Evaluation of Point Mutation Detection in Mycobacterium tuberculosis with Isoniazid Resistance Using Real-Time PCR and TaqMan Probe Assay

Abstract

Rapid methods for diagnosis of Mycobacterium tuberculosis (Mtb) drug resistance and choosing appropriate antibiotic treatment are pivotal. Thirty isoniazid (INH)-resistant and 30 INHsusceptible Mtb isolates were evaluated using minimum inhibitory concentration (MIC) method followed by multiplex real-time PCR (RT-PCR). Amplification refractory mutation system (ARMS) for detection of mutation in 315 codon of katG gene and single-nucleotide polymorphism (SNP) for detection of mutation in −15 (C>T) in the regulatory zone of mabA-inhA were carried out using the TaqMan method. Primers and probe were used for IS6110 region of Mtb as an internal amplification control. The sensitivity and specificity of the RT-PCR TaqMan probe for detection of Mtb complex were 100 %. Detection of INH-resistant Mtb using the ARMS method for KatG had 69 % sensitivity and 100 % specificity. The sensitivity and specificity of SNP in mabA-inhA fragment for detection of INH-resistant Mtb were 53 and 100 %, respectively. Furthermore, considering both regions, the sensitivity of RT-PCR has increased to 75 %. This study revealed that the qPCR-TaqMan method can be used as a standard tool for diagnosis of Mtb. Moreover, ARMS and SNP RT-PCR TaqMan methods can be used as rapid screening methods for detection of INH-resistant Mtb.

Biosensors for Point Mutation Detection

“…The conventional point mutation detection methods include sequencing and PCR based methods ( Manam and Nichols, 1991 ; Riahi et al, 2015 ). The basic mutation site recognition strategies include surface ligation reaction, mismatch binding protein mediated strategy, molecular beacon-based method and fluorescence in situ hybridization (FISH) ( Sun et al, 2018 ; Lu et al, 2020 ).…”

“…PCR based methods, such as PCR based allele detection system (AS-PCR) and fluorescent gene detection technology, allele specific small groove binding probe real-time PCR detection (RT-PCR), PCR restriction fragment length polymorphism (RFLP), polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and heteroduplex mobility analysis (HMA) belong among the most commonly used point mutation detection methods ( Callens and De Clercq, 1999 ; Pozzi et al, 2009 ; Riahi et al, 2015 ; Prykhozhij et al, 2018 ).…”

“…ARMS (amplification refractory mutation system)-PCR is a simple and economical technique for mutation detection. ARMS was used to detect the mutations in katG and fabG genes for rapid diagnosis of Mycobacterium tuberculosis (Mtb) drug resistance ( Riahi et al, 2015 ). Real time quantitative PCR (qPCR) was used to detect and measure the 3243A > G mutation in patients with diabetes ( Singh et al, 2006 ).…”

Herpes Simplex Virus and Tuberculosis-Associated Encephalitis in Northeast of Iran

“…In the present study, molecular diagnosis of encephalitis was confirmed according to HSV-1 and 2 or tuberculosis real-time PCR on CSF. Primers and labeled probes for tuberculosis qRT-PCR, TaqMan method, were used as previously described (20). The standard TaqMan master mix (Takara, Japan) was used for qRT-PCR amplifications.…”

Characteristic and influencing factors of Taqman genotyping calling error

“…There are many methods for SNP genotyping, including MALDI‐TOF mass spectrometry, Taqman probe, restriction fragment length polymorphism (RFLP), microarray, and DNA sequencing . Due to the high cost, MALDI‐TOF mass spectrometry, microarray, and DNA sequencing are not suitable for analysis of large samples.…”