家兔皮肤真菌双重SYBR GreenⅠ定量PCR检测方法的建立
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摘要
为建立家兔皮肤真菌快速检测方法,根据GenBank相关基因,分别设计皮肤真菌和须癣毛癣菌这2对特异引物,建立了皮肤真菌和须癣毛癣菌双重SYBR GreenⅠ定量PCR方法。结果显示,在熔解温度86~86.5℃和88.5~89℃分别出现皮肤真菌和须癣毛癣菌特异波峰,且与其他真菌没有交叉反应;重复试验变异系数为0~0.32%;最低检测浓度为3.3×10/mL;分离菌株样本与病料样本双重SYBR GreenⅠ定量PCR检测结果一致。结果表明,本试验建立的双重SYBR GreenⅠ定量PCR方法特异、稳定、敏感,可以用于临床样品的检测。
To establish rapid detection method of rabbit dermatophyte,two pairs of specific primers were designed for dermatophyte and Trichpohyton mentagrophyte,and the duplex SYBR Green Ⅰquantitative PCR for dermatophyte and Trichpohyton mentagrophyte were also established according to the GenBank related genes.Results showed that in the melting temperature from 86 ℃ to 86.5 ℃ and from 86.5 ℃ to 89 ℃,respectively,specific peaks of dermatophyte and Trichpohyton mentagrophyte appear,and there is no cross reactions with other fungi.The coefficient of variation was 0—0.32% in repeated trials.The minimum test concentration is 3.3×10/mL.The results of duplex SYBR Green Ⅰ quantitative PCR detection were consistent with those of the isolated and diseased samples.These results suggest that the duplex SYBR GreenⅠquantitative real-time PCR is specific,stable and sensitive,and can be used for the detection of clinical samples.
To establish rapid detection method of rabbit dermatophyte,two pairs of specific primers were designed for dermatophyte and Trichpohyton mentagrophyte,and the duplex SYBR Green Ⅰquantitative PCR for dermatophyte and Trichpohyton mentagrophyte were also established according to the GenBank related genes.Results showed that in the melting temperature from 86 ℃ to 86.5 ℃ and from 86.5 ℃ to 89 ℃,respectively,specific peaks of dermatophyte and Trichpohyton mentagrophyte appear,and there is no cross reactions with other fungi.The coefficient of variation was 0—0.32% in repeated trials.The minimum test concentration is 3.3×10/mL.The results of duplex SYBR Green Ⅰ quantitative PCR detection were consistent with those of the isolated and diseased samples.These results suggest that the duplex SYBR GreenⅠquantitative real-time PCR is specific,stable and sensitive,and can be used for the detection of clinical samples.
引文
[1]崔丽娜,姜文学,杨丽萍,等.家兔皮肤真菌病的研究进展[J].家畜生态学报,2011,32(1):102-105.CUI Li-na,JIANG Wen-xue,YANG Li-ping,et al.Research progress of rabbit fungal dermatopathy[J].Acta Ecologiae Animalis Domastici,2011,32(1):102-105.(in Chinese)
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[4]刘月,刘利强,刘娜,等.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[J].中国兽医科学,2015,45(8):832-836.LIU Yue,LIU Li-qiang,LIU Na,et al.Development and application of SYBR GreenⅠreal-time PCR for detecting Trichophyton mentagrophytes in rabbit[J].Chinese Veterinary Science,2015,45(8):832-836.(in Chinese)
[5]董仲生,姜平.云南家兔皮肤真菌病的流行原因及控制结果调查[J].中国养兔,2016(2):33-34.DONG Zhong-sheng,JIANG Ping.Epidemiological reasons and control results of skin mycosis in rabbits in Yunnan[J].Chinese Journal of Rabbit Farming,2016(2):33-34.(in Chinese)
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[7]GRASER Y,CZAIKA V,OHST T.Diagnostic PCR of dermatophytes-an overview[J].J Dtsch Dermatol Ges,2012,10(10):721-725.
[8]韦强,肖琛闻,季权安,等.兔皮肤真菌病病原PCR快速诊断技术研究[J].中国养兔,2017(1):10-12.WEI Qiang,XIAO Shen-wen,JI Quan-an,et al.Study of rapid PCR diagnostic techniques of in rabbit dermatosis[J].Chinese Journal of Rabbit Farming,2017(1):10-12.(in Chinese)
[9]崔丽娜,姜文学,杨丽萍,等.家兔皮肤真菌通用引物PCR检测和培养鉴定的比较研究[J].西南农业学报,2011,24(6):2389-2391.CUI Li-na,JIANG Wen-xue,YANG Li-ping,et al.Comparison of universal primer pcr identification and its culture of rabbit dermatophyte[J].Southwest China Journal of Agricultural Sciences,2011,24(6):2389-2391.(in Chinese)
[10]BERGMANS A M,VAN d EM,KLAASSEN A,et al.Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material[J].Clin Microbiol Infect,2010,16(6):704-710.
[11]桑红.真菌病的诊治概况[J].医学研究生学报,2013,26(8):785-788.SANG Hong.Overview of diagnosis and treatment of fungal diseases[J].Journal of Medical Postgraduates,2013,26(8):785-788.(in Chinese)
[12]GARG J,TILAK R,SINGH A,et al.Evaluation of pandermatophyte nested PCR in diagnosis of onychomycosis[J].J Clin Microbiol,2007,45(10):3443-3445.
[13]刘昕,马彦.皮肤癣菌分子生物学鉴定新进展[J].中国真菌学杂志,2014,9(3):186-188.LIU Xin,MA Yan.Advance on molecular identification of dermatophytes[J].Chinese Journal of Mycology,2014,9(3):186-188.(in Chinese)
[14]胡小平,万喆,王晓红,等.我国代表地区须癣毛癣菌复合体的分子鉴定与分型研究[J].中国真菌学杂志,2011,6(2):70-76.HU Xiao-ping,WAN Zhe,WANG Xiao-hong,et al.Study on molecular identification and typing of Trichophyton mentagrophytes complex from the representative regions in China[J].Chinese Journal of Mycology,2011,6(2):70-76.(in Chinese)
[15]KADHUM O H,NAHER H S,MOHAMMED K,et al.Clinical and histopathological study on dermatophytes infections caused by Trichophyton mentagrophytes using animal model[J].Journal of Natural Sciences Research,2015,16(5):170-176.
[2]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)
[3]PAUGAM A,L'OLLIVIER C,VIGUIE C,et al.Comparison of real-time PCR with conventional methods to detect dermatophytes in samples from patients with suspected dermatophytosis[J].J Microbiol Methods,2013,95(2):218-222.
[4]刘月,刘利强,刘娜,等.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[J].中国兽医科学,2015,45(8):832-836.LIU Yue,LIU Li-qiang,LIU Na,et al.Development and application of SYBR GreenⅠreal-time PCR for detecting Trichophyton mentagrophytes in rabbit[J].Chinese Veterinary Science,2015,45(8):832-836.(in Chinese)
[5]董仲生,姜平.云南家兔皮肤真菌病的流行原因及控制结果调查[J].中国养兔,2016(2):33-34.DONG Zhong-sheng,JIANG Ping.Epidemiological reasons and control results of skin mycosis in rabbits in Yunnan[J].Chinese Journal of Rabbit Farming,2016(2):33-34.(in Chinese)
[6]BEHZADI P,BEHZADI E,RANJBAR R.Dermatophyte fungi:Infections,diagnosis and treatment[J].Medical Journal,2014,1(2):50-62.
[7]GRASER Y,CZAIKA V,OHST T.Diagnostic PCR of dermatophytes-an overview[J].J Dtsch Dermatol Ges,2012,10(10):721-725.
[8]韦强,肖琛闻,季权安,等.兔皮肤真菌病病原PCR快速诊断技术研究[J].中国养兔,2017(1):10-12.WEI Qiang,XIAO Shen-wen,JI Quan-an,et al.Study of rapid PCR diagnostic techniques of in rabbit dermatosis[J].Chinese Journal of Rabbit Farming,2017(1):10-12.(in Chinese)
[9]崔丽娜,姜文学,杨丽萍,等.家兔皮肤真菌通用引物PCR检测和培养鉴定的比较研究[J].西南农业学报,2011,24(6):2389-2391.CUI Li-na,JIANG Wen-xue,YANG Li-ping,et al.Comparison of universal primer pcr identification and its culture of rabbit dermatophyte[J].Southwest China Journal of Agricultural Sciences,2011,24(6):2389-2391.(in Chinese)
[10]BERGMANS A M,VAN d EM,KLAASSEN A,et al.Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material[J].Clin Microbiol Infect,2010,16(6):704-710.
[11]桑红.真菌病的诊治概况[J].医学研究生学报,2013,26(8):785-788.SANG Hong.Overview of diagnosis and treatment of fungal diseases[J].Journal of Medical Postgraduates,2013,26(8):785-788.(in Chinese)
[12]GARG J,TILAK R,SINGH A,et al.Evaluation of pandermatophyte nested PCR in diagnosis of onychomycosis[J].J Clin Microbiol,2007,45(10):3443-3445.
[13]刘昕,马彦.皮肤癣菌分子生物学鉴定新进展[J].中国真菌学杂志,2014,9(3):186-188.LIU Xin,MA Yan.Advance on molecular identification of dermatophytes[J].Chinese Journal of Mycology,2014,9(3):186-188.(in Chinese)
[14]胡小平,万喆,王晓红,等.我国代表地区须癣毛癣菌复合体的分子鉴定与分型研究[J].中国真菌学杂志,2011,6(2):70-76.HU Xiao-ping,WAN Zhe,WANG Xiao-hong,et al.Study on molecular identification and typing of Trichophyton mentagrophytes complex from the representative regions in China[J].Chinese Journal of Mycology,2011,6(2):70-76.(in Chinese)
[15]KADHUM O H,NAHER H S,MOHAMMED K,et al.Clinical and histopathological study on dermatophytes infections caused by Trichophyton mentagrophytes using animal model[J].Journal of Natural Sciences Research,2015,16(5):170-176.