สถาบันวิจัยวิทยาศาสตร์สาธารณสุข

National Institute of Health of Thailand

Genotypic detection of rifampicin resistance by mini-PCR single strand conformational polymorphism

Authors : Benjawan Phetsuksiri*, Sukanya Wattanapokayakit *,Janisara Ruedeeaeksin *, Sopa Srisungngam*, Dhanida Rienthong**, Sang-Nae Cho***, Masanori Matsuoka****, and Patrick J. Brennan*****

 

Affiliations:       *Thai National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Ministry of Public Health, Nonthaburi, Thailand
**Bureau of AIDS, Tuberculosis and Sexual Transmitted Infection, Department of Disease Control, Ministry of Public Health, Thailand
***Department of Microbiology, Yonsei University, College of Medicine, Seoul, Republic of Korea
****Leprosy Research Center, National Institute of Infectious Disease (NIID), Ministry of Health and Welfare, Tokyo, Japan
*****Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
 
Source:             Proceeding: 40th US-Japan Conference on Tuberculosis 2005; 240
                      
Language:       English
 
Abstract:
 
Several techniques use polymerase chain reaction-based strategies to rapidly detect mutation known to confer resistance. One such method is single strand conformational polymorphism (SSCP) analysis. In this study, simple, rapid, and non-radioactive PCR-SSCP was developed and applied to detect mutations associated with rifampicin resistance. Detection of sequence alterations in the region of rpoB gene spanning codons of 500 to 550 for M. tuberculosis and codons of 497 to 629 for M. leprae was targeted. The techniques comprise amplification of the rpoB gene fragment, denaturation, electrophoresis on polyacrylamide gels in conventional mini slab apparatus, and silver staining. The predictive capability of PCR-SSCP was tested in Thai clinical isolates of 37 rifampicin -resistant and 23 -sensitive M. tuberculosis strains. All resistant strains showed SSCP profiles different from that of sensitive controls including M. tuberculosis H37Rv. Among resistant strains, mutations involving single base change in codons of 526 and 531 were frequently found. Further analysis in reference strains of M. leprae revealed that 8 of 8 rifampicin-resistant strains could be identified. The results indicated that recently developed mini-PCR SSCP works efficiently for rapid detection of rifampicin resistance. In addition, rifampicin resistance in M. tuberculosis isolates from Thailand involves alterations in the rpoB gene.