猪附红细胞体单管巢式PCR诊断方法的建立及应用  

Establishment and application of a single-tube nested PCR assay for detection of Eperythrozoon suis

作　　者：郑秀红[1] 贾立军[1] 邢莹[1] 张守发[1] 

机构地区：[1]延边大学农学院动物医学系,吉林龙井133400

出　　处：《中国兽医学报》2011年第2期205-208,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30860203)

摘　　要：根据GenBank上发表的猪附红细胞体16S rRNA基因序列(登录号U88565)设计合成2对引物,建立了猪附红细胞体单管巢式PCR诊断方法,经酶切分析、单管巢式PCR进一步鉴定后进行序列测定,并与血涂片染色镜检、常规PCR进行了比较。结果:扩增的猪附红细胞体基因序列与GenBank中发表的猪附红细胞体基因序列(U88565)同源性为96%;特异性试验表明,设计的引物不能扩增弓形虫、链球菌、大肠杆菌、葡萄球菌及羊附红细胞体等病原体;敏感性试验表明,单管巢式PCR诊断方法最低能够检测出0.116 fg的标准模板DNA。通过对75份血液样本的检测表明,建立的单管巢式PCR方法明显优于血涂片染色镜检法及常规PCR方法,具有较高的敏感性和实用性。本试验建立的单管巢式PCR诊断方法具有特异、敏感、实用等优点,为猪附红细胞体的检测提供了一种新型、可靠的诊断技术。A novel diagnostic method was established to detect the presence of Eperythrozoon suis,two pairs of primers to Eperythrozoon suis were successfully designed according to the 16S rRNA gene sequence of E.suis from GenBank（U88565）,and to develop a single-tube nested PCR assay for detection of E.suis.The cloned genes demonstrated by analysis with enzyme digestion and the single tube nested PCR were proved to be the specific genes for E.suis and the sequence of clone E.suis was compared with that of E.suis published in GenBank （U88565）.Meanwhile,staining of blood smear and conventional PCR were compared with the single-tube nested PCR of E.suis.The results showed that the sequence of the clone E.suis was 96% homology with U88565.As demonstrated by the specificity testing,the PCR primers designed in the present study could not be amplified to form bands from the genomic DNA of Toxoplasma gondii,Streptococcuss species,E.coli,Staphylococcus species and Mycoplasma ovis.The lowest value of sensitivity of the single-tube nested PCR was 0.116 fg DNA of the standard template.The detection of 75 blood samples showed that the a single-tube nested PCR with high sensitivity and practicability is obviously superior to the staining of blood smear and conventional PCR for the detection of Eperythrozoon suis.

关 键 词：猪附红细胞体 单管巢式PCR 诊断技术 

分 类 号：S852.6[农业科学—基础兽医学]