基于RAA-CRISPR/Cas12a牛病毒性腹泻病毒可视化检测方法的建立

doi:10.7668/hbnxb.20196198

华北农学报 ›› 2026, Vol. 41 ›› Issue (5): 218-229. doi: 10.7668/hbnxb.20196198

所属专题： 植物保护；畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

基于RAA-CRISPR/Cas12a牛病毒性腹泻病毒可视化检测方法的建立

张露¹, 陈思旭¹, 张东锦¹, 杨薇¹, 李亚鑫¹, 付玉和², 傲德漠³, 张宇飞¹^,⁴

¹ 内蒙古农业大学兽医学院, 内蒙古呼和浩特 010018
² 浙江领与生物科技有限公司, 浙江杭州 310051
³ 沈阳工学院生命工程学院, 辽宁沈阳 110036
⁴ 农业农村部动物疾病临床诊疗技术重点实验室, 内蒙古呼和浩特 010018

出版日期:2026-11-04
通讯作者:
张宇飞(1988-),男,内蒙古呼和浩特人,讲师,博士,硕士生导师,主要从事病原微生物致病机理及新型疫苗开发研究。
作者简介:
张露(2001-),女,山西朔州人,在读硕士,主要从事病原微生物快速诊断技术开发。
基金资助:
国家自然科学基金项目(32460873); 内蒙古自治区教育厅一流学科科研专项(YLXKZX-NND-025); 内蒙古自治区教育厅一流学科科研专项(YLXKZX-NND-012); 内蒙古自治区自然科学基金项目(2024LHMS03041); 草原英才工程2022年以来新引进人才科研项目(RS2400000096); 内蒙古自治区高层次人才引进科研支持基金项目(DC2300001267); 高校基本科研业务费-科研创新平台能力建设专项-农业农村部动物疾病临床诊疗技术重点实验室能力建设项目(RZ2500001049)

Establishment of a Visual Detection Method for Bovine viral diarrhea virus Based on the RAA-CRISPR/Cas12a

ZHANG Lu¹, CHEN Sixu¹, ZHANG Dongjin¹, YANG Wei¹, LI Yaxin¹, FU Yuhe², AO Demo³, ZHANG Yufei¹^,⁴

¹ College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China
² Zhejiang Lingyu Bio-Sci&Tech Co., Ltd., Hangzhou 310051, China
³ College of Life Engineering, Shenyang Institute of Technology, Shenyang 110036, China
⁴ Key Laboratory of Animal Disease Clinical Diagnosis and Treatment Technology, Ministry of Agriculture and Rural Affairs, Hohhot 010018, China

Published:2026-11-04

摘要/Abstract

摘要：

为建立一种适用基层快速、简单的检测牛病毒性腹泻病毒(BVDV)的方法,将BVDV 5'UTR作为靶标区域,设计重组酶介导等温扩增(RAA)引物、探针及crRNA,构建并优化RAA-CRISPR/Cas12a检测体系,对反应条件、灵敏性、特异性及重复性进行评价,同时用RT-qPCR对100份粪便、30份血液、30份耳缘组织临床样本进行检测。结果显示,引物F3R2在43 ℃条件下RAA扩增效率最佳,CRISPR/Cas12a体系中最佳crRNA与Cas12a浓度分别为25.0,12.5 nmol/L。特异性研究表明,该方法能够特异性检测到BVDV-1a和BVDV-1b亚型,与BVDV-2 及其他常见牛病毒性腹泻的病原均无交叉反应。该方法对重组质粒标准品的最低检测限为2.1 copies/μL。重复性研究表明,批内、批间变异系数分别为 2.6%~5.8% 和 2.5%~5.5%,重复性良好。临床样本检测结果为BVDV-1b亚型感染,RT-qPCR符合率粪便样品为96%,血液样品为100%,耳缘组织样品为100%,一致性良好。综上,建立的BVDV RAA-CRISPR/Cas12a检测方法灵敏性高、特异性强,可为BVDV的现场快速检测提供可靠的技术支撑。

关键词: 牛病毒性腹泻病毒, 重组酶介导等温扩增技术, CRISPR/Cas12a, 现场诊断

Abstract:

This study aimed to establish a rapid,simple assay suitable for on-site detection of Bovine viral diarrhea virus(BVDV)in grassroots settings.The BVDV 5'UTR was selected as the target to design RAA(Recombinase-aided amplification)primers,probes,and crRNA.An RAA-CRISPR/Cas12a detection system was built and optimized,and its reaction conditions,sensitivity,specificity,and repeatability were evaluated.Clinical samples(100 feces,30 blood,30 ear tissue samples)were tested using RT-qPCR for comparison.The results showed that primer F3R2 achieved the optimal amplification efficiency for RAA at 43 ℃.Optimal crRNA and Cas12a concentrations in the CRISPR/Cas12a system were 25.0,12.5 nmol/L.Specificity tests showed the method could detect BVDV-1a and BVDV-1b subtypes without cross-reaction with BVDV-2 or other common bovine viral diarrhea pathogens.The detection limit for plasmid standards was 2.1 copies/μL.Intra- and inter-assay CVs were 2.6%-5.8%,2.5%-5.5%,showing good repeatability.Clinical sample testing revealed infection with the BVDV-1b subtype. The concordance rates relative to the RT-qPCR reference method were 96% for fecal samples, 100% for blood samples, and 100% for ear margin tissue samples, indicating good overall consistency. In summary,the RAA-CRISPR/Cas12a method for BVDV detection shows high sensitivity and excellent specificity,offering reliable technical support for rapid on-site diagnosis of BVDV.

Key words: Bovine viral diarrhea virus, Recombinase-aided amplification, CRISPR/Cas12a, On-site diagnosis

中图分类号:

S823
S852.65

张露, 陈思旭, 张东锦, 杨薇, 李亚鑫, 付玉和, 傲德漠, 张宇飞. 基于RAA-CRISPR/Cas12a牛病毒性腹泻病毒可视化检测方法的建立[J]. 华北农学报, 2026, 41(5): 218-229. doi: 10.7668/hbnxb.20196198.

ZHANG Lu, CHEN Sixu, ZHANG Dongjin, YANG Wei, LI Yaxin, FU Yuhe, AO Demo, ZHANG Yufei. Establishment of a Visual Detection Method for Bovine viral diarrhea virus Based on the RAA-CRISPR/Cas12a[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(5): 218-229. doi: 10.7668/hbnxb.20196198.

http://www.hbnxb.net/CN/Y2026/V41/I5/218

图/表 19

表1 实时荧光RAA引物序列

Tab.1 Primer sequence for real-time fluorescent RAA

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	产物大小/bp Product size
BVDV-RAA-F1	AGGCCGAAAAGAGGCTAGCCATGCCCTTAGTA	297
BVDV-RAA-R1	GTGCCATGTACAGCAGAGATTTTTAGTAGC
BVDV-RAA-F1	AGGCCGAAAAGAGGCTAGCCATGCCCTTAGTA	262
BVDV-RAA-R2	AGTGGGCCTCTGCAGCACCCTATCAGGCTGT
BVDV-RAA-F2	AAGAGGCTAGCCATGCCCTTAGTAGGACTAGC	289
BVDV-RAA-R1	GTGCCATGTACAGCAGAGATTTTTAGTAGC
BVDV-RAA-F2	AAGAGGCTAGCCATGCCCTTAGTAGGACTAGC	254
BVDV-RAA-R2	AGTGGGCCTCTGCAGCACCCTATCAGGCTGT
BVDV-RAA-F3	CCCTGAGTACAGGGTAGTCGTCAGTGGTTCG	210
BVDV-RAA-R1	GTGCCATGTACAGCAGAGATTTTTAGTAGC
BVDV-RAA-F3	CCCTGAGTACAGGGTAGTCGTCAGTGGTTCG	175
BVDV-RAA-R2	AGTGGGCCTCTGCAGCACCCTATCAGGCTGT
BVDV-RAA-F4	CTCGAGATGCCACGTGGACGAGGGCATGCCCA	161
BVDV-RAA-R1	GTGCCATGTACAGCAGAGATTTTTAGTAGC
BVDV-RAA-F4	CTCGAGATGCCACGTGGACGAGGGCATGCCCA	126
BVDV-RAA-R2	AGTGGGCCTCTGCAGCACCCTATCAGGCTGT

表2 crRNA序列

Tab.2 crRNA sequence

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
crRNA1	UAAUUUCUACUAAGUGUAGAUAAGAGGCUAGCCAUGCCCUU
crRNA2	UAAUUUCUACUAAGUGUAGAUCAUGCCCUUAGUAGGACUAG
crRNA3	UAAUUUCUACUAAGUGUAGAUUGUAAUAAGCCUCGAGAUGCCAC

图1 重组质粒标准品鉴定结果 A.重组质粒标准品PCR鉴定结果:M.DNA相对分子质量标准(DL2000),1.标准阳性质粒扩增产物;B.重组质粒标准品测序结果;C.重组质粒标准品序列比对结果。

Fig.1 Identification results of the recombinant plasmid standard A.PCR identification results of recombinant plasmid standards:M.DL2000 DNA Marker,1.Amplification product of the standard positive plasmid;B.Sequencing results of recombinant plasmid standards;C.Sequence alignment results of recombinant plasmid standards.

表3 重组质粒标准品冻融前后Ct值对比

Tab.3 Ct values of recombinant plasmid standards before and after freeze-thaw

未经过冻融的不同浓度的质粒标准品/ (copies/μL) Non-freeze-thawed plasmid standards at different concentrations	Ct值 Ct value	经过冻融的不同浓度的质粒标准品/ (copies/μL) Freeze-thawed plasmid standards at different concentrations	Ct值 Ct value
2.1×10⁹	5.0	2.1×10⁹	4.8
2.1×10⁸	8.5	2.1×10⁸	8.7
2.1×10⁷	11.8	2.1×10⁷	11.9
2.1×10⁶	15.3	2.1×10⁶	15.4
2.1×10⁵	18.9	2.1×10⁵	19.2
2.1×10⁴	22.2	2.1×10⁴	22.2
2.1×10³	25.4	2.1×10³	25.8
2.1×10²	28.7	2.1×10²	28.8
2.1×10¹	31.9	2.1×10¹	32.2
2.1×10⁰	32.9	2.1×10⁰	33.6

图2 实时荧光RAA引物筛选 1-8.F3R2、F3R1、F1R2、F2R2、F1R1、F4R1、F2R1、F4R2;9-16.阴性对照。

Fig.2 Screening of primers for Real-time fluorescent RAA 1-8.F3R2,F3R1,F1R2,F2R2,F1R1,F4R1,F2R1,F4R2;9-16.Negative controls.

图3 实时荧光RAA温度筛选结果 1-4.43,41,39,37 ℃;5-8.阴性对照。

Fig.3 Results of temperature screening for Real-time fluorescent RAA 1-4.43,41,39,37 ℃;5-8.Negative controls.

图4 实时荧光RAA灵敏性试验结果 1-7.2.1×10⁶,2.1×10⁵,2.1×10⁴,2.1×10³,2.1×10²,2.1×10¹,2.1 copies/μL;8.阴性对照。

Fig.4 The sensitivity test for Real-time fluorescent RAA 1-7.2.1×10⁶,2.1×10⁵,2.1×10⁴,2.1×10³,2.1×10²,2.1×10¹,2.1 copies/μL;8.Negative control.

图5 实时荧光RAA特异性试验结果 1-7.BVDV、IBRV、BPIV3、BRV、BCoV、Salmonella、ETEC;8.阴性对照。

Fig.5 The specificity test for Real-time fluorescent RAA 1-7.BVDV,IBRV,BPIV3,BRV,BCoV,Salmonella,ETEC;8.Negative control.

表4 实时荧光RAA重复性评估

Tab.4 Evaluation of Real-time fluorescent RAA reproducibility

质粒标准品浓度/(copies/μL) Plasmid standards concentration	批内重复性 Intra-assay repeatability
2.1×10⁵	4.5
2.1×10³	5.0

图6 crRNA筛选 A.crRNA筛选荧光图;B.crRNA筛选相对荧光强度柱状图。大写字母不同表示Tukey检验结果差异极显著(P<0.000 1)。图7,8,10同。

Fig.6 Screening of crRNA A.Fluorescence profiles of crRNA screening;B.Bar chart of relative fluorescent intensity for crRNA screening.Different capital letters indicate extremely significant differences for Tukey test(P<0.000 1).The same as Fig.7,8,10.

图7 crRNA与Cas12a浓度优化 A.crRNA与Cas12a优化荧光图;B.crRNA与Cas12a相对荧光强度柱状图;C.crRNA与Cas12a热图。

Fig.7 Optimization of crRNA and Cas12a concentration A.Optimal fluorescence images for crRNA and Cas12a;B.Bar chart of relative fluorescence intensity for crRNA and Cas12a;C.Heatmap visualization for crRNA and Cas12a.

图8 RAA-CRISPR/Cas12a反应时间筛选 A.时间筛选荧光图;B.时间筛选相对荧光强度柱状图。

Fig.8 Screening of RAA-CRISPR/Cas12a reaction time A.Time-screening fluorescent images;B.Bar chart of relative fluorescence intensity for time-screening.

图9 RAA-CRISPR/Cas12a 灵敏性试验 A.灵敏性检测荧光图;B.灵敏性检测相对荧光强度柱状图;NC.阴性对照。小写字母不同表示Tukey检验结果差异显著(P<0.05)。

Fig.9 Sensitivity test of RAA-CRISPR/Cas12a A.Sensitivity detection fluorescence images;B.Sensitivity detection bar chart of relative fluorescence intensity;NC.Negative control.Different lowercase letters indicate significant differences for Tukey test(P<0.05).

图10 RAA-CRISPR/Cas12a特异性试验 A.特异性检测荧光图;B.特异性检测相对荧光强度柱状图;C.BVDV不同型的特异性检测;D.BVDV不同型的柱状图;NC.阴性对照。

Fig.10 Specificity test of RAA-CRISPR/Cas12a A.Specificity detection fluorescence profiles;B.Specificity detection bar chart of relative fluorescence intensity;C.Specificity detection against different genotypes of BVDV;D.Bar chart of different BVDV genotypes;NC.Negative control.

表5 RAA-CRISPR/Cas12a重复性检测

Tab.5 Detection reproducibility of RAA/CRISPR-Cas12a

质粒浓度/ (copies/μL) Plasmid concentration	变异系数/% Coefficient of variation
质粒浓度/ (copies/μL) Plasmid concentration	批内重复性 Intra-assay repeatability	批间重复性 Inter-assay reproducibility
2.1×10³	2.6	2.5
2.1×10⁴	5.8	5.5

表6 基于RT-qPCR的RAA-CRISPR/Cas12a粪便样本检测结果

Tab.6 Detection results of RT-qPCR and RAA-CRISPR/Cas12a for fecal samples

检测方法 Testing method		RT-qPCR
检测方法 Testing method		阳性 Positive	阴性 Negative	合计 Total
RAA-	阳性	15	4	19
CRISPR/Cas12a	阴性	0	81	81
	合计	15	85	100

表7 基于RT-qPCR的RAA-CRISPR/Cas12a血液样本检测结果

Tab.7 Detection results of RT-qPCR and RAA-CRISPR/Cas12a for blood samples

检测方法 Testing method		RT-qPCR
检测方法 Testing method		阳性 Positive	阴性 Negative	合计 Total
RAA-	阳性	12	0	12
CRISPR/Cas12a	阴性	0	18	18
	合计	12	18	30

表8 RT-qPCR及RAA-CRISPR/Cas12a对耳缘组织样本检测结果

Tab.8 Detection results of RT-qPCR and RAA-CRISPR/Cas12a for ear tissue samples

检测方法 Testing method		RT-qPCR
检测方法 Testing method		阳性 Positive	阴性 Negative	合计 Total
RAA-	阳性	8	0	8
CRISPR/Cas12a	阴性	0	22	22
	合计	8	22	30

图11 BVDV的鉴定与系统进化分析 A.临床样本10倍梯度稀释检测结果;B.BVDV-E2基因测序结果;C.BVDV-E2基因的系统进化树。红色圆圈表示临床阳性样本。

Fig.11 Identification and phylogenetic analysis of BVDV A.Detection results of 10-fold serial dilution of fecal samples;B.Sequencing result of the BVDV-E2 gene;C.Phylogenetic tree based on the BVDV-E2 gene.Red circles in the figure indicate clinical positive samples.

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基于RAA-CRISPR/Cas12a牛病毒性腹泻病毒可视化检测方法的建立

Establishment of a Visual Detection Method for Bovine viral diarrhea virus Based on the RAA-CRISPR/Cas12a

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基于RAA-CRISPR/Cas12a牛病毒性腹泻病毒可视化检测方法的建立

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