摘要
目的:建立能够同时检测单孢子虫、派琴虫和折光马尔太虫的三重荧光定量PCR方法。方法:根据基因库中单孢子虫、派琴虫和折光马尔太虫的基因序列,设计3对特异性引物和3条用不同荧光基团标记的TaqMan探针,对反应条件和试剂浓度进行优化,建立能够同时检测单孢子虫、派琴虫和折光马尔太虫的三重荧光定量PCR方法。结果:该方法对单孢子虫、派琴虫和折光马尔太虫的检测敏感性分别达到40、400和40个模板拷贝数;此外抗干扰能力强,对单孢子虫、派琴虫和折光马尔太虫不同模板浓度进行组合,仍可有效地同时检测这3种原虫,对嗜水气单胞菌、荧光假单胞菌、副溶血弧菌、溶藻弧菌、河弧菌和拟态弧菌等病原体的检测结果均为阴性。结论:建立的单孢子虫、派琴虫和折光马尔太虫多重荧光定量PCR具有特异、敏感、快速、定量和重复性好等优点,可用于临床上单孢子虫、派琴虫和折光马尔太虫感染的检测。
Objective： To develop a multiplex real-time PCR assay for the rapid detection of Haplosporidium sp.,Perkinsus sp.and Marteilia refringens.Methods： According to the sequences of Haplosporidium sp.,Perkinsus sp.and M.refringens,three sets of specific oligonucleotide primers,along with three TaqMan probes specific for each protozoan parasite were designed.The reaction parameters such as the concentration of three pair of primers,three TaqMan probes and the reaction buffer were optimized to develop a multiplex real-time PCR assay.Results： The sensitivity of multiplex real-time PCR assay was 40,400 and 40 template copies for Haplosporidium sp.,Per kinsus sp.and M.refringens respectively.The multiplex real-time PCR assay was found to be specific and to be able to detect and differentiate Haplosporidium sp.,Perkinsus sp.and M.refringens,and no positive results were ob served when nucleic acid from Aeromonas hydrophila,Pseudomonas fluorescens,Vibrio parahaemolyticu,V.alginolyti cu,V.fluvialis and V.mimicus were used as multiplex real-time PCR templates.Conclusion： This multiplex re al-time PCR assay is a quick,sensitive,and specific test for detection of Haplosporidium sp.,Perkinsus sp.and M.refringens and will be useful for the control of these protozoan parasites in shellfish.
出处
《生物技术通讯》
CAS
2012年第5期713-717,共5页
Letters in Biotechnology
基金
国家百千万人才工程人选专项(945200603)
广西特聘专家专项(2011B020)
广西科技攻关项目(桂科攻0630001-3M)
