Rapid and sensitive detection of Mycobacterium ulcerans by use of a loop-mediated isothermal amplification test

Abstract

This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.

Fig 1

The partial nucleotide sequence of a multicopy insertion sequence, IS2404, showing the most sensitive LAMP primer set. The restriction enzyme HhaI site is shown as a red line. The primers are as follows: F3, forward; B3, backward; LF, loop forward; LB, loop backward; FIP, forward inner (F1c and F2); BIP, backward inner (B2c and B1c).

Fig 2

(A) The visual appearance of the BU LAMP amplification product after addition of a 1:10 dilution of SYBR green I dye. The dye fluoresces strongly when bound to the double-stranded DNA, and the resulting DNA-dye complex gives a green color, while fluorescence is minimal when the dye is free in the solution and gives an orange/brown color. (B) The amplification curves obtained using the ESE-Quant tube scanner. Results can be read using the LCD panel as either positive or negative and/or in real time using a computer with the appropriate software (9). The graph reports the fluorescence in millivolts (mV) on the y axis and time in minutes on the x axis. Sample 1, M. ulcerans-spiked soil; 2, supernatant from positive specimen; 3, DNA from needle aspirate; 4, M. marinum; control (C), M. ulcerans DNA (Agy99); negative control (NC), water.