实验动物泰泽病原位杂交法的建立

Establishment of Molecular Pathological Method for Detection of Tyzzer Disease in Laboratory Animal

目的 建立实验动物泰泽病原位杂交检测方法。方法 用已感染Tyzzer菌小鼠的 3T3细胞 (MT -3T3细胞 )感染 4周龄的ICR小鼠 ,以 4 .0× 10 5细胞量感染动物 ,动物于感染后 4～ 7d发病。根据Tyzzer菌 16srDNA序列设计出两对特异引物 ,以MT - 3T3细胞的DNA提取物为模板 ,进行套式PCR。扩增出Tyzzer菌特有的 196bp条带。以此产物为探针 ,用地高辛PCR一步法进行探针标记。选择感染动物的肝脏 ,采用不同探针浓度进行原位杂交。结果 当探针的最小浓度为 4 0ng μl时 ,在肝细胞的胞质中有阳性结果。 结论 原位杂交方法能直接定位Tyzzer菌的感染位置 ,本研究建立的方法简单 ,易于操作 ,便于普及。

Objective To establish a molecular pathological( in situ hybridization) method for detection of Tyzzer disease in laboratory animals. Methods Four week old ICR mice were infected by injection of 4.0×10 5 MT 3T3 cells through caudal vein. The disease onset occured on the 4th 7th day. Nested PCR was performed with the DNA extract of MT 3T3 cells as the template and two pairs of oligonucleotides designed according to 16s rDNA of bacillus piliformis as primers.The specific 196 bp DNAs were obtained after amplification with internal primers. The 196 bp DNAs were labeled with Digoxin PCR. in situ hybridization was performed with probes of different concentrations on the liver sections of affected animals. Results\ Positive findings appeared in the cytoplasm of hepatocytes at the minimum probe concentration of 40 ng/μl. Conclusion\ The method can localize Tyzzer bacteria directly. It is easy to be popularized and therefore ideal for the early diagnosis.