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

doi:10.7668/hbnxb.20196198

Abstract

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

CLC Number:

S823
S852.65

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.

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URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20196198

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

Figures/Tables 19

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

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

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.

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

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

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

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.

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

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

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.

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.

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

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).

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.

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

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

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

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

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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