C型禽偏肺病毒SYBR Green Ⅰ实时荧光定量PCR方法的建立及应用
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摘要
根据GenBank发表的禽偏肺病毒(aMPV)F基因序列,设计一对特异性引物,建立C亚型aMPV的SYBR GreenⅠ实时荧光定量PCR方法。对该反应体系进行条件优化,建立了标准曲线,并进行特异性、敏感性及重复性试验,然后将建立的方法应用于临床样品和攻毒样品的检测。结果显示:标准曲线循环阈值与模板浓度呈现良好的线性关系;建立的方法只能检测出C亚型aMPV,最低可以检测到0.8×10~1拷贝/μL的核酸模板,重复性试验的变异系数小于4%。应用建立的方法对43份临床样品检测,结果显示均为阴性;对42份35日龄SPF鸡人工感染后1～21 d的气管和肺脏样品进行检测,结果显示攻毒样品均为阳性。因此,研究建立的特异、灵敏、重复性好的C型aMPV实时荧光定量PCR方法,既可适合于该病的早期诊断和流行病学调查,又为该病毒的致病机制研究提供技术支撑。
In order to establish a SYBR Green Ⅰ real-time quantitative PCR method for avian metapneumovirus(aMPV) subtype C, a pair of specific primers was designed based on F gene sequence of aMPV subtype C in GenBank.The reaction system was optimized and the standard curve was established. Specificity, sensitivity, and reproducibility tests were performed. The clinical samples and challenged samples were detected by the developed real-time PCR. The results showed that the standard curve showed a good linear relationship between threshold cycle and template concentration. The established method could only detect aMPV subtype C and had a detection limit of 0.8×10~1 copies/μL of initial templates. The method had a coefficient of variations less than 4% for both intra-and inter-assay. 43 clinical samples were detected by the established method and the results showed that all of clinical samples were negative for aMPV subtype C. 42 trachea and lung samples collected at 1 to 21 days post infection with a MPV subtype C in 35-day-age SPF chickens were detected and the results showed that all the challenged samples were positive. Therefore,a specific, sensitive and reproducible real-time quantitative PCR method for aMPV subtype C was developed in this study. The method can be used for early diagnosis and epidemiological investigation of the disease, and provide technical support for study of the pathogenesis of aMPV.
In order to establish a SYBR Green Ⅰ real-time quantitative PCR method for avian metapneumovirus(aMPV) subtype C, a pair of specific primers was designed based on F gene sequence of aMPV subtype C in GenBank.The reaction system was optimized and the standard curve was established. Specificity, sensitivity, and reproducibility tests were performed. The clinical samples and challenged samples were detected by the developed real-time PCR. The results showed that the standard curve showed a good linear relationship between threshold cycle and template concentration. The established method could only detect aMPV subtype C and had a detection limit of 0.8×10~1 copies/μL of initial templates. The method had a coefficient of variations less than 4% for both intra-and inter-assay. 43 clinical samples were detected by the established method and the results showed that all of clinical samples were negative for aMPV subtype C. 42 trachea and lung samples collected at 1 to 21 days post infection with a MPV subtype C in 35-day-age SPF chickens were detected and the results showed that all the challenged samples were positive. Therefore,a specific, sensitive and reproducible real-time quantitative PCR method for aMPV subtype C was developed in this study. The method can be used for early diagnosis and epidemiological investigation of the disease, and provide technical support for study of the pathogenesis of aMPV.
引文
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[12]胡海霞,赵伟,于庆忠.禽偏肺病毒分子生物学及基因工程疫苗研究进展[J].中国家禽,2012,34(12):1-5.
[2]王艳,庄金秋,梅建国,等.禽偏肺病毒实验室检测方法研究进展[J].中国家禽,2015,37(2):45-49.
[3]鞠小军.B亚型禽偏肺病毒检测方法的建立与应用[D].泰安:山东农业大学,2012.
[4]RAUTENSCHLEIN S,SHEIKH A M,PATNAYAK D P,et al.Effect of an immunomodulator on the efficacy of an attenuated vaccine against avian pneumovirus in turkeys[J].Avian Diseases,2002,46(3):555-561.
[5]张丽华,李和鸣,袁园,等.广西地区规模化鸡场禽偏肺病毒感染情况的血清学调查[J].中国预防兽医学报,2017,39(6):439-442.
[6]张丹俊,戴银,赵瑞宏,等.安徽省部分地区鸡群禽偏肺病毒感染的血清学调查[J].动物医学进展,2017,38(2):126-129.
[7]陈琳,刁有祥,邹金峰.禽偏肺病毒RT-PCR检测方法的建立及应用[J].中国兽医学报,2012,32(7):971-974.
[8]SAIF Y M.禽病学[M].11版.苏敬良,高福,索勋译.北京:中国农业出版社,2005.
[9]阎旭.禽肺病毒F蛋白的原核表达及其间接ELISA方法的建立[D].天津:天津科技大学,2013.
[10]阎旭,韦莉,王菁,等.A型禽偏肺病毒F蛋白的原核表达与抗原性分析[J].中国兽医杂志,2014,50(3):6-9.
[11]COOK J K,CAVANAGH D.Detection and differentiation of avian pneumoviruses(metapneumoviruses)[J].Avian Pathology,2002,31(2):117-132.
[12]胡海霞,赵伟,于庆忠.禽偏肺病毒分子生物学及基因工程疫苗研究进展[J].中国家禽,2012,34(12):1-5.