对虾肝胰腺坏死病副溶血弧菌双重微流控荧光定量PCR快速检测技术的建立与应用

Establishment and application of dual microfluidic fluorescent quantitative PCR for rapid detection of Vibrio parahaemolyticus in shrimp hepatopancreatic necrosis

中文关键词: 急性肝胰腺坏死病  副溶血弧菌  pirA  pirB  微流控芯片  现场检测

英文关键词: AHPND  Vibrio parahaemolyticus  pirA  pirB  Microfluidic chip  Field detection

中文摘要:

      急性肝胰腺坏死病(AHPND)是对虾养殖过程中最常见、最严重的疾病，给对虾养殖造成重大经济损失。AHPND病原种类多、基因型复杂，现有的针对不同病原的检测技术目标导向较弱、检测成本高、时间消耗长，对虾健康养殖亟待开发AHPND的精准快速诊断技术。本研究针对AHPND病原体携带编码一种二元毒素pirA和pirB大型质粒的遗传共性，基于pirA和pirB基因设计特异性引物并建立微流控荧光定量PCR检测方法。该方法对致病基因pirA和pirB特异性强、灵敏度高，最低检测限分别为5.43×100和4.31×101 copies/μL，样品平均检测时间为26 min左右。为进一步评估该方法在实际应用中的准确性，以含有pirA和pirB毒性质粒的副溶血弧菌(Vibrio parahaemolyticus)进行人工感染实验。结果表明，感染后的凡纳滨对虾(Litopenaeus vannamei)鳃丝、肝胰腺、肠道和肌肉等组织随时间的推迟均能检测到pirA和pirB；从感染2 h的结果来看，pirB比pirA检出率更高。此外，致病因子pirA和pirB比toxR的检出率更高，更适合对AHPND致病原的检测。本研究建立的微流控荧光定量PCR检测的方法具有快速、灵敏、高通量、污染少、现场检测、一体化集成等优点。该技术不仅适用于实验室，更符合养殖基层的现场快速检测需求，为及早认知疾病发生风险和病害精准防控提供了新的技术手段与理论支撑。

英文摘要:

      Shrimp has become a highly traded global seafood product, with 8 million tons of shrimp produced annually. Acute hepatopancreatic necrosis disease (AHPND) is the most prevalent and severe disease affecting shrimp aquaculture, resulting in considerable economic losses. The AHPND incidence in shrimp farming was as high as 60%–80% in China, resulting in reduced farming capacity and unstable production. Vibrio parahaemolyticus has been identified as the main causative agent of AHPND. In addition, V. harveyi, V. cambelii, V. algolyticus, and V. owenii are capable of causing similar diseases, demonstrating a distinct pathogenic diversity. Previous studies have indicated that not all of the above-mentioned Vibrios species are capable of causing AHPND, and whole gene sequencing and knockout genes have revealed that pirA and pirB are the primary pathogenic factors responsible for AHPND in shrimp. Specifically, the causative agent for AHPND should be a specific strain of Vibrio carrying the binary toxins pirAVp and pirBVp on the extrachromosomal virulence plasmid pVA1. Among them, the pirB toxin mainly determines the pathogenicity of the bacterium, whereas the pirA virulence is relatively weak. Furthermore, it has been demonstrated that the virulence plasmids encoding the binary genes pirAVp and pirBVp are the main causative agents of AHPND. The virulence gene toxR is prevalent in Vibrio and plays an important role through the genetic diversity of 16S rRNA genes during shrimp infection. Real-time fluorescence quantitative PCR technology has less contamination, more accurate quantification, real-time monitoring, and greater automation than conventional PCR technology, which has been utilized in the fields of transgenic detection, environmental science, and medicine. However, this technique is time-consuming, involves multiple instruments and reagents, and requires personnel with extensive professional skills and experience. As a result of its small size, low sample and reagent consumption, rapid detection speed, miniaturization, and integration, microfluidic chip assay technology has emerged as a new focal point in assay technology. Therefore, in this study, we designed specific primers and established a microfluorescence quantitative PCR assay based on two genes, pirA and pirB, to address the genetic similarity of AHPND pathogens carrying a large plasmid encoding a binary toxin, pirA and pirB. The method was specific for the pathogenic pirA and pirB genes, and only when DNA from AHPND-infected samples was tested could the two genes be successfully amplified, while all other pathogenic bacteria were detected with negative results. The sensitivity was high, and the minimum detection limits for the pirA and pirB genes were 5.43×100 and 4.31×101 copies/μL, respectively. Standard curves for pirA and pirB were constructed and demonstrated good linearity in the concentration range of 5.43×109–5.43×104 copies/μL for pirA (y= –3.145x+6.63, R2=0.999) and 4.31×109–4.31×104 copies/μL for pirB (y= –3.015x+5.45, R2=0.999), with an average sample detection time of approximately 26 min. In order to evaluate the efficacy of the method in practice, artificial infection experiments with V. parahaemolyticus were performed. In this study, artificial infection experiments were induced by both injection and immersion, and samples were collected at different time periods to clinically validate the established method and compare its effectiveness in detecting different shrimp tissues, thereby facilitating a more thorough analysis of the pathogenic pathways of infection. The experimental group with injection as the mode of infection was found to be positive for all tissues in all time periods except the water test, which was negative. The experimental group that used immersion as the infection method showed different results for various time periods and with different genetic tests. In terms of the infection method, the tissues could be infiltrated within 2 h using the injection method, whereas the target genes were not detected in the hepatopancreas at 6 h using the immersion method. This indicated that the injection method infiltrated the tissues more rapidly than the immersion method. According to the comparison results of the three genes, pirB was only negative in the intestine at 2 h and positive in all tissues the rest of the time; pirA was negative in the hepatopancreas and intestine at 2 h, only the intestine was negative at 6 h, and all tissues were positive at 12 h; and toxR was negative in all tissues at 2 h. The rate of infestation from rapid to slow showed that pirB > pirA > toxR. Based on the rate of tissue infestation, pirA and pirB were detected in both cheek filaments and muscles at 2 h, making them the most rapid infiltration agents. Therefore, the strategy of using pirB as the primer and gill filament or muscle as the target tissue is more suitable for the rapid detection of AHPND in the field. In this study, we established a method for microfluidic fluorescent quantitative PCR that has the advantages of being rapid, sensitive, high throughput, less contaminated, on-site detectable, and integrated. The method is not only applicable to the laboratory but also meets the requirements of rapid field detection at hatcheries and farms, and can be used as a new technical method for shrimp fry quality detection and disease control.