核酸内切酶Surveyor酶切法快速检测MTB对吡嗪酰胺耐药性方法的建立及评估

doi:10.3969/j.issn.1000-6621.2021.08.011

摘要/Abstract

摘要：

目的建立应用核酸内切酶Surveyor酶切法快速检测MTB对吡嗪酰胺(pyrazinamide,PZA)耐药性的方法,并对其临床价值进行评估。方法选取来自武汉市肺科医院的耐多药(MDR)MTB菌株91株作为研究对象。分别以上游引物∶下游引物为2∶5的比例扩增所有菌株,同时以上游引物∶下游引物为5∶2的比例扩增结核分枝杆菌标准株H37Rv,然后将PCR产物以1∶1的比例混合杂交,应用核酸内切酶Surveyor酶切反应产物后进行电泳。以杂交条带能够被核酸内切酶Surveyor酶切割作为判定pncA基因及其启动子序列存在基因突变的依据。以BACTEC MGIT 960检测MTB对PZA耐药性方法和MTB的pncA基因及其启动子区基因序列测定法作为对照,评价核酸内切酶Surveyor酶切法的应用价值。结果以BACTEC MGIT 960法检测PZA耐药性作为对照,核酸内切酶Surveyor酶切法检测PZA耐药的敏感度为100.0%(29/29),特异度为87.1%(54/62),准确度为91.2%(83/91)。以MTB的pncA基因及其启动子区的基因测序结果作为对照,核酸内切酶Surveyor酶切法检测MTB对PZA耐药的敏感度为100.0%(37/37),特异度为100.0%(54/54),准确度为100.0%(91/91)。结论核酸内切酶Surveyor酶切法检测pncA基因及其启动子区突变的性能与基因测序高度一致,可以为没有基因测序条件的地区检测MTB对PZA耐药性提供一种新的思路。

关键词: Surveyor酶, 分枝杆菌,结核, 抗药性, 吡嗪酰胺

Abstract:

Objective To establish a probe enzyme digestion method using Surveyor for rapid detection of pyrazinamide (PZA) resistance detection of Mycobacterium tuberculosis (MTB) and evaluate the clinical value. Methods A total of 91 multidrug-resistant (MDR) MTB strains from Wuhan Pulmonary Hospital were collected. All strains were amplified using the above primers∶the downstream primer with the ratio of 2∶5, and the standard strain H37Rv of MTB was amplified using the upstream primer: the downstream primer with the ratio of 5∶2. Then, the PCR products were mixed in a ratio of 1∶1, and the electrophoresis was carried out after being digested by the endonuclease Surveyor enzyme. The gene mutation of pncA gene and its promoter sequence were determined based that the hybridization bands could be cleaved by the endonuclease Surveyor. The application value of endonuclease Surveyor digestion method was evaluated compared to BACTEC MGIT 960 method used to detect PZA resistance and pncA gene and its promoter gene sequencing method. Results For the detection of PZA resistance of MTB, compared to the BACTEC MGIT 960 method, the sensitivity, specificity and accuracy of the probe enzyme digestion method were 100.0% (29/29), 87.1% (54/62) and 91.2% (83/91), respectively. Compared to the sequencing results of pncA gene and its promoter region, the sensitivity, specificity and accuracy of the probe enzyme digestion method were 100.0% (37/37),100.0% (54/54) and 100.0% (91/91), respectively. Conclusion The probe enzyme digestion method demonstrated very high concordance with pncA gene and its promoter region, it might provide a new way for the detection of PZA resistance of MTB in areas without sequencing conditions.

Key words: Surveyor nuclease, Mycobacterium tuberculosis, Drug resistance, Pyrazinamide

胡严杰, 龚世伟, 任易. 核酸内切酶Surveyor酶切法快速检测MTB对吡嗪酰胺耐药性方法的建立及评估[J]. 中国防痨杂志, 2021, 43(8): 813-816. doi: 10.3969/j.issn.1000-6621.2021.08.011

HU Yan-jie, GONG Shi-wei, REN Yi. Establishment and evaluation of rapid detection of pyrazinamide resistance of Mycobacterium tuberculosis by probe enzyme digestion using Surveyor[J]. Chinese Journal of Antituberculosis, 2021, 43(8): 813-816. doi: 10.3969/j.issn.1000-6621.2021.08.011

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