Heidari Leila, Rafiei Dehbidi Gholamreza, Farhadi Ali, Kashkooli Golnar Sami, Zarghampoor Farzaneh, Namdari Sepide, Seyyedi Noorossadat, Fard Saeid Amirzadh, Behzad-Behbahani Abbas
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Shiraz Referral Mycobacteriology Laboratory, Shiraz University of Medical Sciences, Shiraz, Iran.
Iran J Microbiol. 2023 Jun;15(3):401-407. doi: 10.18502/ijm.v15i3.12900.
In clinical diagnostics, molecular methods are used to detect bacilli (MTB) and to distinguish them from non-tuberculous mycobacteria (NTM). They are also used to make the right treatment decision for the patient as soon as possible. The aim of this study was to establish a rapid and novel multiplex PCR (mPCR) assay for the detection and differentiation of MTB and NTM in a single tube.
100 sputum samples positive for acid-fast bacilli (AFB) were included in this study. Mycobacterial culture, biochemical tests, and antibiotic susceptibility testing were performed on samples. After alkaline decontamination, total DNA was extracted from the samples. A primer pair targeting the gene, encoding the beta-subunit of RNA polymerase, was used to detect MTB and NTM, amplifying a 235-bp fragment of MTB and a 136-bp sequence of NTM. A pair of primers targeting a 190-bp fragment of the IS6110 region of MTB was also used to confirm the results. The sensitivity and specificity of the mPCR assay were evaluated using DNA extracted from standard strains. The amplified products were then analyzed by conventional agarose gel electrophoresis.
Of 100 AFB smear-positive sputum samples, 92 MTB DNA, 7 NTM DNA, and one mixed-infection sample were identified in a single tube using mPCR assay. There was no correlation between the AFB degree of smear positivity and PCR results. Of seven NTM isolates, 6 (86%) were resistant to rifampin, isoniazid, and ethambutol, the three first-line anti-tuberculosis drugs.
A single-tube mPCR assay based on the gene provides a rapid and reliable means of detecting and differentiating MTB and NTM in sputum specimens.
在临床诊断中，分子方法用于检测结核杆菌（MTB）并将其与非结核分枝杆菌（NTM）区分开来。这些方法还用于尽快为患者做出正确的治疗决策。本研究的目的是建立一种快速、新颖的多重PCR（mPCR）检测方法，用于在单管中检测和区分MTB和NTM。
本研究纳入了100份抗酸杆菌（AFB）阳性的痰标本。对样本进行了分枝杆菌培养、生化试验和药敏试验。经过碱性去污后，从样本中提取总DNA。使用一对靶向编码RNA聚合酶β亚基基因的引物来检测MTB和NTM，扩增出MTB的235bp片段和NTM的136bp序列。还使用一对靶向MTB IS6110区域190bp片段的引物来确认结果。使用从标准菌株中提取的DNA评估mPCR检测方法的灵敏度和特异性。然后通过常规琼脂糖凝胶电泳分析扩增产物。
在100份AFB涂片阳性的痰标本中，使用mPCR检测方法在单管中鉴定出92份MTB DNA、7份NTM DNA和1份混合感染样本。涂片阳性的AFB程度与PCR结果之间没有相关性。在7株NTM分离株中，6株（86%）对三种一线抗结核药物利福平、异烟肼和乙胺丁醇耐药。
基于该基因的单管mPCR检测方法为痰标本中MTB和NTM的检测和区分提供了一种快速、可靠的手段。
