| 吕若萱,王秀华,王美凤,连新宇,李晨,许华,尹伟力,贾鹏,杨冰.对虾养殖池塘沉积物中IHHNV DNA检测方法的评估与应用.渔业科学进展,2025,46(3):160-169 |
| 对虾养殖池塘沉积物中IHHNV DNA检测方法的评估与应用 |
| Infectious hypodermal and hematopoietic necrosis virus detection in shrimp farming pond sediments |
| 投稿时间：2024-03-28 修订日期：2024-04-16 |
| DOI：10.19663/j.issn2095-9869.20240328001 |
| 中文关键词: IHHNV 环境DNA 沉积物 |
| 英文关键词: Infectious hypodermal and hematopoietic necrosis virus (IHHNV) Environmental DNA (eDNA) Sediment |
| 基金项目:中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金(2024GH02)、现代农业产业技术体系专项资金(CARS48)、国家重点研发计划(2023YFD2400705)、中国水产科学研究院黄海水产研究所基本科研业务费(20603022023025)、海关总署揭榜挂帅项目(2021KH001)和深圳市农业发展专项资金项目：基于eDNA技术的高通量无创渔业病害监测关键技术研发及示范应用(1302)共同资助 |
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| 中文摘要: |
| 对虾传染性皮下和造血组织坏死病毒(IHHNV)是虾类疫病重要病原之一，对对虾养殖业造成危害，其主要检测方法通常通过捕获个体进行分子生物学检测。环境DNA (eDNA)技术可直接从环境样本中快速、经济地监测到目标病原，在水生动物病原检测方面的应用得到快速发展。为了评估eDNA技术检测虾类疾病病原IHHNV的有效性和可行性，本研究以不同粒径底质池塘沉积物作为研究对象，结合3种试剂盒进行提取条件优化，评估不同底质下eDNA的提取效果，利用荧光定量PCR检测方法检测IHHNV最低核酸检出量。结果显示，优化的方法对于沙底沉积物中IHHNV的检测灵敏度可达1.52×102 copies/μL，泥底沉积物中IHHNV的检测灵敏度为1.32×102 copies/μL，该方法灵敏度较高，方便可行，不同的池塘沉积物成分最低检测限浓度相差不到一个数量级，对提取效果差异不明显，可用于沉积物中IHHNV的检测。应用优化的eDNA技术和养殖对虾组织样品qPCR方法对养殖环境中IHHNV的存在情况进行调查。结果显示，调查点养殖池塘沉积物和养殖对虾样品中均有IHHNV检出，且存在一定对应关系。该研究对对虾养殖池塘沉积物中IHHNV DNA检测方法的评估与应用提供了可靠的技术手段，为监测养殖动物健康状态提供了科学依据，同时丰富并完善了eDNA方法在虾类病原监测中的应用。 |
| 英文摘要: |
| Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a shrimp disease that poses a substantial threat to the shrimp farming industry. Owing to the significant economic impact of IHHNV on the global shrimp farming industry, the World Organization for Animal Health (WOAH) has listed IHHNV as a notifiable crustacean pathogen. The primary IHHNV detection method usually involves capturing individual shrimp for molecular biological testing. Using environmental DNA (eDNA) technology, which allows rapid and economical pathogen monitoring directly from environmental samples, has rapidly developed for aquatic animal pathogen detection applications. eDNA is used to detect aquatic pathogens, such as external parasites in fish and crustacean diseases, including white spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), and IHHNV. For instance, for a method has been developed for detecting Cyprinid herpesvirus 3 (CyHV-3) in environmental waters using virus concentration methods and TaqMan polymerase chain reaction (PCR). The concentration-PCR method achieved an average concentration recovery rate of 67.11% for IHHNV detection in environmental waters based on eDNA principles and techniques. Shrimp carrying the IHHNV pathogen can spread the disease to healthy populations, inducing epidemics. Monitoring pathogens in the water environment is a more direct and effective method compared with testing cultivated shrimp for biosecurity investigations and risk assessments. While such eDNA methods are well-studied, research regarding such applications for soil and sediment is limited. Viruses can persist in pond soil and sediments, serving as natural virus reservoirs and providing potential pathways for virus transmission. However, no studies have monitored the presence of IHHNV in natural environment sediments, which is accompanied by a lack of reliable detecting and quantifying IHHNV detection methods for environmental sediments. eDNA methods enable an effective understanding of pathogen transmission mechanisms and the timely establishment of control measures during disease outbreaks. As a promising tool, eDNA detection has significant application prospects in monitoring aquatic animal diseases. This study aimed to evaluate the effectiveness and feasibility of using eDNA technology to detect the shrimp disease pathogen, IHHNV. Different particle size substrates of pond sediments were selected as study subjects. Extraction conditions were optimized using three commercial kits to evaluate the eDNA extraction effects under different substrates and to detect the lowest nucleic acid detection limit of IHHNV using real-time fluorescent quantitative PCR (RT-qPCR). To verify nucleic acid extraction effectiveness from sediments, three different kits were applied to extract DNA from shrimp tissue in both mud and sand substrates, followed by PCR amplification. Considering factors such as kit price, extraction effect, and duration, Kit B and A were selected for nucleic acid extraction from sand and mud sediments, respectively. RT-qPCR amplification of IHHNV in two types of substrate sediments at different addition volumes were observed. As the addition volume of IHHNV-containing shrimp tissue homogenate decreased, the viral load of IHHNV decreased accordingly. The minimum detectable addition volume for IHHNV in sand sediments was 5 μL, with a viral load of 1.52×102 copies/μL; whereas the minimum detectable addition was 10 μL for mud sediments, with a viral load of 1.32×102 copies/μL. The original viral load in 5 μL and 10 μL homogenate volumes were 9.94×102 and 1.72×103 copies/μL, respectively. Compared to the original viral load added, the recovery rate in sand and mud sediments were approximately 15.30% and 7.70%, respectively. The minimum detection limit concentration of different pond sediment components varied by less than an order of magnitude, showing no significant difference in extraction effects, making it suitable for IHHNV detection in sediments. The optimized eDNA technique and RT-qPCR method for cultured shrimp tissue samples were applied to investigate the presence of IHHNV in the farming environment. IHHNV positives were detected in both sediment and shrimp farming ponds between July and September; however, the positive detection rate was lower in sediment than in shrimp (Penaeus vannamei). The study demonstrates that the detection results of pond sediments and cultured P. vannamei samples are consistent, indicating that pond sediments effectively reflect the IHHNV infection status of shrimp farms. IHHNV RT-qPCR detection in pond sediments during the cultivation period from July to September 2022 were observed. IHHNV loads reached 102 copies/μL level in all six farming ponds. Notably, the IHHNV load in the sediment of pond 6-1 reached up to 4.88×102 copies/μL. Viral loads in shrimp tissue samples reached up to 102–103 copies/μL, indicating that IHHNV loads in shrimp tissue samples were higher than those in pond sediments. This study provides a reliable technical method to evaluate IHHNV detection methods in shrimp farming pond sediments, offering a scientific basis for monitoring the health status of cultured animals and supporting the application of eDNA methods for monitoring shrimp pathogens. |
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