犬贾第虫巢式PCR检测方法的建立  

Development of nested PCR assay for Giardia canis in dogs

作　　者：李棕松[1] 高珺珊[1] 翟涛[1] 吴威[1] 宫鹏涛[1] 李建华[1] 杨举[1] 李赫[1] 张国才[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：《中国生物制品学杂志》2013年第11期1668-1671,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(30970322);广州市科技计划项目(2013J4100124)

摘　　要：目的建立犬贾第虫巢式PCR检测方法并进行验证。方法根据犬贾第虫Rnase P基因序列设计两对特异性引物,通过优化Mg2+浓度和退火温度建立巢式PCR检测方法,并进行灵敏度和特异性验证。用优化的巢式PCR方法检测60份犬临床粪样。结果建立的巢式PCR方法的最佳Mg2+浓度为3.0 mmol/L;最佳退火温度为53℃;对犬贾第虫的最低检测量可达1个虫体DNA/ml;对柔嫩艾美尔球虫、伊氏锥虫、弓形虫、阴道毛滴虫、犬心丝虫等寄生虫基因组DNA的扩增结果均为阴性;60份犬临床粪样贾第虫的检出率为61.7%。结论建立的巢式PCR检测方法具有较高的灵敏度和特异性,可用于犬贾第虫感染的临床检测。Objective To develop and verify a nested PCR assay for Giardia canis in dogs. Methods Two pairs of primers were designed based on the sequence of RNase P gene of G. canis, based on which a PCR assay was developed by optimizing the magnesium ion concentration and temperature for annealing, and verified for sensitivity and specificity. Sixty clinical fecal samples of dogs were determined by the developed nested PCR assay. Results The optimal magne- sium ion concentration and temperature for annealing of the developed nested PCR assay were 3. 0 mmol / L and 53 ℃ respectively. The minimum detection limit of G. canis by the developed method was 1 parasitic DNA/ml. All the test re- sults of DNAs of E. tenella, T. evansi, Toxoplasma gondii, Trichomonas vaginalis and Dirofilaria immitis by the method were negative. The detection rate of G. canis in 60 fecal samples of dogs was 61.7%. Conclusion The developed nested PCR assav showed hizh sensitivity and specificity, which might be used for clinical detection of G. canis.

关 键 词：犬 贾第虫 巢式聚合酶链反应 

分 类 号：R382.9[医药卫生—医学寄生虫学] Q789[医药卫生—基础医学]