摘要
目的建立一种能够检测O1群和O139群霍乱弧菌的套式聚合酶链反应方法，并应用于外环境水体标本的监测。方法根据GenBank发表的O1群和O139群霍乱弧菌O抗原编码基因rfb的序列，运用DNAStar Primer Select软件各自设计6条引物，检测不同引物组合的特异性，建立双重套式PCR方法同时检测O1群和O139群霍乱弧菌；并对该方法进行敏感度、特异度、重复性及现场评价。通过比较套式PCR与常规PCR的DNA检出下限分析该方法的敏感度；对32份套式PCR阳性的水体样本（O1群11份，O139群21份）进行重复性评价，挑选部分阳性扩增产物进行测序，分析其与相应基因序列的一致性。结果建立的检测O1群和O139群霍乱弧菌的双重套式PCR体系，根据其扩增的片段大小能够区分O1群和O139群霍乱弧菌，而对其他弧菌DNA样品都未见特异性扩增；敏感度比常规PCR高15000倍，且在多引物或者多模板共存于一个反应管的试验条件下，不会对敏感度产生干扰；32份套式PCR阳性的水样标本，经2次或3次套式PCR试验，结果显示该体系具有较好的重复性和一致性，阴性水样均无任何扩增产物，说明所用引物的特异性较好；序列分析表明扩增产物仅同霍乱弧菌相应的基因序列有100％的一致性。结论本检测相应霍乱弧菌的双重套式PCR方法，具有灵敏、特异、快速、适合基层实验室的特点，可用于监测外环境水体中霍乱弧菌的存在及消长情况。
Objective To establish a duplex nested PCR assay system which is capable for detecting O1 and O139 groups of Vibrio cholerae simultaneously, and is applicable to environmental specimens from routine cholera surveillance. Methods Based on nucleic acid sequences available in GenBank, six sets of primers were designed by PrimerSelect program of DNAStar, targeting the rfb gene that encodes the O antigens of O1 and O139 V. cholerae, respectively. The specificity of several primer combinations was tested. A duplex nested PCR assay system for simultaneously detecting O1 and O139 V. cholerae was established, subsequently, its sensitivity, specificity, reproducibility and field evaluation were tested. The sensitivity of this assay was evaluated by comparing detection limits of nested PCR and conventional PCR. Its reproducibility was tested by 32 positive samples （11 samples positive for O1,21 samples positive for O139） from environmental surveillance. In addition, the selected amplicons from positive samples were sequenced and analyzed with relevant sequences. Results This newly-established duplex nested PCR assay might distinguish O1 V. cholerae from O139 V. cholerae, based on fragment lengths of amplicons,with reliable reproducibility, and no specific amplification was observed as compared with other vibrio species. The sensitivity of this nested PCR was （15 000） higher than conventional PCR,and there was no interference observed with multiple primers and complicated templates in the same vial. In its field evaluation,32 positive DNA samples were detected and be further confirmed with double or triple tests,implying reliable reproducibility and consistency of this system. These results indicated that this assay had reliable reproducibility. No amplification was observed in all negative specimens and also suggested the acceptable specificity of this assay. Sequence analysis of the selected amplification products revealed 100% homogeneous with relevant genes from V. cholerae, indicating that these amplicons were originated from V. cholerae. Conclusion This duplex nested PCR assay system should be rapid, sensitive and especially applicable to small laboratories, and he suitable for dynamic environmental surveillance.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2009年第8期674-679,共6页
Chinese Journal of Preventive Medicine
基金
国家“863”高技术研究发展计划（2006AA02Z425）
国家自然科学基金（30872260）
志谢 陈亢川主任医师对本研究的指导及对论文的修改和完善
关键词
霍乱弧菌
聚合酶链反应
环境监测
Vibrio cholerae
Polymerase chain reaction
Environmental surveillance
