渔业研究 ›› 2024, Vol. 46 ›› Issue (1): 101-110.DOI: 10.14012/j.cnki.fjsc.2024.01.011
• 综述 • 上一篇
收稿日期:
2023-05-11
出版日期:
2024-02-25
发布日期:
2024-02-04
通讯作者:
范晓东
作者简介:
谭念秋(1997—),女,硕士研究生,研究方向:病原微生物。E-mail:2021110513035@stu.cqnu.edu.cn
基金资助:
TAN Nianqiu(), WEI Chunmei, WEN Yusong, FAN Xiaodong()
Received:
2023-05-11
Online:
2024-02-25
Published:
2024-02-04
Contact:
FAN Xiaodong
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。
中图分类号:
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.
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 |
表1 EHP常规的检测技术比较
Tab.1 Comparison of detection techniques for EHP
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[ | |||||||
BIP | GAGGCGTCACAGAAGTAGACATTTTCCCGTATTCTCAA TGTCTACAC | ||||||||||||
F3 | GTGCAATTTAATAGGAGAACATC | ||||||||||||
B3 | GAATGGTAAGCTCCCCAC | ||||||||||||
LF | CGTGAGAATAGTCAGTT | ||||||||||||
LB | ACATACACCTATCATCCCGGAAG | ||||||||||||
EHP-OF | AGGTGGGCAAAGAATGAAAT | — | 60 ℃、 45 min | Cai S X等[ | |||||||||
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[ | |||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||
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[ | |||||||
EHP-R1 | ACCCTGTGAATTGATCATATCTCCTGCCCT |
表2 等温扩增在EHP检测中的靶标基因
Tab.2 Targets for isothermal amplification in EHP detection
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[ | |||||||
BIP | GAGGCGTCACAGAAGTAGACATTTTCCCGTATTCTCAA TGTCTACAC | ||||||||||||
F3 | GTGCAATTTAATAGGAGAACATC | ||||||||||||
B3 | GAATGGTAAGCTCCCCAC | ||||||||||||
LF | CGTGAGAATAGTCAGTT | ||||||||||||
LB | ACATACACCTATCATCCCGGAAG | ||||||||||||
EHP-OF | AGGTGGGCAAAGAATGAAAT | — | 60 ℃、 45 min | Cai S X等[ | |||||||||
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[ | |||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||||
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[ | |||||||
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[ | |||||||
EHP-R1 | ACCCTGTGAATTGATCATATCTCCTGCCCT |
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