家兔须癣毛癣菌SYBR GreenⅠ荧光定量PCR检测方法的建立及初步应用
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摘要
为建立家兔须癣毛癣菌快速检测方法,根据GenBank上已发表的须癣毛癣菌rDNA内转录间隔区核苷酸序列设计了1对特异性引物,建立了检测须癣毛癣菌SYBR GreenⅠ实时荧光定量PCR方法,并用该方法对临床分离的须癣毛癣菌和病料进行检测。结果显示,该方法灵敏度高,对须癣毛癣菌的最低检出量为10copies/μL;特异性强,与犬小孢子菌、絮状表皮癣、红毛癣菌、白色念珠菌、烟曲霉等病原真菌没有交叉反应。该方法操作简单,耗时短,只需2h即可完成整个试验过程。提示该方法可用于须癣毛癣菌的检测。
According to the ITS regions of rDNA of Trichophyton mentagrophytes in GenBank,apair of special primers was designed.Then SYBR Green Ⅰ real-time PCR for detecting T.mentagrophytes in rabbit was established,the fragment of amplification should be 140 bp in length.The developed method was used to detect T.mentagrophytes strains and clinic samples.The result showed that SYBR Green ⅠPCR had many advantages such as rapidness,simple operation,high sensitivity and specificity,so it could be applied to clinic detection of T.mentagrophytes in rabbit.
According to the ITS regions of rDNA of Trichophyton mentagrophytes in GenBank,apair of special primers was designed.Then SYBR Green Ⅰ real-time PCR for detecting T.mentagrophytes in rabbit was established,the fragment of amplification should be 140 bp in length.The developed method was used to detect T.mentagrophytes strains and clinic samples.The result showed that SYBR Green ⅠPCR had many advantages such as rapidness,simple operation,high sensitivity and specificity,so it could be applied to clinic detection of T.mentagrophytes in rabbit.
引文
[1]刘彦威,刘娜,张永英,等.家兔须癣毛癣菌感染快速诊断方法的建立[J].中国兽医科学,2013,43(11):1184-1189.LIU Yan-wei,LIU Na,ZHANG Yong-ying,et al.Establishment of a method for rapid diagnosis of Trichophyton mentagrophytes infection in rabbits[J].Chinese Veterinary Science,2013,43(11):1184-1189.(in Chinese)
[2]吴长龙,魏琴,殷中琼,等.四种中药乙醇提取物对常见皮肤癣菌的体外抗菌活性[J].中国兽医科学,2010,40(11):1189-1193.WU Chang-long,WEI Qin,YIN Zhong-qiong,et al.Antifungal activity in vitro of four alcoholic extracts from Chinese herbs against common dermatophytes[J].Chinese Veterinary Science,2010,40(11):1189-1193.(in Chinese)
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[4]BERGMANS A M C,SCHOULS L M,VANDER ENT M,et al.Validation of PCR-reverse line blot,a method for rapid detection and identification of nine dermatophyte species in nail,skin and hair samples[J].Clin Microbiol Infect,2008,14(8):778-788.
[5]ALEXANDER C L,SHANKLAND G S,CARMAN W,et al.Introduction of a dermatophyte polymerase chain reaction assay to the diagnostic mycology service in Scotland[J].Br J Dermatol,2011,164(5):966-972.
[6]CAFARCHIA C,WEIGL S,FIGUEREDO L A,et al.Molecular identification and phylogenesis of dermatophytes isolated from rabbit farms and rabbit farm workers[J].Vet Microbiol,2012,154(3-4):395-402.
[7]KRAEMER A,MUELLER R S,WERCKENTHIN C,et al.Dermatophytes in pet guinea pigs and rabbits[J].Vet Microbiol,2012,157(1-2):208-213.
[8]CHEN B R,SUN Y,HU X P,et al.Morphology and molecular biological identification of Trichophyton mentagrophytes[J].Chin J Mycol,2010,5(6):321-326.
[9]CAFARCHIA C,CAMARDA A,COCCIOLI C,et al.Epidemiology and risk factors for dermatophytoses in rabbit farms[J].Med Mycol,2010,48(7):975-980.
[10]MEHRABAN F,LAME A,ABDOLAZIZ R L,et al.Epidemiology of dermatophytoses in an area south of Tehran,Iran[J].Mycopathologia,2003,156(4):279-287.
[11]BERGMAN A,HEIMER D,KONDORI N.Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR[J].Clin Microbiol Infect,2013,19(4):205-211.
[12]GARG J,TILAK R,GARG A,et al.Rapid detection of dermatophytes from skin and hair[J].BMC Res Notes,2009,60(2):1-6.
[13]FRALLE E,RODRIGUE M,GANTOIS N,et al.Phylogenetic analysis of Trichophyton mentagrophytes human and animal isolates based on MnSOD and ITS sequence comparison[J].Microbiology,2007,153(10):3466-3477.
[14]BEIFUSS B,BEZOLD G,GOTTLBER P,et al.Direct detection of five common dermatophyte species in clinical samples using a rapid and sensitive 24-h PCR-ELISA technique open to protocol transfer[J].Mycoses,2011,54(2):137-145.
[2]吴长龙,魏琴,殷中琼,等.四种中药乙醇提取物对常见皮肤癣菌的体外抗菌活性[J].中国兽医科学,2010,40(11):1189-1193.WU Chang-long,WEI Qin,YIN Zhong-qiong,et al.Antifungal activity in vitro of four alcoholic extracts from Chinese herbs against common dermatophytes[J].Chinese Veterinary Science,2010,40(11):1189-1193.(in Chinese)
[3]ANNA B D,DITTE M S,MAIKEN C A.Five-hour diagnosis of dermatophyte nail infections with specific detection of Trichophyton rubrum[J].J Clin Microbiol,2007,45(4):1200-1204.
[4]BERGMANS A M C,SCHOULS L M,VANDER ENT M,et al.Validation of PCR-reverse line blot,a method for rapid detection and identification of nine dermatophyte species in nail,skin and hair samples[J].Clin Microbiol Infect,2008,14(8):778-788.
[5]ALEXANDER C L,SHANKLAND G S,CARMAN W,et al.Introduction of a dermatophyte polymerase chain reaction assay to the diagnostic mycology service in Scotland[J].Br J Dermatol,2011,164(5):966-972.
[6]CAFARCHIA C,WEIGL S,FIGUEREDO L A,et al.Molecular identification and phylogenesis of dermatophytes isolated from rabbit farms and rabbit farm workers[J].Vet Microbiol,2012,154(3-4):395-402.
[7]KRAEMER A,MUELLER R S,WERCKENTHIN C,et al.Dermatophytes in pet guinea pigs and rabbits[J].Vet Microbiol,2012,157(1-2):208-213.
[8]CHEN B R,SUN Y,HU X P,et al.Morphology and molecular biological identification of Trichophyton mentagrophytes[J].Chin J Mycol,2010,5(6):321-326.
[9]CAFARCHIA C,CAMARDA A,COCCIOLI C,et al.Epidemiology and risk factors for dermatophytoses in rabbit farms[J].Med Mycol,2010,48(7):975-980.
[10]MEHRABAN F,LAME A,ABDOLAZIZ R L,et al.Epidemiology of dermatophytoses in an area south of Tehran,Iran[J].Mycopathologia,2003,156(4):279-287.
[11]BERGMAN A,HEIMER D,KONDORI N.Fast and specific dermatophyte detection by automated DNA extraction and real-time PCR[J].Clin Microbiol Infect,2013,19(4):205-211.
[12]GARG J,TILAK R,GARG A,et al.Rapid detection of dermatophytes from skin and hair[J].BMC Res Notes,2009,60(2):1-6.
[13]FRALLE E,RODRIGUE M,GANTOIS N,et al.Phylogenetic analysis of Trichophyton mentagrophytes human and animal isolates based on MnSOD and ITS sequence comparison[J].Microbiology,2007,153(10):3466-3477.
[14]BEIFUSS B,BEZOLD G,GOTTLBER P,et al.Direct detection of five common dermatophyte species in clinical samples using a rapid and sensitive 24-h PCR-ELISA technique open to protocol transfer[J].Mycoses,2011,54(2):137-145.