Species Identification of Mycobacteria by PCR-Restriction Fragment Length Polymorphism of the rpoB Gene

Abstract

PCR-restriction fragment length polymorphism analysis (PRA) using the novel region of the rpoB gene was developed for rapid and precise identification of mycobacteria to the species level. A total of 50 mycobacterial reference strains and 3 related bacterial strains were used to amplify the 360-bp region of rpoB, and the amplified DNAs were subsequently digested with restriction enzymes such as MspI and HaeIII. The results from this study clearly show that most of the mycobacterial species were easily differentiated at the species level by this PRA method. In addition, species with several subtypes, such as Mycobacterium gordonae, M. kansasii, M. celatum, andM. fortuitum, were also differentiated by this PRA method. Subsequently, an algorithm was constructed based on the results, and a blinded test was carried out with more than 260 clinical isolates that had been identified on the basis of conventional tests. Comparison of these two sets of results clearly indicates that this new PRA method based on the rpoB gene is more simple, more rapid, and more accurate than conventional procedures for differentiating mycobacterial species.

Severe vitamin D deficiency is associated with non‐tuberculous mycobacterial lung disease: A case‐control study

“…26 NTM species were identified using a polymerase chain reaction and restriction length polymorphism method based on the rpoB gene and confirmed using multilocus sequence analysis based on hsp65, rpoB and 16S ribosomal RNA fragments, as described previously. [27][28][29][30] The aetiological organisms were M. avium complex in 69 (66%) patients (M. avium in 38 and M. intracellulare in 31) and M. abscessus complex in 35 (34%) patients (M. abscessus in 14 and M. massiliense in 21). The study was approved by the Institutional Review Board at our institution, and written informed consent was obtained from each participant.…”

Non‐tuberculous mycobacterial infections of the skin: A retrospective study of 29 cases

“…Mycobacterial isolates were identified according to polymerase chain reaction (PCR) restriction fragment length polymorphisms using the Myco-ID kit. 2 The NTM were cultured from skin biopsy specimens and purulent discharges or exudates from cutaneous lesions. Decontaminated specimens were used to inoculate BACTEC Mycobacteria Growth Indicator Tubes (MGIT; Becton Dickinson, Piscataway, NJ, USA) and 3% Ogawa media (Korean Institute of Tuberculosis, Korean National Tuberculosis Association, Seoul, Korea).…”

An outbreak of skin and soft tissue infection caused by Mycobacterium abscessus following acupuncture

“…Patient specimens homogenized in phosphate-buffered saline and environmental samples as either swabs or water concentrated by filtration through 0.22-lm filters were inoculated on blood agar plates and Middlebrook 7H10 agar plates, and cultured at 37°C for 5-6 days. Colonies were examined for acid-fast bacilli by the Ziehl-Neelsen method [14], and pure culture isolates were subjected to species identification by comparative sequence analyses of portions of the 16S rRNA gene, hsp65 and rpoB, which were amplified by PCR [15][16][17]. Molecular comparisons of the isolates from patient and environmental samples were performed by randomly amplified polymorphic DNA PCR, employing four primers, INS-2, IS986-FP, OPA2, and OPA18, as described by Zhang et al [18], and pulsed-field gel electrophoresis analysis of the large restriction fragments generated by digestion of genomic DNA with AseI, as previously described for M. abscessus [19].…”