PCR方法检测实验动物皮肤病原真菌

Testing Lab Animal Dermatophytes Using PCR Method

目的对用于实验动物皮肤病原真菌检测的分子生物学方法即皮肤病原真菌通用引物PCR与AP-PCR(任意引物聚合酶链式反应)相结合技术的特异性及敏感性进行评价,并通过建立动物模型及实际的皮肤真菌检测来验证此方法的可行性。方法特异性测定:用皮肤病原真菌通用引物进行PCR,扩增大肠杆菌DNA、犬肝炎病毒DNA及小鼠肝癌H22细胞DNA。敏感性测定:以紫外分光光度法测定DNA质量浓度,根据测定值将DNA进行10倍系列质量浓度稀释,得到100 ng^10 fg的8个质量浓度,然后对其进行PCR扩增。建立实验动物皮肤病原真菌感染的动物模型。用皮肤病原真菌通用引物PCR与AP-PCR相结合技术检测从动物模型及河北省各地的实验动物单位采集的动物皮肤标本。结果用皮肤病原真菌通用引物扩增实验动物三种皮肤病原真菌标准菌株,均扩增出大小为440 bp的条带,而大肠杆菌、犬肝炎病毒、小鼠肝癌H22细胞均未扩增出条带。皮肤病原真菌通用引物PCR敏感性测定,模板DNA 100 ng^100 fg的7个系列浓度均扩增出了阳性条带,10 fg为阴性。建立了实验动物皮肤病原真菌感染的动物模型。对河北省各地的实验动物皮肤真菌检测过程中出现一例通用引物阳性标本。结论皮肤病原真菌通用引物(CHS1 1S,CHS1 1R)具有较强的特异性,敏感度为100 fg;将皮肤病原真菌通用引物PCR与AP-PCR技术相结合可用于检测实验动物皮肤病原真菌。

Objective To evaluate the sensitivity and specificity of the molecular biology method which combining the universal primer PCR and arbitrary primer PCR for the detection of pathogenic dermatophytes in laboratory animals, and validate the feasibility of the method through building animal models of pathogenic dermatophytes in laboratory animals and practical detection of dermatophytes. Methods The determination of the specificity： E. coli DNA, Canine hepatitis virus DNA and mouse hepatoma carcinoma H22 cell DNA was amplified by universal primers, The determination of the sensitivity： DNA weight concentration was determined by UV-vis spectroscopy and it was further diluted by series of ten times weight concentration based upon the former measurement. The resulting eight weight concentrations ranging from 100 ng ~ 10 fg were used for further PCR amplification. Then build the animal models and identify some skin samples of laboratory animals from Hebei province. Results Amplification of the three standard strains DNA with universal primers yielded the same band （about 440 bp）. While E. coli, Canine hepatitis virus, mouse hepatoma carcinoma H22 cell were not amplificated with strains. Sensitivity determination of universal primer PCR： Seven samples ranging from 100 ng to 100 fg shown positive, except the 10 fg sample yielding negative band. We founded the animal models of pathogenic dermatophytes in laboratory animals. For one of the skin samples of laboratory animals from Hebei province, the universal primer PCR amplification yielded a 440 bp band. Conclusions The universal primer （CHS1 IS, CHS1 1R）is regarded as a specificity primer. The result showed that the sensitivity of universal primer PCR is 100 fg. Combining universal primer PCR and AP-PCR could differentiate the three familiar dermatophytes in laboratory animals.