Early detection of multidrug resistant (MDR) Mycobacterium tuberculosis in a single tube with in-house designed fluorescence resonance energy transfer (FRET) probes using real-time PCR.

Rapid and correct diagnosis is crucial for the management of multidrug resistance (MDR) in Mycobacterium tuberculosis (MTB). The present study aims at rapid diagnosis for identification of multidrug resistance tuberculosis (MDR-TB) using real-time PCR. FRET hybridization probes targeting most prominent four selected codons for rpoB526 and 531 and for katG314 and 315 genes were designed and evaluated on 143 clinical MTB isolates and paired sputa for rapid detection of MDR-TB. The results of real-time PCR were compared with gold standard L-J proportion method and further validated by DNA sequencing. Of the 143 MTB positive cultures, 85 and 58 isolates were found to be ‘MDR’ and ‘pan susceptible’, respectively by proportion L-J method. The sensitivity of real-time PCR for the detection of rifampicin (RIF) and isoniazid (INH) were 85.88 and 94.11%, respectively, and the specificity of method was found to be 98.27%. DNA sequencing of 31 MTB isolates having distinct melting temperature (Tm) as compared to the standard drug susceptible H37Rv strain showed 100% concordance with real-time PCR results. DNA sequencing revealed the mutations at Ser531Leu, His526Asp of rpoB gene and Ser315Thr, Thr314Pro of katG gene in RIF and INH resistance cases. This real-time PCR assay that targets limited number of loci in a selected range ensures direct and rapid detection of MDR-TB in Indian settings. However, future studies for revalidation as well as refinement are required to break the limitations of MDR-TB detection.

Quick Detecting Mutation Site of 315 Codon in katG Gene of INH resistant MTB by Stem-ring Molecular Probe in Liquid / 中华医院感染学杂志

OBJECTIVE To detect 315 codon of mutation site in katG of isoniazid(INH)-resistant Mycobacterium tyberculosis(MTB) by stem-ring molecular probe quickly and detect out the fluorescence sign of hybridization between amplified products of katG 315 codon and probe in liquid by fluorescence spectrophotometer.The results were confirmed by sequencing.METHODS The software,Beacon designer,was used to design the katG 315 codon stem-ring molecular probe and the amplification system,and the relationship between the way and sequencing of the amplification products were compared.RESULTS The difference between PCR products from standard strain and INH-resistant one was significant in detecting the fluorescent light by use of fluorescence spectrophotometer.We detected fluorescent light signal between the 16 INH resistant strains and 10 H37RV standard strains.The resistant rate to INH detected was about 44%,and the rate of coincidence was about 97.5%.CONCLUSIONS The stem-ring molecular probe technology show high sensitivety in detecting mutation site of nucleic acid.The rate of coincidence is good between fluorescence spectrophotometer way and sequencing.

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis from Korea / 대한임상병리학회지

BACKGROUND: The genetic basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has been attributed to at least four genes. Mutations of the katG gene encoding catalase-eroxidase have been shown to cause resistance to INH. Among these mutations, the point mutation of codons 315 and 463 are frequently found. To determine whether simple screening method could potentially be useful for the detection of INH-esistant strains, we investigated the presence of mutation of codons 315 and 463 of katG gene in INH-esistant M. tuberculosis from Korea. METHODS: We used the polymerase chain reaction (PCR) and direct sequencing analysis to detect the point mutation of codons 315 and 463 of katG gene in 48 strains of INH-esistant and 10 strains of INH-usceptible M. tuberculosis. RESULTS: No amplification product was generated from 2 of 48 INH-esistant strains. Among the remaining 46 isolates, point mutations at codons 315 and 463 were identified in 19 isolates (41.3%) and 40 isolates (87.0%), respectively. Among the 10 INH-usceptible strains, the codon 463 point mutation was identified in 7 isolates, however, no point mutation of the codon 315 was found. CONCLUSIONS: These results suggest that the point mutation of codon 463 of katG gene of M. tuberculosis may be a polymorphism not related with INH resistance. Although the mutation of codon 315 of katG gene was limited to 41.3% of INH-esistant isolates, further investigation of the codon 315 of katG gene should lead to increased understanding of resistance genes and the deveolpment of rapid molecular detection methods.

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis from Korea / 대한임상병리학회지

BACKGROUND: The genetic basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has been attributed to at least four genes. Mutations of the katG gene encoding catalase-eroxidase have been shown to cause resistance to INH. Among these mutations, the point mutation of codons 315 and 463 are frequently found. To determine whether simple screening method could potentially be useful for the detection of INH-esistant strains, we investigated the presence of mutation of codons 315 and 463 of katG gene in INH-esistant M. tuberculosis from Korea. METHODS: We used the polymerase chain reaction (PCR) and direct sequencing analysis to detect the point mutation of codons 315 and 463 of katG gene in 48 strains of INH-esistant and 10 strains of INH-usceptible M. tuberculosis. RESULTS: No amplification product was generated from 2 of 48 INH-esistant strains. Among the remaining 46 isolates, point mutations at codons 315 and 463 were identified in 19 isolates (41.3%) and 40 isolates (87.0%), respectively. Among the 10 INH-usceptible strains, the codon 463 point mutation was identified in 7 isolates, however, no point mutation of the codon 315 was found. CONCLUSIONS: These results suggest that the point mutation of codon 463 of katG gene of M. tuberculosis may be a polymorphism not related with INH resistance. Although the mutation of codon 315 of katG gene was limited to 41.3% of INH-esistant isolates, further investigation of the codon 315 of katG gene should lead to increased understanding of resistance genes and the deveolpment of rapid molecular detection methods.

The Relationship between Isoniazid Resistance and 463 CodonMutation of katG Gne in Mycobacterium Tuberculosis / 결핵

BACKGROUND: The 463 codon mutation of katG gene has been reported as an useful marker for the detection of isoniazid(INH) resistant strains of M. tuberculosis. This study aimed to elucidate relationship between 463 mutation in katG gene and INH resistance in M. tuberculosis. METHOD: DNA was extracted from 28 INH susceptible strains(MIC > or = 0.2microg/ml on the Lowenstein Jensen media) and used for amplification of 189bp fragment containing 463 codon by PCR. Amplified fragments were digested by restriction enzyme Msp I, analyzed by single strand conformation polymorphism(SSCP) in the MDE gel and sequenced to prove mutation. RESULT: Only 7(25%) out of 28 were digestible by restriction enzyme Msp I. The SSCP pattern of 21 strains were distinctly different from that of M. tuberculosis H37Rv. Msp I undigestible PCR fragment was substituted at 463 codon from Arg(CGG) to Leu(CTG). CONCLUSION: This finding clearly indicate no relationship between 463 codon mutation of the katG gene and INH resistance.