Development of a Peptide Nucleic Acid-Based Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation among Mycobacterium abscessus Strains

Abstract

c Recently, the need to distinguish between members of the Mycobacterium abscessus group has gained increasing attention. Here, we introduced a novel peptide nucleic acid (PNA) real-time PCR method targeting the hsp65 gene in order to distinguish between four subspecies within the M. abscessus group (M. abscessus and 3 types of M. massiliense).T he taxonomic status of the Mycobacterium abscessus group remains undetermined. The recent development of genetic investigation tools has revealed that the M. abscessus group can be further divided into three closely related taxa, i.e., M. abscessus, M. massiliense and M. bolletii (1-3). Recently, it was reported that M. massiliense can be further subdivided into three genotypes (here referred to as types I, II-1, and II-2) based on analyses of the hsp65 sequence (4, 5). Because M. massiliense differs from M. abscessus in its susceptibility to clarithromycin, distinguishing between the two organisms has clinical importance (6, 7). Furthermore, phylogenetic separation between three types of M. massiliense has also been reported (4,8), highlighting the epidemiological importance of their separation (7,9,10).Peptide nucleic acids (PNAs) are artificially synthesized DNA analogues with an uncharged peptide backbone (11,12). A PNA probe-based real-time PCR assay has been developed for the diagnosis of mycobacterial infections, particularly for distinguishing between Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) in clinical specimens (13,14 T , and M. xenopi ATCC 19250 T ), were used in this study. All clinical isolates were collected from the Asan Medical Center (Seoul, Republic of Korea) during the period from January 2004 to June 2011. This work was approved by the institutional review board of Seoul National University Hospital (C-1202-057-398) and the Asan Medical Center (2012-0170). The mycobacteria were maintained as a subculture of a frozen stock before use in molecular assays. Bacterial chromosomal DNA was extracted from the clinical isolates by the bead-beater-phenol extraction method, as described previously (15).The primers were designed using the Oligo software (version 6.5; Molecular Biology Insights). These primers produced 377-bp hsp65 amplicons (from nucleotides 380 to 756 in the M. abscessus hsp65 gene). We designed a set of three PNA probes for specific simultaneous detection in a single reaction of the four subspecies of the M. abscessus group (M. abscessus and specific types I, II-1, and II-2) using the LC-PDS (version 2.0) software. The probes were further developed according to the design guidelines of the PNA manufacturer, using three different single nucleotide polymorphisms (SNPs) within 604-bp hsp65 sequences (see Fig. S1 in the supplemental material). We used these reporter dyes to detect different sequences: 6-carboxyfluorescein (FAM) for the detection of M. abscessus and M. massiliense at the subspecies level, Hex for discriminating M. massiliense type I and type II, and Texas Red for discriminating M. massiliense types II-1 and I...

Separation of Mycobacterium abscessus into subspecies or genotype level by direct application of peptide nucleic acid multi-probe- real-time PCR method into sputa samples

“…Notably, the T m s measured from sputum samples showed small decreasing shifts (1–2.7 °C) compared to our previously reported T m s of clinical isolates of the M. abscessus genotypes [ 22 ] without compromising the discrimination of the genotypes. This outcome may be due to the presence of PCR-inhibitory substances in the DNA extracts from the sputum specimens because inhibitory substances can cause T m shifts [ 35 , 36 ].…”

“…In a previous study, we developed an hsp65 PNA RT-PCR targeting three signature SNPs in the 604-bp hsp65 gene that enabled the separation of the four types of M. abscessus ( M. abscessus and three types of M. massiliense ) at the cultured isolate level. The intra-genotypic conservation and inter-genotypic variation for each SNP of members of the M. abscessus group were confirmed by successful separation in 227 out of 228 clinical isolates (99.6 %) [ 22 ]. In this study, the application of the hsp65 PNA RT-PCR method to sputum samples demonstrated that this method could directly identify members of the M. abscessus group at the subspecies or genotypic level from sputum samples with 96.7 % sensitivity (29/30 samples) and 100 % specificity (Table 2 ), thereby demonstrating its feasibility in identifying the M. abscessus group not only from cultured isolates but also directly from sputum samples.…”

“…PNA molecular probes are significantly shorter than the TaqMan or LightCycler probes, making probe design and genotype discrimination easier [ 33 ]. Despite the short sequence length (13–15 bp) of the probes, our hsp65 PNA RT-PCR could be successfully applied to the separation of members of the M. abscessus group from sputum samples (Table 1 ) as well as clinical isolates [ 22 ]. The presence of a mismatch in a PNA/DNA duplex is reported to be more destabilizing than a mismatch in a DNA/DNA duplex, suggesting that the use of PNA probes may lead to more pronounced difference in the average T m in SNP detection compared to the use of DNA probes [ 34 ].…”

“…For the detection of M. abscessus from sputum samples, the hsp65 PNA RT-PCR method was applied to 60 sputum DNA samples, as previously described [ 22 ]. Briefly, a total of three reporter dyes were used for the specific simultaneous detection of the four types of the M. abscessus group ( M. abscessus , Type I, Type II-1, and Type II-2) in a single reaction.…”

“…With the exception of one clinical isolate, most of the samples (227/228 isolates, 99.6 % sensitivity) were clearly separated at the subspecies or genotype levels, thereby demonstrating the technique’s feasibility for the detection of M. abscessus at the mycobacterial culture level. To the best of our knowledge, this was the first report to use a PNA- based multi-probe approach for bacterial diagnosis [ 22 ].…”