摘要
为建立猪囊孢球虫和毕氏肠微孢子虫双重nest-PCR快速检测方法,参考文献引物,合成针对猪囊孢球虫SSU rRNA、毕氏肠微孢子虫ITS位点的特异性序列,优化双重nest-PCR反应中的引物比、退火温度和循环数等各项反应条件,建立快速检测2种肠道原虫的双重nest-PCR方法.针对该方法的特异性、敏感性和重复性进行检验。结果表明,猪囊孢球虫、毕氏肠微孢子虫最低可检测DNA量分别为209×10^(-6)和527×10^(-9) mg/L;特异性和重复性良好。用建立的双重nest-PCR方法对48份猪粪样DNA进行检测,用单病原检测nest-PCR扩增猪囊孢球虫和毕氏肠微孢子虫,阳性率分别为12.50%(6/48)和22.92%(11/48),混合感染阳性率为4.17%(2/48);双重nest-PCR猪囊孢球虫和毕氏肠微孢子虫的阳性率分别为8.33%(4/48)和20.83%(10/48),混合感染阳性率为4.17%(2/48);单重nest-PCR和双重nest-PCR检测结果无统计学差异(P>0.05)。表明该方法可用于猪肠道2种原虫流行病学调查和临床检测。
To establish a double nest-PCR method for rapid detection of Cystoisospora suis and Enterocytozoon bieneusi(E.bieneusi),the primers which could specifically amplify the sequences of the SSU rRNA of Cystoisospora suis,and the ITS of the E.bieneusi were synthesized according to the literature, the reacion influence factors such as the primers ratio of two pathogens, annealing temperature, reaction cycle number was examined and optimized, and then the specificity, sensitivity and repeatability of the method were tested.The results showed that the minimum detectable DNA of Cystoisospora suis and E.bieneusi were 209×10^(-6) and 527×10^(-9) mg/L,respectively.The specificity and repeatability test results were good.Forty-eight DNA samples of pig feces were amplified by existing simplex PCR and duplex PCR established in this experiment, the positive rates of simplex nest-PCR amplification of Cystoisospora suis,E.bieneusi,and mixed infection were 12.50%(6/48) and 22.92%(11/48) and 4.17%(2/48),respectively;the positive rates of duplex nest-PCR for Cystoisospora suis,E.bieneusi,and mixed infection were 20.83%(10/48),8.33%(4/48) and 4.17%(2/48),respectively.There was no significant difference between simplex and duplex nest-PCR(P>0.05).These results show that this method can be used for epidemiological investigation and clinical detection.
作者
王朋林
陈凯丽
郑玲
刘林科
王荣军
张龙现
宁长申
菅复春
WANG Penglin;CHEN Kaili;ZHENG Ling;LIU Linke;WANG Rongjun;ZHANG Longxian;NING Changshen;JIAN Fuchun(College of Animal Science and Veterianry Medicine,Henan Agricultural University,Zhengzhou 450046,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第3期500-504,共5页
Chinese Journal of Veterinary Science
基金
猪重要疫病的诊断与检测新技术研究资助项目(2016YFD0500707)。
