinia pseudotuberculosis by loop-mediated isothermal amplification

Abstract

We developed a loop-mediated isothermal amplification method able to detect Yersinia pseudotuberculosis strains in 30 min by using six primers designed by targeting the inv gene. This method is more sensitive than PCR and might be a useful tool for detecting and identifying Y. pseudotuberculosis.

FIG. 1.

Detection of the LAMP amplification signals. A total of 10⁵ CFU of template DNA of Y. pseudotuberculosis 1b, 2a, 3, 4b, 5a, and 6 was used for the LAMP reaction.

FIG. 2.

Electrophoretic analysis of LAMP (A) and PCR (B) products. The numbers above each lane represent 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ CFU per reaction tube of template DNA of Y. pseudotuberculosis 1b. Lane D, LAMP product after digestion with BssHII; lane N, LAMP or PCR in the absence of template DNA; lane M, 1-kb ladder DNA size marker.

FIG. 3.

LAMP detection of the inv gene in liver samples from Y. pseudotuberculosis-infected monkeys and uninfected monkeys. The samples of each lane and the number of bacteria isolated, in log CFU/gram, from each sample are the following: lanes 1 to 7, squirrel monkey, 5.1, 6.8, 6.4, 6.8, 5.1, 2.2, and 5.0, respectively; lane 8, orangutan, 5.2; lanes 9 to 12, squirrel monkey, 4.9, 6.3, 6.7, and 5.6, respectively; lane 13, dark-handed gibbon, 5.2. Lanes 14 and 15, squirrel monkeys from which no Yersinia species were isolated. Lane N, LAMP in the absence of template DNA. Lane M, 1-kb ladder DNA size marker.