Molecular technique for rapid identification of mycobacteria

Abstract

Identification of mycobacteria through conventional microbiological methods is cumbersome and time-consuming. Recently we have developed a novel bacterial identification method to accurately and rapidly identify different mycobacteria directly from water and clinical isolates. The method utilizes the PCR to amplify a portion of the small subunit rRNA from mycobacteria. The 5' PCR primer has a fluorescent label to allow detection of the amplified product. The PCR product is digested with restriction endonucleases, and an automated DNA sequencer is employed to determine the size of the labeled restriction fragments. Since the PCR product is labeled only at the 5' end, the analysis identifies only the restriction fragment proximal to the 5' end. Each mycobacterial species has a unique 5' restriction fragment length for each specific endonuclease. However, frequently the 5' restriction fragments from different species have similar or identical lengths for a given endonuclease. A set of judiciously chosen restriction enzymes produces a unique set of fragments for each species, providing us with an identification signature. Using this method, we produced a library of 5' restriction fragment sizes corresponding to different clinically important mycobacteria. We have characterized mycobacterial isolates which had been previously identified by biochemical test and/or nucleic acid probes. An analysis of these data demonstrates that this protocol is effective in identifying 13 different mycobacterial species accurately. This protocol has the potential of rapidly (less than 36 h) identifying mycobacterial species directly from clinical specimens. In addition, this protocol is accurate, sensitive, and capable of identifying multiple organisms in a single sample.

Assessment of fungal diversity using terminal restriction fragment (TRF) pattern analysis: comparison of 18S and ITS ribosomal regions

“…One advantage of using the TRF method to analyze complex microbial communities is that individual community members may be identi¢ed based on the presence of individual peaks in the TRF pattern [4,7,9]. In order to investigate whether the ITS or 18S regions would better facilitate this kind of analysis, observed TRF pro¢les (both 18S and ITS) from four pure fungal isolates were compared against those predicted from DNA sequence database information.…”

“…Patterns can be compared from di¡erent communities over time or under different environmental or treatment conditions (for examples, see [4^8]). In addition, TRF pro¢les or ribotypes (ribosomal genotypes, in this case characterized by combinations of TRF lengths across di¡erent enzymes for the same analyzed sample) may be used to identify speci¢c community members [4,7,9].…”

Validating T-RFLP as a sensitive and high-throughput approach to assess bacterial diversity patterns in human anterior nares

“…Terminal restriction fragment length polymorphism (T-RFLP) was originally developed to identify mycobacteria (Avaniss-Aghajani et al, 1996) and has since been commonly used for comparing differences in the microbial communities of a diverse range of environments (Osborn et al, 2000;Marsh & Jared, 2005;Tiquia, 2009;Joo et al, 2010;Nakano et al, 2010). The generated terminal restriction fragment abundances (peaks corresponding to species or groups of related species) are presented as the relative proportion to the total abundance of the sample, as do these other aforementioned profiling methods.…”

Protistan Diversity Estimates Based on 18S rDNA from Seawater Incubations in the Western North Atlantic1

“…This study was designed to examine microbial eukaryote assemblage diversity (largely protistan), focusing on small subunit (18S) ribosomal RNA genes. Two culture-independent PCRbased methods including terminal restriction fragment length polymorphism (T-RFLP) (Avaniss-Aghajani et al 1996;Liu et al 1997) and cloning/sequencing (Giovannoni et al 1990;Moon-van der Staay, De Wachter, and Vaulot 2001;Rappe, Kemp, and Giovannoni 1995;Schmidt, Delong, and Pace 1991) were used to assess protistan diversity in a coastal ecosystem and to examine the response of the community to containment during 72-h incubations. Overall, both approaches yielded comparable results with respect to the general response of the community to containment.…”