[1]安乐乐,罗迅,杨宣叶,等.猪繁殖与呼吸综合征病毒ORF7基因实时荧光定量PCR检测方法的建立[J].江苏农业学报,2024,(09):1681-1688.[doi:doi:10.3969/j.issn.1000-4440.2024.09.012]
　AN Lele,LUO Xun,YANG Xuanye,et al.Establishment of real-time fluorescence quantitative PCR method for detection of ORF7 gene of porcine reproductive and respiratory syndrome virus[J].,2024,(09):1681-1688.[doi:doi:10.3969/j.issn.1000-4440.2024.09.012]
点击复制

猪繁殖与呼吸综合征病毒ORF7基因实时荧光定量PCR检测方法的建立()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2024年09期
页码:
1681-1688
栏目:
畜牧兽医·水产养殖·益虫饲养
出版日期:
2024-09-30

文章信息/Info

Title:
Establishment of real-time fluorescence quantitative PCR method for detection of ORF7 gene of porcine reproductive and respiratory syndrome virus
作者:
安乐乐1; 2; 罗迅2; 杨宣叶1; 2; 胡欣妍1; 2; 赵永清1; 2
(1.西北民族大学生物医学研究中心,甘肃兰州730030；2.西北民族大学生命科学与工程学院,甘肃兰州730100)
Author(s):
AN Lele1; 2; LUO Xun2; YANG Xuanye1; 2; HU Xinyan1; 2; ZHAO Yongqing1; 2
(1.Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China；2.College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730100, China)
关键词:
猪繁殖与呼吸综合征;  ORF7基因;  TaqMan实时荧光定量PCR;  早期诊断
Keywords:
porcine reproductive and respiratory syndrome; ORF7 gene; TaqMan real-time fluorescence quantitative PCR; early diagnosis
分类号:
S855.3
DOI:
doi:10.3969/j.issn.1000-4440.2024.09.012
文献标志码:
A
摘要:
为建立一种快速、高效检测猪繁殖与呼吸综合征病毒(PRRSV)的实时荧光定量PCR方法,本研究根据GenBank公布的中国流行PRRSV病毒株基因序列,制备包含PRRSV ORF7基因的重组质粒标准品,同时,针对PRRSV保守区域ORF7基因序列设计特异性引物和探针,并优化引物和探针终浓度，建立PRRSV TaqMan实时荧光定量PCR检测方法。以质粒标准品为模板构建检测方法线性模型,评估灵敏度、重复性和特异性,并在疑似临床样品中初步应用。本研究建立的PRRSV TaqMan荧光定量PCR检测方法线性关系良好,线性决定系数为0.997 5；病毒载量检测限度为 1 μL 3.32×101拷贝；变异系数在组内和组间重复中均小于1.500%；仅与PRRSV发生特异性反应,未与其他病毒发生交叉反应。临床样品qPCR检测阳性率为36.92%,高于PCR检测阳性率(26.15%),阳性符合率为100%。基于PRRSV ORF7基因构建的TaqMan荧光定量PCR检测方法线性关系良好、灵敏度高、重复性好、特异性强,可快速、高效检测临床样品PRRSV,为PRRSV早期诊断、及时防控和流行病学调查提供了科学的技术支撑。
Abstract:
To establish a rapid and efficient qPCR method for the detection of porcine reproductive and respiratory syndrome virus (PRRSV), standard recombinant plasmid samples containing the ORF7 gene of PRRSV were prepared, according to the gene sequences of popular PRRSV strains in China annunciated by GenBank. Meanwhile, specific primers and probes were designed according to the ORF7 gene sequence of the conserved region of PRRSV, and the final concentrations of primers and probes were optimized to construct a real-time fluorescence quantitative PCR detection method for PRRSV TaqMan. The linear model of the assay was constructed by using the plasmid standard sample as the template to evaluate the sensitivity, repeatability and specificity, and the preliminary application was made in suspected clinical samples. The linear determination coefficient of the established TaqMan qPCR detection method for PRRSV was 0.997 5 and the linear relationship was good. The minimum detection limit of viral load was 3.32×101 copies/μL. The coefficient of variation was less than 1.500% in both intra-group and inter-group replicates. It only reacted specifically with PRRSV, and did not cross-react with other viruses. The positive detection rate of qPCR in clinical samples was 36.92%, which was higher than that of PCR (26.15%), and the positive coincidence rate was 100%. The TaqMan fluorescence quantitative PCR method based on PRRSV ORF7 gene had good linear relationship, high sensitivity, good repeatability and strong specificity. It can quickly and efficiently detect PRRSV in clinical samples, which provides scientific technical support for PRRSV early diagnosis, timely prevention and control as well as epidemiological investigation.

参考文献/References:

[1]ZHOU L, KANG R M, ZHANG Y, et al. Emergence of two novel recombinant porcine reproductive and respiratory syndrome viruses 2 (lineage 3) in Southwestern China[J]. Veterinary Microbiology,2019,232:30-41.
[2]PEI Y L, LIN C H, LI H, et al. Genetic background influences pig responses to porcine reproductive and respiratory syndrome virus[J]. Frontiers in Veterinary Science,2023,10:1289570.
[3]FANG K, LIU S D, LI X M, et al. Epidemiological and genetic characteristics of porcine reproductive and respiratory syndrome virus in South China between 2017 and 2021[J]. Frontiers in Veterinary Science,2022,9:853044.
[4]GUO Z H, CHEN X X, LI R, et al. The prevalent status and genetic diversity of porcine reproductive and respiratory syndrome virus in China: a molecular epidemiological perspective[J]. Virology Journal,2018,15:2.
[5]DOKLAND T. The structural biology of PRRSV[J]. Virus Research,2010,154(1):86-97.
[6]张喜懿, 温贵兰, 管国丹, 等. 猪繁殖与呼吸综合征病毒贵州株GZ-RNSP2基因的克隆与遗传变异分析[J]. 中国畜牧兽医,2020,47(11):3659-3666.
[7]ZHENG Y J, LI G, LUO Q, et al. Research progress on the N protein of porcine reproductive and respiratory syndrome virus[J]. Frontiers in Microbiology,2024,15:1391697.
[8]杨峰,周宏超,许信刚,等. 猪繁殖与呼吸综合征病毒实时荧光定量PCR检测方法的建立及初步应用[J]. 动物医学进展,2018,39(9):25-29.
[9]GAO J C, XIONG J Y, YE C, et al. Genotypic and geographical distribution of porcine reproductive and respiratory syndrome viruses in mainland China in 1996-2016[J]. Veterinary Microbiology, Elsevier,2017,208:164-172.
[10]XU H, LI C, GONG B J, et al. Protective efficacy of a candidate live-attenuated vaccine derived from the SD-R strain against NADC34-like porcine reproductive and respiratory syndrome virus[J]. Vaccines,2023,11(8):1349.
[11]TIAN K G, YU X L, ZHAO T Z, et al. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J]. PLoS One,2007,2(6):e526.
[12]贺亮,贺峻琳,徐玉,等. 猪繁殖与呼吸综合征病毒研究进展[J]. 黑龙江畜牧兽医,2017(17):104-107.
[13]杨晓宇,陈世界,林华,等. 多重TaqMan荧光定量PCR检测仔猪先天性震颤相关病毒方法的建立与应用[J]. 江苏农业学报,2020,36(4):971-977.
[14]门旭坤,孙婷婷,罗画叶,等. CSFV-ASFV-PRRSV多重PCR检测方法的建立[J]. 中国动物传染病学报,2023：1-8. DOI：10.19958/j.cnki.cn31-2031/s.20230606.002.
[15]张磊,华利忠,郝飞,等. 后非瘟时代猪场PRRSV类NADC30感染防控案例分析[J]. 江苏农业科学,2023,51(21):182-187.
[16]周宇骏,龙明英,孟芳,等. 2021-2022年湖南省PRRSV流行病学调查及基因组特征分析[J]. 南方农业学报,2023,54(12):3748-3756.
[17]陈玉红,徐琦,刘玉良,等. 猪繁殖与呼吸综合征病毒通用微芯片荧光定量RT-PCR方法的建立和初步应用[J]. 中国兽医科学,2021,51(11):1361-1368.
[18]郭振华,陈鑫鑫,李睿,等. 中国猪繁殖与呼吸综合征病毒流行历史及现状[J]. 畜牧兽医学报,2018,49(1):1-9.
[19]HAN J, ZHOU L, GE X N, et al. Pathogenesis and control of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus[J]. Veterinary Microbiology,Elsevier,2017, 209:30-47.
[20]LI Y F, WANG X L, BO K T, et al. Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China[J]. Veterinary Journal,2007,174(3):577-584.
[21]GAUGER P C, FAABERG K S, GUO B Q, et al. Genetic and phenotypic characterization of a 2006 United States porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field[J]. Virus Research,2012,163(1):98-107.
[22]张宇鑫,吴艳,张伟,等. 猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪圆环病毒2型和猪流感病毒四重PCR检测方法的建立[J]. 动物医学进展,2023,44(8):22-27.
[23]王方洲,张婧,马雪青,等. 猪繁殖与呼吸综合征病毒一步法多重RT-PCR检测方法的建立和应用[J]. 中国兽医科学,2022,52(7):805-814.
[24]王新港,周峰,王傲杰,等. 猪繁殖与呼吸综合征病毒和盖他病毒双重RT-PCR检测方法的建立与应用[J]. 病毒学报,2018,34(2):232-238.
[25]吴双,张聪,袁慧莎,等. 血清4型禽腺病毒TaqMan探针实时荧光定量PCR检测方法的建立与应用[J]. 江苏农业学报,2023,39(1):134-141.
[26]张杰,刘雪威,白娟,等. 猪繁殖与呼吸综合征病毒SYBR GreenⅠ荧光定量PCR方法的建立与应用[J]. 中国兽医科学,2019,49(12):1505-1513.

相似文献/References:

[1]徐雷,赵军,杨晓宇,等.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[J].江苏农业学报,2019,(01):109.[doi:doi:10.3969/j.issn.1000-4440.2019.01.016]
　XU Lei,ZHAO Jun,YANG Xiao-yu,et al.Establishment and application of RT-PCR methods for identifying porcine reproductive and respiratory syndrome virus(PRRSV) NADC30-like strain and HP-PRRSV strain[J].,2019,(09):109.[doi:doi:10.3969/j.issn.1000-4440.2019.01.016]

备注/Memo

备注/Memo:
收稿日期：2024-02-29基金项目：甘肃省自然科学基金项目(21JR1RA221)；兰州市科技计划项目(2023-1-31)；中央高校基本科研项目(31920220050)作者简介：安乐乐(1997-),男,河南周口人,硕士研究生,主要从事病毒基因工程研究。(Tel)17836954272；(E-mail)1803180130@qq.com通讯作者：赵永清,(E-mail)450883800@qq.com
更新日期/Last Update: 2024-11-17