淋病奈瑟菌大观霉素耐药基因检测方法的建立与评价

杨桂琴; 李梦欢; 王有为; 雍刚; 王红仁; 别明江

doi:10.12182/20250160402

淋病奈瑟菌大观霉素耐药基因检测方法的建立与评价

Establishment and Evaluation of a Nucleic Acid Amplification Test for Spectinomycin-Resistant Neisseria gonorrhoeae

摘要

摘要:
目的建立淋病奈瑟菌大观霉素耐药基因的检测方法并对其进行评价。
方法设计淋病奈瑟菌特异性引物NG1/NG2以及淋病奈瑟菌rpsE基因突变（80_82 delTTA）特异性引物，通过PCR和实时荧光PCR（quantitative real-time PCR, qPCR）技术，分别以大观霉素敏感和耐药淋病奈瑟菌、大肠埃希菌、铜绿假单胞菌和伤寒沙门菌等基因组核酸为待检样本，以评价该方法的敏感度及特异度。
结果 NG1/NG2引物能够有效扩增淋病奈瑟菌特异性片段，其余细菌扩增结果为阴性；E64/E175R和E-87/E95R能够有效区分待测样本的rpsE基因是否带有突变（80_82 delTTA）。利用PCR方法，NG1/NG2、E64/E175R和E87/E95R检测目的基因的最低检测限分别为414.8、414.8、4.1拷贝/μL，而qPCR检测方法的最低检测限分别为41.5、41.5、4.1×10^－2拷贝/μL。
结论本研究成功建立了一种高特异度和高敏感度的淋病奈瑟菌大观霉素耐药性的核酸检测方法，有望为临床快速诊断淋病感染和治疗决策提供指导。

Abstract:
Objective To develop and evaluate a nucleic acid amplification test for spectinomycin-resistant Neisseria gonorrhoeae (N. gonorrhoeae).
Methods N. gonorrhoeae-specific primers NG1/NG2 and primers specific to the N. gonorrhoeae rpsE gene mutation (80_82 delTTA) were designed. Genomic nucleic acids of spectinomycin-sensitive and resistant N. gonorrhoeae, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi were used as templates to be amplified by PCR and quantitative real-time PCR (qPCR). The sensitivity and specificity of the method were evaluated accordingly.
Results The NG1/NG2 primers could effectively amplify specific fragments of N. gonorrhoeae, yielding negative results for the nucleic acid amplification test of the other types of bacteria tested. E64/E175R and E-87/E95R could effectively differentiate the wild type and mutant (80_82 delTTA) rpsE genes. In PCR reactions, the minimum limits of NG1/NG2, E64/E175R, and E87/E95R for the target genes were 414.8 copies, 414.8 copies, and 4.1 copies /μL, respectively, while those for qPCR reactions were 41.5, 41.5, and 4.1×10^－2 copies /μL, respectively.
Conclusion A nucleic acid amplification test for spectinomycin-resistant N. gonorrhoeae with high specificity and sensitivity was successfully established in this study, which is expected to provide support for the rapid diagnosis of N. gonorrhoeae infection and treatment decision-making in clinical settings.

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