牛病毒性腹泻病毒和牛肠道病毒双重RAA-LFD快速检测方法的建立及应用

doi:10.13560/j.cnki.biotech.bull.1985.2026-0234

摘要/Abstract

摘要：

目的建立快速、简便、可视化的牛病毒性腹泻病毒（Bovine viral diarrhea virus, BVDV）和牛肠道病毒（Bovine enterovirus, BEV）双重快速检测方法。方法利用重组酶介导恒温扩增技术（recombinase aided amplification, RAA），针对BVDV 5'UTR和BEV VP1保守区设计并筛选出特异性好、灵敏度高的引物和探针，结合侧流层析试纸条（lateral flow dipstick, LFD）建立并优化双重RAA-LFD反应体系，并对反应时间、温度、特异性、灵敏度和重复性进行评估，用建立的双重RAA-LFD检测方法与国家标准和文献报道的qPCR法分别对新疆各地区113份疑似犊牛腹泻的临床样品进行检测，完成不同检测方法间的准确率比对分析。结果建立的双重RAA-LFD检测方法在42 ℃反应20 min可完成检测，BVDV、BEV的最低检出限分别为10⁰ copies/μL和10¹ copies/μL，且与大肠杆菌（Escherichia coli, E.coli）、沙门氏菌（Salmonella）和牛冠状病毒（Bovine coronavirus, BCoV）等犊牛腹泻重要病原无交叉反应。检测113份临床样品，其中BVDV阳性检出率为18.6%，BEV阳性检出率为22.1%，混合感染率为8.6%，与qPCR总体阳性符合率为100%。结论建立了操作简单、快速、灵敏度高、特异性强、准确率高的BVDV和BEV双重RAA-LFD检测方法，为实现犊牛腹泻的早期预警和综合防控提供重要手段。

关键词: 牛病毒性腹泻病毒, 牛肠道病毒, 重组酶介导恒温扩增, 侧流层析试纸条

Abstract:

Objective This study aimed to establish a rapid, simple, and visual dual detection method for Bovine viral diarrhea virus (BVDV) and Bovine enterovirus (BEV). Method Based on recombinase aided amplification (RAA), specific primers and probes with high sensitivity and specificity were designed and screened for the conserved regions of the BVDV 5'UTR gene and the BEV VP1 gene. A dual RAA-lateral flow dipstick (LFD) system was established and optimized, and its reaction time, temperature, specificity, sensitivity, and repeatability were evaluated. A total of 113 clinical samples suspected of calf diarrhea from various regions of Xinjiang were tested blindly using both the developed dual RAA-LFD method and qPCR and enabling an accuracy comparision analysis among different testing methods. Result The established dual RAA-LFD detection method can complete the test within 20 min at 42 ℃，with detection limits of 10⁰ copies/μL for BVDV and 10¹ copies/μL for BEV. No cross-reaction was observed with Escherichia coli (E.coli), Salmonella orBovine coronavirus (BCoV). The 113 clinical samples collected, the positive detection rates for BVDV and BEV were 18.6% and 22.1%, respectively, with a co-infection rate of 8.6%. The overall positive agreement rate was 100%. Conclusion A simple, rapid, sensitive, specific, and accurate dual RAA-LFD method was established for the simultaneous detection of BVDV and BEV. This method provides an important tool for the early warning and comprehensive prevention-and-control of calf diarrhea.

Key words: Bovine viral diarrhea virus, Bovine enterovirus, recombinase aided amplification, lateral flow dipstick

刘宇辰, 刘芯怡, 刘心愿, 靳征, 莫沙·衣米提, 关团, 丁剑, 史慧君, 付强. 牛病毒性腹泻病毒和牛肠道病毒双重RAA-LFD快速检测方法的建立及应用[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0234.

LIU Yu-chen, LIU Xin-yi, LIU Xin-yuan, JIN Zheng, MOSA Yimiti, GUAN Tuan, DING Jian, SHI Hui-jun, FU Qiang. Establishment and Application of Rapid Detection Method for Bovine Viral Diarrhea Virus and Bovine Enterovirus Dual RAA-LFD[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0234.

图/表 11

表1 BVDV和BEV双重RAA-LFD与qPCR检测结果比较分析

Table 1 Comparative analysis of BVDV and BEV dual RAA-LFD and qPCR detection methods

样品名称 Sample name	RAA-LFD检测结果 RAA-LFD detection results （No. of samples）	qPCR检测结果 qPCR detection results （No. of samples）
BVDV阳性	21	21
BVDV阴性	92	92
BEV阳性	25	25
BEV阴性	88	88

图1 目的基因的PCR扩增结果M：DL2000 DNA marker；NC：阴性对照

Fig. 1 PCR amplification results of target geneM: DL2000 DNA marker; NC: Negative control

图2 RAA引物筛选结果A：BVDV阴性正常引物对（F1R2、F2R2）；B：BEV阴性正常引物对（F4R4、F5R5）；C：BVDV最佳引物对（F2R2）；D：BEV最佳引物对（F5R5）

Fig. 2 RAA primer screening resultsA: BVDV-negative normal primer pairs (F1R2, F2R2). B: BEV-negative normal primer pairs (F4R4, F5R5). C: Optimal BVDV primer pair (F2R2). D: Optimal BEV primer pair (F5R5)

图3 甜菜碱浓度优化结果M：DL2000 DNA marker；NC：阴性对照；A：BVDV最佳甜菜碱浓度（1.0 mol/L）；B：BEV最佳甜菜碱浓度（1.0 mol/L）

Fig. 3 Betaine concentration optimization resultsM: DL2000 DNA marker. NC: Negative control. A: Optimal betaine concentration for BVDV (1.0 mol/L). B: Optimal betaine concentration for BEV (1.0 mol/L)

图4 探针浓度和引物浓度优化结果A：BVDV最佳探针浓度（40 nmol/L）；B：BEV最佳探针浓度（40 nmol/L）；C：BVDV和BEV最佳引物浓度配比（300 nmol/L∶100 nmol/L）

Fig. 4 Optimization of probe and primer concentrationsA: Optimal probe concentration for BVDV (40 nmol/L). B: Optimal probe concentration for BEV (40 nmol/L). C: Optimal primer concentration ratio of BVDV to BEV (300 nmol/L∶100 nmol/L)

图5 反应时间和温度优化结果A：最佳反应时间（20 min）；B：最佳反应温度（42 ℃）

Fig. 5 Optimization of reaction time and temperatureA: Optimal reaction time (20 min). B: Optimal reaction temperature (42 ℃)

图6 特异性、灵敏度和重复性试验结果A：特异性试验；B：BVDV灵敏度（1×100 copies/μL）和BEV灵敏度（1×101 copies/μL）；C：重复性试验

Fig. 6 Results of specificity, sensitivity and repeatability testsA: Specificity test. B: Sensitivity of BVDV (1×100 copies/μL) and BEV (1×101 copies/μL). C: Repeatability test

图7 单盲测试结果

Fig. 7 Single-blind test results

图8 BVDV和BEV双重RAA-LFD可视化检测

Fig. 8 Visual detection of BVDV and BEV by duplex RAA-LFD

图9 BVDV和BEV qPCR检测阳性结果

Fig. 9 Positive qPCR results for BVDV and BEV

图10 临床样品检测对比结果

Fig. 10 Comparison of detection results for clinical samples

参考文献 33

[1]	丁鼎, 丁晓军, 穆萨·热合曼, 等. 新疆部分规模化牛场犊牛腹泻病毒性病原检测与流行特点分析 [J]. 中国畜牧兽医, 2025(3): 1272-1280.
	Ding D, Ding XJ, Musa R, et al. Analysis on viral pathogen detection and epidemic characteristics of calf diarrhea in some large-scale cattle farms in Xinjiang [J]. China Anim Husb Vet Med, 2025(3): 1272-1280.
[2]	陈伯祥, 王佳, 赵子惠, 等. 牛病毒性腹泻/黏膜病病原学特征及其检测方法研究进展 [J]. 畜牧兽医杂志, 2025, 44(2): 97-102.
	Chen BX, Wang J, Zhao ZH, et al. Research progress on etiological characteristics and pathogen detection methods of bovine viral diarrhea-mucosal disease [J]. J Anim Sci Vet Med, 2025, 44(2): 97-102.
[3]	庄金秋,梅建国,刘吉山,等.牛肠道病毒的病原学特征及检测技术研究进展 [J].中国奶牛, 2018, (6): 29-31.
	Zhuang JQ, Mei JG, Liu JS, et al. Research progress on the pathogenic characteristics and detection techniques of the Bovine enterovirus [J]. China Dairy Cattle, 2018, (06): 29-31.
[4]	Chang XR, Zhang ZY, Cui XY, et al. Genetic diversity and recombination of bovine enterovirus strains in China [J]. Microbiol Spectr, 2024, 12(3): e02800-e02823.
[5]	Baker JC. The clinical manifestations of bovine viral diarrhea infection [J]. Vet Clin N Am Food Anim Pract, 1995, 11(3): 425-445.
[6]	刘泽余, 刘占悝, 李智杰, 等. 吉林省部分牛场BVDV、IBRV、BRSV的感染情况调查 [J]. 畜牧与兽医, 2019, 51(12): 101-105.
	Liu ZY, Liu ZK, Li ZJ, et al. Investigation of BVDV, IBRV and BRSV infection in some cattle farms of Jilin Province [J]. Anim Husb Vet Med, 2019, 51(12): 101-105.
[7]	董坤, 胡俊英, 张芷源, 等. 吉林省牛病毒性腹泻病毒与牛肠道病毒混合感染的流行病学调查 [J]. 中国兽医学报, 2022, 42(6): 1143-1148.
	Dong K, Hu JY, Zhang ZY, et al. Co-infection of bovine viral diarrhea virus with bovine enterovirus among cattle herds in Jilin Province [J]. Chin J Vet Sci, 2022, 42(6): 1143-1148.
[8]	常丽云, 刘志勇, 秦建华, 等. 河北省唐山市奶犊牛病毒性腹泻流行病学调查 [J]. 中国畜牧兽医, 2021, 48(11): 4213-4219.
	Chang LY, Liu ZY, Qin JH, et al. Epidemiological investigation of viral diarrhea of dairy calves in Tangshan City, Hebei Province [J]. China Anim Husb Vet Med, 2021, 48(11): 4213-4219.
[9]	张颖慧, 岳华, 马艳君, 等. 肉牛呼吸道疾病综合征的病原学检测 [J]. 西南民族大学学报: 自然科学版, 2019, 45(5): 467-473.
	Zhang YH, Yue H, Ma YJ, et al. Pathogens detection of bovine respiratory disease complex in beef cattle [J]. J Southwest Minzu Univ Nat Sci Ed, 2019, 45(5): 467-473.
[10]	古丽巴哈尔·图尔荪, 张福慧, 张群, 等. 新疆地区牛肠道病毒的病毒分离鉴定及毒株差异 [J]. 中国兽医学报, 2022, 42(5): 873-878.
	Gulbahar·T, Zhang FH, Zhang Q, et al. Characterization of bovine enterovirus strains and molecular difference from Xinjiang autonomous region [J]. Chin J Vet Sci, 2022, 42(5): 873-878.
[11]	李英利, 常继涛, 于力. 牛肠道病毒的分离鉴定 [J]. 中国预防兽医学报, 2011, 33(5): 388-390.
	Li YL, Chang JT, Yu L. Isolation and identification of bovine enterovirus in China [J]. Chin J Prev Vet Med, 2011, 33(5): 388-390.
[12]	王郅杰, 白云龙, 夏成, 等. 黑龙江省规模化牧场奶牛常见病调查分析 [J]. 中国奶牛, 2023(4): 27-32.
	Wang ZJ, Bai YL, Xia C, et al. Investigation and analysis on the occurrence of common diseases of dairy cows in scale pasture in Heilongjiang Province [J]. China Dairy Cattle, 2023(4): 27-32.
[13]	Oliveira TES, Scuisato GS, Pelaquim IF, et al. The participation of a malignant catarrhal fever virus and Mycoplasma bovis in the development of single and mixed infections in beef and dairy cattle with bovine respiratory disease [J]. Front Vet Sci, 2021, 8: 691448.
[14]	MacKay IM. Real-time PCR in the microbiology laboratory [J]. Clin Microbiol Infect, 2004, 10(3): 190-212.
[15]	Basera SS, Singh R, Vaid N, et al. Detection of rotavirus infection in bovine calves by RNA-PAGE and RT-PCR [J]. Indian J Virol, 2010, 21(2): 144-147.
[16]	Monteiro FL, Cargnelutti JF, Martins B, et al. Detection of bovine pestiviruses in sera of beef calves by a RT-PCR based on a newly designed set of pan-bovine pestivirus primers [J]. J VET Diagn Invest, 2019, 31(2): 255-258.
[17]	金美君, 胡云皓, 辛凌翔, 等. 11种致牛腹泻病原的一步法多重PCR/RT-PCR检测方法的建立 [J]. 微生物学通报, 2025, 52(1): 383-396.
	Jin MJ, Hu YH, Xin LX, et al. A one-step multiplex PCR/RT-PCR method for detecting 11 pathogens causing bovine diarrhea [J]. Microbiol China, 2025, 52(1): 383-396.
[18]	高睿, 徐伟, 罗艳, 等. 4种致牛腹泻病毒多重荧光定量PCR检测方法的建立及应用 [J]. 西北农林科技大学学报: 自然科学版, 2023, 51(10): 20-28.
	Gao R, Xu W, Luo Y, et al. Establishment and application of multiple fluorescent quantitative PCR for detection of 4 bovine diarrhea viruses [J]. J Northwest A&F Univ Nat Sci Ed, 2023, 51(10): 20-28.
[19]	孙吉, 岳华, 汤承. 检测牛5种腹泻病毒的多重RT-PCR方法的建立及应用 [J]. 畜牧兽医学报, 2022, 53(1): 209-218.
	Sun J, Yue H, Tang C. Establishment and application of multiplex RT-PCR for detection of five bovine diarrhea viruses [J]. Acta Vet Zootechnica Sin, 2022, 53(1): 209-218.
[20]	Guo YN, Leil X, Wan JS, et al. Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for detection of bovine viral diarrhea virus [J]. J Biotech Res, 2023, 15: 410-418.
[21]	毛迎雪, 刘蒙达, 张皓博, 等. 重组酶介导等温扩增技术(RAA)在病原微生物检测中的应用进展 [J]. 中国动物检疫, 2024, 41(1): 60-66.
	Mao YX, Liu MD, Zhang HB, et al. Advances on application of recombinase aided amplification in detection of pathogenic microorganisms [J]. China Anim Health Insp, 2024, 41(1): 60-66.
[22]	Piepenburg O, Williams CH, Stemple DL, et al. DNA detection using recombination proteins [J]. PLoS Biol, 2006, 4(7): e204.
[23]	国家市场监督管理总局, 国家标准化管理委员会. 牛病毒性腹泻/粘膜病诊断技术规范: [S]. 北京: 中国标准出版社, 2018.
	State Administration for Market Regulation, Standardization Administration of the People’s Republic of China. Technical specifications for diagnosis of bovine viral diarrhea/mucosal disease: [S]. Beijing: Standards Press of China, 2018.
[24]	陆泳锟, 张德雄, 王晓虎, 等. 基于5'UTR基因的牛肠道病毒TaqMan探针实时荧光定量PCR检测方法的建立 [J]. 病毒学报, 2025, 41(5): 1488-1498.
	Lu YK, Zhang DX, Wang XH, et al. Development of a TaqMan probe based real-time quantitative PCR assay for the detection of bovine enterovirus targeting the 5'UTR gene [J]. Chin J Virol, 2025, 41(5): 1488-1498.
[25]	尹柳祎, 王建龙, 郭宇, 等. 牛肠道病毒Syber Green Ⅰ荧光定量PCR检测方法的建立 [J]. 中国病原生物学杂志, 2024, 19(10): 1177-1181.
	Yin LY, Wang JL, Guo Y, et al. Establishment of a Syber Green I fluorescence quantitative PCR method for the detection of bovine enterovirus [J]. Journal of Pathogen Biology, 2024, 19(10): 1177-1181.
[26]	闫志浩, 杨柳菲, 曹馨月, 等. 牛病毒性腹泻病毒和牛肠道病毒二重PCR检测方法的建立及病原学分析 [J]. 养殖与饲料, 2025, 24(10): 52-56.
	Yan ZH, Yang LF, Cao XY, et al. Establishment of a duplex PCR detection method for bovine viral diarrhea virus and bovine enterovirus and pathogeny analysis [J]. Anim Breed Feed, 2025, 24(10): 52-56.
[27]	张福慧, 郑学博, 崔续媛, 等. 检测牛肠道病毒RT-RAA-LFD方法的建立与初步应用 [J]. 中国兽医学报, 2024, 44(11): 2348-2355.
	Zhang FH, Zheng XB, Cui XY, et al. Establishment and preliminary application of RT-RAA-LFD method for the detectionof bovine Enteroviruses [J]. Chin J Vet Sci, 2024, 44(11): 2348-2355.
[28]	李惠惠, 董可儿, 刘馨博, 等. 牛诺瓦病毒和牛轮状病毒双重RAA-LFD快速检测方法的建立及应用 [J]. 畜牧兽医学报, 2024, 55(1): 406-412.
	Li HH, Dong KE, Liu XB, et al. Establishment and application of rapid detection method for bovine norovirus and bovine rotavirus dual RAA-LFD [J]. Acta Vet Zootechnica Sin, 2024, 55(1): 406-412.
[29]	Craw P, Balachandran W. Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review [J]. Lab Chip, 2012, 12(14): 2469.
[30]	Li J, MacDonald J, von Stetten F. Review: a comprehensive summary of a decade development of the recombinase polymerase amplification [J]. Analyst, 2019, 144(1): 31-67.
[31]	Wang Y, Wang Y, Lan RT, et al. Multiple endonuclease restriction real-time loop-mediated isothermal amplification a novel analytically rapid, sensitive, multiplex loop-mediated isothermal amplification detection technique [J]. J Mol Diagn, 2015, 17(4): 392-401.
[32]	Zhang N, Hou LW, Li DR, et al. Establishment and application of duplex recombinase-aided amplification combined with lateral flow dipsticks for rapid and simultaneous visual detection of Klebsiella pneumoniae and Staphylococcus aureus in milk [J]. Foods, 2025, 14(4): 573.
[33]	田兴苗, 王健霖, 郭磊, 等. 鸡毒支原体和滑液囊支原体双重LFD-RPA快速检测方法的建立 [J]. 生物技术通报, 2024(7): 117-124.
	Tian XM, Wang JL, Guo L, et al. Establishment of dual LFD-RPA rapid detection method for Mycoplasma gallisepticum and Mycoplasma synoviae [J]. Biotechnol Bull, 2024(7): 117-124.