猪繁殖与呼吸综合征类NADC30与HP-PRRSV毒株RT-PCR鉴别方法的建立与应用
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摘要
猪繁殖与呼吸综合征病毒(PRRSV)是一种能够引起大面积呼吸道疾病并快速传播,导致母猪流产和仔猪死亡的RNA病毒,其特性为变异快,存在毒株间的重组。因此,病原的快速检测和鉴定对于该病的防控十分重要。本试验建立了一种能够区分类高致病猪繁殖与呼吸综合征病毒类NADC30与HP-PRRSV毒株的检测方法,根据nsp2基因的高变区的比对结果,选择其保守序列,设计一对检测引物,类NADC30扩增片段大小为334 bp,HP-PRRSV毒株扩增片段大小为514 bp,通过优化反应条件,最终建立了一种一对引物就可以区分检测类NADC30与HP-PRRSV毒株的RT-PCR检测方法。应用本试验所建立的RT-PCR方法对2017年12月至2018年2月采至四川多地区的疑似PRRSV感染发病的46份肺脏进行检测。结果显示,类NADC30检出率为32. 6%,高于HP-PRRSV毒株的检出率(10. 9%)。该方法的建立,为类NADC30和HP-PRRSV毒株的快速鉴别诊断提供了方法,具有重要意义。
The porcine reproductive and respiratory syndrome virus( PRRSV) is a RNA virus that can cause a large area of respiratory diseases and spread rapidly,resulting in a large number of abortions and piglet deaths in sows. Its characteristics include rapid variation and recombination between strains. Therefore,the rapid detection and identification of pathogens is very important for the prevention and control of the disease. To establish a RT-PCR detection method for distinguishing the PRRSV NADC30-like strain from HP-PRRSV strain,the conserved sequence was selected and a pair of detection primers were designed based on the comparison of the hypervariable regions of the nsp2 gene. The amplified fragments of the NADC30-like strain and HP-PRRSV strain were 334 bp and 514 bp. By optimizing the reaction conditions,a RT-PCR detection method using a pair of primers to distinguish NADC30-like strain and the HPPRRSV was established. Using the RT-PCR method established in this experiment,46 cases of suspected PRRSV infections collected from many areas in Sichuan from December2017 to February 2018 were tested. The detection rate of NADC30-like was 32. 6%,which was higher than that of HPPRRSV strain( 10. 9%). In conclusion,these results provide a method for rapid differential diagnosis of NADC30-like and HP-PRRSV strains and have great significance.
The porcine reproductive and respiratory syndrome virus( PRRSV) is a RNA virus that can cause a large area of respiratory diseases and spread rapidly,resulting in a large number of abortions and piglet deaths in sows. Its characteristics include rapid variation and recombination between strains. Therefore,the rapid detection and identification of pathogens is very important for the prevention and control of the disease. To establish a RT-PCR detection method for distinguishing the PRRSV NADC30-like strain from HP-PRRSV strain,the conserved sequence was selected and a pair of detection primers were designed based on the comparison of the hypervariable regions of the nsp2 gene. The amplified fragments of the NADC30-like strain and HP-PRRSV strain were 334 bp and 514 bp. By optimizing the reaction conditions,a RT-PCR detection method using a pair of primers to distinguish NADC30-like strain and the HPPRRSV was established. Using the RT-PCR method established in this experiment,46 cases of suspected PRRSV infections collected from many areas in Sichuan from December2017 to February 2018 were tested. The detection rate of NADC30-like was 32. 6%,which was higher than that of HPPRRSV strain( 10. 9%). In conclusion,these results provide a method for rapid differential diagnosis of NADC30-like and HP-PRRSV strains and have great significance.
引文
[1]吴加强,姜平.猪繁殖与呼吸综合征[J].畜牧与兽医,1999,3(S1):42-46.
[2]郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国禽畜传染病,1996,18(2):1-4.
[3]童光志,周艳君,郝晓芳,等.高致病性猪繁殖与呼吸综合征病毒的分离鉴定及其分子流行病学分析[J].中国预防兽医学报,2007,29(5):323-327.
[4]彭红.高致病性猪蓝耳病的流行病学调查报告[J].湖南畜牧兽医,2009(6):37-39.
[5]周峰,常洪涛,赵军,等. 2012-2013年猪繁殖与呼吸综合征病毒河南流行株的分离鉴定及分子流行病学调查[J].中国兽医学报,2014,34(9):1398-1404,1410.
[6]卢艳敏.猪感染PRRSV后Cathelicidins抗菌肽基因的表达情况[J].江苏农业科学,2018,46(2):24-27.
[7]张栋,王善辉,王振勇.猪繁殖与呼吸综合征的临床症状与鉴别诊断[J].中国畜牧兽医文摘,2006,21(1):17-19.
[8]张宁,刘德清,洪绍峰,等. PRRSV阳性血清驯化对后备母猪繁殖性能的影响[J].南方农业学报,2017,48(6):1113-1118.
[9]李小璟,龚双燕,陈瑛琪,等.猪繁殖与呼吸综合征病毒与猪流行性腹泻病毒双重RT-PCR检测方法的建立与应用[J].中国兽医科学,2017,47(5):544-550.
[10]孙英峰.天津地区PRRSV类NADC30毒株的监测、分离毒株的基因组特征与致病性分析[D].北京:中国农业大学,2017.
[11] ROVIRA A,BALASCH M,SEGAL S J,et al.Experimental inoc-ulation of conventional pigs with porcine reproductive and respira-tory syndrome virus and porcine circovirus 2[J].Journal of Virolo-gy,2002,76(7):3232-3239.
[12]吴佳俊,遇秀玲,蒋桃珍,等.猪繁殖与呼吸综合征灭活疫苗研究进展[J].中国预防兽医学报,2010,32(8):655-658.
[13]周磊,丁玉平.猪繁殖与呼吸综合征病毒类NADC30毒株的新近流行[J].中国猪业,2017,12(11):20-23.
[2]郭宝清,陈章水,刘文兴,等.从疑似PRRS流产胎儿分离PRRSV的研究[J].中国禽畜传染病,1996,18(2):1-4.
[3]童光志,周艳君,郝晓芳,等.高致病性猪繁殖与呼吸综合征病毒的分离鉴定及其分子流行病学分析[J].中国预防兽医学报,2007,29(5):323-327.
[4]彭红.高致病性猪蓝耳病的流行病学调查报告[J].湖南畜牧兽医,2009(6):37-39.
[5]周峰,常洪涛,赵军,等. 2012-2013年猪繁殖与呼吸综合征病毒河南流行株的分离鉴定及分子流行病学调查[J].中国兽医学报,2014,34(9):1398-1404,1410.
[6]卢艳敏.猪感染PRRSV后Cathelicidins抗菌肽基因的表达情况[J].江苏农业科学,2018,46(2):24-27.
[7]张栋,王善辉,王振勇.猪繁殖与呼吸综合征的临床症状与鉴别诊断[J].中国畜牧兽医文摘,2006,21(1):17-19.
[8]张宁,刘德清,洪绍峰,等. PRRSV阳性血清驯化对后备母猪繁殖性能的影响[J].南方农业学报,2017,48(6):1113-1118.
[9]李小璟,龚双燕,陈瑛琪,等.猪繁殖与呼吸综合征病毒与猪流行性腹泻病毒双重RT-PCR检测方法的建立与应用[J].中国兽医科学,2017,47(5):544-550.
[10]孙英峰.天津地区PRRSV类NADC30毒株的监测、分离毒株的基因组特征与致病性分析[D].北京:中国农业大学,2017.
[11] ROVIRA A,BALASCH M,SEGAL S J,et al.Experimental inoc-ulation of conventional pigs with porcine reproductive and respira-tory syndrome virus and porcine circovirus 2[J].Journal of Virolo-gy,2002,76(7):3232-3239.
[12]吴佳俊,遇秀玲,蒋桃珍,等.猪繁殖与呼吸综合征灭活疫苗研究进展[J].中国预防兽医学报,2010,32(8):655-658.
[13]周磊,丁玉平.猪繁殖与呼吸综合征病毒类NADC30毒株的新近流行[J].中国猪业,2017,12(11):20-23.