Use of PCR and Reverse Line Blot Hybridization Macroarray Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences for Rapid Identification of 34 Mycobacterium Species

Abstract

The aim of this study was to develop a PCR and reverse line blot hybridization (PCR-RLB) macroarray assay based on 16S-23S rRNA gene internal transcribed spacer sequences for the identification and differentiation of 34 mycobacterial species or subspecies. The performance of the PCR-RLB assay was assessed and validated by using 78 reference strains belonging to 55 Mycobacterium species, 219 clinical isolates which had been identified as mycobacteria by high-performance liquid chromatography or gas chromatography, three skin biopsy specimens from patients with suspected leprosy which had been shown to contain acid-fast bacilli, and isolates of 14 nonmycobacterial species. All mycobacteria were amplified in the PCR and hybridized with a genus-specific probe (probe MYC). The 34 species-specific probes designed in this study hybridized only with the corresponding Mycobacterium species. The mycobacterial PCR-RLB assay is an efficient tool for the identification of clinical isolates of mycobacteria; it can reliably identify mixed mycobacterial cultures and M. leprae in skin biopsy specimens.Mycobacterium tuberculosis infection continues to increase worldwide at an estimated annual rate of 3.5% (http://www .who.int/tb/publications/global_report/2005/pdf/Full.pdf). The rapidly spreading human immunodeficiency virus (HIV) epidemic in many parts of the world will further increase the number of HIV-related cases of tuberculosis as well as the number of nontuberculous mycobacterial infections. The rapid and accurate identification of clinically significant mycobacterial species is necessary for optimal medical and public health interventions (3, 21).The results of traditional methods for the identification of closely related Mycobacterium species by growth characteristics (pigment, growth rate, colonial morphology) and biochemical tests are often difficult to interpret, even for some very common species. The tests are cumbersome and time-consuming, require expertise, lack sensitivity and reproducibility (21), and often use nonstandardized reagents. Rapid biochemical methods, such as high-performance liquid chromatography (HPLC) of mycolic acids and gas chromatography (GC) of fatty acids, are available only in specialized laboratories (2).The rapid identification of mycobacteria to the species level is recommended in clinical laboratories to ensure accurate diagnosis and effective therapy. This has stimulated the development in recent years (1, 4-9, 11-20, 22, 25, 29, 30) of a number of rapid and more accurate identification tools based on molecular technology.Several DNA sequences or genes have been targeted for the differentiation of Mycobacterium species, including the 16S rRNA gene (8, 9), the 23S rRNA gene (13, 15), the 16S-23S rRNA gene spacer region (15), the 32-kDa protein gene (22), dnaA (14), dnaJ (25), gyrB (4), recA (1), rpoB (6, 17), secA1 (29), and sod (30). There are at least two commercially available systems for the identification of Mycobacterium spp. based on line probe hybridization, namely, the INNO-LiPA...

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Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

“…, typing of methicillin-resistant Staphylococcus aureus [3][4][5] and Salmonella sp 6 , molecular serotyping of Streptococcus pneumoniae 7,8 , Streptococcus agalactiae 9 and enteroviruses 10,11 , identification of Mycobacterium sp 12 , detection of genital [13][14][15] and respiratory tract 16 and other 17 pathogens and detection and identification of mollicutes 18 . However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.…”