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Application of Deletion- Targeted Multiplex PCR technique for detection of Mycobacterium tuberculosis Beijing strains in samples from tuberculosis patients.

Khosravi AD, Goodarzi H, Alavi SM, Akhond MR - Iran J Microbiol (2014)

Bottom Line: From 160 PCR-confirmed MTB isolates, 18 (11.25%) showed mutation in katG gene related to INH resistance and 20 (12.5%), associated with mutation in rpoB gene related to RIF resistance, and 8 (5%) were detected as Beijing strain using multiplex PCR.Using DTM- PCR, the rate of Beijing strains in the region of study was determined as 5%.Although for detection of MTB antimicrobial resistance, it is advised to use a combination of conventional antimicrobial susceptibility testing and molecular techniques, however for time saving, it seems that DTM-PCR, is a simple technique for use in areas of the world where Beijing strains are highly prevalent.

View Article: PubMed Central - PubMed

Affiliation: Health Research Institute, Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran ; Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

ABSTRACT

Background and objective: Molecular epidemiological studies have shown that certain genotypes of Mycobacterium tuberculosis (MTB) are over-represented in limited geographical regions, suggesting of evolution of certain genotypes with increasing virulence and pathogenicity. Beijing strain of MTB was initially described by its potential to cause outbreaks worldwide and its association with drug resistance. Due to tuberculosis (TB)-related mortality which is associated with Beijing genotype, this study was designed with the aim to detect the MTB Beijing genotype in the region of study.

Materials and methods: A total of 170 clinical isolates of MTB were collected from the TB reference laboratory of Khuzestan province, Iran, over one year period from February 2010 to February 2011. Phenotypic tests were used for preliminary detection of MTB. Culture positive MTB isolates were confirmed by multiplex PCR based on IS6110 gene with subsequent screening for resistance to isoniazid (INH), and rifampin (RIF) by PCR using relevant primers. Three set of primers were used to differentiate Beijing from non-Beijing strains by using Deletion- Targeted Multiplex (DTM) PCR.

Results: From 160 PCR-confirmed MTB isolates, 18 (11.25%) showed mutation in katG gene related to INH resistance and 20 (12.5%), associated with mutation in rpoB gene related to RIF resistance, and 8 (5%) were detected as Beijing strain using multiplex PCR. The majority of detected Beijing strains (6/8[75%]) comprised mutation in katG gene with the prevalent mutation specifically in codon 315. In 4 Beijing strains (2.5%), mutation in rpoB gene were also detected.

Conclusion: Using DTM- PCR, the rate of Beijing strains in the region of study was determined as 5%. Although for detection of MTB antimicrobial resistance, it is advised to use a combination of conventional antimicrobial susceptibility testing and molecular techniques, however for time saving, it seems that DTM-PCR, is a simple technique for use in areas of the world where Beijing strains are highly prevalent.

No MeSH data available.


Related in: MedlinePlus

Multiplex polymerase chain reaction (PCR) products on a 2% agarose gel. Beijing strains generated 129-bp and 523-bp fragments. Non-Beijing strains generated 95-bp and 523-bp fragments.Lanes 1: 100-bp DNA ladder;2: Mycobacterium tuberculosis H37Rv;3, 6, 7, 9, 11, 12 and 14: non-Beijing strain;4, 5, 8 and10: Beijing strain; Lane 13: Distilled water blank control
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Figure 1: Multiplex polymerase chain reaction (PCR) products on a 2% agarose gel. Beijing strains generated 129-bp and 523-bp fragments. Non-Beijing strains generated 95-bp and 523-bp fragments.Lanes 1: 100-bp DNA ladder;2: Mycobacterium tuberculosis H37Rv;3, 6, 7, 9, 11, 12 and 14: non-Beijing strain;4, 5, 8 and10: Beijing strain; Lane 13: Distilled water blank control

Mentions: The 160 PCR-confirmed MTB isolates, were screened for resistance to INH and RIF later. There were 18 tuberculosis patients (11.25%) whose isolate showed mutation in katG gene related to INH resistance, 20 (12.5%) associated with mutation in rpoB gene related to RIF resistance, and 8(5 %) were identified as MDR. The DNA extracts were then investigated for the presence of Beijing and non Beijing strains by DTM PCR (Fig. 1).


Application of Deletion- Targeted Multiplex PCR technique for detection of Mycobacterium tuberculosis Beijing strains in samples from tuberculosis patients.

Khosravi AD, Goodarzi H, Alavi SM, Akhond MR - Iran J Microbiol (2014)

Multiplex polymerase chain reaction (PCR) products on a 2% agarose gel. Beijing strains generated 129-bp and 523-bp fragments. Non-Beijing strains generated 95-bp and 523-bp fragments.Lanes 1: 100-bp DNA ladder;2: Mycobacterium tuberculosis H37Rv;3, 6, 7, 9, 11, 12 and 14: non-Beijing strain;4, 5, 8 and10: Beijing strain; Lane 13: Distilled water blank control
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4385573&req=5

Figure 1: Multiplex polymerase chain reaction (PCR) products on a 2% agarose gel. Beijing strains generated 129-bp and 523-bp fragments. Non-Beijing strains generated 95-bp and 523-bp fragments.Lanes 1: 100-bp DNA ladder;2: Mycobacterium tuberculosis H37Rv;3, 6, 7, 9, 11, 12 and 14: non-Beijing strain;4, 5, 8 and10: Beijing strain; Lane 13: Distilled water blank control
Mentions: The 160 PCR-confirmed MTB isolates, were screened for resistance to INH and RIF later. There were 18 tuberculosis patients (11.25%) whose isolate showed mutation in katG gene related to INH resistance, 20 (12.5%) associated with mutation in rpoB gene related to RIF resistance, and 8(5 %) were identified as MDR. The DNA extracts were then investigated for the presence of Beijing and non Beijing strains by DTM PCR (Fig. 1).

Bottom Line: From 160 PCR-confirmed MTB isolates, 18 (11.25%) showed mutation in katG gene related to INH resistance and 20 (12.5%), associated with mutation in rpoB gene related to RIF resistance, and 8 (5%) were detected as Beijing strain using multiplex PCR.Using DTM- PCR, the rate of Beijing strains in the region of study was determined as 5%.Although for detection of MTB antimicrobial resistance, it is advised to use a combination of conventional antimicrobial susceptibility testing and molecular techniques, however for time saving, it seems that DTM-PCR, is a simple technique for use in areas of the world where Beijing strains are highly prevalent.

View Article: PubMed Central - PubMed

Affiliation: Health Research Institute, Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran ; Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

ABSTRACT

Background and objective: Molecular epidemiological studies have shown that certain genotypes of Mycobacterium tuberculosis (MTB) are over-represented in limited geographical regions, suggesting of evolution of certain genotypes with increasing virulence and pathogenicity. Beijing strain of MTB was initially described by its potential to cause outbreaks worldwide and its association with drug resistance. Due to tuberculosis (TB)-related mortality which is associated with Beijing genotype, this study was designed with the aim to detect the MTB Beijing genotype in the region of study.

Materials and methods: A total of 170 clinical isolates of MTB were collected from the TB reference laboratory of Khuzestan province, Iran, over one year period from February 2010 to February 2011. Phenotypic tests were used for preliminary detection of MTB. Culture positive MTB isolates were confirmed by multiplex PCR based on IS6110 gene with subsequent screening for resistance to isoniazid (INH), and rifampin (RIF) by PCR using relevant primers. Three set of primers were used to differentiate Beijing from non-Beijing strains by using Deletion- Targeted Multiplex (DTM) PCR.

Results: From 160 PCR-confirmed MTB isolates, 18 (11.25%) showed mutation in katG gene related to INH resistance and 20 (12.5%), associated with mutation in rpoB gene related to RIF resistance, and 8 (5%) were detected as Beijing strain using multiplex PCR. The majority of detected Beijing strains (6/8[75%]) comprised mutation in katG gene with the prevalent mutation specifically in codon 315. In 4 Beijing strains (2.5%), mutation in rpoB gene were also detected.

Conclusion: Using DTM- PCR, the rate of Beijing strains in the region of study was determined as 5%. Although for detection of MTB antimicrobial resistance, it is advised to use a combination of conventional antimicrobial susceptibility testing and molecular techniques, however for time saving, it seems that DTM-PCR, is a simple technique for use in areas of the world where Beijing strains are highly prevalent.

No MeSH data available.


Related in: MedlinePlus