鹿人五毛滴虫巢式PCR检测方法的建立  

Development of the Nested PCR Assay for Detection of Pentatrichomonas hominis in Deers

作　　者：董兵[1] 余裕强[1] 李显赫[1] 李智鹏[1] 宫鹏涛[1] 李建华[1] 张西臣[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《天津农业科学》2015年第5期7-10,22,共5页Tianjin Agricultural Sciences

基　　金：国家自然科学基金(30970322)

摘　　要：为了建立鹿人五毛滴虫巢式PCR检测方法并进行验证,根据人五毛滴虫18S r RNA基因的特异性片段设计巢式PCR特异性引物,对两轮PCR反应的退火温度和反应体系中Mg2+浓度进行探索和优化,在此基础上,进一步通过灵敏性和特异性的检测。采用建立的巢式PCR检测方法对68份鹿粪便样品进行检测。结果表明,建立的巢式PCR方法,第一轮PCR最适退火温度为53℃,第二轮PCR最适退火温度为53℃;镁离子浓度为2.5 mmol·L-1。对鹿人五毛滴虫的检测精度可达每毫升10个虫体DNA;对犬贾第虫、阴道毛滴虫、弓形虫、柔嫩艾美尔球虫、大肠杆菌、旋毛虫基因组DNA的扩增的结果均为阴性;68份鹿粪便样品鹿人五毛滴虫的检出率为50%。由此可见,建立的巢式PCR检测方法具有较高的灵敏度和特异性,可用于鹿人五毛滴虫感染的临床检测。To develop a nested PCR for detection of Pentatrichomonas hominis in deers, designed two pairs of primers based on the sequence of Pentatrichomonas hominis 18 S r RNA published on Gen Bank, through optimized PCR reaction conditions including the annealing temperature, the concentration of magnesium ions, the nested PCR was established. And the specificity, sensitivity and 68 clinical samples were assayed. The results showed that nested PCR method established best for the first reaction： Mg2 ＋concentration was 2.5 mmol·L^-1 and optimal annealing temperature was 53 ℃; for the second reaction： optimal annealing temperature was 55 ℃.The DNA of 10 parasites could be detected by the assay, there were no detection with the DNA of Giardia canis, Trichomonas vaginalis, Toxoplasma gondii, Eimeria tenella, Escherichia coli and Trichinella spiralis. The infection rate of these 68 samples was 50%.The nested PCR was sensitive and specific, it could be used for detection of clinical sample.

关 键 词：鹿 肠道毛滴虫 巢式PCR 

分 类 号：S852.72[农业科学—基础兽医学]