虾肝肠胞虫重组酶聚合酶侧向流试纸条快速检测方法的建立

doi:10.11924/j.issn.1000-6850.casb2024-0775

摘要/Abstract

摘要：

虾肝肠胞虫(EHP)是一种可感染多种虾类的寄生虫，为提高该病原检测效率，本研究结合重组酶聚合酶扩增(RPA)和侧向流试纸条(LFS)技术，建立针对EHP的现场快速检测方法。根据EHP spore wall protein 1(SWP)基因保守区域，遵循RPA技术的引物探针设计原则，设计了3套RPA引物探针。通过RPA-LFS技术筛选扩增效率最高的引物探针，再优化反应温度和反应时间，建立针对EHP的快速检测方法，并对该方法的检测限、特异性、临床样品检测结果可靠性进行验证。经过优化，RPA-LFS反应体系在37℃恒温下反应15 min即可完成目的基因扩增，再使用试纸条对扩增产物进行检测，整体检测时间可控制在20 min内，检测限可达10 copies的EHP DNA，且特异性强，与白斑综合征病毒(WSSV)等水产病原核酸无交叉反应。临床样本检测结果与WOAH推荐的荧光PCR检测方法的结果一致。试验结果表明，本研究建立的EHP RPA-LFS检测方法，相对于荧光PCR和nested PCR检测方法，拥有耗时短、检测结果可见、不依赖贵重仪器、检测成本低等优势，可以实现EHP的现场快速检测，非常适合基层、小型实验室推广应用，为EHP的检测监测工作提供了新的方法。

关键词: 虾肝肠胞虫, 现场快速检测, 重组酶聚合酶扩增, 侧向流试纸条, 检测限, 特异性

Abstract:

Enterocytozoon hepatopenaei (EHP) is an intracellular parasite that can infect various shrimp species. To develop an on-site rapid detection method of EHP, recombinant polymerase chain reaction (RPA) and lateral flow strip (LFS) techniques were combined in this study. 3 pairs of primers and probes were designed targeting the highly conserved region of EHP spore wall protein 1(SWP) gene according to primer design principle of RPA. By optimizing reaction conditions and screening the best primers and probes, an RPA with lateral flow dipstick (RPA-LFS) detection method for EHP was developed in this study. Then, detection limit, specificity and reliability were detected. After optimization, the target gene was successfully amplified in reaction temperature of 37℃ for only 15 minutes. With LFS detection, the overall detection time was within 20 minutes. The detection limit of RPA-LFS was 10 copies of EHP DNA. The results of specificity test showed that this method just specifically reacted with the EHP DNA, and had no cross-reaction with other aquatic animal virus such as white spot syndrome virus (WSSV). The clinical samples detection results of the RPA-LFS method were the same as the results of real time PCR method recommended by WOAH. This EHP RPA-LFS detection method established in this study has the advantages of shorter time, visible results, no dependence on expensive instruments, and lower detection costs, compared with qPCR and nested PCR detection methods. This method can achieve on-site rapid detection of EHP and is very suitable for small laboratories, providing a new assay for EHP detection.

Key words: Enterocytozoon hepatopenaei, on-site rapid detection, recombinase polymerase amplification, lateral flow strip, detection limit, specificity

张旻, 景宏丽, 王娜, 吴绍强. 虾肝肠胞虫重组酶聚合酶侧向流试纸条快速检测方法的建立[J]. 中国农学通报, 2025, 41(20): 157-164.

ZHANG Min, JING Hongli, WANG Na, WU Shaoqiang. Development of A Recombinase Polymerase Amplification Method Combined with Lateral Flow Strip for Rapid Detection of the Enterocytozoon hepatopenaei[J]. Chinese Agricultural Science Bulletin, 2025, 41(20): 157-164.

图/表 8

编号	名称	基因序列(5′—3′)
1	RPA-LFS-1F	CCCATATGAAAGGCTTTTGGAGTTGAGCAAG
	RPA-LFS-1R	Biotin-GCCTCCAATGGTAGGGTTACTGCACTGTTAT
	RPA-LFS-1probe	5’FAM- CAGGATTTGAGGAGCGAGTAAGACCATCTATG [THF]CTGAACAGCTAGATAG-3'C3 Spacer
2	RPA-LFS-2F	CCTTACGCGGGTGGTTTTTTATCAACG
	RPA-LFS-2R	Biotin- CCAATGTAATCATCTTTTGCCGCAGCATAAC
	RPA-LFS-2probe	5'FAM- CCAAGATTTAGTAGATGAATCGATTATA [THF]ATGCCATCAATGGAATATTGG -3'C3 Spacer
3	RPA-LFS-3F	CAGAGTGTTGTTAAGGGTTTAAGTAATTACG
	RPA-LFS-3R	Biotin- GAAAAATCATTGCAAACTTCTTTAAGCTG
	RPA-LFS-3probe	5'FAM- CAAGGGAACTGTCGCTTCAGCCATGCCAGCCGTC-[THF]-TCCAGGCAACCGATG -3'C3 Spacer

表1. RPA引物探针设计与序列

图1. 引物探针的筛选 N：阴性对照；P：pUC-EHP；1：RPA-LFS-1F/1R/1probe；2：RPA-LFS-2F/2R/2probe；3：RPA-LFS-3F/3R/3probe

图2. EHP RPA-LFS检测方法反应温度的优化 N：阴性对照；1：17℃；2：22℃；3：27℃；4：32℃；5：37℃；6：42℃

图3. EHP RPA-LFS检测方法反应时间的优化

. N：阴性对照；1：5 min；2：10 min；3：15 min；4：20 min；5：25 min

图4. EHP RPA-LFS检测方法特异性检测结果 N：阴性对照；P：阳性对照（重组质粒pUC-EHP）；1：WSSV；2：CEV；3：KHV；4：GFHNV；5：EHP

图5. EHP RPA-LFS方法和荧光PCR方法检测限结果 N：阴性对照；7~0：107~100 copies模板量

图6. EHP RPA-LFS检测方法重复性结果 N：阴性对照；1~5：5份平行样品

项目	阳性样品数量/份	阴性样品数量/份
EHP RPA-LFS检测方法	7	22
EHP荧光PCR检测方法	7	22
符合率/%	100	100

表2. 临床样品检测EHP结果

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