Establishment and Application of Dual RPA-LFD Detection Method for Capripox Virus and Orf Virus

doi:10.13560/j.cnki.biotech.bull.1985.2023-1106

Abstract

Abstract:

【Objective】Based on recombinase polymerase amplification（RPA）detection technology combined with（lateral flow dipstick, LFD），it is aimed to establish dual RPA-LFD method for the rapid identification of capripox virus（CaPV）and orf virus（ORFV）. 【Method】The conserved fragments of CaPV P32 gene and ORFV 011 gene were selected for the amplification of target fragments and the construction of recombinant plasmids. Three pairs of primers for CaPV and ORFV RPA, and one probe for CAPV-HP and ORFV-HP were designed. Single base RPA primer screening test was performed. The reaction time and temperature of double RPA-LFD were optimized. The optimal primers and probe matching systems of dual RPA-LFD were explored. Dual RPA-LFD sensitivity, specificity and repeatability tests were performed. The method was used to detect 55 clinical samples. 【Result】The results of primer screening test showed that the primers had the strongest specificity and the highest amplification efficiency for CaPV-RPA-F3/R3 and ORFV-RPA-F1/R1. The method had the best amplification efficiency at reaction temperature of 39℃ and reaction time of 11 min. The optimal primer ratio of CaPV-RPA-F3/R3 and ORFV-RPA-F1/R1 was 0.8 μL∶1.6 μL, and the optimal probe ratio of CaPV-HP and ORFV-HP was 0.1 μL∶0.9 μL. The minimum detection limit of dual RPA-LFD sensitivity test was CaPV/ORFV 4.65/3.94×10⁰ copies/μL. The specific test results showed no cross-reaction with PPRV, IBRV, Sau and SPN. The positive rate of RPA-LFD was consistent with that of PCR. 【Conclusion】This study successfully established a dual RPA-LFD detection method for CaPV and ORFV, which can be used for the rapid differential diagnosis of CaPV and ORFV mixed infection in clinic.

Key words: CaPV, ORFV, RPA-LFD, differential diagnosis, mixed infection

WANG Ling-ling, MA Ai-hong, SONG Qian-jin, WANG Xiao-long, CAO Xiao-zhen, LIU Zhi-feng, CHEN Ya-fei, LI Ji-dong. Establishment and Application of Dual RPA-LFD Detection Method for Capripox Virus and Orf Virus[J]. Biotechnology Bulletin, 2024, 40(5): 103-111.

Figures/Tables 7

Table 1 Primers and probe sequences

引物 Primer	上游和下游引物 Upstream and downstream primers	引物序列 Primer sequence（5'-3'）	目标长度 Target length/bp
CaPV-PCR	F1	GGTCGCGAAATTTCAGATGTAG	503
CaPV-PCR	R1	TCATATCCCCCTGTGTACGAAT	503
ORFV-PCR	F1	TCCACTATCAAGAACCTCGGGC	709
ORFV-PCR	R1	TTATTGGCTTGCAGAACTCCGA	709
CaPV-RPA	F1	TTATATGGGAAAAGGTAGAAAAATCAGGAGG	287
CaPV-RPA	R1	CTATTATGAGAAGTTTCACGTAATTGGAAAATG	287
CaPV-RPA	F2	CAAAACACTTTAGTTTATGGAAATCGTATG	184
CaPV-RPA	R2	CTATTATGAGAAGTTTCACGTAATTGGAAA	184
CaPV-RPA	F3	CTTATATGGGAAAAGGTAGAAAAATCAGGAGG	289
CaPV-RPA	R3	Biotin-TTATGAGAAGTTTCACGTAATTGGAAAATGTC	289
ORFV-RPA	F1	CACCAACAAGCACCTGGCCTGGGACCTCATGAAC	170
ORFV-RPA	R1	Biotin-GCGAGTCAGAGAAGAATACGCCGCCCCCGGAGTG	170
ORFV-RPA	F2	CCACCAACAAGCACCTGGCCTGGGACCTCATGAAC	198
ORFV-RPA	R2	TGCGGTAGAAGCCTAAGAAGCGCTCCGGCGAGTCA	198
ORFV-RPA	F3	GCΑCCGCΑTΑGΑGΑΑCGCCΑΑGΑΑCΑGCΑTC	176
ORFV-RPA	R3	CCGCGTTCTTCCACTCGGTGATGATCACG	176
CaPV-HP	Probe	Digoxin-CTΑTTGCΑΑΑΑCΑCTTTΑGTTTΑTGGΑΑΑT/idsp/GTΑTGCCGΑTGCGGΑT-C3-spacer	47
ORFV-HP	Probe	FΑM-GCTCTGCTGCGCCGTCGTCΑCGCCCΑCGGC/idsp/ ΑCGΑΑCTTCCΑCCTCΑ-C3-spacer	47

Fig. 1 Results of recombinant plasmid construction a: Results of CaPV P32 gene（M: DNA marker DL1000; 1: P32 gene）. b: Results of ORFV 011 gene（M: DNA marker DL1000; 1: 011 gene）

Fig. 2 Primer screening of CaPV and ORFV a: Primer screening of CaPV（M: DNA marker DL500; 1-6: CaPV-RPA-F1/R1; 8-13: CaPV-RPA-F2/R2; 15-20: CaPV-RPA-F3/R3; 7, 14, 21: negative control）.b: Primer screening of ORFV（M: DNA marker DL500; 1-6: ORFV-RPA-F1/R1; 8-13: ORFV-RPA-F2/R2; 15-20: ORFV-RPA-F3/R3; 7, 14, 21: negative control）

Fig. 3 Results of reaction condition optimization a: Optimization of reaction time（1-7: 5, 7, 9, 11, 13, 15 min, negative control）. b: Optimization of reaction temperature（1-9: 25, 30, 35, 37, 39, 42, 47, 52℃, negative control）. c: Optimization of primer ratio（1-5: CaPV-RPA-F3/R3: ORFV-RPA-F1/R1: 0.8∶1.6; 1∶1.4; 1.2∶1.2; 1.4∶1; 1.6∶0.8）. d: Optimization of probe ratio（1-5: CaPV-HP: ORFV-HP: 0.1∶0.9; 0.3∶0.7; 0.5∶0.5; 0.7∶0.3; 0.9∶0.1）. C: Quality control line; T1: CaPV; T2: ORFV. The same below

Fig. 4 Sensitivity（a）and specificity（b）test results a: 1-9（CaPV/ORFV）: 4.65/3.94×107 copies/μL; 4.65/3.94×106 copies/μL; 4.65/3.94×105 copies/μL; 4.65/3.94×104 copies/μL; 4.65/3.94×103 copies/μL; 4.65/3.94×102 copies/μL; 4.65/3.94×101 copies/μL; 4.65/3.94×100; negative control. b: 1-8: CaPV ORFV; CaPV; ORFV; PPRV; IBRV; Sau; SPN; negative control

Fig. 5 Experiment for repeatability a: Repeated experiment within the group（1-3: CaPV/ORFV: 4.65/3.94×105 copies/μL; 5-7: CaPV/ORFV: 4.65/3.94×106 copies/μL; 9-11: CaPV/ORFV: 4.65/3.94×107 copies/μL; 4, 8, 12: negative control）. b: Repeated experiments between groups（1: CaPV/ORFV: 4.65/3.94×107 copies/μL; 2: CaPV/ORFV: 4.65/3.94×106 copies/μL; 3: CaPV/ORFV: 4.65/3.94×105 copies/μL; 4: negative control）

Table 2 Analysis of PCR and RPA-LFD results of 55 clinical samples

检测方法 Detection method	CaPV阳性病例数 Number of CaPV positive cases	ORFV阳性病例数 Number of ORFV positive cases	CaPV、ORFV阳性病例数 Number of CaPV and ORFV positive cases
PCR	5	3	3
RPA-LFD	5	3	3

References 30

[1]	刘芳. 一起疑似羊痘病毒感染的实验室诊断及病毒分离鉴定[D]. 长春: 吉林大学, 2020.
	Liu F. Laboratory diagnosis and virus isolation and identification of a suspected sheep pox virus infection[D]. Changchun: Jilin University, 2020.
[2]	Aregahagn S, Tadesse B, Tegegne B, et al. Spatiotemporal distributions of sheep and goat pox disease outbreaks in the period 2013-2019 in eastern Amhara region, Ethiopia[J]. Vet Med Int, 2021, 2021: 6629510.
[3]	刘宇馨. 中国及周边地区绵羊痘和山羊痘的时空流行特征及环境风险因素研究[D]. 哈尔滨: 东北农业大学, 2022.
	Liu YX. Temporal and spatial epidemic characteristics and environmental risk factors of sheep pox and goat pox in China and its surrounding areas[D]. Harbin: Northeast Agricultural University, 2022.
[4]	Hamdi J, Bamouh Z, Jazouli M, et al. Experimental infection of indigenous North African goats with goatpox virus[J]. Acta Vet Scand, 2021, 63(1): 9.
[5]	Hamdi J, Munyanduki H, Tadlaoui KO, et al. Capripoxvirus infections in ruminants: a review[J]. Microorganisms, 2021, 9(5): 902.
[6]	Muhsen M, Protschka M, Schneider LE, et al. Orf virus(ORFV)infection in a three-dimensional human skin model: characteristic cellular alterations and interference with keratinocyte differentiation[J]. PLoS One, 2019, 14(1): e0210504.
[7]	张成. 羊传染性脓疱病毒感染新西兰兔模型的建立[D]. 阿拉尔: 塔里木大学, 2022.
	Zhang C. Establishment of A New Zealand rabbit model infected with infectious goat impetigo virus[D]. Ala'er: Tarim University, 2022.
[8]	王改丽. ORFV感染宿主细胞的mRNA表达谱变化及其诱导细胞自噬机制的研究[D]. 长春: 吉林大学, 2015.
	Wang GL. The changes of mRNA expression profile in host cells infected with ORFV and the mechanism of ORFV-induced cell autophagy[D]. Changchun: Jilin University, 2015.
[9]	姚晓婷. 羊口疮病毒的遗传演化及其在与山羊细胞互作中的双向转录组研究[D]. 杨凌: 西北农林科技大学, 2022.
	Yao XT. Genetic evolution of sheep mouth sore virus and its two-way transcriptome study in interaction with goat cells[D]. Yangling: Northwest A & F University, 2022.
[10]	Bukar AM, Jesse FFA, Abdullah CAC, et al. Immunomodulatory strategies for parapoxvirus: current status and future approaches for the development of vaccines against orf virus infection[J]. Vaccines, 2021, 9(11): 1341.
[11]	何师师. 羊传染性脓疱病毒ORF120基因缺失株的免疫效果评价[D]. 长春: 吉林大学, 2023.
	He SS. Evaluation of immune effect of ORF120 gene deletion strain of infectious pustular virus in sheep[D]. Changchun: Jilin University, 2023.
[12]	刘昂, 程逸文, 安琪, 等. 羊源多杀性巴氏杆菌重组酶聚合酶扩增诊断方法的建立[J]. 中国畜牧兽医, 2021, 48(10): 3752-3760. doi: 10.16431/j.cnki.1671-7236.2021.10.027
	Liu A, Cheng YW, An Q, et al. Establishment of a rapid diagnostic method of RPA for Pasteurella multocida from goat[J]. China Anim Husb Vet Med, 2021, 48(10): 3752-3760.
[13]	Tan MY, Liao C, Liang LN, et al. Recent advances in recombinase polymerase amplification: principle, advantages, disadvantages and applications[J]. Front Cell Infect Microbiol, 2022, 12: 1019071.
[14]	Wang F, Ge DB, Wang L, et al. Rapid and sensitive recombinase polymerase amplification combined with lateral flow strips for detecting Candida albicans[J]. Anal Biochem, 2021, 633: 114428.
[15]	Liu LB, Wang JF, Nie FP, et al. Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus[J]. J Virol Methods, 2023, 320: 114788.
[16]	周峰, 王志宇, 张梅, 等. 绵羊痘病毒和山羊痘病毒通用RPA检测方法的建立与优化[J]. 中国兽医科学, 2022, 52(7): 830-836.
	Zhou F, Wang ZY, Zhang M, et al. Establishment and optimization of a RPA diganostic method for sheep pox virus and goat pox virus[J]. Chin Vet Sci, 2022, 52(7): 830-836.
[17]	Onchan W, Ritbamrung O, Changtor P, et al. Sensitive and rapid detection of Babesia species in dogs by recombinase polymerase amplification with lateral flow dipstick(RPA-LFD)[J]. Sci Rep, 2022, 12(1): 20560.
[18]	张珊珊, 李楠, 郝镯, 等. 布鲁氏菌RPA-LFD快速检测方法的建立与应用[J]. 中国兽医科学, 2021, 51(10): 1215-1220.
	Zhang SS, Li N, Hao Z, et al. Development and evaluation of a rapid visualization detection method for Brucella based on RPA-LFD[J]. Chin Vet Sci, 2021, 51(10): 1215-1220.
[19]	Haegeman A, De Vleeschauwer A, De Leeuw I, et al. Overview of diagnostic tools for capripox virus infections[J]. Prev Vet Med, 2020, 181: 104704.
[20]	岳怀宁, 李艳蕊, 张亚楠, 等. 非洲猪瘟病毒可视化重组酶辅助扩增检测方法的建立与初步应用[J]. 中国兽医杂志, 2023, 59(5): 19-25.
	Yue HN, Li YR, Zhang YN, et al. Establishment and preliminary application of visual recombinase aided amplification method for detection of African swine fever virus[J]. Chin J Vet Med, 2023, 59(5): 19-25.
[21]	王姝然, 范厚勇, 徐嘉楠, 等. 弗氏柠檬酸杆菌RPA-LFD快速检测方法的建立及应用[J]. 大连海洋大学学报, 2024, 39(2):241-249.
	Wang SR, Fan HY, Xu JN, et al. Establishment and application of rapid detection method for Citrobacter Frei RPA-LFD[J]. J Dalian Ocean Univ, 2024, 39(2):241-249.
[22]	Pang F, Long QQ. Recent advances in diagnostic approaches for orf virus[J]. Appl Microbiol Biotechnol, 2023, 107(5/6): 1515-1523.
[23]	Murray L, Edwards L, Tuppurainen ESM, et al. Detection of capripoxvirus DNA using a novel loop-mediated isothermal amplification assay[J]. BMC Vet Res, 2013, 9: 90. doi: 10.1186/1746-6148-9-90 pmid: 23634704
[24]	Das A, Babiuk S, McIntosh MT. Development of a loop-mediated isothermal amplification assay for rapid detection of capripoxviruses[J]. J Clin Microbiol, 2012, 50(5): 1613-1620. doi: 10.1128/JCM.06796-11 pmid: 22357504
[25]	Yang Y, Qin XD, Wang GX, et al. Development of a fluorescent probe-based recombinase polymerase amplification assay for rapid detection of orf virus[J]. Virol J, 2015, 12: 206. doi: 10.1186/s12985-015-0440-z pmid: 26631157
[26]	朱宇翔, 秦嘉超, 季英华, 等. 菜豆黄花叶病毒RPA-LFD技术快速检测方法的建立与应用[J]. 江苏农业科学, 2023, 51(14): 70-75.
	Zhu YX, Qin JC, Ji YH, et al. Establishment and application of rapid detection method of bean yellow mosaic virus RPA-LFD technology[J]. Jiangsu Agric Sci, 2023, 51(14): 70-75.
[27]	陈思楠, 王金凤, 张倩, 等. 猪圆环病毒2型可视化RPA快速检测方法的建立[J]. 中国兽医科学, 2022, 52(3): 304-309.
	Chen SN, Wang JF, Zhang Q, et al. Development of the visual RPA assay for rapid detection of porcine circovirus 2[J]. Chin Vet Sci, 2022, 52(3): 304-309.
[28]	Zheng M, Liu Q, Jin NY, et al. A duplex PCR assay for simultaneous detection and differentiation of capripox virus and orf virus[J]. Mol Cell Probes, 2007, 21(4): 276-281.
[29]	Venkatesan G, Balamurugan V, Bhanuprakash V. TaqMan based real-time duplex PCR for simultaneous detection and quantitation of capripox and orf virus genomes in clinical samples[J]. J Virol Methods, 2014, 201: 44-50. doi: 10.1016/j.jviromet.2014.02.007 pmid: 24552953
[30]	Liao L, Shi W, Ma C, et al. Duplex detection of Vibrio cholerae and Vibrio parahaemolyticus by real-time recombinase polymerase amplification[J]. Biomed Environ Sci, 2022, 35(12): 1161-1165. doi: 10.3967/bes2022.148 pmid: 36597298