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| 对虾养殖池塘沉积物中IHHNV DNA检测方法的评估与应用 |
| Evaluation and application of infectious hypodermal and hematopoietic necrosis virus (IHHNV) detection in shrimp farming pond sediments |
| 投稿时间：2024-03-28 修订日期：2024-04-16 |
| DOI： |
| 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 (CARS 48),国家重点研发计划项目(2023YFD2400705),中国水产科学研究院黄海水产研究所基本科研业务费项目(20603022023025) |
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| 中文摘要: |
| 对虾传染性皮下及造血组织坏死病毒(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,结合三种试剂盒进行提取条件优化,评估不同底质下eDNA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检测灵敏度可达1.52×102 copies/μL,泥底沉积物中IHHNV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科学依据,同时丰富并完善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 one of the significant pathogens of shrimp diseases, posing a threat to the shrimp farming industry. The significant economic impact of IHHNV on the global shrimp farming industry has led the World Organization for Animal Health (WOAH) to list it as a notifiable crustacean pathogen.Its primary detection method usually involves capturing individuals for molecular biology testing. The application of environmental DNA (eDNA) technology, which allows for the rapid and economical monitoring of target pathogens directly from environmental samples, has rapidly developed in the detection of aquatic animal pathogens. Researchers have utilized eDNA methods to detect aquatic pathogens, such as external parasites in fish and pathogens in crustacean diseases including WSSV, EHP, and IHHNV. A developed method for detecting CyHV-3 in environmental waters using virus concentration methods and TaqMan PCR . It 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, leading to epidemics. Monitoring pathogens in the water environment is more direct and effective for biosecurity investigations and risk assessments than testing cultivated shrimp.While eDNA methods for detecting aquatic pathogens in water bodies are well-studied, research on 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, and there is a lack of reliable methods for detecting and quantifying IHHNV in 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 the monitoring of 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 the study subjects. Optimization of extraction conditions was conducted using three commercial products of kits to evaluate the eDNA extraction effects under different substrates and to detect the lowest nucleic acid detection limit of IHHNV using quantitative PCR. To verify the effectiveness of nucleic acid extraction 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 was selected for nucleic acid extraction from sand sediments, and Kit A was used for mud sediments. The results of the fluorescent quantitative PCR amplification of IHHNV in two types of substrate sediments at different addition volumes are shown. The results indicated that as the addition volume of shrimp tissue homogenate containing IHHNV decreased, the viral load of IHHNV also showed a decreasing trend. The minimum detectable addition volume for IHHNV in sand sediments was 5μL, with a viral load of 1.52×102 copies/μL; for mud sediments, it was 10μL, with a viral load of 1.32×102 copies/μL. The original viral load in 5μL and 10μL volumes of homogenate was 9.94×102 copies/μL and 1.72×103 copies/μL, respectively. Compared to the original viral load added, the recovery rate in sand sediments was approximately 15.30%, and in mud sediments, it was about 7.70%. 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 the detection of IHHNV in sediments. The optimized eDNA technique and qPCR method for cultured shrimp tissue samples were applied to investigate the presence of IHHNV in the farming environment. The results showed that IHHNV positives were detected in both sediment and P. vannamei in shrimp farming ponds between July and September, but the detection rate of positives was lower in sediment than in P. 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. The results of IHHNV fluorescent quantitative PCR detection in pond sediments during the cultivation period from July to September are presented . The results indicate that IHHNV loads reached 102 copies/μL level in all six farming ponds; notably, the IHHNV load in pond 6-1 sediment reached up to 4.88×102 copies/μL. The results of IHHNV fluorescent quantitative PCR detection in P. vannamei samples are shown in Table 3, with viral loads in shrimp tissue samples reaching 102 to 103 copies/μL level, indicating that IHHNV loads in shrimp tissue samples were higher than those in pond sediments. This study provides a reliable technical means for the evaluation and application of IHHNV detection methods in shrimp farming pond sediments, offering a scientific basis for monitoring the health status of cultured animals and enriching and improving the application of eDNA methods in the monitoring of shrimp pathogens. |
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