鸡传染性支气管炎病毒纳米PCR检测方法的建立及初步应用
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摘要
根据鸡传染性支气管炎病毒(IBV)的3′端非编码区保守区域,设计并合成一对特异性引物,建立IBV纳米PCR检测方法,并进行了特异性、灵敏性和重复性试验,然后将建立的方法进行初步应用。结果显示:该方法特异性良好,能从4种不同基因型IBV核酸样品中检测到约346 bp的目的条带,而对禽偏肺病毒、H9亚型禽流感病毒、新城疫病毒、传染性喉气管炎病毒、传染性法氏囊病毒、马立克氏病病毒、禽白血病病毒、鸭肝炎病毒等其他病原检测结果均为阴性;敏感性试验表明,纳米PCR的最低检测浓度为1.01×10~(-4)ng/μL,其敏感性是普通PCR的10倍;重复性试验显示3次均能检出IBV核酸。纳米PCR方法的临床应用表明,送检样品及攻毒鸡样品的检出率分别达到50%(10/20)和60%(30/50),与普通PCR方法检测结果符合率为100%。研究建立的IBV纳米PCR检测方法具有较高的敏感性和良好的特异性,为IBV的临床诊断、病原检测和流行病学调查提供了新的技术手段。
According to the conserved region of the 3′ non-coding region of avian infectious bronchitis virus(IBV),a pair of specific primers were designed and synthesized to develop an Nano-PCR detection method for IBV, and the specificity, sensitivity and reproducibility of the Nano-PCR method were evaluated. Also, the developed Nano-PCR method was applied to detected samples of the clinical cases and the challenged birds. The results showed that the developed Nano-PCR method was specific and a band of about 346 bp was only detected from the nucleic acid sample of IBV, but not from avian metapneumovirus, infectious bursal disease virus, Marek′s disease virus, Newcastle disease virus, avain leucosis virus, H9 subtype avian influenza virus, infectious laryngotracheitis virus, duck hepatitis virus and other pathogens. Sensitivity tests showed that the minimum detection concentration of Nano-PCR was 1.01×10~(-4) ng/μL,and that was 10 times of the conventional PCR. Repeatability test shows that IBV nucleic acid could be detected three times. The application test showed that the positive rate of the clinical and challenged samples were 50%(10/20) and60%(30/50), respectively, and the coincidence rate with the conventional PCR method was 100%. The developed IBV Nano-PCR detection method had higher sensitivity and good specificity. This method provided a new technical means for clinical diagnosis, pathogen detection and epidemiological investigation of IBV.
According to the conserved region of the 3′ non-coding region of avian infectious bronchitis virus(IBV),a pair of specific primers were designed and synthesized to develop an Nano-PCR detection method for IBV, and the specificity, sensitivity and reproducibility of the Nano-PCR method were evaluated. Also, the developed Nano-PCR method was applied to detected samples of the clinical cases and the challenged birds. The results showed that the developed Nano-PCR method was specific and a band of about 346 bp was only detected from the nucleic acid sample of IBV, but not from avian metapneumovirus, infectious bursal disease virus, Marek′s disease virus, Newcastle disease virus, avain leucosis virus, H9 subtype avian influenza virus, infectious laryngotracheitis virus, duck hepatitis virus and other pathogens. Sensitivity tests showed that the minimum detection concentration of Nano-PCR was 1.01×10~(-4) ng/μL,and that was 10 times of the conventional PCR. Repeatability test shows that IBV nucleic acid could be detected three times. The application test showed that the positive rate of the clinical and challenged samples were 50%(10/20) and60%(30/50), respectively, and the coincidence rate with the conventional PCR method was 100%. The developed IBV Nano-PCR detection method had higher sensitivity and good specificity. This method provided a new technical means for clinical diagnosis, pathogen detection and epidemiological investigation of IBV.
引文
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[14]柴瑞影，话荣虹，袁万哲，等.猪流行性乙型脑炎病毒纳米PCR检测方法的建立[J].中国兽医学报，2017,37(3):471-474.
[15]崔尚金，胡泉博，刘业兵，等.非洲猪瘟病毒高效纳米PCR检测方法的建立与初步应用[J].中国预防兽医学报，2012,34(10):807-809.
[16]范文胜，唐宁，董志华，等.一株鸭源传染性支气管炎病毒的分离鉴定及结构蛋白基因和血清型分析[J].微生物学报，2019,59(3):523-532.
[17]舒端阳. TRIzol——一把同时提取核酸和蛋白质的利器[J].生物学通报，2012,47(9):9-10.
[18]边葶苈，朱林文思，雷静，等.鸡传染性支气管炎病毒变异株的分离及分子特征研究[J].中国预防兽医学报，2013,35(12):960-963.
[19] LI H K, HUANG J H, LüJ H, et al. Nanoparticle PCR:Nanogold-assisted PCR with enhanced specificity[J]. Angewandte Chemie-international Edition, 2005, 44(32):5100-5103.
[20]梁冰，顾大勇，鲁卫平，等.纳米金新型基因芯片检测系统结合限制性酶切非PCR法同时检测多种病原性微生物的研究[J].解放军医学杂志，2009,34(5):511-514.
[21]郑丽，任卫科，路超，等.禽呼肠孤病毒纳米PCR检测方法的建立与应用[J].中国兽医科学，2018,47(6):700-706.
[22] MA X, CUI Y, QIU Z, et al. A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains[J]. Journal of Virological Methods, 2013, 193(2):374-378.
[2] HE Y, LI Z, TANG N, et al. Prevention and treatment effects of Jiebai granule on chickens infected with avian infectious bronchitis[J]. Journal of Southern Argiculture,2016, 47(9):1602-1607.
[3]童琴英.一例鸡传染性支气管炎与大肠杆菌病混合感染的诊治[J].畜牧兽医科学，2017,61(2):51.
[4] CALLISON S A, HILT D A, JACKWOOD M W, et al.Rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using reale-time reverse transcriptase-polymerase chain reaction[J].Journal of Virological Methods, 2005, 124(1-2):183-190.
[5]马利.禽呼肠孤病毒和传染性支气管炎病毒的环介导等温扩增检测方法的研究[D].泰安：山东农业大学，2012.
[6] MO M L, LI M, HUANG B C, et al. Molecular characterization of major structural protein genes of avian coronavirus infectious bronchitis virus isolates in southern China[J].Viruses, 2013, 5(12):3007-3020.
[7]张丽华，吴翠兰，张志鹏，等. 2012~2013年鸡传染性支气管炎病毒广西分离株S1基因序列分析与血清型鉴定[J].病毒学报，2016,32(1):62-69
[8] MO M L, HUANG B C, WEI P, et al. Complete genome sequences of two china virulent avian coronavirus infectious bronchitis virus variants[J]. Journal of Virology,2012, 86(19):10903-10904.
[9]王秀英，韦平，陈秋英，等.鸡传染性支气管炎病毒广西分离株血清型的分析[J].中国兽医学报，2010,30(2):183-187.
[10]庄金秋，阮震，张颖，等.鸡传染性支气管炎病毒实验室检测方法研究进展[J].家禽科学，2018,39(7):50-58.
[11]刘清岱，王金菊，王勇.纳米粒子PCR研究进展[J].生物学通报，2010,45(2):1-3.
[12] KEBLINSKI P, PHILLPOT S R, CHOI S, et al. Mechanisms of heat flow in suspensions of nano-sized particles(nanofluids)[J]. International Journal of Heat and Mass Transfer, 2002, 45(4):855-863.
[13]袁万哲，邹云婧，孙继国，等.检测新城疫病毒的纳米PCR技术建立与初步应用[J].畜牧与兽医，2016,48(1):104-106.
[14]柴瑞影，话荣虹，袁万哲，等.猪流行性乙型脑炎病毒纳米PCR检测方法的建立[J].中国兽医学报，2017,37(3):471-474.
[15]崔尚金，胡泉博，刘业兵，等.非洲猪瘟病毒高效纳米PCR检测方法的建立与初步应用[J].中国预防兽医学报，2012,34(10):807-809.
[16]范文胜，唐宁，董志华，等.一株鸭源传染性支气管炎病毒的分离鉴定及结构蛋白基因和血清型分析[J].微生物学报，2019,59(3):523-532.
[17]舒端阳. TRIzol——一把同时提取核酸和蛋白质的利器[J].生物学通报，2012,47(9):9-10.
[18]边葶苈，朱林文思，雷静，等.鸡传染性支气管炎病毒变异株的分离及分子特征研究[J].中国预防兽医学报，2013,35(12):960-963.
[19] LI H K, HUANG J H, LüJ H, et al. Nanoparticle PCR:Nanogold-assisted PCR with enhanced specificity[J]. Angewandte Chemie-international Edition, 2005, 44(32):5100-5103.
[20]梁冰，顾大勇，鲁卫平，等.纳米金新型基因芯片检测系统结合限制性酶切非PCR法同时检测多种病原性微生物的研究[J].解放军医学杂志，2009,34(5):511-514.
[21]郑丽，任卫科，路超，等.禽呼肠孤病毒纳米PCR检测方法的建立与应用[J].中国兽医科学，2018,47(6):700-706.
[22] MA X, CUI Y, QIU Z, et al. A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains[J]. Journal of Virological Methods, 2013, 193(2):374-378.
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