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Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods.

Abstract：

:Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

journal_name

Mem Inst Oswaldo Cruz

journal_title

Memorias do Instituto Oswaldo Cruz

authors

Isfahani BN,Tavakoli A,Salehi M,Tazhibi M

doi

10.1590/s0074-02762006000600004

subject

Has Abstract

pub_date

2006-09-01 00:00:00

pages

597-602

issue

eissn

0074-0276

issn

1678-8060

pii

S0074-02762006000600004

journal_volume

101

pub_type

杂志文章

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Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods.