One-tube loop-mediated isothermal amplification combined with restriction endonuclease digestion and ELISA for colorimetric detection of resistance to isoniazid, ethambutol and streptomycin in Mycobacterium tuberculosis isolates.

In this study, we designed a simple and rapid colorimetric detection method, a one-tube loop-mediated isothermal amplification (LAMP)-PCR-hybridization-restriction endonuclease-ELISA [one-tube LAMP-PCR-HY-RE-ELISA] system, to detect resistance to isoniazid, ethambutol and streptomycin in strains of Mycobacterium tuberculosis isolated from clinical specimens. The clinical performance of this method for detecting isoniazid-resistant, ethambutol-resistant and streptomycin-resistant isolates of M. tuberculosis showed 98.9%, 94.3% and 93.8%, respectively. This assay is rapid and convenient that can be performed within one working day. One-tube LAMP-PCR-HY-RE-ELISA system was designed based on hot spot point mutations in target drug-resistant genes, using LAMP-PCR, hybridization, digestion with restriction endonuclease and colorimetric method of ELISA. In this study, LAMP assay was used to amplify DNA from drug-resistant M. tuberculosis, and ELISA was used for colorimetrical determination. This assay will be a useful tool for rapid diagnosis of mutant codons in strains of M. tuberculosis for isoniazid at katG 315 and katG 463, ethambutol at embB 306 and embB 497, and streptomycin at rpsL 43.

[1]  J. Farrar,et al.  PCR-Restriction Fragment Length Polymorphism for Rapid, Low-Cost Identification of Isoniazid-Resistant Mycobacterium tuberculosis , 2007, Journal of Clinical Microbiology.

[2]  J. Tercero,et al.  Mutations in the rpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Isolates in Spain and Their Rapid Detection by PCR–Enzyme-Linked Immunosorbent Assay , 2001, Journal of Clinical Microbiology.

[3]  J. Palomino.  Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis. , 2009, FEMS immunology and medical microbiology.

[4]  L. Mirabello,et al.  Phenotypic and Molecular Characterization of Mycobacterium tuberculosis Isolates Resistant to both Isoniazid and Ethambutol , 2005, Antimicrobial Agents and Chemotherapy.

[5]  F. Goñi,et al.  Prevalence and dissemination of the Ser315Thr substitution within the KatG enzyme in isoniazid-resistant strains of Mycobacterium tuberculosis isolated in Uruguay. , 2008, Journal of medical microbiology.

[6]  Eun Kyu Lee,et al.  Loop Mediated Isothermal Amplification of DNA , 2008 .

[7]  Hojoong Kim,et al.  Treatment of isoniazid-resistant pulmonary tuberculosis , 2008, BMC infectious diseases.

[8]  Yen-Hsu Chen,et al.  Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA-hybridization assay for molecular detection of Mycobacterium tuberculosis. , 2009, Journal of microbiological methods.

[9]  K. Feldmann,et al.  Rapid, simple, and culture-independent detection of rpsL codon 43 mutations that are highly predictive of streptomycin resistance in Mycobacterium tuberculosis. , 2000, The American journal of tropical medicine and hygiene.

[10]  K. Feldmann,et al.  Usefulness of Mycobacterium tuberculosis genomic mutations in the genes katG and inhA for the prediction of isoniazid resistance. , 1997, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[11]  F. Cockerill,et al.  Rapid identification of a point mutation of the Mycobacterium tuberculosis catalase-peroxidase (katG) gene associated with isoniazid resistance. , 1995, The Journal of infectious diseases.

[12]  M. Guerrero-Olazarán,et al.  Frequency of mutations in rpoB and codons 315 and 463 of katG in rifampin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates from northeast Mexico. , 2003, Microbial drug resistance.

[13]  T. Victor,et al.  Analysis of isoniazid, streptomycin and ethambutol resistance in Mycobacterium tuberculosis isolates from Morocco. , 2009, Journal of infection in developing countries.

[14]  G. Woods,et al.  Susceptibility Test Methods: Mycobacteria, Nocardia, and Other Actinomycetes* , 2011 .

[15]  G. Sotgiu,et al.  Multidrug-resistant and extensively drug-resistant tuberculosis in the West. Europe and United States: epidemiology, surveillance, and control. , 2009, Clinics in chest medicine.

[16]  R. Sarin,et al.  Seven-year DOTS-Plus pilot experience in India: results, constraints and issues. , 2009, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[17]  W. Yew,et al.  Multidrug-resistant and extensively drug-resistant tuberculosis. , 2009, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[18]  S T Cole,et al.  Mechanisms of drug resistance in Mycobacterium tuberculosis. , 1996, Current topics in microbiology and immunology.

[19]  E. Mokaddas,et al.  Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations. , 2004, Molecular and cellular probes.

[20]  Suhail Ahmad,et al.  Recent advances in the diagnosis and treatment of multidrug-resistant tuberculosis. , 2009, Respiratory medicine.

[21]  Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis , 2009, Antimicrobial Agents and Chemotherapy.

[22]  Ellen Jo Baron,et al.  Manual of clinical microbiology , 1975 .

[23]  H. Grewal,et al.  Isoniazid and rifampicin resistance-associated mutations in Mycobacterium tuberculosis isolates from Yangon, Myanmar: implications for rapid molecular testing. , 2009, The Journal of antimicrobial chemotherapy.

[24]  E. Mokaddas,et al.  Frequency of embB codon 306 mutations in ethambutol-susceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait. , 2007, Tuberculosis.

[25]  V. Robison,et al.  Extensively drug-resistant tuberculosis in the United States, 1993-2007. , 2008, JAMA.

[26]  M. Hazbón,et al.  Transfer of embB Codon 306 Mutations into Clinical Mycobacterium tuberculosis Strains Alters Susceptibility to Ethambutol, Isoniazid, and Rifampin , 2008, Antimicrobial Agents and Chemotherapy.

[27]  Toru Mori,et al.  Detection of Multidrug Resistance in Mycobacterium tuberculosis , 2006, Journal of Clinical Microbiology.

[28]  A. Velayati,et al.  Molecular analysis of isoniazid resistance in different genotypes of Mycobacterium tuberculosis isolates from Iran. , 2008, Microbial drug resistance.

[29]  I. Azam,et al.  Trends in Mycobacterium tuberculosis resistance, Pakistan, 1990-2007. , 2009, International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases.

[30]  V. Cheng,et al.  Direct detection of Mycobacterium tuberculosis in clinical specimens using single-tube biotinylated nested polymerase chain reaction-enzyme linked immunoassay (PCR-ELISA). , 2004, Diagnostic microbiology and infectious disease.