螺旋杆菌PCR检测方法的建立及初步应用

Establishment of Polymerase Chain Reaction for Helicobactor sp.and PCR Assay in SD Rats and Field Voles

根据螺旋杆菌rRNA保守区设计出 4对引物 ,分别为P5、P6 ;P7、P8;P9、P10和H1、H2。从人工免疫抑制SD大鼠中分离到螺旋杆菌纯化的DNA为模板 ,按照各对引物的反应条件 ,筛选出最佳引物P7、P8和H1、H2 ,扩增片段长度分别为 374bp和 375bp ,并用这二对引物对大肠埃希菌、溶血性链球菌、嗜肺巴氏杆菌进行PCR扩增 ,以分析其特异性。将PCR方法应用于东方田鼠的检测 ,从几十份东方田鼠粪便中检测出 10份螺旋杆菌为阳性。PCR检测方法的建立 。

Oligonucleotide primers were desi gned from Helicobactor sp. regions of the 16 s rRNA gene that are conserved a mong members of the Helicobactor genus.The assay amplified the expected 374 bp product with P7P8 and 375 bp with H1H2,but no fragment was observed with prim ers P5P6 and P9P10.The specificity of the reaction was determined by testing DNA extracted from Helicobactor of experimentally immunosuppressive rats and Escherichia coli.,Hemolyticus streptococcus,Pasteurella pneumotropica. A produc t of the expected size was generated with Helicobactor, but not with other ba cteria.PCR assay was used to detect Hilicobactor infections in Field Voles,w ith positive 10 samples out of 57 fecal pellets.In conclusion,16S rRNA-based PC R established here is useful for sensitive and specific identification of Heli cobacter in infected animals.