小反刍兽疫病毒环介导等温扩增检测方法的建立
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摘要
为了建立适用于基层养殖场快速、可视化的小反刍兽疫病毒(PPRV)环介导等温扩增(LAMP)检测方法,根据GenBank公布的PPRV F基因保守区核苷酸序列为靶序列,设计一组特异性LAMP引物,优化反应条件(时间、温度)和反应体系(dNTP、Mg2+、甜菜碱浓度),并进行特异性和灵敏性试验,最后用建立的方法对临床样品RNA进行检测。结果表明,所建立的方法在65℃时反应45min即可获得清晰的梯状条带;扩增物经10×SYBR GreenⅠ荧光染料染色,紫外线照射之后即可直接判定结果;灵敏性试验表明,该方法能够检出的PPRV核酸的最低浓度为8.63ng/mL,与常规RT-PCR方法相比,灵敏度增高100倍;该方法特异性良好,与山羊痘病毒(GTPV)、羊支原体(Mccp)、羊口疮病毒(ORFV)均无交叉反应;对16份临床样品RNA检测结果显示,该方法与传统RT-PCR检测结果一致。建立的LAMP检测方法的特异性、灵敏度均好,可用于临床小反刍兽疫病毒的检测。
In order to establish a quick and visual LAMP molecular detection method that can be used for grassroots farm,regarding PPRV F gene conserved sequence region published in GenBank as the target,a new set of specific LAMP primers were designed.The method was established by optimizing the reaction conditions(such as time and temperature),reaction system(such as dNTP,Mg2+and betaine concentration),and then made a test about clinical RNA samples.A clear ladder can be obtained at 65 ℃for 45 min using this method.Tinted with 10×SYBR Green I fluorescent dyes,the amplified products were determined directly after UV irradiation.The sensitivity test showed that the lowest PPRV nucleic acid concentration was 8.63 ng/mL,which was 100 times more sensitive than that of reverse transcriptional-polymerase chain reaction.The results of specificity assay demonstrated that there was no cross reaction between goatpoxvirus(GTPV),Mycoplasma capricolum subsp.capripneumonia(Mccp)and orf virus(ORFV),which came to a conclusion that the method established was specific.16 cases of clinical samples RNA detection results showed it accords with the results of reverse transcriptional-polymerase chain reaction.This method can be applied to clinical detection of PPRV because of good performances in specificity and sensitivity.
In order to establish a quick and visual LAMP molecular detection method that can be used for grassroots farm,regarding PPRV F gene conserved sequence region published in GenBank as the target,a new set of specific LAMP primers were designed.The method was established by optimizing the reaction conditions(such as time and temperature),reaction system(such as dNTP,Mg2+and betaine concentration),and then made a test about clinical RNA samples.A clear ladder can be obtained at 65 ℃for 45 min using this method.Tinted with 10×SYBR Green I fluorescent dyes,the amplified products were determined directly after UV irradiation.The sensitivity test showed that the lowest PPRV nucleic acid concentration was 8.63 ng/mL,which was 100 times more sensitive than that of reverse transcriptional-polymerase chain reaction.The results of specificity assay demonstrated that there was no cross reaction between goatpoxvirus(GTPV),Mycoplasma capricolum subsp.capripneumonia(Mccp)and orf virus(ORFV),which came to a conclusion that the method established was specific.16 cases of clinical samples RNA detection results showed it accords with the results of reverse transcriptional-polymerase chain reaction.This method can be applied to clinical detection of PPRV because of good performances in specificity and sensitivity.
引文
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[11]欧新华,张如胜,宋克云,等.逆转录-环介导等温扩增方法检测甲型H1N1流感病毒[J].中华检验医学杂志,2010,33(5):443-445.
[12]Parida M M,Santhosh S R,Dash P K,et al.Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus[J].J Clin Microbiol,2006,44(11):4172-4178.
[13]Mori Y,Nagamine K,Tomita N,et al.Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[J].Biochem Bioph Res Co,2001,289(1):150-154.
[2]Peste des petits ruminants[OL]http://www.oie.int.
[3]Notomi T,Okayama H,Masubuchi H,et al.Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res,2000,28(12):E63.
[4]陈兵,胡运发,孙洁,等.新城疫病毒实时荧光RT-LAMP检测方法的建立[J].动物医学进展,2014,35(7):11-14.
[5]胡嘉欣,温永俊,霍志云,等.犬瘟热病毒RT-LAMP检测方法的建立及初步应用[J].中国畜牧兽医,2012,39(10):45-49.
[6]Zhang Y,Yi Y,Du W,et al.Development of loop-mediated isothermal amplification with Plasmodium falciparum unique genes for molecular diagnosis of human malaria[J].Pathogens&Global Health,2017(5):1-9.
[7]Higa Y,Uemura R,Yamazaki W,et al.An improved loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis[J].J Vet Med Sci,2016,78(8):1343.
[8]郝玉青,刘健鹏,杨增岐.陕北白绒山羊小反当兽疫病毒RT-PCR方法的建立[J].动物医学进展,2017,38(7):12-16.
[9]赵柏林,孔冬妮,孙雨,等.我国小反刍兽疫流行情况及防控措施[J].畜牧与兽医,2017,49(3):108-110.
[10]何亚鹏,鲍长磊,张琪,等.小反刍兽疫病毒疫苗株N和M基因的克隆、序列分析及原核表达[J].西北农业学报,2017,26(2):179-184.
[11]欧新华,张如胜,宋克云,等.逆转录-环介导等温扩增方法检测甲型H1N1流感病毒[J].中华检验医学杂志,2010,33(5):443-445.
[12]Parida M M,Santhosh S R,Dash P K,et al.Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus[J].J Clin Microbiol,2006,44(11):4172-4178.
[13]Mori Y,Nagamine K,Tomita N,et al.Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation[J].Biochem Bioph Res Co,2001,289(1):150-154.