摘要
目的建立石膏样毛癣菌(Tm)、石膏样小孢子菌(Mg)、犬小孢子菌(Mc)和猴类毛癣菌(Am)的多重PCR检测方法,用于实验动物皮肤病原真菌的快速检测。方法根据Genbank公布的四种皮肤病原真菌的18S-28S rRNA序列设计特异性引物,通过引物、d NTP、Taq DNA聚合酶浓度及退火温度和时间的优化,建立四种皮肤病原真菌的多重PCR体系。经特异性和敏感性检验后,对15份人工感染真菌大鼠毛和260份实验动物毛发样本进行检测。结果所建多重PCR方法能够有效区分四种皮肤病原真菌,产生192 bp(Tm)、460 bp(Mg)、290 bp(Mc)和602 bp(As)的目的片段,最低检测限分别为5.9 pg/μL、6.6 pg/μL、9.5 pg/μL和5.1 pg/μL。被检的15份人工感染样本扩增结果准确,260份毛发样本中未检测出四种真菌。结论所建立的多重PCR方法能够同时对实验动物中的四种皮肤病原真菌进行快速的检测,具有较高的应用价值。
Objective To develop a multiplex polymerase chain reaction（ m PCR） assay for detection of four pathogenic dermatophytes [Trichophyton mentagrophytes（ Tm）,Microsporum gypseum（ Mg）,Microsporum canis（ Mc）,and Arthroderma simii（ As） ] in laboratory animals,which could be used rapidly and simultaneously for direct detection of those four pathogens. Methods We designed 5 specific primers according to 18S-28 S rRNA sequences of the four pathogenic dermatophytes reported in Genbank. The four m PCR assays were established through optimizing the concentration of primers,d NTP,Taq DNA polymerase and the annealing temperature. After verifying the specificity and sensibility,this method was used to detect 15 hair samples with artificial infection and 260 samples taken from laboratory animals. Results This m PCR technique can distinguish the four dermatophytes by producing 192 bp（ Tm）,460 bp（ Mg）,290 bp（ Mc） and 602 bp（ As） fragments. The sensibility for detection of the four dermatophytes was 5. 9 pg / μL,6. 6 pg /μL,9. 5 pg /μL and 5. 1 pg /μL,respectively. The results of 15 artificial infection samples were accurate,and the results of 260 hairs samples were negative for the four fungi. Conclusions Our results suggest that the m PCR assay developed inthis study can efficiently detect the four dermatophytes,is a useful and rapid technique for rapid detection of the pathogenic dermatophytes in laboratory animals.
出处
《中国比较医学杂志》
CAS
北大核心
2015年第12期65-70,共6页
Chinese Journal of Comparative Medicine
基金
国家科技支撑计划项目:实验动物新品种的种群建立与质量标准化研究(编号:2011BAI15B01)
