王李宝　黎慧　于志君　史文军　冯卫　赵然　沈辉　成婕　管小平　秦亚兵　万夕和

摘要： 為评估微流控芯片法在虾肝肠胞虫（EHP）、致急性肝胰腺坏死病副溶血性弧菌（VpAHPND）、十足目虹彩病毒1（DIV1）和白斑综合征病毒（WSSV）4种南美白对虾常见病原联检中的各项性能，设计针对上述4种病原扩增反应的引物，对已有芯片进行新的功能划分，同时优化该基因芯片的反应体系及恒温扩增条件。结果表明，优化的联检方法可同时检测上述4种病原，特异性好，30 min反应时间即可给出病原联检结果。EHP、 DIV1、WSSV、VpAHPND指标检出限分别为1 μl 0.57拷贝、0.43拷贝、2.12拷贝 、3.18拷贝，已达到甚至超过了环介导等温扩增技术（LAMP）检测的灵敏性。本次测试中的南美白对虾4种病原微流控芯片联检方法所用的检测试剂盒具有操作简便、检测灵敏、特异性强、检测时间短、设备要求简单等特点，适合整个养殖过程中对南美白对虾EHP、VpAHPND、DIV1和WSSV的监测与筛查工作。

关键词： 南美白对虾；病原；微流控芯片；联检

中图分类号： S945.4 文献标识码： A 文章编号： 1000-4440（2023）02-0471-08

Testing of microfluidic chip for joint detection of four common pathogens of Litopenaeus vannamei

WANG Li-bao¹， LI Hui¹， YU Zhi-jun^1，2， SHI Wen-jun¹， FENG Wei¹， ZHAO Ran^1，2， SHEN Hui¹， CHENG Jie¹， GUAN Xiao-ping³， QIN Ya-bing⁴， WAN Xi-he¹

（1.Jiangsu Institute of Oceanology and Marine Fisheries， Nantong 226007 China;2.National Demonstration Center for Experimental Fisheries Science Education， Shanghai Ocean University， Shanghai 201306， China;3.Yili Yueran Ecological Agriculture Co.， Ltd. Yili  835300， China;4.Comprehensive Service Center of Dayu Town， Rudong County， Nantong 226412， China）

Abstract： To evaluate the performance of microfluidic chip method in the joint detection of four common pathogens of Litopenaeus vannamei including Enterocytozoon hepatopenaei （EHP）， acute hepatopancreas necrosis disease caused by Vibrio parahaemolyticus （VpAHPND）， Decapod iridescent virus 1 （DIV1） and white spot syndrome virus （WSSV）， primers for the amplification reactions of the above four pathogens were designed， new functional divisions were made for the existing chips， and the reaction system and constant temperature amplification conditions of the microfluidic chip were optimized. The results showed that， the optimized combined detection method could detect the above four pathogens simultaneously with good specificity， and the joint detection results could be obtained within 30 min reaction time. The detection limits of EHP， DIV1， WSSV and VpAHPND were 0.57 copies/μl， 0.43 copies/μl， 2.12 copies/μl and 3.18 copies/μl， respectively. Some of the limits had reached or even exceeded the testing sensitivity of loop-mediated isothermal amplification （LAMP） method. The detection kit used in the joint detection method of microfluidic chip for the four pathogens of L. vannamei in this test is characterized by simple operation， sensitive detection， strong specificity， short detection time and simple equipment requirements， and is suitable for the monitoring and screening of EHP， VpAHPND， DIV1 and WSSV of L. vannamei in the whole breeding process.

Key words： Litopenaeus vannamei;pathogens;microfluidic chip;joint detection

病害对南美白对虾养殖业的影响与台风、洪涝等自然灾害产生的影响不相上下^[1]。对虾肝肠胞虫（Enterocytozoon hepatopenaei，EHP）、对虾急性肝胰腺坏死病副溶血性弧菌（AHPND-causing Vibrio parahaemolyticus，VpAHPND）、十足目虹彩病毒1（Decapod iridescent virus 1，DIV1）和白斑综合征病毒（White spot syndrome virus，WSSV）等病原成为许多地区对虾大批量死亡或生长延滞的主要原因之一^[2]。简化传统分子检测过程中繁琐的样品处理程序和扩增步骤，研发采样现场适用的便携式分析仪器及配套试剂，实现现场快速即时检测（Point-of-care testing，POCT）是当务之急。该联检方法的研发，将为在养殖各个环节实时监控病原体的携带情况，并有效防控扩散、减少经济损失提供理论基础与技术支撑。……