论文部分内容阅读
目的:建立一种快速检测志贺菌的环介导恒温核酸扩增(LAMP)技术,为志贺菌食源性疾病监测、诊断提供技术支撑。方法:在对志贺菌基因序列进行分析、对比基础上,根据志贺菌ipaH基因序列保守区域设计6条特异性引物;通过对LAMP技术反应条件优化,实现对志贺菌的快速检测;用不同来源志贺菌及沙门菌、大肠杆菌、副溶血性弧菌、柠檬酸杆菌、金黄色葡萄球菌、乙型溶血性链球菌等菌株验证方法的敏感性和特异性。结果:当25μl反应体系中含1.6μmol/L内引物(FIP、BIP)、0.2μmol/L外引物(F3、B3)、0.2μmol/L环引物(LF、LB)、1 mmol/L dNTP、6 mmol/L Mg-SO4,0.8 mol/L甜菜碱、1×反应缓冲液、DNA模版,8 U Bst DNA聚合酶,扩增条件为60℃,60 min时,LAMP技术对志贺菌最低检测浓度为200 CFU/ml,经8 h增菌,最低检测浓度可达到20 CFU/ml。对32株不同来源的志贺菌检测结果,所有反应管内肉眼观察有混浊,为阳性,而13种139株其它非志贺菌株反应结果均呈透明为阴性。对50件食品检测结果与常规培养方法及实时荧光PCR方法相比无显著性差异(P>0.05,χ2=0.27)。结论:本研究建立的志贺菌LAMP检测方法具有特异性强、灵敏度高、方便快捷、成本低等特点,适合在基层、小型实验室以及无条件购买荧光定量PCR仪的病原微生物检验检测机构推广使用。
OBJECTIVE: To establish a loop-mediated constant temperature nucleic acid amplification (LAMP) technique for rapid detection of Shigella to provide technical support for the monitoring and diagnosis of Shigella foodborne diseases. Methods: Based on the analysis and comparison of Shigella gene sequences, six specific primers were designed based on the conserved region of ipaH gene in Shigella. The reaction conditions of LAMP were optimized to achieve rapid detection of Shigella. Different sources of Shigella and Salmonella, Escherichia coli, Vibrio parahaemolyticus, Citrobacter, Staphylococcus aureus, Streptococcus hemolyticus and other strains validation method sensitivity and specificity. Results: When 25 μl reaction system contained 1.6 μmol / L of internal primers (FIP, BIP), 0.2 μmol / L of external primers (F3 and B3), 0.2 μmol / L of loop primers (LF and LB) 6 mmol / L Mg-SO4, 0.8 mol / L betaine, 1 × reaction buffer, DNA template and 8 U Bst DNA polymerase. The amplification conditions were 60 ℃ and 60 min. Concentration of 200 CFU / ml, after 8 h enrichment, the minimum detection concentration of up to 20 CFU / ml. The results of Shigella from 32 different sources showed that all reaction tubes were cloudy and positive for all eyes, while the results of 13 139 strains of other non-Shigella strains were transparent and negative. There was no significant difference (P> 0.05, χ2 = 0.27) between the detection results of 50 foods and conventional culture methods and real-time fluorescence PCR methods. Conclusion: The detection method of Shigella LAMP established in this study has the characteristics of high specificity, high sensitivity, convenience and quickness, low cost and so on. It is suitable for the popularization and application of pathogenic microorganism testing and testing institutions in grassroots, small laboratories and unconditional purchase of fluorescence quantitative PCR instrument .