建立基于SYBR Green I的实时荧光定量PCR法检测十足目虹彩病毒1型

Establishment of a SYBR Green I-based real-time PCR for the detection of decapod iridescent virus 1.

作者信息

Xu Ting, Tan Rongxiang, Zhu Yutao, Ye Jian

机构信息

School of Life and Environmental Sciences, Shaoxing University, Shaoxing 312000, China.

School of Life and Environmental Sciences, Shaoxing University, Shaoxing 312000, China.

出版信息

J Invertebr Pathol. 2023 Nov;201:107998. doi: 10.1016/j.jip.2023.107998. Epub 2023 Oct 5.

Abstract

Decapod iridescent virus 1 (DIV1) is an emerging pathogen that mainly threatens decapod crustaceans, causing high mortalities and leading to huge economic losses. In this study, a pair of specific primers were designed for the major capsid protein (MCP) gene of DIV1, and a SYBR Green I-based real-time PCR method was developed. The method displayed good linearity (R = 1.000) and good repeatability in detecting standards of DIV1 MCP fragments ranging from 6.2 × 10 to 6.2 × 10 DNA copies/μl. Specificity analysis revealed that the real-time PCR was specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. Sensitivity analysis revealed that the real-time PCR could efficiently detect DIV1 DNA as low as 62 copies/μl within 35 cycles. In summary, the established real-time PCR provides an efficient, sensitive, and reliable detection method for DIV1.

摘要

十足目虹彩病毒1(DIV1)是一种新兴病原体,主要威胁十足目甲壳类动物,可导致高死亡率并造成巨大经济损失。在本研究中,针对DIV1的主要衣壳蛋白(MCP)基因设计了一对特异性引物,并建立了基于SYBR Green I的实时荧光定量PCR方法。该方法在检测浓度范围为6.2×10至6.2×10个DNA拷贝/微升的DIV1 MCP片段标准品时,显示出良好的线性关系(R = 1.000)和重复性。特异性分析表明,该实时荧光定量PCR对DIV1具有特异性,不与其他常见虾类病原体或健康虾的DNA发生反应。敏感性分析表明,该实时荧光定量PCR能够在35个循环内有效检测低至62拷贝/微升的DIV1 DNA。总之,所建立的实时荧光定量PCR为DIV1提供了一种高效、灵敏且可靠的检测方法。

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