| 摘要: |
| 由哈氏弧菌(Vibrio harveyi)、副溶血弧菌(Vibrio parahaemolyticus)及溶藻弧菌(Vibrio 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变得越来越重要。在普通两轮巢式PCR引物的基础上,对这三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,确定了能精准检测三种弧菌的方法,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年度中国浙江、福建一带大黄鱼养殖样本,所有样本均检测出哈氏弧菌和溶藻弧菌,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。 |
| 关键词: 巢式PCR 大黄鱼 弧菌病 病原检测 |
| DOI： |
| 分类号: |
| 基金项目:浙江省省属高校基本科研业务费项目（2021J005）；浙江省自然科学基金重点项目（Z23D060004） |
|
| Establishment of three nested PCR detection methods for pathogenic Vibrio species and application of pathogen detection in Larimichthys crocea |
|
wei xuelian1, li jiji1, shi hui2, tao zhen3, duan xinbing4, ye yingying1, guo baoying4
|
|
1.National Engineering Research Center for Marine Aquaculture, Zhejiang Ocean University;2.Key Laboratory of Mariculture and Enhancement of Zhejiang Province, Zhejiang Marine Fisheries Research Institute;3.School of fishery, Zhejiang Ocean University,;4.Marine Science and Technology College, Zhejiang Ocean University
|
| Abstract: |
| Vibrio disease caused by pathogenic Vibrio species such as Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio 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 Larimichthys crocea. |
| Key words: Nested PCR Larimichthys crocea Vibrio disease Pathogen detection |