进口对虾急性肝胰腺坏死病病原分离鉴定及环介导等温扩增(LAMP)检测方法的建立

Isolation and Identification of the Pathogen Associated with Acute Hepatopancreatic Necrosis Disease in Imported Prawns and the Establishment of A Loop-mediated Isothermal Amplification (LAMP) Assay

摘要:

该研究抽取了453批在2017~2018年自越南、泰国、马来西亚进境的对虾样品进行对虾急性肝胰腺坏死病（Acute Hepatopancreatic Necrosis Disease，AHPND）病原的分离鉴定，并对结果进行分析。结果显示：直接取对虾肝胰腺组织进行病原分离检出率只有2.87%，而肝胰腺组织经过富集培养后，检出率则达到16.11%；来自越南进境的对虾样品阳性检出率最高，312批次的样品阳性检出率为21.79%，分离出11株阳性菌株，分离成功率为15.07%。为了快速、灵敏和可靠的鉴定AHPND病原，该研究建立了一种快速检测AHPND病原的环介导等温扩增（LAMP）方法。结果表明：仅AHPND病原具有明显的扩增曲线和颜色变化，质粒的检测低限为1 fg/μL，比普通PCR方法高100倍，且稳定性强；对37个实际样本检测显示，阳性率为35.13%，阳性检出率高于普通PCR检测结果。该研究建立的LAMP快速检测方法适用于AHPND病原特异性检测，病原的分离鉴定分析也可为进口对虾的实验室检测提供参考数据。

Abstract:

In this study, 453 batches of samples were collected from prawns imported from Vietnam, Thailand and Malaysia during 2017~2018, to isolate and identify the pathogens of acute hepatopancreatic necrosis disease (AHPND). The analysis of the obtained results showed that the detection rate of the pathogen isolated from the hepatopancreas tissues of prawns was only 2.87%, whilst the detection rate reached 16.11% after enrichment culture. The prawn samples imported from Vietnam had the highest positive detection rate, with the positive detection rate of 312 batches of samples being 21.79%, and 11 positive strains were isolated with the rate of separation success being 15.07%. In order to identify quickly, sensitively and reliably the AHPND pathogens, a loop-mediated isothermal amplification (LAMP) assay was established for rapid detection of AHPND pathogens in this research. The results showed that only the AHPND pathogen had obvious amplification curve and color change, and the limit of plasmid detection was 1 fg/μL, which was 100 times higher than that of the conventional PCR method, and had high stability. The detection of 37 actual samples showed that the positive rate was 35.13%, which was higher than that of conventional PCR test. The LAMP rapid detection method established in this study is suitable for the specific detection of AHPND pathogens, and the isolation, identification and analysis of pathogens can also provide reference data for laboratory detection of imported prawns.