Direct Application of AvaII PCR Restriction Fragment Length Polymorphism Analysis (AvaII PRA) Targeting 644 bp Heat Shock Protein 65 (hsp65) Gene to Sputum Samples

Abstract

Of the validated species in the genus Mycobacterium, M. tuberculosis is the most common and important pathogen and causes 2 million deaths and over 8 million cases of tuberculosis annually worldwide (2-4, 7). In addition to the multidrug resistant strains of M. tuberculosis, NTM (non-tuberculosis mycobacteria) infection can cause other serious clinical problems. Moreover, because the drugs of choice for the treatment of NTM infection are heavily dependent on taxonomic statuses (15,19,20), it is of importance that we are able to differentiate these at the species level during early diagnostic procedures.The classical identification of mycobacteria based on cultural and biochemical tests may take several weeks, and sometimes, even then, may fail to provide a precise identification. To resolve such disadvantages of conventional methods, various PCR-mediated methods have been applied for the rapid detection and identification of mycobacteria species. Among these, PCRrestriction analysis (PRA) is preferred, since it offers an easy, rapid, and inexpensive means of identifying mycobacteria species. Thus, a number of PRA methods targeting several chronometers, such as, 16S rDNA (6, 8), hsp65 (5, 17), dnaJ (16), and rpoB (11), have been developed. However, although these PRA techniques have been successfully applied to mycobacteria identification at the culture level, problems are encountered when they are applied directly to clinical specimens, especially sputum samples, which also contain numbers of commensal bacteria from the respiratory tract, because these bacteria produce complex PRA patterns that are difficult to interpret.Previously, we reported that a novel PRA method that targets 644 bp hsp65 DNA is useful for the differentiation of mycobacterial species at the culture level (12). In particular, by only the use of a single restriction enzyme, AvaII, it can differentiate the three most clinically important mycobacteria species, i.e., M. tuberculosis and two MACs, M. avium and M. intracellulare. Moreover, the simplicity of this PRA method might enable the direct detection of pathogenic mycobacteria from clinical specimens such as sputum.In the present study, to evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 sequence Abstract: To evaluate the usefulness of the AvaII PRA method targeting 644-bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS6110 PCR. Although this method showed a sensitivity slightly lower than IS6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.

In the Wake of the Ongoing Mass Mortality Events: Co-occurrence of Mycobacterium, Haplosporidium and Other Pathogens in Pinna nobilis Collected in Italy and Spain (Mediterranean Sea)

“…Molecular markers have been used to elucidate the taxonomy of the Mycobacterium of P. nobilis. Although the hsp65 gene has been reported to be the most suitable target for the differentiation of mycobacteria species (Kim et al, 2005b;Mun et al, 2007), it has also been reported that some discrepancies can exist between hsp65, internal transcribed spacer 1 (ITS1) and 16S rDNA results for species differentiation. When comparing the hsp65 and ITS, we noticed that the 16S-23S internal transcribed spacer region (ITS) had better identification ability.…”

Molecular Evidence of Lateral Gene Transfer in rpoB Gene of Mycobacterium yongonense Strains via Multilocus Sequence Analysis

“…In order to verify the above hypothesis, a phylogenetic analysis of the three M. yongonense strains was performed using two other genes (16S rRNA and hsp65 genes), which have been used widely for mycobacteria taxonomies and diagnostics [13], [19], [20], [21]. Despite some problems in the bacteria taxonomy, the 16S rRNA gene sequence-based comparisons have been and remain invaluable in describing the prokaryotic diversity; they are indispensable in the delineation of bacterial species [22].…”

Detection of the rs10250202 polymorphism in protection of telomeres 1 gene through introducing a new restriction enzyme site for PCR–RFLP assay

“…PCR–RFLP is a traditional method to detect gene polymorphisms (Mun et al 2007 ; Wardak et al 2005 ). The advantage of this method is simple, fast and effective, and many restriction enzymes are cheap and commercially available.…”