一步法PCR检测大鼠肺支原体核酸的研究

一步法体外扩增结合Ｓｏｕｔｈｅｒｎ杂交检测Ｍ５３鼠肺支原体标准株，设计一对特异寡核苷酸引物及探针，合成、纯化、建立了特异、敏感、快速的检测手段。扩增产物经琼脂糖凝胶电泳鉴定，结果显示鼠肺支原体Ｍ５３株基因组ＤＮＡ７１０ｂｐ特异谱带。对５０只ＳＤ大鼠进行检测，结果ＰＣＲ方法检出率高于分离培养法，扩增产物行Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交验证，采用碱性磷酸酶标记寡核苷酸探针，可与膜上特异靶ＤＮＡ序列杂交，而阴性对照无杂交信号。特异性实验检出１０ｐｇ的ＤＮＡ。充分说明一步法ＰＣＲ，具有高度、特异、灵敏、快速等优势，适应与大、小鼠监测中应用。

Studies on Detecting Nuclectide of Mycoplasma Pulmonis One-Step Methods by Polymerase Chain Reaction

M53 stardand strains were detected using one-step method amplipifiction and southern boltting a pair of oligonuoleotide primers and probe, were designed, synthesized and purified a specific, sensitiv and rapid detected meothod on agarose gel eletrophoresis products showed the 710bp specific band of M53 genomice DNAs. of 50 subjects were detected from SD rats, the results suggest that test-rate of PCR was higher than that of isolates cultivate method. The amplified products were confirmed by southern boltting, using alkaline phophatase (AP) labeled oligonuoeleotide probe and the specificity DNAs sequence by southern blotting onto Nylon Hybond-N membranes, and the negtive cotrans were no signals. By PCR specific detection, as little as 10pg of Mycoplasma Pulmonis DNAs yielded positive results. These results indicated that one-step PCR assay was rapid, specific, sensitive, and suitable for the detection of rats and mice.