卡氏肺孢子虫PCR方法敏感性研究

目的从3种PCR方法中选择出一种检测卡氏肺孢子虫(Pc)敏感性最高的PCR方法,供以后的实验中应用。方法Wistar大鼠30只,皮下注射地塞米松,每周二次,制备PCP动物模型,8周后全部剖杀作大鼠肺印片,六亚甲基四胺银染色。各鼠取肺组织02g,均浆后提取DNA,用三种PCR方法作PCR试验。选取六亚甲基四胺银染色和三种PCR检测均为阳性的大鼠DNA标本20份,对每份标本作对倍稀释1/2、1/4、1/8、1/16……至1/2048,同时用三种PCR方法做扩增试验直到某一稀释度时PCR试验阴性为止,选择出最为敏感的一组PCR方法,并做统计学分析。结果三种方法最低稀释度的均数DHFR-PCT为1/7879±174;TS-PCR为1/4371±207;MT-PCR为1/10975±136。结论三种检测Pc的PCR方法中以MT-PCR方法最为敏感。

Selection of the most sensitive method of PCR for the detection of Pneumocystis carinii

To choice the most sensitive method of PCR to detect the presence of Pneumocystis carinii, the PCR animal model was developed by biweekly subcutaneous injections of dexamethasone into Wistar rats for 8 weeks, and the animals were killed to obtain the lung specimen. The lung specimens were smeared and stained with Gomori's methenamine silver nitrate technic (GMS). 0.2 g of the lung tissue suspension from each rat was used to extract DNA, and three PCR methods were used for amplification of nucleic acid.20 specimens were chosen from the rats with which both the GMS and PCR were positive. Each specimen was serially diluted ( from 1∶2, 1∶4, 1∶8……to1∶2 048) and subjected to PCR amplification with 3 methods of PCR till the point of most tenuous concentration showed negative result. It was found that the average values of the lowest dilution undertaken in these 3 PCR methods were 1∶78.79± 1.74 for DHFR PCR;1∶43.71±2.07 for TS PCR and 1∶1097.5 ±1.36 for MT PCR. It concludes that the MT PCR seems the most sensitive one among these 3 PCR methods.