Detection of Mycobacteriosis in guppy, Poecilia reticulata, by loop-mediated isothermal amplification method

Mycobacterium spp. were isolated from diseased guppies Poecilia reticulata from Nakorn Pathom province, Thailand, during a period of severe disease loss. Two mycobacterial isolates, Thai 3 and Thai 4, were closely related to M. gordonae by phylogenetic analyses. A loop-mediated isothermal amplification (LAMP) technique was designed for M. gordonae identification in guppies. The 16S rRNA gene of the M. gordonae of Thai 4 was used to construct specific primers for LAMP. The LAMP reaction condition was tested and optimized at 65C for 60 min. The sensitivity of the LAMP was about 2.5 x 10 super(2) CFU. The LAMP method was applied to examine those guppies challenged with M. gordonae and healthy guppies from farms. The LAMP test detected M. gordonae in 66.6% of the experimentally inoculated fish whereas 11.1% was detected by microbiological culture and both of the techniques detected 37.8% of diseased fish among the healthy fish. The results suggest that the LAMP test is a rapid and reliable method for the detection of M. gordonae infected guppies.