聚合酶链反应检测肺孢子菌的实验研究

摘要

目的 探讨应用改良传统三步聚合酶链反应(PCR)方法检测肺孢子菌DNA的意义.方法 选择Wistar大鼠40只,随机分成实验组和对照组各20只.实验组每周2次皮下注射地塞米松,诱导产生肺孢子菌;对照组注射等剂量的生理盐水.8周后,收集大鼠肺组织和支气管肺泡灌洗液(BALF),分别用三步法与两步法PCR技术检测肺孢子菌DNA,并与Giemsa染色法比较.结果 PCR方法检测肺泡灌洗液中肺孢子菌DNA的方法敏感性(70％)明显高于常规染色法(30％)(P＜0.05),且敏感度93％、特异度100％、阳性预测值100％、阴性预测值83％.实验组中PCR检测肺组织和支气管肺泡灌洗液的肺孢子菌DNA阴性者,Giemsa病原染色法亦为阴性.实验组大鼠的肺组织与支气管肺泡灌洗液分别行传统三步法与二步法PCR检测,2种检测方法阳性率相同,分别为80％和70％,差异无统计学意义(P＞0.05).对照组大鼠各种方法检测均为阴性.结论 两步法PCR可作为早期诊断肺孢子菌肺炎(PCP)的方法,可用于BALF中检测肺孢子菌DNA,其敏感度、特异度、阳性预测值和阴性预测值均很高,易于临床推广应用.%Objective To investigate the significance of the application of improved conventional three-step polymerase chain reaction (PCR) to detect pneumocystis jiroveci (PJ). Methods 40 Wistar rats were selected and randomized into experimental and control groups (n=20 each). The experimental group was subcutaneously injected dexamethasone twice a week for induction of PJ, while the control group was injected the same dose of saline. Bron-chio-alveolar lavage fluid (BALF) of the two groups was collected after 8 weeks and detected DNA of PJ using three-step PCR and two-step PCR, respectively. Thereafter, the PCR was compared with Giemsa stain. Results The sensitivity of PCR (70%) was significantly higher than that of the conventional stain method (30%) in detection of DNA of PJ in BALF (F<0.05), with the sensitivity being 93%, specificity 100%, positive prediction value 100% and negative prediction value 83%. In experimental group, the DNA of PJ in BALF was found negative by PCR as well as Giemsa stain. Furthermore, BALF of the experimental group was detected by conventional three-step and two-step methods, respectively. The positive rates of those two methods were found nearly same (80% vs 70%, respectively) (P＞0.05). For the control group, the results were all found negative by different detective methods. Conclusion The two-step PCR can be used for the early diagnosis of PCP as well as the detection of DNA of PJ, with high sensitivity, specificity, positive prediction value and negative prediction value. Thus, two-step PCR was favorable in clinical promoton and application.