等温扩增技术应用于虾肝肠孢虫检测的研究进展

doi:10.14012/j.cnki.fjsc.2024.01.011

摘要/Abstract

摘要：

虾肝肠孢虫(Enterocytozoon hepatopenaei,EHP)是一种专性细胞内寄生虫,其大面积的暴发使对虾养殖业遭受巨大的经济损失,且目前尚未发现有效治疗EHP感染的特效药。EHP的检测手段主要包括组织病理学和分子生物学,其中属于分子生物学领域的等温扩增技术因具有简便性、低成本、较高的灵敏度和特异性等特点而成为潜在的田间检测方法。本文对EHP的常规检测方法进行综述,对环介导等温扩增技术和重组酶等温扩增技术及其在EHP检测中的应用进行重点介绍,以期为EHP感染的早期检测及高效防控提供参考,促进对虾养殖业的健康发展。

关键词: 虾肝肠孢虫, 等温扩增, 检测, 环介导等温扩增, 重组酶等温扩增

Abstract:

Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasite that has caused huge economic losses in shrimp aquaculture due to its large outbreaks,and no specific drugs have been found to effectively treat EHP infection.Histopathology and molecular biology are the main methods used to detect EHP.The isothermal amplification method,a key technique in molecular biology,has gained significant attention as a potential field detection method owing to its simplicity,cost-effectiveness,high sensitivity,and specificity.This paper provides a comprehensive review of conventional detection methods for EHP,including loop-mediated isothermal amplification (LAMP) and recombinase isothermal amplification (RPA),and discusses their applications in EHP detection.The aim is to offer valuable insights for the early detection,efficient prevention,and control of EHP infection,ultimately promoting the sustainable development of shrimp aquaculture.

Key words: Enterocytozoon hepatopenaei (EHP), isothermal amplification, detection, LAMP, RPA

中图分类号:

S945.4

谭念秋, 魏春梅, 文钰淞, 范晓东. 等温扩增技术应用于虾肝肠孢虫检测的研究进展[J]. 渔业研究, 2024, 46(1): 101-110.

TAN Nianqiu, WEI Chunmei, WEN Yusong, FAN Xiaodong. Research progress of isothermal amplification technique applied to the detection of Enterocytozoon hepatopenaei[J]. Journal of Fisheries Research, 2024, 46(1): 101-110.

图/表 2

参考文献 59

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检测技术 Detection techniques	优点 Advantages	缺点 Disadvantages	检测环境 Detection environment
组织病理学 Histopathology	易观察、直观	操作繁琐、灵敏度差、易漏检、易误诊	实验室
聚合酶链式反应(PCR) Polymerase chain reaction	操作简单、金标准、成本低、适合大样本检测	检测下限较高	实验室
巢氏PCR Nested PCR	灵敏度较高、特异性较好、操作简单	假阳性	实验室
实时荧光定量PCR Quantitative real-time PCR	定量分析、可重复、可分析感染程度	成本较高	实验室
数字PCR Digital PCR	精确检测、定量	成本高、仪器昂贵	实验室
原位杂交 Insitu hybridization	直观、反映感染位置及组织感染程度	费时、繁琐	实验室
环介导等温扩增 Loop-mediated isothermal amplification	简便、快速	引物设计难、易假阳性	实验室、田间
重组酶等温扩增 Recombinase polymerase amplification	简便、快速、检测下限低	易出现非特异性扩增	实验室、田间

检测技术 Detection techniques	优点 Advantages	缺点 Disadvantages	检测环境 Detection environment
组织病理学 Histopathology	易观察、直观	操作繁琐、灵敏度差、易漏检、易误诊	实验室
聚合酶链式反应(PCR) Polymerase chain reaction	操作简单、金标准、成本低、适合大样本检测	检测下限较高	实验室
巢氏PCR Nested PCR	灵敏度较高、特异性较好、操作简单	假阳性	实验室
实时荧光定量PCR Quantitative real-time PCR	定量分析、可重复、可分析感染程度	成本较高	实验室
数字PCR Digital PCR	精确检测、定量	成本高、仪器昂贵	实验室
原位杂交 Insitu hybridization	直观、反映感染位置及组织感染程度	费时、繁琐	实验室
环介导等温扩增 Loop-mediated isothermal amplification	简便、快速	引物设计难、易假阳性	实验室、田间
重组酶等温扩增 Recombinase polymerase amplification	简便、快速、检测下限低	易出现非特异性扩增	实验室、田间

基因 Genes	靶标特异性 Target specificity	引物 Primers	引物序列5’-3’ Primer sequences	检测限 Limit of detection	反应温度和时间 Reaction temperature and time		文献 Literatures
Unknow	—	FIP	CGTTTGGTTCGACAGTCCAATTTTTATGAGTAACAATA TAAAACATGA	10 copies/反应	63 ℃、 60 min		李英瑕等^[48]
		BIP	GAGGCGTCACAGAAGTAGACATTTTCCCGTATTCTCAA TGTCTACAC
		F3	GTGCAATTTAATAGGAGAACATC
		B3	GAATGGTAAGCTCCCCAC
		LF	CGTGAGAATAGTCAGTT
		LB	ACATACACCTATCATCCCGGAAG
		EHP-OF	AGGTGGGCAAAGAATGAAAT	—	60 ℃、 45 min		Cai S X等^[55]
		EHP-OB	AAGCAGCACAATCCACTC
		EHP-IF	CCCAGCATTGTCGGCATAGTATCAAGGACGAAGGCTAGA
		EHP-IB	TGTTGCGAGAGCGATGCTCCTTGCGAGCGTACTATC
		EHP-LF	AGAACTACAGCGGTGTCTAATC
SWP	特异性强	EHP-F1	TTGCAGAGTGTTGTTAAGGGTTTAAGTA	10 copies/μL	42 ℃、 20 min		Li J B等^[56]
		EHP-R1	CTTCTTTAAGCTGTTTGTCTCCAACTGTATT
		EHP-F2	TTTGGCGGCACAATTCTCAAACATTTTCACC
		EHP-R2	ACTTCTTTAAGCTGTTTGTCTCCAACTGTAT
		probe	AGAGACGATATTTACACAGACACAGCATT(i6FAM-dTa)(THFb)(iBHQ1-dTc )AGGATATGAGCTTTC-(C3-SPACERd )
		RPA-F	ACAATTTCAAACACTGTAAACCTTAAAGCA	13 copies/μL	39 ℃、 3~7 min		Ma C等^[52]
		RPA-R	TCATTCATTTTCCTTTTATCTTCTGATATG
		RPA-P	TAAAAAGAGACGATATTTACACAGACACAG[FAM-dT][THF][BHQ1-dT]ATTTGTAGGATATGAGCT
		RPA-F	CAGCTTAAAGAAGTTTGCAATG	1 copy/μL	37 ℃、1.5 h		Yang L H 等^[53]
		RPA-R	CATTTTCCTTTTATCTTCTGATATGC
		PfAgo-g1	PHO-CATTCATATGCTTTAG
		PfAgo-g2	PHO-ATATGCATTCATATGC
		PfAgo-g3	PHO-CTGATATGCATTCATA
		PfAgo-g4	PHO-CTTCTGATATGCATTC
		PfAgo-g5	PHO-TATCTTCTGATATGCA
		MB	FAM-ggtgcgTTTTATCTTCTGATATcgcacc-BHQ1
		EHP7-F3	GTAATTACGAGTTTGGCGG	10 copies/μL	60 ℃、 40 min		Kumar T S 等^[57]
		EHP7-B3	TTTCCTTTTATCTTCTGATATGCA
		EHP7-BIP	GCTGTGTCTGTGTAAATATCGTCTCTTTTAACA TTTTCACCATTGGTCA
		EHP7-FIP	ATGAGCTTTCAAATACAGTTGGAGATTTTCATATGCTTT AGAAAAATCATTGC
		EHP7 LF	ATGCTTTAAGGTTTACAGTGTTTGA
		EHP7 LR	CAAACAGCTTAAAGAAGTTT
SSU rRNA	特异性较差、易交叉污染	EHP-F3	TTTCGGGCTCTGGGGATA	10 copies/μL	65 ℃、 40 min		Ma B等^[45]
		EHP-B3	CCCCCATCAATTTCCAACGG
		EHP-FIP	AAGCAGCACAATCCACTCCTGGTTTTGCTCGCAAGGG TGAAACT
		EHP-BIP	AACGCGGGAAAACTTACCAGGGTTTTGCACCACTCTT GTCTACCTC
		EHP-LF	GTCCTTCCGTCAATTTCGCTT
		EHP-LB	TCAAGTCTATCGTAGATTGGAGACA
		EHP2-F3	TGGAGGGCAAGTTTTGGTG	10 copies/μL	65 ℃、 45 min	Kumar T S 等^[46]
		EHP2-B3	GAGCATCGCTTTCGCCTC
		EHP2-BIP	TGCATCTACGACTACGGACCC/TTTT/GCCGCGGTAAT TCCAACTC
		EHP2-FIP	AGTAGCGGAACGGATAGGGAGC/TTTT/CCAGGTGGGG TCTTGAG
		EHP2-LF	ACTGCAGCATCCACCATA
		EHP2-LR	ATGGTATAGGTGGGCAAAGAATG
		FIP	AAGCAGCACAATCCACTCCTGGGCTCGCAAGGGTGAA ACT	3.71 copies/μL	63 ℃、 40 min	孙妍等^[58]
		BIP	AACGCGGGAAAACTTACCAGGGGCACCACTCTTGTCT ACCTC
		F3	TTTCGGGCTCTGGGGATA
		B3	CCCCCATCAATTTCCAACGG
		LF	GTGTCCTTCCGTCAATTTCGC
		LB	TCAAGTCTATCGTAGATTGGAGACA
		EHP-F3	GATGCTTGGTGTGGGAGAA	1.78×101 copies/μL	65 ℃、 40 min	俞焕腾^[59]
		EHP-B3	CCCCCCATCAATTTCCAACG
		EHP-FIP	TCCTGGTAGTGTCCTTCCGTCATTTTTTTTCGGGCTCTGG GGATA
		EHP-BIP	AACGCGGGAAAACTTACCAGGGTTTTGCACCACTCTTG TCTACCTC
		EHP-LB	GTTTCACCCTTGCGAGCG
		EHP-2-F	CGTGAAGCAATTGGAGGGCAAGTTTTGGTG	10 copies/μL	37 ℃、 20 min	黄纪徽等^[26]
		EHP-2-R	CTATCCGTTCCGCTACTCTCAACAAACTCAAT
		EHP-1-P	[FAM-dT]G[THF]C[BHQ-dT] GCAGTTAAAGGG TCC
18S	特异性强	F3	ACTATGCCGACAATGCTG	440 fg/μL	63 ℃、 30 min	陈进会等^[25]
		B3	TAGAAGATCTCACTGGTTCCA
		FIP	TTAAGCAGCACAATCCACTCCTGATAGTACGCTCGCAAGC
		BIP	GCGGGAAAACTTACCAGGGTCCATGCACCACTCTTGTC
		LF	GGTAGTGTCCTTCCGTCAATT
		LB	ATCGTAGATTGGAGACATGAGG
PTP2	特异性强	EHP-F1	CACTCAAGGAATGGCTCAAGGGTTCAAAAT	50 copies/反应	37 ℃、 1.0 h(RPA)	Kanitchinda S 等^[50]
PTP2	特异性强	EHP-R1	ACCCTGTGAATTGATCATATCTCCTGCCCT	50 copies/反应	37 ℃、 1.0 h(RPA)	Kanitchinda S 等^[50]

基因 Genes	靶标特异性 Target specificity	引物 Primers	引物序列5’-3’ Primer sequences	检测限 Limit of detection	反应温度和时间 Reaction temperature and time		文献 Literatures
Unknow	—	FIP	CGTTTGGTTCGACAGTCCAATTTTTATGAGTAACAATA TAAAACATGA	10 copies/反应	63 ℃、 60 min		李英瑕等^[48]
		BIP	GAGGCGTCACAGAAGTAGACATTTTCCCGTATTCTCAA TGTCTACAC
		F3	GTGCAATTTAATAGGAGAACATC
		B3	GAATGGTAAGCTCCCCAC
		LF	CGTGAGAATAGTCAGTT
		LB	ACATACACCTATCATCCCGGAAG
		EHP-OF	AGGTGGGCAAAGAATGAAAT	—	60 ℃、 45 min		Cai S X等^[55]
		EHP-OB	AAGCAGCACAATCCACTC
		EHP-IF	CCCAGCATTGTCGGCATAGTATCAAGGACGAAGGCTAGA
		EHP-IB	TGTTGCGAGAGCGATGCTCCTTGCGAGCGTACTATC
		EHP-LF	AGAACTACAGCGGTGTCTAATC
SWP	特异性强	EHP-F1	TTGCAGAGTGTTGTTAAGGGTTTAAGTA	10 copies/μL	42 ℃、 20 min		Li J B等^[56]
		EHP-R1	CTTCTTTAAGCTGTTTGTCTCCAACTGTATT
		EHP-F2	TTTGGCGGCACAATTCTCAAACATTTTCACC
		EHP-R2	ACTTCTTTAAGCTGTTTGTCTCCAACTGTAT
		probe	AGAGACGATATTTACACAGACACAGCATT(i6FAM-dTa)(THFb)(iBHQ1-dTc )AGGATATGAGCTTTC-(C3-SPACERd )
		RPA-F	ACAATTTCAAACACTGTAAACCTTAAAGCA	13 copies/μL	39 ℃、 3~7 min		Ma C等^[52]
		RPA-R	TCATTCATTTTCCTTTTATCTTCTGATATG
		RPA-P	TAAAAAGAGACGATATTTACACAGACACAG[FAM-dT][THF][BHQ1-dT]ATTTGTAGGATATGAGCT
		RPA-F	CAGCTTAAAGAAGTTTGCAATG	1 copy/μL	37 ℃、1.5 h		Yang L H 等^[53]
		RPA-R	CATTTTCCTTTTATCTTCTGATATGC
		PfAgo-g1	PHO-CATTCATATGCTTTAG
		PfAgo-g2	PHO-ATATGCATTCATATGC
		PfAgo-g3	PHO-CTGATATGCATTCATA
		PfAgo-g4	PHO-CTTCTGATATGCATTC
		PfAgo-g5	PHO-TATCTTCTGATATGCA
		MB	FAM-ggtgcgTTTTATCTTCTGATATcgcacc-BHQ1
		EHP7-F3	GTAATTACGAGTTTGGCGG	10 copies/μL	60 ℃、 40 min		Kumar T S 等^[57]
		EHP7-B3	TTTCCTTTTATCTTCTGATATGCA
		EHP7-BIP	GCTGTGTCTGTGTAAATATCGTCTCTTTTAACA TTTTCACCATTGGTCA
		EHP7-FIP	ATGAGCTTTCAAATACAGTTGGAGATTTTCATATGCTTT AGAAAAATCATTGC
		EHP7 LF	ATGCTTTAAGGTTTACAGTGTTTGA
		EHP7 LR	CAAACAGCTTAAAGAAGTTT
SSU rRNA	特异性较差、易交叉污染	EHP-F3	TTTCGGGCTCTGGGGATA	10 copies/μL	65 ℃、 40 min		Ma B等^[45]
		EHP-B3	CCCCCATCAATTTCCAACGG
		EHP-FIP	AAGCAGCACAATCCACTCCTGGTTTTGCTCGCAAGGG TGAAACT
		EHP-BIP	AACGCGGGAAAACTTACCAGGGTTTTGCACCACTCTT GTCTACCTC
		EHP-LF	GTCCTTCCGTCAATTTCGCTT
		EHP-LB	TCAAGTCTATCGTAGATTGGAGACA
		EHP2-F3	TGGAGGGCAAGTTTTGGTG	10 copies/μL	65 ℃、 45 min	Kumar T S 等^[46]
		EHP2-B3	GAGCATCGCTTTCGCCTC
		EHP2-BIP	TGCATCTACGACTACGGACCC/TTTT/GCCGCGGTAAT TCCAACTC
		EHP2-FIP	AGTAGCGGAACGGATAGGGAGC/TTTT/CCAGGTGGGG TCTTGAG
		EHP2-LF	ACTGCAGCATCCACCATA
		EHP2-LR	ATGGTATAGGTGGGCAAAGAATG
		FIP	AAGCAGCACAATCCACTCCTGGGCTCGCAAGGGTGAA ACT	3.71 copies/μL	63 ℃、 40 min	孙妍等^[58]
		BIP	AACGCGGGAAAACTTACCAGGGGCACCACTCTTGTCT ACCTC
		F3	TTTCGGGCTCTGGGGATA
		B3	CCCCCATCAATTTCCAACGG
		LF	GTGTCCTTCCGTCAATTTCGC
		LB	TCAAGTCTATCGTAGATTGGAGACA
		EHP-F3	GATGCTTGGTGTGGGAGAA	1.78×101 copies/μL	65 ℃、 40 min	俞焕腾^[59]
		EHP-B3	CCCCCCATCAATTTCCAACG
		EHP-FIP	TCCTGGTAGTGTCCTTCCGTCATTTTTTTTCGGGCTCTGG GGATA
		EHP-BIP	AACGCGGGAAAACTTACCAGGGTTTTGCACCACTCTTG TCTACCTC
		EHP-LB	GTTTCACCCTTGCGAGCG
		EHP-2-F	CGTGAAGCAATTGGAGGGCAAGTTTTGGTG	10 copies/μL	37 ℃、 20 min	黄纪徽等^[26]
		EHP-2-R	CTATCCGTTCCGCTACTCTCAACAAACTCAAT
		EHP-1-P	[FAM-dT]G[THF]C[BHQ-dT] GCAGTTAAAGGG TCC
18S	特异性强	F3	ACTATGCCGACAATGCTG	440 fg/μL	63 ℃、 30 min	陈进会等^[25]
		B3	TAGAAGATCTCACTGGTTCCA
		FIP	TTAAGCAGCACAATCCACTCCTGATAGTACGCTCGCAAGC
		BIP	GCGGGAAAACTTACCAGGGTCCATGCACCACTCTTGTC
		LF	GGTAGTGTCCTTCCGTCAATT
		LB	ATCGTAGATTGGAGACATGAGG
PTP2	特异性强	EHP-F1	CACTCAAGGAATGGCTCAAGGGTTCAAAAT	50 copies/反应	37 ℃、 1.0 h(RPA)	Kanitchinda S 等^[50]
PTP2	特异性强	EHP-R1	ACCCTGTGAATTGATCATATCTCCTGCCCT	50 copies/反应	37 ℃、 1.0 h(RPA)	Kanitchinda S 等^[50]