羊痘病毒与羊口疮病毒双重RPA-LFD检测方法的建立与应用

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

摘要/Abstract

摘要：

【目的】旨在建立一种基于重组聚合酶（recombinase polymerase amplification, RPA）检测技术结合测流层析试纸条（lateral flow dipstick, LFD）快速鉴别检测羊痘病毒（capripox virus, CaPV）与羊口疮病毒（orf virus, ORFV）的双重RPA-LFD检测方法。【方法】选取CaPV P32基因及ORFV 011基因的保守片段，进行目的片段扩增并构建重组质粒；设计CaPV及ORFV RPA引物各3对，CaPV-HP、ORFV-HP探针各1条；进行单重基础RPA引物筛选试验；对双重RPA-LFD的反应时间及反应温度进行优化；探索双重RPA-LFD最佳引物配比体系及最佳探针配比体系；进行双重RPA-LFD灵敏度、特异性及重复性试验；用所建立的方法对采集的55份临床样品进行检测。【结果】引物筛选试验结果显示，引物对CaPV-RPA-F3/R3、ORFV-RPA-F1/R1的特异性最强、扩增效率最高；该方法在反应温度为39℃、反应时间为11 min的反应条件下扩增效率最佳；CaPV-RPA-F3/R3、ORFV-RPA-F1/R1的最佳引物配比为0.8 μL∶1.6 μL，CaPV-HP、ORFV-HP的最佳探针配比为0.1 μL∶0.9 μL；双重RPA-LFD灵敏性试验最低检出限为CaPV/ORFV 4.65/3.94×10⁰ copies/μL；特异性试验结果显示与PPRV、IBRV、Sau、SPN均无交叉反应；临床样品检测结果显示RPA-LFD检测阳性率与PCR检测阳性率相符合。【结论】成功建立了CaPV与ORFV双重RPA-LFD快速检测方法，可用于临床中CaPV与ORFV混合感染的快速鉴别诊断。

关键词: 羊痘病毒, 羊口疮病毒, RPA-LFD, 鉴别诊断, 混合感染

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

王玲玲, 马爱红, 宋前进, 王小龙, 曹晓真, 刘治凤, 陈亚飞, 李继东. 羊痘病毒与羊口疮病毒双重RPA-LFD检测方法的建立与应用[J]. 生物技术通报, 2024, 40(5): 103-111.

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.

图/表 7

表1 引物及探针序列

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

图1 重组质粒构建结果 a：CaPV P32基因扩增结果（M：DNA DL1000；1：P32基因）；b：ORFV 011基因扩增结果（M：DNA DL1000；1：011基因）

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）

图2 CaPV和ORFV引物筛选 a：CaPV引物筛选（M：DNA DL500；1-6：CaPV-RPA-F1/R1；8-13：CaPV-RPA-F2/R2；15-20：CaPV-RPA-F3/R3；7、14、21：阴性对照）；b：ORFV引物筛选（M：DNA DL500；1-6：ORFV-RPA-F1/R1；8-13：ORFV-RPA-F2/R2；15-20：ORFV-RPA-F3/R3；7、14、21：阴性对照）

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）

图3 反应条件优化结果 a：反应时间优化（1-7：5、7、9、11、13、15 min、阴性对照）；b：反应温度优化（1-9：25、30、35、37、39、42、47、52℃、阴性对照）；c：引物配比优化（1-5：CaPV-RPA-F3/R3∶ORFV-RPA-F1/R1（μL）：0.8∶1.6；1∶1.4；1.2∶1.2；1.4∶1；1.6∶0.8）；d：探针配比优化（1-5：CaPV-HP∶ORFV-HP（μL）：0.1∶0.9；0.3∶0.7；0.5∶0.5；0.7∶0.3；0.9∶0.1）。C：质控线；T1：CaPV；T2：ORFV；下同

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

图4 灵敏度（a）和特异性（b）试验结果

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

图5 重复性试验 a：组内重复试验（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：阴性对照）；b：组间重复试验（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：阴性对照）

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）

表2 PCR与RPA-LFD对临床55个样本检测结果分析

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

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