An improved loop mediated isothermal amplification based assay for the rapid identification of genomic DNA of Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei, the causative agent of the potentially life-threatening infectious disease, melioidosis, is endemic in Bangladesh. The spatial clustering of clinical incidence and the fatal outcome of melioidosis necessitates unequivocal and rapid detection and identification. Culture constitutes the gold standard test for the diagnosis of melioidosis, but identification of B. pseudomallei by culture is time consuming. In the view of the above, the present study has been designed to develop a Loop Mediated Isothermal Amplification based assay (LAMP) for the rapid and accurate detection of B. pseudomallei.

Twenty-two culture confirmed B. pseudomallei isolates and 18 non B. pseudomallei bacterial species were included in the study. DNA were extracted and amplified by in-house LAMP assay, utilizing a set of newly designed primers at optimized conditions. The LAMP product was visualized by various methods. The limit of detection (LOD) of LAMP was determined and detection of B. pseudomallei directly from sterile pre-inoculated urine by the LAMP technique was evaluated.

All 22 B pseudomallei were positive by the in-house LAMP while the 18 heterogeneous bacterial species were LAMP negative. The target sequence amplified by the LAMP outer primers showed the highest similarity with B. pseudomallei isolate UKMH10, confirming the specificity of the newly designed primers. The LOD of the in-house LAMP assay was found to be 0.78 pg, which is much lower than the LOD of PCR (0.78 ng). The in-house LAMP was also found positive for B. pseudomallei using direct sample such as pus and pre-inoculated urine. A closed tube LAMP assay using agar dye capsule was adopted to minimize contamination with LAMP amplification product and thus to increase the overall precision of the test.

Rapid and accurate detection of B. pseudomallei was developed using LAMP-based assay with a set of in-house designed primers. This detection procedure can be established for a cost-effective diagnosis of melioidosis at the rural level.