EHP、SHIV双重TaqMan实时荧光定量PCR检测方法的构建及应用

Development of a Duplex TaqMan Real-time PCR Assay for Detection of Enterocy Tozoon Hepatopenaei and Shrimp Hemocyte Iridovirus

本研究根据GenBank中已有的虾肝肠胞虫(EHP)和虾血细胞虹彩病毒(SHIV)基因的保守序列,设计特异的引物和探针。建立了快速诊断EHP和SHIV的双重TaqMan实时荧光定量PCR检测方法,并对其特异性、敏感性和稳定性进行检测。结果表明:该方法检测限可达10 copies/μL,其敏感性是SYBR Green real-time PCR的10倍,普通PCR法的100倍;对白斑综合征病毒、传染性皮下及造血组织坏死病毒、高致病性副溶血弧菌,以及桃拉综合征病毒的检测结果均为阴性,表明无交叉反应,具有良好特异性;重复性试验结果表明,该方法Ct值的变异系数小于4%,具有良好的稳定性。对37份已知检测结果的样品进行检测,结果符合率为100%。本研究建立的EHP和SHIV检测方法具有快速、特异性强、灵敏度高等优点,适用于对虾隐性感染的早期监测。

In this study,specific probes and primers were designed according to Enterocy Tozoon Hepatopenaei(EHP)and Shrimp Hemocyte Iridovirus(SHIV)gene sequences in GenBank.A duplex TaqMan-based real-time quantitative PCR assay was then developed by optimizing the reaction conditions.The results demonstrated that this PCR assay was more sensitive for EHP and SHIV with the detection limit of 1×10 copies/μL,which was 10-fold higher than SYBR Green real-time PCR and 100-fold higher than conventional PCR.Specifi city tests showed no cross reactions with White spot syndrome virus,Infectious hypodermal and hematopoietic necrosis virus,High pathogenicity ibrio parahaemolyticus and Taura syndrome virus.Repeatability tests demonstrated that the threshold cycle of the method gave a good stability with the coeffi cient of variations were less than 4%.Then 37 samples were tested by using a duplex TaqMan real-time PCR and conventional PCR and the results reached 100%agreement.In conclusion,the duplex TaqMan-based real-time quantitative PCR assay developed here were specifi c and sensitive for rapid detection of EHP and SHIV.Therefore,it could be suitable for early duplex TaqMan-based real-time quantitative PCR assay of latent infection of shrimp.