Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis.
Mycobacterium tuberculosis and Mycobacterium bovis are pathogenic bacterial species in the genus Mycobacterium and the causative agents of most cases of tuberculosis (TB). Detection of M. tuberculosis and M. bovis using conventional culture- and biochemical-based assays is time-consuming and laborious. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. In the present study, a visual loop-mediated isothermal amplification (LAMP) assay was designed from the rimM (encoding 16S rRNA-processing protein) gene sequence and used to rapidly detect M. tuberculosis and M. bovis from clinical samples in South China. The visual LAMP reaction was performed by adding calcein and manganous ion, allowing the results to be read by simple visual observation of color change in a closed-tube system, and which takes less than 1 h at 65 degrees C. The assay correctly identified 84 M. tuberculosis isolates, 3 M. bovis strains and 1 M. bovis BCG samples, but did not detect 51 non-tuberculous mycobacteria (NTM) isolates and 8 other bacterial species. Sensitivity of this assay for detection of genomic DNA was 1 pg. Specific amplification was confirmed by the ladder-like pattern of gel electrophoresis and restriction enzyme HhaI digestion. The assay successfully detected M. tuberculosis and M. bovis not only in pure bacterial culture but also in clinical samples of sputum, pleural fluid and blood. The speed, specificity, sensitivity of the rimM LAMP, the lack of a need for expensive equipment, and the visual readout show great potential for clinical detection of M. tuberculosis and M. bovis.
Zhu
Ru-Yi
RY
College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
Zhang
Kou-Xing
KX
Zhao
Ming-Qiu
MQ
Liu
Ya-Hong
YH
Xu
Yun-Yi
YY
Ju
Chun-Mei
CM
Li
Bing
B
Chen
Jin-Ding
JD
D001769:Blood / Q000382:microbiology; D002681:China; D003116:Color; D005260:Female; D005452:Fluoresceins / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008297:Male; D008345:Manganese / Q000378:metabolism; D009163:Mycobacterium bovis / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010996:Pleural Effusion / Q000382:microbiology; D012269:Ribosomal Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis*
D005452:Fluoresceins; D012269:Ribosomal Proteins; D008345:Manganese; C007740:fluorexon