白斑综合征病毒和对虾内参基因双重荧光定量PCR检测方法的建立OA

[Introduction]The white spot syndrome virus(WSSV)is a highly infectious and lethal pathogen that poses a significant threat to the global shrimp farming industry.WSSV can spread rapidly through both horizontal and vertical transmission,leading to mortality rates of up to 100%within 3-10 days post-infection.Due to the absence of effective prevention and treatment measures,developing rapid,accurate,and sensitive detection technologies for WSSV is crucial for timely diagnosis and implementation of control measures to prevent disease spread.[Objective]This study aimes to establish a sensitive and specific duplex real-time fluorescent quantitative PCR(qPCR)method for the simultaneous detection of WSSV and an endogenous con-trol gene in shrimp.[Methods]First,specific primers and TaqMan probes were designed based on the con-served sequences of the WSSV genome,establishing a single WSSV qPCR detection system.Specificity and re-peatability tests confirmed that the method had excellent specificity for WSSV without cross-reactivity,with the coefficient of variation for different gradients of plasmid standards being less than 1.5%,indicating high repeat-ability.Subsequently,primers and probes for the endogenous control gene of Litopenaeus vannamei were de-signed and synthesized,and the primer sequences were optimized to establish a duplex qPCR detection system.[Results]Sensitivity tests revealed that the detection limit of the duplex system for WSSV was 15 copies·µL-1,with a strong linear relationship between the logarithmic value of the initial template amount and the cycle threshold(Ct)value of WSSV plasmid standards within the range of 6.6 to 6.6×105 copies·µL-1(R²=0.998).Fur-thermore,the incorporation of the endogenous control gene effectively distinguished false-negative results caused by nucleic acid extraction or operational issues in the samples.[Conclusion]In conclusion,the estab-lished duplex qPCR detection method for WSSV and the shrimp endogenous control gene exhibits high sensitiv-ity,strong specificity,and good repeatability.This method provides a reliable technical approach for the rapid diagnosis and epidemiological investigation of WSSV,which is crucial for controlling WSSV outbreaks and en-suring the sustainable development of the shrimp farming industry.