PCR技术检测RH株弓形虫组织内感染的实验研究  

Experimental study on PCR technique for detecting the infection of Toxoplasma gondii

作　　者：单连玉[1] 杨秀珍[1] 刘佩梅[1] 

机构地区：[1]天津医科大学人体寄生虫学教研室,天津300070

出　　处：《天津医科大学学报》2002年第4期416-419,共4页Journal of Tianjin Medical University

基　　金：天津市自然科学基金资助 (编号 :973609311)

摘　　要：目的 :建立敏感、特异的聚合酶链反应 (PCR) ,以检测组织内弓形虫感染。方法 :优化PCR反应关键步骤的条件 ,以相同的B1引物扩增相近的病原体判断其特异性 ,扩增倍比稀释的DNA检测其灵敏度 ,并以PCR法检测实验鼠肝、血中DNA的动态变化 ,结果与免疫组化法进行比较 ,评价其检测生物学样本的可行性。结果 :实验建立的PCR技术的灵敏度能达到检测出一个虫体的DNA ,且特异性强 ,不扩增鼠DNA、人DNA、隐孢子虫、卡氏肺孢子虫、鼠疟原虫的DNA。检测实验感染小鼠血、肝脏样本 ,在感染后2天 ,3只鼠血样本PCR结果阳性 ,3只小鼠肝标本2只阳性 ;感染后3天至濒死前所有小鼠检测结果均阳性 ;血样本阳性检出率100 % (11/11) ,肝脏组织样本阳性检出率81.8 % (9/11)。结论 :PCR方法是一种敏感、特异、快速的诊断方法 。Objective:To establish a sensitive and specific PCR technique for detecting DNA of T. gondii in experimentall infected mice.Methods:RH strain of T. gondii was pured from intraperitoneally infected mice. DNA of T. gondii was extracted and used for PCR test. Key steps of PCR test were opitimized and the target was B1 gene. In order to determine specificity of B1-PCR, amplification using the same primer pairs was performed with DNA from Cryptosporidium, Pneumocystis carinii and Plasmodium berghei. B1-PCR was also used to detect the DNA from blood and liver tissue of experimentall infected mice with T. gondii.Results:The optimized annealing temperature of B1-PCR was 55 ℃ and the concentration of Mg2+ was 1.5 mmol/L or 2.0 mmol/L. DNA in a single organism can be amplified and detected with the PCR test. No amplification was seen with DNA from Cryptosporidium,Pneumocystis carinii and Plasmodium berghei. DNA of T. gondii from whole blood and liver tissues of infected mice could be detected as early as day 2 post infection. The total positive rate reached to the highest value 100%(11/11) when detecting blood samples and 81.8%(9/11) when detecting liver samples.Conclusion:PCR technique is a sensitive, specific, rapid diagnostic method.

关 键 词：PCR技术 实验研究 弓形虫 聚合酶链反应 弓形虫感染 

分 类 号：R531.8[医药卫生—内科学] R446.[医药卫生—临床医学]