PCR Restriction Analysis Research Articles

Age- and Disease-Related Methylation of Multiple Genes in Nonneoplastic Duodenum and in Duodenal Juice

Methylation of CpG islands contributes to gene silencing during cancer development, and although some methylation alterations are promising diagnostic markers of cancer, some CpG islands are also methylated in normal tissues. We have previously observed that some normally unmethylated CpG islands that undergo methylation in pancreatic cancers are normally methylated in the adjacent duodenum. Because duodenal methylation patterns are an important consideration when sampling pancreatic tissues for pancreatic cancer methylation alterations, we determined the DNA methylation patterns of 24 genes in the normal duodenum of patients with pancreatic disease and related these patterns to demographic factors. The nonneoplastic duodenal mucosa of 158 patients with pancreatic carcinoma and 41 patients with chronic pancreatitis was analyzed using methylation-specific PCR and combined bisulfite restriction analysis. Secretin-stimulated pancreatic/duodenal juice from 15 individuals undergoing endoscopic investigation for upper gastrointestinal disease was also analyzed. Low-level methylation was detectable by methylation-specific PCR in the nonneoplastic duodenum of many patients with pancreatic cancer and chronic pancreatitis as well as in the pancreaticoduodenal secretions of patients without pancreaticobiliary disease. For many genes, the prevalence of methylation increased with age and was more prevalent in patients with pancreatic cancer than in age-matched patients with chronic pancreatitis. Our results indicate that strategies to detect pancreatic cancer using aberrantly methylated genes should rely on analysis of pure pancreatic juice rather than on pancreatic juice collected within the duodenal lumen. Patients who develop pancreatic cancer may have a greater propensity to methylate CpG islands than age-matched controls.

Rare variant of apolipoprotein E (Arg136→Ser) in two normolipidemic individuals.

Through the analysis of the common apolipoprotein (apo) E gene polymorphism in large Caucasian population study with the PCR and subsequent restriction analysis, we have identified carriers of mutant allele Arg136-->Ser. Both of them (71-years-old female and her 43-years-old son) have normal lipid parameters. We suggest that Arg136-->Ser mutation in apoE is not necessarily connected with elevated lipid levels in all cases. Furthermore, so far unidentified factors (environmental and/or genetic) are important for the development of lipid metabolism disorders in apoE Arg136-->Ser mutation carriers.

A new method for species identification and differentiation of Mycobacterium chelonae complex based on amplified hsp65 restriction analysis (AHSPRA)

Members of the Mycobacterium chelonae complex (MCC), namely M. chelonae, Mycobacterium abscessus and Mycobacterium immunogenum, have been implicated in nosocomial infections and occupational respiratory illnesses like hypersensitivity pneumonitis (HP) associated with contaminated metalworking fluid (MWF) exposures. Close relationship among these member species makes their differentiation cumbersome using the existing methods. Here we report a simple and rapid method for unambiguous identification and differentiation of the three-member species of the MCC group with PCR-restriction analysis targeting a 667-bp segment of a variable region of the 65-kDa-heat shock protein (hsp65) gene. This assay, described as Amplified hsp65 Restriction Analysis (AHSPRA), can discriminate all the three individual species using a one-step restriction digestion using either BbvI or Eco0109I. The enzyme NarI can differentiate M. immunogenum from the other two MCC species (M. chelonae and M. abscessus). The developed method was validated using several non-MCC reference species of other rapidly growing mycobacteria (RGM) and MCC field isolates from MWF samples. Direct cell-lysis was used instead of the conventional DNA template preparation, which improved the rapidity, simplicity and adaptability of the developed method. The results suggest that the developed method can unambiguously differentiate species of the M. chelonae complex from other RGM species and from one another.

Methylation of the Tumor Suppressor Gene PTPRO (Receptor-Type Protein Tyrosine Phosphatase) Is Associated with Expression of Important Apoptosis-Related Proteins in Chronic Lymphocytic Leukemia (CLL).

We previously demonstrated that apoptosis-inhibiting proteins (e.g., Bcl-2, Mcl-1, and XIAP) are important in predicting response to chemotherapy and antibody therapy for CLL. Additionally, genetic abnormalities and epigenetic control of gene expression have become increasingly important in understanding the prognosis and potential therapeutic opportunities in this disease. Greater than 80% of patients with CLL have genetic abnormalities detectable by FISH. Important subsets include those with a less favorable prognosis (deletions of 17p, 11q), an intermediate prognosis (trisomy 12), or a favorable outcome (deletion of 13q). PTPRO, encoding a receptor-type tyrosine phosphatase, is a specific tumor suppressor gene located in chromosomal region 12p12.3. PTPRO has been highly characterized as inhibiting anchorage-independent tumor growth in human lung carcinomas. This gene has been found to be silenced by promoter methylation, and can be re-expressed by hypomethylating agents in human lung tumor models. Thus, we examined the methylation profile of the promoter region of PTPRO in primary leukemic cells from patients with CLL, and in B cells obtained from normal volunteers. Analysis of the PTPRO CpG island by methylation-specific PCR and combined bisulfite restriction analysis (COBRA) demonstrated that this region was methylated in 91% of samples from 46 patients, whereas it was unmethylated in control B cells from normal volunteers. Specific quantitative determinations of important proteins related to apoptosis in the leukemic cells from 20 of these patients showed that, in the group with methylation of the PTPRO promoter, there was a significant elevation of Bcl-2 (p=0.079), Mcl-1 (p=0.004), XIAP (p=0.048), Bcl-2/BAX ratio (p=0.0024), and Mcl-1/BAX ratio (p=0.0017). There was no apparent relationship with FISH category or immunoglobulin VH mutational status. Thus, PTPRO promoter methylation analysis may identify a subset of CLL patients less likely to respond to therapy, and provide a potential reversible target for new therapeutic approaches to this disease. Finally, analysis of methylation of CLL samples by restriction landmark genome scanning also revealed methylation of genes for three additional protein tyrosine phosphatases: PTPRN2, PTPRZ2, and PTPN11. Increases in methylation of two of these (PTPRN2 and PTPRZ2) in CLL samples relative to control B cells was confirmed by COBRA. These results provide additional potential molecular targets for CLL therapy.

Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis.

Genome-wide losses of DNA methylation have been regarded as a common epigenetic event in malignancies and may play crucial roles in carcinogenesis. Limited information is available on the global methylation status in normal tissues and other cancer types beyond colonic carcinoma. Here we applied the combined bisulfite restriction analysis PCR to evaluate the methylation status of LINE-1 repetitive sequences in genomic DNA derived from microdissected samples from several human normal and neoplastic tissues. We found that methylation of LINE-1 in leukocytes was independent of age and gender. In contrast, normal tissues from different organs showed tissue-specific levels of methylated LINE-1. Globally, most carcinomas including breast, colon, lung, head and neck, bladder, esophagus, liver, prostate, and stomach, revealed a greater percentage of hypomethylation than their normal tissue counterparts. Furthermore, DNA derived from sera of patients with carcinoma displayed more LINE-1 hypomethylation than those of noncarcinoma individuals. Finally, in a colonic carcinogenesis model, we detected significantly greater hypomethylation in carcinoma than those of dysplastic polyp and histological normal colonic epithelium. Thus, the methylation status is a unique feature of a specific tissue type and the global hypomethylation is a common epigenetic process in cancer, which may progressively evolve during multistage carcinogenesis.

Use of DNA from Human Stools to Detect Aberrant CpG Island Methylation of Genes Implicated in Colorectal Cancer

Abstract Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16INK4a, APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.

Distribución de patrones PRA en aislamientos clínicos del complejo Mycobacterium avium procedentes de España y Suramérica.

Mycobacterium avium complex (MAC) infections are the most frequent systemic infections associated with advanced AIDS. DNA probes for accurate identification of mycobacteria are available but are very expensive in many Latin American settings. Consequently, most Latin American diagnostic laboratories employ inaccurate and outdated tests for mycobacteria identification. Therefore, PCR restriction analysis (PRA) of the hsp65 gene was evaluated for the identification of 163 MAC human isolates originated from Spain and South America. The predominant PRA type in each country was: M. avium type I in Argentina (23/42, 55%) and Brazil (48/72, 67%), M. avium type II in Spain (18/26, 69%) and M. avium type III in Colombia (10/23, 43%). The Colombia frequency is noteworthy, since the PRA type III was quite infrequent in the other three countries. Furthermore, its presence has not been reported outside the Americas. The advantages and disadvantages of PRA in diagnostic mycobacteriology are discussed.

Multicenter evaluation of the fully automated bactec mgit 960 system and three molecular methods for the isolation and the identification of mycobacteria from clinical specimens

A combination of Bactec MGIT 960 system, a PCR-based assay and a PCR-Restriction Analysis procedure (PRA) was assessed for the detection and identification of mycobacteria from clinical samples. The MGIT recovered 243 from 266 mycobacterial isolates. The PCR-based assay correctly identified all (234) Mycobacterium tuberculosis isolates, while 35 nontuberculous mycobacterial isolates were identified by PRA.

Genotypic Differentiation of Twelve Clostridium Species by Polymorphism Analysis of the Triosephosphate Isomerase (tpi) Gene

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.

Novel allelic variants of Mycobacteria isolated in Brazil as determined by PCR-restriction enzyme analysis of hsp65.

Human isolates of Mycobacterium collected in 16 different states of Brazil were submitted to PCR-restriction analysis (PRA) of a 439-bp fragment of the hsp65 gene with HaeIII and BstEII. Fourteen allelic variants not described in clinical isolates so far were observed among 36 (10%) of 356 Brazilian strains, including a new pattern for Mycobacterium scrofulaceum, M. intracellulare, and M. flavescens, two new patterns for M. fortuitum, three new patterns each for M. gordonae and M. terrae, and one new pattern for M. avium complex-like strains. Two unidentified strains each also presented a new pattern, strongly suggesting that Mycobacterium genotypes are distributed biogeographically. The PRA procedure was also performed with 43 reference isolates belonging to 34 species, adding a further six new patterns to the identification algorithm. A database containing the normalized restriction patterns of both enzymes was constructed. Patterns available on the Internet can be introduced into this database, which will make possible the comparison of genotypes from isolates from different parts of the world.

PLA1/A2 polymorphism of the platelet glycoprotein receptor IIb/IIIa in young patients with cryptogenic TIA or ischemic stroke

Background: The relationship between the PIA2 allele of the Leu 33Pro polymorphism of glycoprotein IIb/IIIa receptor (GPIIb/IIIa) and ischemic stroke is uncertain. The purpose of this study was to investigate a possible association between the GPIIb/IIIa PIA1/A2 polymorphism and the occurrence of cryptogenic stroke in young patients. Methods: From a consecutive series of 80 patients aged 45 or less with a recent ischemic stroke or TIA, we selected 45 patients with stroke due to small vessel occlusion or stroke of undetermined etiology (according to the TOAST criteria). Controls were 60 healthy blood donors with a similar age distribution. All patients underwent CT of the brain and were screened for cardiovascular risk factors, cardiac disorders and large vessel disease. The frequency of the PIA2 allele was determined by PCR and Msp1 restriction analysis. Results: Eight patients (16%) and 16 controls (27%) were heterozygous for PIA2 allele. Two patients (4%) were homozygous for PIA2.The relative risk of ischemic stroke associated with PIA2 allele was estimated at 0.8 (95% CI: 0.3–1.9). Conclusion: This study does not support the association between the PIA1/A2 polymorphism and cryptogenic stroke or TIA in patients aged 45 or less.

The Arg allele in position 192 of PON1 is associated with carotid atherosclerosis in subjects with elevated HDLs

Human serum paraoxonase (PON1) is an HDL-associated enzyme involved in the protection of lipoproteins from oxidation. A polymorphism at position 192 (Gln/Arg) influences its activity in a substrate-dependent manner. The aim of the present study was to evaluate, in vivo, the contribution of the PON1-192 polymorphism to the protective effect of HDLs. Three hundred and forty seven subjects in the upper and lower decile of sex-specific HDL cholesterol distribution were selected from participants in a cardiovascular disease prevention study. PON1 genotypes were determined by PCR amplification and restriction analysis. Blood pressure, height, weight, smoking and alcohol habits, as well as the presence of familial or personal history of CHD were recorded. Plasma lipids and blood glucose were measured by routine enzymatic methods. As a measure of antiatherogenic HDL effect, carotid atherosclerosis was assessed by ultrasonography. Allele and genotype frequencies did not differ significantly between low- and high-HDL groups. Similarly, no significant difference was observed among genotypes in all variables studied. Subjects with Gln/Arg or Arg/Arg had more carotid abnormalities than Gln/Gln with an adjusted odds ratio (OR) of 3.27 (95% CI, 1.61–6.64, P=0.001) for abnormal carotid score. Stepwise logistic regression analysis showed that in the whole population age and presence of low-HDL were the only independent predictors for abnormal carotid score. In low-HDL, age was the only independent predictor entered into the model (OR 1.09/year, P<0.0001); in high-HDL, age entered first (OR, 1.07/year, P=0.001), followed by the presence of Gln/Arg or Arg/Arg (OR, 2.94, 95% CI, 1.47–5.91, P=0.002). In conclusion, in subjects with low levels of HDL cholesterol, carotid atherosclerosis is not related to PON1-192 polymorphism. On the other hand, in subjects with elevated concentration of HDL cholesterol, the presence of carotid atherosclerosis is significantly associated with the arginine variant in position 192 of the PON1 gene.

Rare Variant of Apolipoprotein E (Arg136 ® Cys) in Subject with Normal Lipid Values

During the screening of apolipoprotein (apo) E gene polymorphism with PCR and subsequent restriction analysis, we have identified a female carrier with a mutant allele Arg136 ® Cys. This proband had normal lipid parameters and no history of coronary artery disease (CAD). We did not confirm the previously described connection between apo E Arg136 ® Cys mutation and elevated lipid levels. In the case of this mutation, other factors (environmental and/or genetic) are important for the development of lipid metabolism disorders.

No association between T102C polymorphism of serotonin-2A receptor gene and clinical phenotypes of Chinese schizophrenic patients

Serotonin dysfunction has been implicated in the pathogenesis of schizophrenia. Previous studies have shown an association between the T102C polymorphism of the 5HT2a receptor gene and schizophrenia. However, negative findings have also been reported. One possible explanation for such discrepancy is disease heterogeneity due to the current limitations in the diagnosis of schizophrenia. We conducted a case–control study of the T102C polymorphism with detailed characterisation of the clinical phenotypes to investigate the possible association with schizophrenia not only at the diagnostic level, but also with reference to other clinical phenotypes potentially related to serotonin dysfunction. Four hundred and seventy-one biologically unrelated schizophrenic patients and 523 unrelated healthy controls of Han Chinese descent in Hong Kong were compared for genotypes and allele frequencies of the T102C polymorphism by PCR amplification and restriction analysis. No evidence of association was detected at the diagnostic level and various clinical phenotypes. However, we found a trend association with small effect size between genotype 102T/102C and patients with better verbal fluency and less motor co-ordination soft neurological signs. There is a need for future large-scale studies on the possible associations between genetic polymorphisms and neurocognitive function impairments in schizophrenia.

Genes coding for polymerase proteins are essential for attenuation of the cold-adapted A/Leningrad/134/17/57 (H2N2) influenza virus

Background: The A/Leningrad/134/17/57 (H2N2) (Len/17) cold-adapted (ca) master strain used as a donor of attenuation for preparing live attenuated influenza vaccine was derived from the A/Leningrad/134/57 wild-type virus (Len/wt) after 17 passages in eggs at 25 °C. In contrast to Len/wt, the Len/17 virus is sensitive to high (40 °C) temperature ( ts), is able to grow at low (25 °C) temperature ( ca), and is attenuated ( att) for humans. Len/17 is different from Len/wt by eight coding mutations in internal genes PB2 (V-478-L), PB1 (K-265-N, V-591-I), PA (L-28-P, V-341-L), M1 (I-15-V), M2 (A-86-T) and NS2 (M-100-I). However, the role of individual mutant genes in the manifestation of the ts, ca, and att phenotypes was not established. To evaluate this, single gene reassortants (SGRs) with one mutated gene in the background of the Len/wt genome were studied. Methods: Since heterogeneity (A- or T-86 in the M2 gene) was noted in Len/17, the ts and ca clone 28 of this donor (Len/17-cl28) that has A-86 in M2 was used to prepare the SGRs. Preservation of mutations in SGRs was monitored by PCR-restriction (RFLP) analysis. Virus replication in ferret lungs was observed to evaluate the att phenotype of SGRs. Results: PB2- and PB1-SGRs each manifested the ts phenotype, and PB2-SGR demonstrated the ca phenotype as well. PA-SGR was ca, but not ts. Mutations in the M1 and NS2 genes did not affect the ts or ca phenotypes of corresponding SGRs. Ferrets infected with Len/17-cl28, Len/wt and PB2-, PB1- or PA-SGRs demonstrated comparable titers in nasal turbinates on the third day, while infectious virus was found only in the lungs of ferrets infected with Len/wt and PA-SGR. Conclusion: These data suggest that mutations in the PB2 (V-478-L) and PB1 (K-265-N, V-591-I) genes play a critical role in the attenuation of the ca Len/17 vaccine donor strain.

Live attenuated reassortant influenza vaccine prepared using A/Leningrad/134/17/57 (H2N2) donor strain is genetically stable after replication in children 3-6 years of age

Background: Live influenza A vaccines (LIVs) for adults and children are prepared in Russia by reassorting current epidemic strains with cold-adapted (ca) attenuated donor viruses A/Leningrad/134/17/57 (H2N2) (Len/17) and A/Leningrad/134/47/57 (H2N2) (Len/47), respectively. Len/17 and Len/47 were derived from the A/Leningrad/134/57 (H2N2) wild-type virus after 17 and 47 passages in eggs at 25 °C. Two doses of Len/47-based vaccine have been used for vaccinating children. In a recent study, children were vaccinated with the Len/17-based LIV in order to determine if this vaccine is safe and immunogenic after a single dose vaccination. As part of this study, type A isolates obtained from children 3–6 years of age on the third day after vaccination were analyzed for genetic stability. Methods: PCR-restriction (RFLP) analysis was used to detect mutations in genomes of isolates. Results: Internal genes of Len/17 contain eight coding mutations in the PB2 (1), PB1 (2), PA (2), M1 (1), M2 (1) and NS (1) genes. All of these mutations, except one, were conserved in the genomes of all 28 strains studied. Similar to other studies with Len/17, the A-86-T change in the M2 protein, which was shown to be heterogeneous in the parent donor strain, was absent in 13 isolates. Since it was shown that mutations in polymerase genes play a leading role in the attenuation of Len/17, it is important to emphasize their conservation in all of the isolates. Moreover, an additional mutation in the PB2 protein (S-478-R), previously known only for the more attenuated Len/47 ca donor strain, was detected in 9 of 28 isolates, indicating that this mutation may also be heterogeneous in the donor virus. Conclusion: The Len/17-based LIV is genetically stable in children.

Rapid differentiation of "Mycobacterium canettii" from other Mycobacterium tuberculosis complex organisms by PCR-restriction analysis of the hsp65 gene.

A total of 102 isolates of the Mycobacterium tuberculosis complex, including available "M. canettii" isolates, were studied by PCR-restriction analysis of a 441-bp fragment of the hsp65 gene. PRA upon HhaI enzyme digestion (GCGC) allowed easy differentiation of "M. canettii" from other members of the M. tuberculosis complex (three bands of 260, 105, and 60 bp for "M. canetti," compared to four bands of 185, 105, 75, and 60 bp for other members of the M. tuberculosis complex). Sequencing of the 441-bp hsp65 fragment of "M. canettii" isolates showed the disappearance of an HhaI site at position 235 due to a C-to-T transition that corresponded to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv. Considering that "M. canettii" may also exist as a stable rough morphotype, we suggest that the true number of "M. canettii" isolates may be underestimated in clinical microbiology laboratories.

Some probiotic properties of yeast isolates from infant faeces and Feta cheese

Yeast isolates from infant faeces and Feta cheese were characterized to species level by phenotypic criteria, Randomly Amplified Polymorphic DNA (RAPD)-PCR and mitochondrial DNA (mt-DNA) restriction analysis. Results suggested that there is a good agreement between phenotypic characterization of yeasts and RAPD-PCR at species level; in addition, RAPD-PCR as well as mt-DNA restriction analysis provided good discrimination at strain level. Some technological and probiotic properties of selected strains were also investigated. The test strains exhibited lipolytic and proteolytic activities. They also tolerated low pH and survived satisfactory in gastric juice in vitro as well as in the presence of bile. In general, the isolates from faeces were more resistant to low pH and bile than those from Feta cheese. Selected strains could be used as starter supplements for industrial fermentations.

The frequency of C282Y and H63D mutations in Hemochromatosis gene in native Estonians.

Four hundred and forty two adult individuals of Estonian nationality were examined in different regions of Estonia for the C282Y and H63D HFE mutations to determine the allele and genotype frequencies. The sample consisted only of those people whose at least four grandparents were born in Estonia, and have lived settled in the same region. The study was carried out using the PCR technique and restriction analysis for C282Y and H63D mutations respectively. For the C282Y mutation the frequency of heterozygotes was 6.6% and homozygotes 0.2%, giving allele frequency 0.035. The allele frequency for the H63D mutation was 0.136, and the frequency of homo- and hetero-zygotes 1.6% and 24.0% respectively.

Description of a novel Mycobacterium simiae allelic variant isolated from Caribbean AIDS patients by PCR-restriction enzyme analysis and sequencing of hsp 65 gene

A sudden upsurge in the isolation of Mycobacterium simiae from terminally ill AIDS patients was recently reported on the Caribbean island of Guadeloupe. Identification of these M. simiae isolates was achieved using biochemical tests and further confirmed by PCR-restriction analysis (PRA) of a 439-bp fragment of hsp 65. A novel PRA profile III (three Bst EII fragments of 240/125/80 bp and four Hae III fragments of 145/125/40/25 bp) was observed in four blood isolates from two patients. The 16 S rRNA gene sequencing of the hypervariable A region confirmed that all the pattern III isolates were indeed M. simiae species, and the hsp 65 sequencing confirmed the existence of a new hsp 65 allele in these caribbean isolates. A hsp 65 sequence-based phylogenetic tree was also created for 39 species including M. simiae and related mycobacterial species as well as other rapid and slow growing mycobacteria, and may serve as an useful tool for identification of mycobacteria to species level.