食源性沙门氏菌携带质粒介导的喹诺酮类耐药基因的液相芯片检测技术研究

杜强; 黎俊宏; 严程; 赵莹; 屠博文; 毛旭健; 董昕; 张露杰

doi:10.13386/j.issn1002-0306.2022050067

食源性沙门氏菌携带质粒介导的喹诺酮类耐药基因的液相芯片检测技术研究

Applied Research for Liquid-phase Chip Detection of Foodborne Salmonella Carrying Plasmid-mediated Quinolone Resistance Genes

摘要

摘要: 目的：建立应用新型液相芯片技术检测食源性沙门氏菌携带的质粒介导喹诺酮类耐药（PMQR）基因中4种基因：qnrS、aac（6'）-Ib-cr、oqxA、oqxB的方法。方法：针对食源性沙门氏菌携带的qnrS、aac（6'）-Ib-cr、oqxA、oqxB四种PMQR基因，设计对应引物和微球，采用液相芯片技术对7株标准菌株进行特异性实验；对4株各含1种PMQR基因的食源性沙门氏菌进行重复性和灵敏度实验；然后检测来自食源性风险监测的71株耐喹诺酮类沙门氏菌，和普通PCR进行对比实验。结果：成功建立了液相芯片技术检测食源性沙门氏菌携带的qnrS、aac（6'）-Ib-cr、oqxA、oqxB四种PMQR基因的方法，携带qnrS基因的耐药株检出限为5 CFU/mL、携带aac（6'）-Ib-cr基因的耐药株检出限为25 CFU/mL、携带oqxA和oqxB基因的耐药株检出限为10 CFU/mL。所有阳性判定结果荧光中位值（MFI）均≥5倍阴性对照组。重复性实验变异系数（CV）均小于5%，特异性实验结果特异性100%，阴性菌株无阳性信号反应。qnrS检出率29.6%（21/71）、aac（6'）-Ib-cr 35.2%（25/71）、oqxA 28.2%（20/71）、oqxB 23.9%（17/71），方法比对的结果符合率为100%。结论：实验建立的液相芯片技术检测食源性沙门氏菌质粒介导喹诺酮类耐药基因qnrS、aac（6'）-Ib-cr、oqxA、oqxB的方法具有灵敏度高、特异性好、稳定性强、结果准确的特点，可以为食源性沙门氏菌PMQR基因的检测以及耐药性的监控提供技术支撑。

Abstract: Objective: To establish a method to detect four genes: qnrS, aac(6')-Ib-cr, oqxA, oqxB, among the plasmid-mediated quinolone resistance (PMQR) genes carried by foodborne Salmonella by applying a novel liquid-phase chip technique. Methods: For the four PMQR genes carried by foodborne Salmonella: qnrS, aac(6')-Ib-cr, oqxA, and oqxB, corresponding primers and microspheres were designed, and liquid-phase microarray technology was used to perform specificity experiments on seven standard strains. Reproducibility and sensitivity experiments were performed on four foodborne Salmonella strains containing one PMQR gene each. 71 quinolone resistant Salmonella strains from foodborne risk monitoring were tested, and a comparison experiment with ordinary PCR was carried out. Results: A method for the detection of four PMQR genes, qnrS, aac(6')-Ib-cr, oqxA and oqxB, in foodborne Salmonella was successfully established by liquid-phase microarray technology. The LOD (limit of detection) of qnrS, aac(6')-Ib-cr, oqxA and oqxB were 5, 25, 10 and 10 CFU/mL respectively. The median fluorescence values (MFI) of all positive determinations were ≥5 times those of the negative control group. The coefficients of variation (CV) of the repeatability experiments were less than 5%. In the specificity experiments, all quinolone resistant Salmonella strains were detected, and no cross-reactivity with other non-target bacteria was observed. The detection rate of qnrS, aac(6')-Ib-cr, oqxA and oqxB were 29.6% (21/71), 35.2% (25/71), 28.2% (20/71), 23.9% (17/71) respectively. The coincidence rate between the liquid chip technology and PCR was 100%. Conclusion: The experimentally established liquid-phase chip technique for the detection of foodborne Salmonella plasmid-mediated quinolone resistance genes qnrS, aac(6')-Ib-cr, oqxA and oqxB is characterized by high sensitivity, good specificity, stability, and accurate results, which can provide technical support for the detection of foodborne Salmonella PMQR genes and the monitoring of drug resistance.