微生物农药耳霉菌急性经呼吸道感染性及致病性的PCR检测法

PCR method for detection of acute pulmonary toxicity and pathogenicity of microbial pesticide-conidioblous thromboides

目的应用PCR法检测微生物农药耳霉菌经呼吸道急性染毒对大鼠的感染性及致病性,探讨PCR法在真菌类微生物农药感染性及致病性研究中的应用。方法将50只健康7-9周龄SPF级Wistar大鼠按体重随机分为2组,分别为实验组（40只）和对照组（10只）,雌雄各半。将2.0×108个/ml耳霉菌悬浮剂按0.5 ml/只经气管一次性注入,对照组不作处理;连续观察21 d。记录动物的体重、中毒症状、死亡情况,并对死亡动物进行大体解剖。于染毒后第0、3、7、14、21天剖解动物的脑、血液、淋巴结、肝、脾、肺、肾等组织及盲肠内容物提取基因组DNA,利用耳霉菌nuc-LSU基因特异性引物进行PCR检测。结果实验组动物染毒耳霉菌后立即取样肺组织中耳霉菌的PCR扩增结果为阳性,肝、脾、肾、脑、淋巴结、血液、盲肠内容物等组织中均未检出耳霉菌;染毒后第3、7、14、21天,肝、脾、肺、肾、脑、淋巴结、血液、盲肠内容物等组织中均未检出耳霉菌。实验动物染毒后,未见明显中毒症状,观察期内无大鼠死亡,大体检查及病理检查均未见明显异常。结论耳霉菌经呼吸道急性染毒后对大鼠无感染性及致病性。本检测方法可快速、有效地检测出耳霉菌的侵染及清除情况,且具有良好的特异性、可重复性,可做为微生物农药感染性、致病性的检测方法。

Objective PCR method was applied to detect the acute pulmonary toxicity and pathogenicity of conidioblous thromboides（C. thromboides） in this research,to explore the application of PCR method in the pathogenicity of microbial pesticides. Methods A total of 50 healthy SPF Wistar rats（7-9 weeks old） were randomly divided to two groups, the experimental group（n=40） and control group（10 rats）, half male and half female. The 2×108units/ml suspending agent of C.thromboides was given at 0.5 ml per rat through intratracheal instillation and control group without any treatment, with continuous observation of 21 d. The weight, poisoning symptom and death were recorded. At the 3rd,7th,14 th and 21 st day after treatment,the brain,blood,lymph nodes,liver,spleen,lung,kidney and cecum contents were collected,genome DNA of these tissue samples were extracted,then amplified by PCR reaction with nuc-LSU gene specific primers of C. thromboides.Results The PCR amplification products of lung tissue in experimental group appeared the positive stripe in agarose gel,the remaining tissues and organs were negative. The organs of experimental group at the 3rd,7th,14 th and 21 st day after exposure showed up the negative results in PCR reaction. No dead and obvious poisoning symptoms in experimental group were observed after exposure, no pathological changes were seen. Conclusion C.thromboides may not infect the animal＇s tissues and organs through respiratory tract infected, which suggests that the test samples have no pathogenicity to rats. The PCR method can effectively detect the removal of test substance and can be used as a detection method with good specificity for acute pulmonary toxicity and pathogenicity of microbial pesticides.