十足目虹彩病毒1和虾肝肠胞虫双重荧光定量PCR检测方法的建立

doi:10.3969/j.issn.1004-1524.20240730

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (12): 2468-2478.DOI: 10.3969/j.issn.1004-1524.20240730

十足目虹彩病毒1和虾肝肠胞虫双重荧光定量PCR检测方法的建立

谈荣想¹(), 斯广杰², 孙海涛², 许婷¹^,^*()

1.绍兴文理学院生命与环境科学学院,浙江绍兴 312000
2.浙江华珍科技有限公司,浙江诸暨 311804

收稿日期:2024-08-13 出版日期:2025-12-25 发布日期:2026-01-09
作者简介:谈荣想(1999—),男,江西瑞昌人,硕士研究生,研究方向为水生动物病害与免疫学。E-mail:2395774885@qq.com
通讯作者: *许婷,E-mail:xuting@usx.edu.cn

Establishment of a double quantitative PCR method for the detection of Decapod iridescent virus 1 and Enterocytozoon hepatopenaei

TAN Rongxiang¹(), SI Guangjie², SUN Haitao², XU Ting¹^,^*()

1. School of Life and Environmental Sciences, Shaoxing University, Shaoxing 312000, Zhejiang, China
2. Zhejiang Huazhen Sci & Tech Co., Ltd., Zhuji 311804, Zhejiang, China

Received:2024-08-13 Online:2025-12-25 Published:2026-01-09

摘要/Abstract

摘要： 为建立一种可同时检测十足目虹彩病毒1(Decapod iridescent virus 1, DIV1)与虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)的方法,本研究在已有DIV1实时荧光定量PCR(quantitative real-time PCR, qPCR)检测方法的基础上,开发了一种基于SYBR Green I的双重qPCR方法,分别靶向DIV1的主要衣壳蛋白(major capsid protein, MCP)基因和EHP的孢壁蛋白(spore wall protein, SWP)基因。结果显示,该方法中DIV1与EHP的熔解温度(Tm)分别为(82.0±0.5)℃和(77.0±0.5)℃,能够特异性检出DIV1和EHP,而对其他常见虾病原和健康虾样本的检测结果均为阴性;在35个循环内,DIV1与EHP的最低检测限分别为75 copies·μL^-1和15 copies·μL^-1;重复性试验显示,组内和组间变异系数均低于2%。综上,本研究建立的双重qPCR检测方法具有高度的特异性、灵敏度与重复性,适用于虾中DIV1和EHP感染的快速监测。

关键词: 双重SYBR Green I实时荧光定量PCR, 十足目虹彩病毒1, 虾肝肠胞虫, 虾

Abstract:

To establish a method for simultaneous detection of Decapod iridescent virus 1 (DIV1) and Enterocytozoon hepatopenaei (EHP), this study developed a double SYBR Green I-based quantitative real-time PCR (qPCR) assay, building upon an established qPCR method for DIV1. The assay targets the major capsid protein (MCP) gene of DIV1 and the spore wall protein (SWP) gene of EHP. The results showed that the melting temperatures (Tm) for DIV1 and EHP were (82.0±0.5)℃ and (77.0±0.5)℃, respectively. The method specifically detected DIV1 and EHP, while showing negative results for other common shrimp pathogens and healthy shrimp samples. Within 35 amplification cycles, the limits of detection were 75 copies·μL^-1 for DIV1 and 15 copies·μL^-1 for EHP. Reproducibility tests showed that both intra- and inter-assay coefficients of variation were below 2%. In conclusion, the established double qPCR method exhibits high specificity, sensitivity, and reproducibility, and is suitable for rapid monitoring of DIV1 and EHP infections in shrimp.

Key words: double SYBR Green I quantitative real-time PCR, Decapod iridescent virus 1, Enterocytozoon hepatopenaei, shrimp

中图分类号:

S945.4

谈荣想, 斯广杰, 孙海涛, 许婷. 十足目虹彩病毒1和虾肝肠胞虫双重荧光定量PCR检测方法的建立[J]. 浙江农业学报, 2025, 37(12): 2468-2478.

TAN Rongxiang, SI Guangjie, SUN Haitao, XU Ting. Establishment of a double quantitative PCR method for the detection of Decapod iridescent virus 1 and Enterocytozoon hepatopenaei[J]. Acta Agriculturae Zhejiangensis, 2025, 37(12): 2468-2478.

图/表 11

表1 本研究的引物序列信息

Table 1 Sequences of primers used in this study

引物名称 Primer name	引物序列(5'→3') Primer sequence(5'→3')	扩增片段长度 Product length/bp	目的 Purpose
DIV1-QF	GCCATTCCCGAACTCACC	154	双重qPCR方法检测DIV1
DIV1-QR	CTTCACCCTTTGCCGCTT		Detection of DIV1 by double qPCR method
SWP-stdF	TTAAGTAATTACGAGTTTGGC	318	构建pMD-SWP标准质粒
SWP-stdR	GTTATTTACAGTTTTGCGTTG		Construction of pMD-SWP standard plasmid
EHP-QF	TGGCGGCACAATTCTCAAACAT	102	双重qPCR方法检测EHP
EHP-QR	GCTGTGTCTGTGTAAATATCGTC		Detection of EHP by double qPCR method

图1 虾肝肠胞虫SWP基因的PCR扩增结果 M, DL2000 DNA marker;1, SWP基因扩增产物。

Fig.1 PCR amplification results of SWP gene from Enterocytozoon hepatopenaei M, DL2000 DNA marker; 1, PCR amplification products of SWP gene.

图2 十足目虹彩病毒1与虾肝肠胞虫的双重SYBR Green I qPCR熔解曲线 EHP,EHP标准质粒pMD-SWP的熔解峰;DIV1,DIV1标准质粒pMD-MCP的熔解峰;NTC,阴性对照。

Fig.2 Melting curve of the double SYBR Green I qPCR for Decapod iridescent virus 1 (DIV1) and Enterocytozoon hepatopenaei(EHP) EHP, The melting curve of EHP standard plasmid pMD-SWP; DIV1,The melting curve of DIV1 standard plasmid pMD-MCP; NTC,Negative control.

图3 DIV1双重SYBR Green I qPCR扩增曲线和灵敏性试验结果 1~7,7.5×107~7.5×101 copies·μL-1的pMD-MCP系列标准品;NTC,阴性对照。

Fig.3 Amplification curve and sensitivity of the double SYBR Green I qPCR for DIV1 1-7, 7.5×107-7.5×101 copies·μL-1 of the standard plasmids pMD-MCP; NTC, Negative control.

图4 EHP双重SYBR Green I qPCR扩增曲线和灵敏性试验结果 1~7,1.5×107~1.5×101 copies·μL-1的pMD-SWP系列标准品;NTC,阴性对照。

Fig.4 Amplification curve and sensitivity of the double SYBR Green I qPCR for EHP 1-7, 1.5×107-1.5×101 copies·μL-1 of the standard plasmids pMD-SWP; NTC, Negative control.

图5 DIV1和EHP的双重SYBR Green I qPCR检测标准曲线

Fig.5 The double SYBR Green I qPCR standard curves generated for DIV1 and EHP

图6 DIV1和EHP双重SYBR Green I qPCR检测的熔解曲线 EHP,梯度稀释的pMD-SWP系列标准品;DIV1,梯度稀释的pMD-MCP系列标准品;NTC,阴性对照。

Fig.6 Melting curve for the double SYBR Green I qPCR for DIV1 and EHP EHP, Serial dilution of standard plasmid pMD-SWP; DIV1, Serial dilution of standard plasmid pMD-MCP; NTC, Negative control.

图7 双重SYBR Green I qPCR特异性试验结果 A,扩增曲线;B,熔解曲线。DIV1,DIV1阳性样本;EHP,EHP阳性样本;DIV1+EHP,混合感染样本;others,其他虾病阳性样本、健康凡纳滨对虾幼苗和阴性对照。

Fig.7 Analysis of the specificity of the double SYBR Green I qPCR A, Amplification curve; B, Melting curve. DIV1, DIV1 positive sample; EHP, EHP positive sample; DIV1+EHP, DIV1 and EHP co-infection sample; Others, Samples infected with other shrimp pathogens, healthy L. vannamei juveniles and negative control.

表2 DIV1双重SYBR Green I qPCR检测的组内和组间重复性

Table 2 Intra-assay and inter-assay variability of the double SYBR Green I qPCR detection assay for DIV1

pMD-MCP标准品拷贝数 Copies of the standard plasmid	组内重复Intra-assay variability						组间重复 Inter-assay variability
pMD-MCP标准品拷贝数 Copies of the standard plasmid	第1天Day 1		第5天Day 5		第10天Day 10		组间重复 Inter-assay variability
pMD-MCP/(copies·μL^-1)	Cq	CV/%	Cq	CV/%	Cq	CV/%	Cq	CV/%
7.5×10⁷	13.43±0.12	0.92	13.77±0.06	0.46	13.35±0.03	0.19	13.52±0.21	1.52
7.5×10⁶	16.80±0.02	0.10	17.04±0.12	0.68	17.00±0.06	0.35	16.94±0.13	0.76
7.5×10⁵	20.46±0.06	0.31	20.52±0.02	0.09	20.40±0.02	0.10	20.46±0.06	0.30
7.5×10⁴	23.90±0.05	0.21	24.35±0.08	0.33	24.04±0.07	0.30	24.09±0.21	0.86
7.5×10³	27.21±0.08	0.31	27.55±0.06	0.21	27.54±0.08	0.27	27.43±0.18	0.64
7.5×10²	30.65±0.15	0.50	31.12±0.02	0.06	30.94±0.02	0.05	30.90±0.22	0.71
7.5×10¹	33.75±0.04	0.12	34.12±0.02	0.05	33.62±0.08	0.25	33.83±0.23	0.68

表3 EHP双重SYBR Green I qPCR检测的组内和组间重复性

Table 3 Intra-assay and inter-assay variability of the double SYBR Green I qPCR detection assay for EHP

pMD-SWP标准品拷贝数 Copies of the standard plasmid	组内重复Intra-assay variability						组间重复 Inter-assay variability
pMD-SWP标准品拷贝数 Copies of the standard plasmid	第1天Day 1		第5天Day 5		第10天Day 10		组间重复 Inter-assay variability
pMD-SWP/(copies·μL^-1)	Cq	CV/%	Cq	CV/%	Cq	CV/%	Cq	CV/%
1.5×10⁷	14.05±0.03	0.23	14.13±0.04	0.30	14.17±0.02	0.13	14.11±0.06	0.41
1.5×10⁶	17.40±0.04	0.23	17.48±0.05	0.26	17.45±0.05	0.29	17.45±0.05	0.31
1.5×10⁵	20.62±0.07	0.32	20.75±0.10	0.46	20.80±0.11	0.53	20.72±0.12	0.55
1.5×10⁴	24.15±0.06	0.24	24.33±0.13	0.55	24.47±0.04	0.18	24.32±0.16	0.65
1.5×10³	27.64±0.03	0.12	27.83±0.03	0.10	27.83±0.02	0.05	27.77±0.10	0.35
1.5×10²	31.09±0.07	0.21	31.31±0.14	0.45	31.16±0.14	0.45	31.19±0.14	0.45
1.5×10¹	33.91±0.11	0.32	33.89±0.20	0.58	34.60±0.15	0.43	34.13±0.37	1.09

表4 临床样本使用巢式PCR和双重SYBR Green I qPCR检测的结果

Table 4 Detection results of clinical samples by nested-PCR and double SYBR Green I qPCR

巢式PCR检测 Detection by nested-PCR		双重SYBR Green I qPCR检测Detection by double SYBR Green I qPCR
巢式PCR检测 Detection by nested-PCR		阳性Positive	阴性Negative	总计Total
DIV1	阳性Positive	4	0	4
	阴性Negative	2	49	51
	总计Total	6	49	55
EHP	阳性Positive	9	0	9
	阴性Negative	0	46	46
	总计Total	9	46	55

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十足目虹彩病毒1和虾肝肠胞虫双重荧光定量PCR检测方法的建立

Establishment of a double quantitative PCR method for the detection of Decapod iridescent virus 1 and Enterocytozoon hepatopenaei

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