耳霉菌急性注射对大鼠的感染性及致病性
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摘要
目的应用PCR扩增技术检测微生物农药耳霉菌急性注射感染性及致病性,探讨PCR方法在真菌类微生物农药感染性及致病性评价中的应用。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,分别为耳霉菌染毒组(40只)和对照组(10只),雌雄各半。将2.0×107 CFU/ml耳霉菌悬浮剂按0.5 ml/只经腹腔一次性注射,对照组不作处理;连续观察21 d。记录动物中毒症状、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、淋巴结、肝、脾、肺、肾等组织,采集血液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。结果耳霉菌染毒组动物注射耳霉菌后第0、3、7、14、21天,脑、血液、淋巴结、肝、脾、肺、肾、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。结论耳霉菌经腹腔注射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 To explore the application of PCR method in the infectivity and pathogenicity evaluation of microbial pesticides,PCR method is applied to detect infectivity and pathogenicity of microbial pesticide Conidioblous thromboides(C.thromboides) by acute intraperitoneal injection in this research. 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(n=10),half male and half female,and for each rat in the experimental group,0.5 ml 2 ×107 CFU/ml suspending agent of C.thromboides was given through intraperitoneal injection for 21 consecutive days,the control group received no treatment. The weight,poisoning symptom and death were recorded. At the 3 rd,7 th,14 th and 21 st day and after exposure immediately,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 organs of experimental group at the 3 rd,7 th,14 th,21 st day and after exposure immediately showed 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 can not infect the rat's tissues and organs through intraperitoneal injection,which suggests that the test samples have no infectivity and pathogenicity. On the other hand,PCR method can effectively detect the removal of test substance and be used as a detection method for infectivity and pathogenicity of microbial pesticides by acute intraperitoneal injection with good specificity.
Objective To explore the application of PCR method in the infectivity and pathogenicity evaluation of microbial pesticides,PCR method is applied to detect infectivity and pathogenicity of microbial pesticide Conidioblous thromboides(C.thromboides) by acute intraperitoneal injection in this research. 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(n=10),half male and half female,and for each rat in the experimental group,0.5 ml 2 ×107 CFU/ml suspending agent of C.thromboides was given through intraperitoneal injection for 21 consecutive days,the control group received no treatment. The weight,poisoning symptom and death were recorded. At the 3 rd,7 th,14 th and 21 st day and after exposure immediately,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 organs of experimental group at the 3 rd,7 th,14 th,21 st day and after exposure immediately showed 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 can not infect the rat's tissues and organs through intraperitoneal injection,which suggests that the test samples have no infectivity and pathogenicity. On the other hand,PCR method can effectively detect the removal of test substance and be used as a detection method for infectivity and pathogenicity of microbial pesticides by acute intraperitoneal injection with good specificity.
引文
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[7]赵淼,何宁,周殿明,等.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[J].环境与健康杂志,2016,33(6):501-504.
[8]周殿明,赵淼,何宁,等.微生物农药耳霉菌致病性的PCR检测[J].环境与健康杂志,2016,33(9):797-800.
[2]段永兰,侯金丽,邢文会.我国微生物农药的研究与展望[J].安徽农业科学,2010,38(8):4135-4138.
[3]何永梅.真菌杀虫剂-块状耳霉菌[J].农药市场信息,2011(17):41.
[4]Humber RA.Entomophthoromycota:a new phylum and reclassification for entomophthoroid fungi[J].Mycotaxon,2012,120:477-492.
[5]Jahnke KD,Bahnweg G.Activity and structural characteristics of steroid and non-steroid lipid growth factors for Haliphthoros milfordensis,a fungal parasite of marine crustaceans[J].Trans Br Mycol Soc,1984,83:447-454.
[6]黄原.分子系统学---原理、方法及应用[M].北京:中国农业出版社,1998:209-210.
[7]赵淼,何宁,周殿明,等.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[J].环境与健康杂志,2016,33(6):501-504.
[8]周殿明,赵淼,何宁,等.微生物农药耳霉菌致病性的PCR检测[J].环境与健康杂志,2016,33(9):797-800.