产单核李斯特菌的改良分子信标荧光PCR-磁捕获快速检测体系的建立  

ESTABLISHMENT OF RAPID DETECTION SYSTEM OF LISTERIA MONOCYTOGENES WITH MODIFIED MOLECULAR BEACONS PCR_ IMMUNOMAGNETIC BEADS CAPTURE

作　　者：王冰[1] 扈庆华[1] 石晓路[1] 李庆阁[2] 郑琳琳[2] 林一曼[1] 段永翔[3] 

机构地区：[1]深圳市疾病预防控制中心,深圳518020 [2]厦门大学生命科学学院 [3]深圳市南山区疾病预防控制中心

出　　处：《现代预防医学》2009年第13期2512-2514,共3页Modern Preventive Medicine

基　　金：国家自然科学基金资助项目(30300281);广东省卫生厅资助项目(A2003709);深圳市科技局资助项目(200404139)

摘　　要：[目的]建立改良分子信标荧光PCR-磁捕获快速检测产单核李斯特菌(LMO)的体系,应用于食品中LMO的污染状况调查及食物中毒快速诊断。[方法]根据GenBank公布的LMOhlyA基因的保守序列,设计引物和改良分子信标探针,建立改良分子信标荧光PCR检测体系。运用磁捕获材料对PCR检测阳性的增菌液进一步处理后划平皿做分离培养。[结果]改良分子信标-实时PCR反应体系DNA灵敏度为110fg,菌液灵敏度为99cfu/ml或4cfu/PCR反应体系,无交叉反应。以此反应体系检测28株LMO,均出现特异的荧光信号。对228份食品进行LMO检测,8份增菌液LMO实时荧光PCR阳性。用磁捕获材料处理此8份增菌液,全部细菌培养阳性,高于国标法的6份阳性。[结论]改良分子信标-实时PCR反应体系快速、灵敏度高,特异性强,运用磁捕获试剂能有效提高培养阳性率。应用该检测系统能提高LMO的检出率和准确性。该系统可应用于LMO食品污染状况调查及食物中毒的快速诊断。[ Objective ] To establish rapid detection system of Lsteria monocytogenes using modified molecular beacons and real-time PCR immunomagnetic beads capture. The established method was applied to investigate the infectious status of listeria monocytogenes in food and LMO food poisoning. [Methods] One set of primer was designed based on the core sequence of hlyA gene published on GenBank to detect LMO. The corresponding modified molecular beacons labeled with FAM were designed. The modified molecular beacons and real-time PCR assay were applied to detect the LMO in food. On the basis of the above experiments, immunomagnetic beads capture was used to isolate and culture the LMO in the above positive samples. [ Results] For the modified molecular beacons based real time PCR assay, the sensitivity achieved 110 fg or 4 cfu/PCR reaction. There was no cross reaction with other bacteria as control. The real-time PCR assay was used to detect 28 LMO strains, no false signals were observed, The detection time was reduced from at least five days to only one day. A total of 228 food poisoning samples were tested, 8 were found LMO positive by realtime PCR. Among the tested positive samples, all were LMO positive by immunomagnetie beads capture method, 6 were LMO positive by modified traditional method. [ Conclusion ] The modified molecular beacons based real-time PCR assay is rapid, sensitive and specific. Immunomagnetic beads capture method can effectively enhance positive rate in the culture. Using this system in LMO detection can improve the detection rate and veracity. It could be applied in the rapid diagnosis of LMO food poisoning and investigation of the infectious status in food.

关 键 词：产单核李斯特菌 改良分子信标 实时PCR 检测 免疫磁珠捕获 

分 类 号：R446.5[医药卫生—诊断学]