基于孢子壁蛋白基因靶点的虾肝肠胞虫实时荧光定量PCR体系的构建及应用

Establishment and Application of A Real-Time PCR Assay Targeted on the Spore Wall Protein Gene for Detecting Microsporidian Enterocytozoon hepatopenaei

基于GenBank中公布的虾肝肠胞虫(EHP)孢子壁蛋白(spore wall protein,SWP)基因序列设计一对特异性引物,构建并优化了基于EHP-SWP靶点的SYBR实时荧光定量PCR(qPCR)检测方法。试验结果显示,该引物在退火温度为60℃时具有最佳的扩增效果,扩增效率达98.8%,扩增产物的溶解曲线为1个单峰。本研究构建的qPCR体系对5.02×10^(1)~5.02×10^(8)copies·μL^(-1)的EHP-SWP DNA片段的检测响应有良好线性关系,扩增产物的阈值循环数(Ct)与模板起始拷贝数对数值[log(Copies)]的关系为:Ct=-0.2844 log(Copies)+10.45,(R^(2)=0.992),检测灵敏度达5.02×10^(1)copies·μL^(-1)。引物特异性检测结果显示,本研究设计的检测引物仅能特异的扩增EHP模板,而对其他多种虾病原如传染性皮下及造血组织坏死病毒(IHHNV)、白斑综合征病毒(WSSV)、虾虹彩病毒(SHIV)、副溶血弧菌、哈维氏弧菌的扩增结果均为阴性,表明引物无交叉反应,具有良好特异性。重复性实验表明,该方法检测结果变异系数小于2%,可重复性高。应用本研究构建的检测体系对浙江海洋大学西闪岛试验基地虾塘中出现生长缓慢症状的51尾虾的EHP携带量进行检测,结果显示,EHP的感染指数与体长呈现负相关,并且,随着体长的增大,负相关性逐渐减弱。本研究为EHP的定量检测提供了一种新的具有高特异性和灵敏度的方法。

In this study we designed a pair of specific primers according to the spore wall protein(SWP)sequences of Enterocytozoon hepatopenaei(EHP)published in GenBank,established and optimized a fluorescence SYBR GreenⅡreal-time quantitative PCR(qPCR)assay targeted on the SWP gene for detecting EHP.The result shows that:when the annealing temperature is 60℃,the pair primers reached an optimum amplification effect with an amplification efficiency of 98.8%,and the melting curve of amplified products exhibited only one specific peak.Between 5.02×10^(1)-5.02×10^(8) copies·μL^(-1) the detection response were linearly correlated with the DNA fragments of EHP-SWP.The cycles of amplification threshold(Ct)and the logarithmic of the initial template copies[log(Copies)]conformed to Ct=-0.2844 log(Copies)+10.45,of which the coefficient of association R^(2 )was 0.992.The method has a detection sensitivity of 5.02×10^(1) copies·μL^(-1),10 times lower than that of conventional PCR.Primer matching test shows that the primers designed in this study could only specifically amplify the EHP template,for many other shrimp pathogens(such as infectious hypodermal and hematopoietic necrosis virus(IHHNV),white spot syndrome virus(WSSV),shrimp iridovirus(SHIV),Vibrio parahaemolyticus,V.harveyi),the amplification result is negative,indicating that the primers have a high specificity with no cross matching or false amplification.Duplication test showed that the coefficient variation of this assay was less than 2%,and has a good repeatability.The established qPCR system was applied to detect the EHP infection of 51 shrimps with slow growth symptoms collected from a shrimp pond of Zhejiang Ocean University Xishan experiment base.The results showed that:the relative exponential copies of EHP infection is negatively correlated with the shrimp body length,and this correlation tends to decay with the body length increases.This study provides a new assay for the quantitative detection of EHP with high specificity and sensitivity.