血红素加氧酶-1显性负性突变体转基因小鼠模型的建立与鉴定

目的对泰泽病原体nested—PCR检测方法进行优化和应用研究。方法以感染泰泽病原体RJ株的大鼠肝组织作为阳性对照，按国标GB/T 14926．10—2008《实验动物泰泽病原体检测方法》间接免疫荧光检测fIFA）呈阳性的上海地区大鼠肝组织为材料，对本室已建立的泰泽病原体nested—PCR检测方法中的DNA抽提方法和PCR过程进行优化，并应用于22只免疫抑制实验动物的泰泽病原体检测。结果肝脏组织DNA以酚／氯仿抽提法为好；nested—PCR出现196bp的特异性条带，此方法的敏感性达1pg；产物纯化后进行测序比对分析显示，上海地区实验大鼠感染样品以及RJ株感染样品的序列均与Genbank中泰泽病原体16S rRNA（Genbank D14638．1）的同源性为100％。应用研究表明，22个样品中IFA检测阳性样品8个，阳性率为36．4％；Nested-PCR检测阳性样品13个，包含所有的IFA阳性样品，阳性率为59．1％。结论本方法灵敏度高，特异性强，经进一步应用改进后，可用于泰泽病原体的检测。

Establishment of a Transgenic Mouse Model of Heme Oxygenase-1 Dominant Negative Mutation

Objective To optimize nested-PCR of Bacillus piliformis detection and to explore its application. Method The liver from rats infected by Bacillus piliformis RJ was used as the positive control. This sample was positive for Bacillus piliformis detected by indirect immunofluorescence（IFA） according to the GB/T 14926.10-2008 《laboratory animal- Method for examination of Tyzzer＇s organism GB/T14926.10-2008》. Nested-PCR of Bacillus piliformis established previously by our lab was optimized, including DNA extraction and PCR conditions. This optimized nested-PCR was applied to detect the Bacillus piliformis of 22 immunosuppressive rats. Results Phenol/chloroform DNA extraction method of liver was optimal; A specific 196 bp DNA fragment was obtained, the sensitivity of this method reached to 1 pg; Blast results showed that all the sequences of the specific DNA fragment shared 100% homology with the 16 S rRNA of Bacillus piliformis presented in Genbank （D14638.1）. Twenty-two immunosuppressive rats were detected for Bacillus piliformis. The positive rate by IFA is 36.4%（8/22）; The positive rate by nested-PCR is 59.1% （13/22indicating nested-PCR is more sensitive than IFA. Meanwhile all the positive samples detected by IFA were also positive detected by nested PCR. Condusion This optimized nested-PCR method has high sensitivity and specificity and can be applied to Bacillus piliformis detection.