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

doi:10.3969/j.issn.1004-1524.20240730

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (12): 2468-2478.DOI: 10.3969/j.issn.1004-1524.20240730

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S945.4

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.

Figures/Tables 11

References 33

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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

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

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

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

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

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

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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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