环形泰勒虫SYBR Green I荧光定量PCR方法的建立及应用
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摘要
为建立一种快速、敏感检测牛环形泰勒虫的方法,本研究根据GenBank中登录的牛环形泰勒虫Tams1基因序列,设计合成1对特异性引物,通过优化反应条件,建立了检测牛环形泰勒虫SYBR Green I荧光定量PCR检测方法。研究结果显示,该方法可以特异地检测牛环形泰勒虫,而对牛巴贝斯虫、反刍动物艾立希体和弓形虫检测均为阴性;该方法的灵敏度可达到180拷贝/μL,比常规PCR敏感10倍;组内和组间变异系数均小于1.0%。对15份临床血液样品和20只璃眼蜱进行检测,SYBR Green I荧光定量PCR和常规PCR的阳性检出率分别为42.86%和28.57%。本研究建立的SYBR Green I荧光定量PCR检测方法具有特异性强、敏感性高的特点,可准确、高效检测牛环形泰勒虫,为牛环形泰勒虫病防控提供技术支持。
A SYBR Green I based real-time PCR assay was established for the detection of Theileria annulata with a pair of specific primers designed according to the T.annulata Tams1 gene.The result showed that the assay was specific for detecting T.annulata,but not for Babesia bovis,Ehrlichia ruminantium and Toxoplasma gondii.The sensitivity of the method was180 copies/μL,which was 10 times more sensitive than that of the conventional PCR.The coefficients of variations were less than1.0%for both intra-assay and inter-assay.Among 15 clinical blood samples and 20 ticks of Hyalomma detections,the positive rate was 42.86%by the SYBR Green I based real-time PCR and 28.57%by conventional PCR.These results showed that the SYBR Green I based real-time PCR established in this study was a fast,sensitive,specific and reliable method for detecting T.annulata,which will be a technical support for T.annulata prevention and control.
A SYBR Green I based real-time PCR assay was established for the detection of Theileria annulata with a pair of specific primers designed according to the T.annulata Tams1 gene.The result showed that the assay was specific for detecting T.annulata,but not for Babesia bovis,Ehrlichia ruminantium and Toxoplasma gondii.The sensitivity of the method was180 copies/μL,which was 10 times more sensitive than that of the conventional PCR.The coefficients of variations were less than1.0%for both intra-assay and inter-assay.Among 15 clinical blood samples and 20 ticks of Hyalomma detections,the positive rate was 42.86%by the SYBR Green I based real-time PCR and 28.57%by conventional PCR.These results showed that the SYBR Green I based real-time PCR established in this study was a fast,sensitive,specific and reliable method for detecting T.annulata,which will be a technical support for T.annulata prevention and control.
引文
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[11]D'Oliveira C,Vandermerve M,Habela M,et al.Detection of Theileria annulata in blood samples of carrier cattle by PCR[J].J Clin Microbiol,1995,33(10):2665-2669.
[12]Majidiani H,Nabavi R,Ganjali M,et al.Detection of Theileria annulata carriers in Holstein-Friesian(Bos taurus taurus)and Sistani(Bos taurus indicus)cattle breeds by polymerase chain reaction in Sistan region,Iran[J].J Parasit Dis,2016,40(4):1184-1188.
[13]Sallemi S,Rjeibi M R,Rouatbi M,et al.Molecular prevalence and phylogenetic analysis of Theileria annulata and Trypanosoma evansi in cattle in Northern Tunisia[J].Vet Med Sci,2017,4(1):17-25.
[14]Nourollahifard S R,Khalili M,Ghalekhani N.Detection of Theileria annulata in blood samples of native cattle by PCR and smear method in Southeast of Iran[J].J Parasit Dis,2015,39(2):249-252.
[2]Ferrer R,Artigas A,Suarez D,et al.Prevalence of Theileria annulata infection in the salivary glands of ticks(Hyalomma anatolicum)[J].Indian J Vet Res,2009,48(10):1487-1488.
[3]Sallemi S,Rjeibi M R,Rouatbi M,et al.Molecular prevalence and phylogenetic analysis of Theileria annulata and Trypanosoma evansi in cattle in Northern Tunisia[J].Vet Med Sci,2018,4(1):17-25.
[4]Abaker I A,Salih D A,Haj L M,et al.Prevalence of Theileria annulatain dairy cattle in Nyala,South Darfur State,Sudan[J].Vet World,2017,10(12):1475-1480.
[5]Weir W,Karagenc T,Gharbi M,et al.Population diversity and multiplicity of infection in Theileria annulata[J].Int J Parasitol,2011,41(2):193-203.
[6]马腾,周金林.梨形虫病的分子生物学研究进展[J].中国动物传染病学报,2006,14(2):22-27.
[7]党志胜,罗建勋,殷宏.环形泰勒虫病诊断技术的发展概况[J].青海畜牧兽医杂志,2004,34(1):37-39.
[8]Gomes J,Inacio J.Direct detection of Theileria annulata in bovine blood samples using standard and isothermal DNA amplification approaches[J].Methods Mol Biol,2015,1247(1247):175-182.
[9]Santhosh S R,Parida M M,Dash P K,et al.Development and evaluation of SYBR Green I-based one-step real-time RT-PCRassay for detection and quantification of Chikungunya virus[J].J Virol Methods,2007,39(3):73-80.
[10]Kundave V R,Ram H,Rafqi S I,et al.Comparative evaluation of microscopy and PCR assay for detection of Theileria annulata infection in ruminants[J].J Anim Res,2017,7(4):699-703.
[11]D'Oliveira C,Vandermerve M,Habela M,et al.Detection of Theileria annulata in blood samples of carrier cattle by PCR[J].J Clin Microbiol,1995,33(10):2665-2669.
[12]Majidiani H,Nabavi R,Ganjali M,et al.Detection of Theileria annulata carriers in Holstein-Friesian(Bos taurus taurus)and Sistani(Bos taurus indicus)cattle breeds by polymerase chain reaction in Sistan region,Iran[J].J Parasit Dis,2016,40(4):1184-1188.
[13]Sallemi S,Rjeibi M R,Rouatbi M,et al.Molecular prevalence and phylogenetic analysis of Theileria annulata and Trypanosoma evansi in cattle in Northern Tunisia[J].Vet Med Sci,2017,4(1):17-25.
[14]Nourollahifard S R,Khalili M,Ghalekhani N.Detection of Theileria annulata in blood samples of native cattle by PCR and smear method in Southeast of Iran[J].J Parasit Dis,2015,39(2):249-252.