摘要
目的针对香港海鸥形菌,利用同源加尾系统(Homo-Tag Assisted Non-Dimer,简称HAND)建立一种基于改良分子信标的荧光PCR检测方法,并对这种方法进行条件优化和评价。方法根据香港海鸥形菌的基因保守序列16SrDNA基因设计探针和特异性引物,并在引物的5’端加上同源尾巴序列,减少或避免引物二聚体的形成,通过优化通用尾巴引物和特异性加尾引物的浓度与反应温度等PCR参数构建荧光PCR的反应体系。结果建立的基于HAND系统的改良分子信标PCR的检测体系引物二聚体明显减少,检出限达到20CFU/ml。结论建立的基于HAND系统的改良分子信标荧光PCR检测方法具有检测时间短、灵敏度高、特异性好的优点,能够应用于香港海鸥形菌的快速筛查。
Objective To develop a modified molecular beacon real - time PCR assay based on Homo - Tag Assisted Non - Dimer （HAND） system for detection of Laribacter hongkongensis, and optimize and evaluate the assay. Methods Based on the consensus sequences of 16SrDNA gene published on GeneBank, primers and molecular beacon probe of Laribacter hongteon- gensis were designed, the homologous tail sequences were added to the 5＇ end to prevent or reduce the formation of primer dimer. The fluorescence PCR system was constructed by optimizing the PCR parameters like concentrations of homologous tail primers and specific primers as well as reaction temperature. Results The modified molecular beacon real - time PCR assay based on HAND system was developed, in which the primer dimer was significantly reduced. The sensitivity of the assay was 20 CUF/ml. Conclusions The real- time PCR assay based on HAND system has the advantage of rapidity, sensitivity and specificity. It can be applied to the rapid detection of Laribacter hongkongensis.
出处
《实用预防医学》
CAS
2015年第1期37-40,共4页
Practical Preventive Medicine
基金
湖南省科技厅科研基金项目(2013SK3232)
