耐甲氧西林金黄色葡萄球菌核酸快速检测方法的建立与评价

Establishment and evaluation of a rapid nucleic acid detection method for methicillin-resistant Staphylococcus aureus

摘要:

为建立一种快速、准确的耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)核酸即时检测方法，本研究首先针对16S rRNA、nuc和mecA基因设计特异性引物与荧光探针；以MRSA标准菌株制备梯度核酸标准品，优化qPCR体系并测序验证，完成方法构建。随后系统评估该体系的灵敏度、线性范围、重复性、特异性及抗干扰性能。最后，将优化后的方法应用于临床样本，并以临床实验室常用的鉴定方法(质谱法)为参比方法，验证其实用价值。本研究建立的MRSA核酸快速检测体系，可在30 min内完成对MRSA核酸的检测，对16S rRNA、nuc和mecA基因的灵敏度分别为1.0×10²、1.0×10²、1.0×10³ CFU/mL；线性范围分别为1.0×10²?1.0×10⁹、1.0×10²?1.0×10⁹、1.0×10³?1.0×10⁹ CFU/mL；批内和批间的变异系数均小于5%；与多种病原体之间无交叉反应；2 g/L的血红蛋白和100 μg /mL头孢曲松钠对检测无干扰；与质谱法相比，具有较好的一致性，灵敏度和特异性较高。结果表明，本研究构建的MRSA核酸快速检测方法兼具高灵敏度与操作简便性，为病原微生物核酸即时检测提供了新策略，可作为临床微生物快速筛查的可靠技术选项。

Abstract:

To establish a rapid and accurate nucleic acid-based point-of-care detection assay for methicillin-resistant Staphylococcus aureus (MRSA). We designed specific primers and fluorescent probes targeting 16S rRNA, nuc, and mecA genes. Gradient DNA (deoxyribonucleic acid) standards prepared from an MRSA reference strain were used to optimize the qPCR system, and amplicons and T-A cloning plasmids were sequenced for verification. Subsequently, the established multiplex real-time PCR assay was systematically evaluated for sensitivity, linearity, precision, specificity, and interference tolerance. Finally, the optimized method was applied to clinical samples, with mass spectrometry—a commonly used clinical laboratory identification method—serving as the reference standard to validate its practical value. The assay completed detection within 30 min, with limits of detection of 1.0×10², 1.0×10², and 1.0×10³ CFU/mL and linear ranges of 1.0×10²?1.0×10⁹, 1.0×10²?1.0×10⁹, and 1.0×10³?1.0×10⁹ CFU/mL for 16S rRNA, nuc, and mecA, respectively. Intra- and inter-batch coefficients of variation were <5%, and no cross-reactivity was observed. Neither hemoglobin (2 g/L) nor 100 μg/mL ceftriaxone sodium interfered with the detection results. The method showed high concordance, sensitivity, and specificity versus MALDI-TOF MS. In conclusion, the MRSA nucleic-acid detection assay developed in this study combines high sensitivity with operational simplicity, providing a new strategy for point-of-care pathogen nucleic-acid testing and serving as a reliable tool for rapid clinical microbial screening.