ESTABLISHMENT OF ULTRA-SENSITIVE AND HIGHLY SPECIFIC NESTED PCR DETECTION METHODS AND THEIR APPLICATION IN EARLY WARNING OF VIBRIO INFECTIONS IN LARIMICHTHYS CROCEA AQUACULTURE
Vibrio disease caused by pathogenic Vibrio species such as Vibrio harveyi,V.parahaemolyticus,and V.alginolyticus is one of the most common and harmful diseases in the cultivation of Larimichthys crocea.However,the current means of control after the occurrence of the disease are limited.Therefore,it has become increasingly important to establish a high sensitivity and high specificity pathogen detection technology for early warning of diseases.In this study,based on the ordinary two-round nested PCR primers,a set of three-round nested PCR primers were designed for each of these three Vibrio species.Through optimization of amplification conditions,specificity testing,and sensitivity determination,a method capable of accurately detecting the three Vibrio species was established,achieving a single amplification product,a false positive rate of 0 for reference strains,and a lowest detectable sensitivity of 2.50 copies/μL(V.harveyi),3.12 copies/μL(V.parahaemolyticus),and 2.53 copies/μL(V.alginolyticus)for target sequences.Using the developed detection method,disease samples from the breeding populations in Zhejiang and Fujian provinces in China from 2021 to 2023 were tested.All samples were found to be positive for V.harveyi and V.alginolyticus,while V.parahaemolyticus was not detected.The results indicate that Vibrio disease in the Zhejiang and Fujian provinces is mainly caused by V.harveyi and V.alginolyticus.The detection method developed in this study has the potential to be applied for early warning and routine detection of Vibrio disease in species such as L.crocea.
万方数据
维普
