摘要
目的建立一种检测俄亥俄等孢球虫的巢式PCR检测方法并初步用于犬球虫病的诊断。方法依据GenBank上俄亥俄等孢球虫ITS-1基因序列设计两对特异性引物。收集俄亥俄等孢球虫卵囊,采用饱和盐水漂浮法进行纯化,利用液氮-热水浴反复冻融、研磨,然后采用粪便基因组提取试剂盒提取DNA。建立巢式PCR方法,优化退火温度,并进行特异性和灵敏度检验。利用构建的巢式PCR方法对109份犬粪便样品(长春地区65份,哈尔滨地区44份)进行犬俄亥俄等孢球虫检测。结果构建的巢式PCR方法第二轮PCR最佳退火温度为53℃;用该方法检测犬贾第虫、阴道毛滴虫、刚地弓形虫、新孢子虫、微小隐孢子虫基因组标本均无交叉反应,检测目的核酸的最低浓度为2.05×10-12 ng/μl,较普通PCR灵敏度提高106倍。使用构建的巢式PCR方法对长春地区的65份犬粪便样品和哈尔滨地区44份犬粪便样品进行检测,阳性率分别为38.46%和6.81%。结论基于俄亥俄等孢球虫ITS-1基因序列建立的巢式PCR方法具有较高的灵敏度和特异度,可用于犬俄亥俄等孢球虫病的诊断和流行病学调查。
Objective To establish a nested PCR technique as an efficient method of detecting Isospora ohioensis.Methods Two pairs of primers were designed based on the partial sequence of the ITS-1gene of Isospora ohioensis in GenBank.Isospora ohioensis samples were collected and purified using the saturated solution floating method.Oocysts were subjected to three cycles of freezing in liquid nitrogen and thawing at 60℃ and then ground to release nucleic acids.Total nucleic acids were extracted with the TIANamp Stool DNA Kit.A nested PCR technique was established by optimizing the temperature for annealing,and verifying the technique＇s sensitivity and specificity.A total of 109 fecal samples of dogs（65fecal samples from dogs in Changchun and 44 fecal samples from dogs in Haerbin）were tested. Results The optimal annealing temperature for the second round of PCR was 53℃.Nested PCR was highly specific and there were no cross-reactions with DNA fromGiardia,Trichomonas vaginalis,Toxoplasma gondii,Neospora caninum,or Cryptosporidium parvum.The assay detected 2.05×10^-12 ng/μL of DNA,and nested PCR had a level of sensitivity 1×10^6times that of conventional PCR.Nest PCR was used to test 65 fecal samples from dogs in Changchun and 44 fecal samples from dogs in Haerbin.Dogs in,Changchun had a rate of infection of 38.46% and dogs in Haerbin had a rate of infection of 6.81%. Conclusion Nested PCR based on the ITS-1gene of Isospora ohioensis had a high level of specificity and sensitivity.This technique has laid the foundation for the detection and epidemiological study of Isospora ohioensis infection in dogs.
出处
《中国病原生物学杂志》
CSCD
北大核心
2016年第9期795-798,共4页
Journal of Pathogen Biology
基金
国家科技支撑计划项目(No.2015BAI07B02)
国家公益性行业(农业)科研专项(No.201303042)
