Multiplex PCR and reverse hybridization to detect and identify imycobacterium i species

Abstract

The human immunodeficiency virus (HIV) epidemic increases in the incidence of tuberculosis and other infection caused by nontuberculous mycobacteria (NTM). It is therefore important to rapidly and accurately identify species of mycobacteria for treatment guidelines. In this study, multiplex PCR and reverse hybridization were developed to identify species of mycobacteria. Multiplex PCR could identify M. tuberculosis complex , M. avium, M. intracellulare, and genus Mycobacterium in only one test. The sensitivity of multiplex PCR to detect DNA of genus Mycobacterium, M. tuberculosis complex , M. avium, and M. intracellulare was found to be 10 pg, 10 pg, 10 pg, and 1 ng, respectively. Multiplex PCR could not amplify DNA of other bacteria and fungi except Nocardia. The multiplex PCR collectly identified 85 clinical isolates of mycobacteria, detected and identified mycobacteria in 50 AFB-positive clinical specimens and 50 signal-positive hemoculture samples. The developed reverse dot blot hybridization was able to detect the additional Mycobacterium species from the amplified product of multiplex PCR, i.e., M. chelonae or M. abscessus, M. flavescens, M. fortuitum, M. gordonae, M. kansasii or M. gastri or M. scrofulaceum or M. simiae, and M. xenopi. The sensitivity of reverse dot blot hybridization was the same as that of multiplex PCR. Species-specific probes did not cross-react with DNA of other bacteria and fungi. This study concludes that multiplex PCR and reverse hybridization to detect and identify Mycobacterium species are rapid (within 6-10 h), accurate method and might be applicable for identification of mycobacteria in routine laboratories