犬小孢子菌、石膏样小孢子菌、须毛癣菌多重PCR检测方法的建立及初步应用
|
摘要
宠物浅表皮肤真菌病主要由犬小孢子菌、石膏样小孢子菌、须毛癣菌感染引起。本研究根据3种真菌基因内转录间隔区(internal transcribed spacer,ITS)区域的序列差异设计4条引物,通过优化多重PCR反应条件,建立了一种可同时快速检测这3种真菌的多重PCR,同时对其特异性、灵敏性、重复性进行分析。结果显示,建立的多重PCR方法对犬小孢子菌、石膏样小孢子菌、须毛癣菌基因组DNA均可扩增出特异性预期片段,条带大小分别为286、596、188 bp;最低检测限(灵敏性)分别为12.6、8.8、13.6pg/μL;被检测的6份人工模拟样品扩增结果准确。应用该多重PCR方法检测27份人临床皮屑样品和43份宠物犬、猫毛发样品,结果显示阳性检出率分别为74.07%(20/27)和83.72%(36/43);采用常规真菌分离培养法从阳性样品中获得10株致病性真菌,分离率为17.86%(10/56);两种检测方法的种属鉴定结果表明多重PCR法优于常规方法。本研究建立的多重PCR检测方法简便、特异、灵敏,可同时对宠物浅表真菌病原作出快速诊断与鉴别。
Superficial skin fungal disease in pet animals is mainly caused by Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. The aim of this study was to develop a multiplex PCR method for rapid detection of these three fungal pathogens. Four primers were designed in accordance with the differences of fungal ITS region sequences and the conditions of this multiplex PCR were optimized for its speci?c, sensitive and reproducible performance. The results showed that the multiplex PCR method ampli?ed the speci?c expected fragments of 286 bp, 596 bp and 188 bp for Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. The minimum detection limits were 12.6 pg/μL for Microsporum canis, 8.8 pg/μL for Microsporum gypseum and13.6 pg/μL for Trichophyton mentagrophytes, respectively. Six spiked samples were tested and results were accurate. Subsequently,clinical samples were collected and tested with the multiplex PCR method. The positive rates were 74.07%(20/27) for human samples and 83.72%(36/43) for pet samples. In addition, 10 pathogenic fungus isolates were obtained from the above positive samples by conventional isolation method. The results showed that the multiplex PCR method was simple, speci?c and sensitive and might be used for rapid diagnosis and identi?cation of pet super?cial fungal pathogens.
Superficial skin fungal disease in pet animals is mainly caused by Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. The aim of this study was to develop a multiplex PCR method for rapid detection of these three fungal pathogens. Four primers were designed in accordance with the differences of fungal ITS region sequences and the conditions of this multiplex PCR were optimized for its speci?c, sensitive and reproducible performance. The results showed that the multiplex PCR method ampli?ed the speci?c expected fragments of 286 bp, 596 bp and 188 bp for Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. The minimum detection limits were 12.6 pg/μL for Microsporum canis, 8.8 pg/μL for Microsporum gypseum and13.6 pg/μL for Trichophyton mentagrophytes, respectively. Six spiked samples were tested and results were accurate. Subsequently,clinical samples were collected and tested with the multiplex PCR method. The positive rates were 74.07%(20/27) for human samples and 83.72%(36/43) for pet samples. In addition, 10 pathogenic fungus isolates were obtained from the above positive samples by conventional isolation method. The results showed that the multiplex PCR method was simple, speci?c and sensitive and might be used for rapid diagnosis and identi?cation of pet super?cial fungal pathogens.
引文
[1]张淼洁,王立林,曲萍,等.我国人与犬、猫共患病防控存在的主要问题与对策建议[J].中国比较医学杂志,2012,22(4):82-86.
[2]刘伟,金艺鹏,林德贵.犬猫皮肤真菌病的诊断及药物治疗[J].中国兽医杂志,2006,42(4):37-38.
[3]刘国芳.犬猫皮肤真菌病流行病学及其病原菌分子生物鉴定[D].南京:南京农业大学,2010.
[4]Mathy A,Baldo A,Schoofs L,et al.Fungalysin and dipeptidyl-peptidase gene transcription in Microsporum canis strains isolated from symptomatic and asymptomatic cats[J].Vet Microbiol,2010,146(1-2):179-182.
[5]Krakhecke A G,Afonso E,Ferreira J C,et al.In vitro susceptibility testing of Microsporum gypseum isolated from healthy cattle and soil samples against itraconazole,terbinafine,fluconazole and topical veterinarian drugs[J].Mycopathologia,2005,159(3):377-380.
[6]夏咸柱,才学鹏,林德贵,等.宠物源人兽共患病防控战略研究[J].中国动物检疫,2017,34(2):34-37,41.
[7]周莎桑,戴方伟,宋晓明,等.实验动物皮肤病原真菌检测方法的改良[J].中国比较医学杂志,2010,20(6):58-60,84.
[8]Shafiee S,Khosravi A R,Ashrafi Tamai I.Comparative study of Microsporum canis isolates by DNAfingerprinting[J].Mycoses,2014,57(8):507-512.
[9]Kawasaki M,Aoki M,Ishizaki H.Phylogenetic relationships of some Microsporum and Arthroderma species inferred from mitochondrial DNA analysis[J].Mycopathologia,1995,130(1):11-21.
[10]Leibner-Ciszak J,Dobrowolska A,Krawczyk B,et al.Evaluation of a PCR melting profile method for intraspecies differentiation of Trichophyton rubrum and Trichophyton interdigitale[J].J Med Microbiol,2010,59(Pt2):185-192.
[11]Kanbe T,Suzuki Y,Kamiya A,et al.Speciesidentification of dermatophytes Trichophyton,Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes[J].JDermatol Sci,2003,33(1):41-54.
[12]Liu D,Coloe S,Baird R,et al.PCR identification of Trichophyton mentagrophytes var.interdigitale and T.mentagrophytes var.mentagrophytes dermatophytes with a random primer[J].J Med Microbiol,1997,46(12):1043-1046.
[13]De Hoog G S,Bowman B,Graser Y,et al.Molecular phylogeny and taxonomy of medically important fungi[J].Med Mycol,1998,36 Suppl 1:52-56.
[14]刘彦威,柳焕章,刘建钗,等.河北兔场皮肤病原真菌rDNA-ITS序列分析[J].中国兽医学报,2013,33(9):1383-1386.
[15]中国实验动物学会.实验动物微生物学检测方法(2):实验动物皮肤病原真菌检测方法:GB/T14926.4-2001[S].北京:中华人民共和国国家质量监督检验检疫总局,2002.
[16]喻芙蓉,王丽萍.畜禽皮肤病的诊断和防治[J].兽医导刊,2015(20):154.
[17]王萍,禹泽中.宠物真菌性皮肤病临床研究[J].中国动物传染病学报,2008,16(3):53-55.
[18]杨虹,高志琴,陈健,等.犬小孢子菌致皮肤癣菌病162例回顾分析[J].中国真菌学杂志,2013,8(4):217-219.
[19]黄英河,匡玉宝,赵伟峰,等.79例家庭宠物致浅部真菌皮肤病的病原学分析[J].医学检验与临床,2016,27(4):40-42.
[20]付相钰,邹学敏.头癣病与家养宠物的相关性及健康教育效果分析[J].护理实践与研究,2008,5(3):4-5.
[21]王再兴,王绥,徐钰.伍德灯和共聚焦激光扫描显微镜在部分皮肤浅部真菌感染诊断中的应用价值评估[J].中国皮肤性病学杂志,2015(7):752-755.
[22]王有为,罗才会,李红霞,等.47766例拟诊皮肤真菌病患者病原菌分离培养结果分析[J].中国真菌学杂志,2016,11(1):37-40.
[2]刘伟,金艺鹏,林德贵.犬猫皮肤真菌病的诊断及药物治疗[J].中国兽医杂志,2006,42(4):37-38.
[3]刘国芳.犬猫皮肤真菌病流行病学及其病原菌分子生物鉴定[D].南京:南京农业大学,2010.
[4]Mathy A,Baldo A,Schoofs L,et al.Fungalysin and dipeptidyl-peptidase gene transcription in Microsporum canis strains isolated from symptomatic and asymptomatic cats[J].Vet Microbiol,2010,146(1-2):179-182.
[5]Krakhecke A G,Afonso E,Ferreira J C,et al.In vitro susceptibility testing of Microsporum gypseum isolated from healthy cattle and soil samples against itraconazole,terbinafine,fluconazole and topical veterinarian drugs[J].Mycopathologia,2005,159(3):377-380.
[6]夏咸柱,才学鹏,林德贵,等.宠物源人兽共患病防控战略研究[J].中国动物检疫,2017,34(2):34-37,41.
[7]周莎桑,戴方伟,宋晓明,等.实验动物皮肤病原真菌检测方法的改良[J].中国比较医学杂志,2010,20(6):58-60,84.
[8]Shafiee S,Khosravi A R,Ashrafi Tamai I.Comparative study of Microsporum canis isolates by DNAfingerprinting[J].Mycoses,2014,57(8):507-512.
[9]Kawasaki M,Aoki M,Ishizaki H.Phylogenetic relationships of some Microsporum and Arthroderma species inferred from mitochondrial DNA analysis[J].Mycopathologia,1995,130(1):11-21.
[10]Leibner-Ciszak J,Dobrowolska A,Krawczyk B,et al.Evaluation of a PCR melting profile method for intraspecies differentiation of Trichophyton rubrum and Trichophyton interdigitale[J].J Med Microbiol,2010,59(Pt2):185-192.
[11]Kanbe T,Suzuki Y,Kamiya A,et al.Speciesidentification of dermatophytes Trichophyton,Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes[J].JDermatol Sci,2003,33(1):41-54.
[12]Liu D,Coloe S,Baird R,et al.PCR identification of Trichophyton mentagrophytes var.interdigitale and T.mentagrophytes var.mentagrophytes dermatophytes with a random primer[J].J Med Microbiol,1997,46(12):1043-1046.
[13]De Hoog G S,Bowman B,Graser Y,et al.Molecular phylogeny and taxonomy of medically important fungi[J].Med Mycol,1998,36 Suppl 1:52-56.
[14]刘彦威,柳焕章,刘建钗,等.河北兔场皮肤病原真菌rDNA-ITS序列分析[J].中国兽医学报,2013,33(9):1383-1386.
[15]中国实验动物学会.实验动物微生物学检测方法(2):实验动物皮肤病原真菌检测方法:GB/T14926.4-2001[S].北京:中华人民共和国国家质量监督检验检疫总局,2002.
[16]喻芙蓉,王丽萍.畜禽皮肤病的诊断和防治[J].兽医导刊,2015(20):154.
[17]王萍,禹泽中.宠物真菌性皮肤病临床研究[J].中国动物传染病学报,2008,16(3):53-55.
[18]杨虹,高志琴,陈健,等.犬小孢子菌致皮肤癣菌病162例回顾分析[J].中国真菌学杂志,2013,8(4):217-219.
[19]黄英河,匡玉宝,赵伟峰,等.79例家庭宠物致浅部真菌皮肤病的病原学分析[J].医学检验与临床,2016,27(4):40-42.
[20]付相钰,邹学敏.头癣病与家养宠物的相关性及健康教育效果分析[J].护理实践与研究,2008,5(3):4-5.
[21]王再兴,王绥,徐钰.伍德灯和共聚焦激光扫描显微镜在部分皮肤浅部真菌感染诊断中的应用价值评估[J].中国皮肤性病学杂志,2015(7):752-755.
[22]王有为,罗才会,李红霞,等.47766例拟诊皮肤真菌病患者病原菌分离培养结果分析[J].中国真菌学杂志,2016,11(1):37-40.