摘要
由哈维氏弧菌(Vibrio harveyi)、副溶血弧菌(V.parahaemolyticus)及溶藻弧菌(V.alginolyticus)等病菌感染导致的弧菌病是大黄鱼养殖中最常见、最具危害性的疾病之一,然而目前针对发病后的遏制手段有限,因此亟待建立高灵敏度、高特异性的病原检测技术以实现病害早期预警.在普通两轮巢式PCR引物的基础上,对这3种弧菌分别设计一套三轮巢式PCR引物,通过扩增条件优化、特异性检验及灵敏度测定,确定了能精准检测3种弧菌的方法,达到了扩增产物单一、参考菌假阳性检出率为0、靶序列最低检出灵敏度分别为2.50 copies/μL(哈维氏弧菌)、3.12 copies/μL(副溶血弧菌)、2.53 copies/μL(溶藻弧菌)等效果.利用开发的检测方法,检测了 2021～2023年度浙江、福建一带大黄鱼养殖样本,所有样本均检测出哈维氏弧菌和溶藻弧菌,副溶血弧菌未检出.结果显示浙江、福建一带大黄鱼弧菌病主要是由哈维氏弧菌和溶藻弧菌引起.开发的检测方法有望应用于大黄鱼等养殖物种弧菌病的早期预警和常规检测.
Abstract
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.
基金项目
浙江省省属高校基本科研业务费项目(2021J005号)
浙江省自然科学基金重点项目(Z23D060004号)
维普
万方数据