Identification of Mycobacterium Species by PCR-Restriction Fragment Length Polymorphism Analyses Using Fluorescence Capillary Electrophoresis

Abstract

We developed a scheme for the rapid identification ofMycobacterium species based upon PCR amplification of polymorphic genetic regions with fluorescent primers followed by restriction and analysis by fluorescence capillary electrophoresis.Mycobacterium species were identified by restriction enzyme analysis of a 439-bp segment of the 65-kDa heat shock protein gene (labeled [both strands] at the 5′ end with 4,7,2′,7′-tetrachloro-6-carboxyfluorescein) using HaeIII and BstEII and of a 475-bp hypervariable region of the 16S rRNA gene (labeled [both strands] at the 5′ end with 6-carboxyfluorescein) using HaeIII and CfoI. Samples were analyzed on an automated fluorescence capillary electrophoresis instrument, and labeled fragments were sized by comparison with an internal standard. DNA templates were prepared with pure cultures of type strains. In all, we analyzed 180 strains, representing 22 Mycobacterium species, and obtained distinctive restriction fragment length polymorphism (RFLP) patterns for 19 species. Three members of the Mycobacterium tuberculosis complex had a common RFLP pattern. A computerized algorithm which eliminates subjectivity from pattern interpretation and which is capable of identifying the species within a sample was developed. The convenience and short preparatory time of this assay make it comparable to conventional methodologies such as high-performance liquid chromatography and hybridization assays for identification of mycobacteria.

Identification of Medically Important Yeasts Using PCR-Based Detection of DNA Sequence Polymorphisms in the Internal Transcribed Spacer 2 Region of the rRNA Genes

“…2, inset). Although similar discrepancies have been observed by others using capillary electrophoresis (9), our data demonstrate both the precision and accuracy of this technique for determining length polymorphisms among PCR products.…”

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].…”

hsp65 PCR-restriction analysis (PRA) with capillary electrophoresis for species identification and differentiation of Mycobacterium kansasii and Mycobacterium chelonae–Mycobacterium abscessus group

“…2,[15][16][17] Specifically, PRA with capillary electrophoresis can be used to increase the accuracy and rapidity of the identification process. 21,22 In this study, we aimed to identify and differentiate M. kansasii and M. chelonae-M. abscessus group isolates with the use of automated capillary electrophoresis-based PRA and our previously constructed diagnostic scheme. 20 The results obtained using the automated fluorescence capillary electrophoresis instrument agreed with the results obtained with the same strains using conventional agarose gel electrophoresis.…”