猪精液提取蓝耳病病毒核酸的方法比较
|
摘要
为筛选从猪精液中提取微量蓝耳病病毒(PRRSV)核酸的有效方法,将蓝耳病(变异株)弱毒苗稀释成不同滴度(TCID_(50)/mL)的病毒液,与健康猪常温精液混合作用。分别用Trizol法、磁珠法、离心柱法提取含毒精液中的病毒RNA,用荧光RT-PCR试验检测病毒RNA,结果表明,当精液中病毒滴度高于0.1 TCID_(50)/mL时,3种病毒RNA提取方法均可检出。离心柱法对猪精液中PRRSV核酸提取灵敏度最高,可检出0.01 TCID_(50)/mL滴度的病毒,稳定性好。
In order to Screening effective methods for extracting trace porcine reproductive and respiratory syndrome virus(PRRSV) RNA from boar semen, the attenuated vaccine of PRRSV(mutant strain) was diluted into different titers(TCID_(50)/mL) and added to fresh semen samples of healthy boars. Trizol, magnetic bead and spin column method were respectively used to extract virus RNA from semen containing PRRSV and virus RNA was detected by Fluorescent RT-PCR. The results showed that virus RNA could be detected by using all three extraction methods when the titers of virus in semen was higher than 0.1 TCID_(50)/mL. Spin column method for extracting PRRSV RNA from boar semen has the highest sensitivity, and the virus titers of 0.01 TCID_(50)/mL can be detected with good stability.
In order to Screening effective methods for extracting trace porcine reproductive and respiratory syndrome virus(PRRSV) RNA from boar semen, the attenuated vaccine of PRRSV(mutant strain) was diluted into different titers(TCID_(50)/mL) and added to fresh semen samples of healthy boars. Trizol, magnetic bead and spin column method were respectively used to extract virus RNA from semen containing PRRSV and virus RNA was detected by Fluorescent RT-PCR. The results showed that virus RNA could be detected by using all three extraction methods when the titers of virus in semen was higher than 0.1 TCID_(50)/mL. Spin column method for extracting PRRSV RNA from boar semen has the highest sensitivity, and the virus titers of 0.01 TCID_(50)/mL can be detected with good stability.
引文
[1]Jeffrey J.Zimmerman.猪病学[M].10版.赵德民,张中秋,沈建忠,等译.北京:中国农业出版社,2014.
[2]田克恭.公猪精液传播的疫病及其控制[J].中国猪业,2012,(8):6-8.
[3]李建玲,杨小亮.高致病性猪蓝耳病的研究进展[J].中国兽医杂志,2014,50(6):61-63.
[4]高许雷,吴发兴,朱紫祥,等.我国五省区部分猪场高致病性猪蓝耳病毒感染情况检测与分析[J].动物医学进展,2009,30(11):32-36.
[5]李桃梅,赖志,崔立.猪繁殖与呼吸综合征病毒垂直传播及其危害研究进展[J].中国畜牧兽医,2015,42(5):1311-1317.
[6]赵爱华,李玉文,齐欣.关于辽宁省规模猪场高致病性猪蓝耳病流行及防控情况的调研报告[J].现代畜牧兽医,2013,(2):34-35.
[7]杨国丽,王军,高志峰,等.2015-2016年辽宁省高致病性猪蓝耳病分子生物学调查分析[J].现代畜牧兽医,2017,(10):46-50.
[8]周斌,吴增坚,贾赟,等.种公猪精液中猪瘟和蓝耳病病毒混合感染的快速检测[J].畜牧与兽医,2007,37(5):1-3.
[9]李正甫,赵谦,刘佳,等.种公猪精液携带猪繁殖与呼吸障碍综合征病毒调查[J].上海畜牧兽医通讯,2016,(4):53-55.
[10]吴永刚.四川省部分地区公猪精液六种病毒性疾病的分子检测[D].雅安:四川农业大学,2014.
[11]姜佳佳,李美珍,覃绍敏,等.种公猪精液中与繁殖障碍有关的5种垂直传播病毒的检测[J].黑龙江畜牧兽医,2016,(2):102-104.
[2]田克恭.公猪精液传播的疫病及其控制[J].中国猪业,2012,(8):6-8.
[3]李建玲,杨小亮.高致病性猪蓝耳病的研究进展[J].中国兽医杂志,2014,50(6):61-63.
[4]高许雷,吴发兴,朱紫祥,等.我国五省区部分猪场高致病性猪蓝耳病毒感染情况检测与分析[J].动物医学进展,2009,30(11):32-36.
[5]李桃梅,赖志,崔立.猪繁殖与呼吸综合征病毒垂直传播及其危害研究进展[J].中国畜牧兽医,2015,42(5):1311-1317.
[6]赵爱华,李玉文,齐欣.关于辽宁省规模猪场高致病性猪蓝耳病流行及防控情况的调研报告[J].现代畜牧兽医,2013,(2):34-35.
[7]杨国丽,王军,高志峰,等.2015-2016年辽宁省高致病性猪蓝耳病分子生物学调查分析[J].现代畜牧兽医,2017,(10):46-50.
[8]周斌,吴增坚,贾赟,等.种公猪精液中猪瘟和蓝耳病病毒混合感染的快速检测[J].畜牧与兽医,2007,37(5):1-3.
[9]李正甫,赵谦,刘佳,等.种公猪精液携带猪繁殖与呼吸障碍综合征病毒调查[J].上海畜牧兽医通讯,2016,(4):53-55.
[10]吴永刚.四川省部分地区公猪精液六种病毒性疾病的分子检测[D].雅安:四川农业大学,2014.
[11]姜佳佳,李美珍,覃绍敏,等.种公猪精液中与繁殖障碍有关的5种垂直传播病毒的检测[J].黑龙江畜牧兽医,2016,(2):102-104.