犬和猫皮肤癣菌三重荧光定量PCR检测方法的建立及应用  

Establishment and Application of Triplex Fluorescent PCR for Detection of Dermatophytes in Dogs and Cats

作　　者：徐博 郭思宇 纪佳豪 李梓豪 刘文扬 勾永志 白二钢 李睿文[1] XU Bo;GUO Si-yu;JI Jia-hao;LI Zi-hao;LIU Wen-yang;GOU Yong-zhi;BAI Er-gang;LI Rui-wen(College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei,071000,Chian;Baoding Animal Disease Prevention and Control Center,Baoding,Hebei,071000,Chian;Hebei Chuang'an Biotechnology Co.Ltd,Baoding,Hebei,071000,Chian)

机构地区：[1]河北农业大学动物医学院,河北保定071000 [2]保定市动物疾病预防控制中心,河北保定071000 [3]河北创安生物科技有限公司,河北保定071000

出　　处：《动物医学进展》2024年第9期56-60,共5页Progress In Veterinary Medicine

基　　金：河北省高等学校科学技术研究项目(ZC2023056)。

摘　　要：建立鉴别犬小孢子菌(Microsporum canis,MC)、须癣毛癣菌(Trichophyton mentagrophytes,TM)和秕糠马拉色菌(Malassezia furfur,MF)的三重荧光PCR检测方法,根据MC、TM和MF 3种真菌的ITS基因保守序列设计引物和探针,建立三重荧光PCR检测方法。所建立的三重荧光PCR方法仅对MC、TM和MF出现阳性扩增,与大肠埃希氏菌、产色葡萄球菌和铜绿假单胞菌等犬、猫皮肤病常见病原无交叉反应,对MC、TM和MF的最低检出限分别为1.36×10^(1)、1.35×10^(1)、1.33×10^(1)拷贝;组内和组间变异系数均小于2%,重复性好。应用建立三重荧光PCR方法对88份犬、猫临床样品进行检测,结果检出1份MC阳性、21份TM阳性、20份MF阳性,检测结果与单一荧光PCR方法一致。该方法可对MC、TM和MF同时快速检测,可用于犬、猫真菌性皮肤病的快速诊断。A triplex fluorescent PCR method was established to identify Microsporum canis(MC),Trichophyton mentagrophytes(TM)and Mala ssezia furfur(MF),which was based on the conserved sequences of the ITS genes of the three common dermatopathogenic fungi,namely MC,TM and MF.The primers and probes were designed based on the conserved sequences of the ITS genes of three common dermatopathogenic fungi,MC,TM and MF,and the triplex fluorescence PCR assay was established.The established triplex fluorescence PCR method showed positive amplification only for MC,TM and MF.There was no crossreactivity with Escherichia coli,Staphylococcus chromogenes and Pseudomonas aeruginosa,which are the common pathogens of canine and feline dermatosis,and the lowest detection limits of MC,TM and MF were 1.36×10^(1),1.35×10^(1) and 1.33×10^(1) copies,respectively.The coefficients of variation of intra-and inter-groups were both less than 2%,and the reproducibility was good.The established triplex fluorescent PCR method for MC,TM and MF was applied to 88 clinical samples of dogs and cats,and the results showed that 1 MC,21 TM and 20 MF were detected positively,which was consistent with the single fluorescent PCR method.This method can rapidly detect MC,TM and MF at the same time,and can be used for the rapid diagnosis of fungal dermatosis in dogs and cats.

关 键 词：犬和猫皮肤病 犬小孢子菌 须癣毛癣菌 秕糠马拉色菌 三重荧光PCR 

分 类 号：S853.5[农业科学—临床兽医学]