摘要
创伤弧菌(VV)是一种重要的人畜共患和水生动物病原体,能引起人类和水生动物的弧菌病,严重影响人身健康和水产养殖业发展。快速、灵敏、准确的VV检测方法对于保障人民生命健康、降低水产养殖业损失至关重要。为建立一种新型VV检测方法,基于重组酶辅助扩增(RAA)技术和基于成簇的规律间隔的短回文重复序列和CRISPR相关蛋白12a(CRISPR/Cas12a)系统,构建了一种通过试纸条(LFS)读取VV检测结果的方法RAA-CRISPR/Cas12a-LFS。该方法不依赖精密仪器,通过肉眼读取LFS结果便可快速、灵敏、准确检测VV,检测限为10 copies/μL VV基因组DNA,检测时间约为60 min。此外,该方法具有高特异性,并可准确检出血液、粪便、虾类等加标样品中的VV。因此,所构建的VV检测方法RAA-CRISPR/Cas12a-LFS兼具简便、快速、灵敏、准确的特性,有望用于弧菌病早期诊断以及水产品VV感染/污染情况的现场检测。
Vibrio vulnificus(VV),a zoonotic and aquatic pathogen found worldwide,causes vibriosis in aquatic animals and humans,which can bring heavy economic losses to aquaculture and seriously affect the personal safety of fishermen and consumers,respectively.Rapid,sensitive and accurate methods for the detection of VV are essential to protect human health and reduce economic losses of aquaculture.In order to establish a novel method for the detection of VV,this study constructed a VV detection method,RAA-CRISPR/Cas12a-LFS,based on recombinase aided amplification assay(RAA),CRISPR/Cas12a system and lateral flow strip(LFS).This method does not rely on a sophisticated instrument,can rapidly,sensitively and accurately detect VV through reading the results of LFS with the naked eye,and the limit of detection is 10 copies/μL VV genomic DNA within 60 min for the detection time.Moreover,the method shows satisfactory specificity toward non-target bacteria and high accuracy in the spiked blood,stool,and shrimp samples.Therefore,our constructed VV detection method,RAA-CRISPR/Cas12a-LFS,simultaneously satisfies simplicity,speed,sensitivity,and accuracy,and shows great potential for the early diagnosis of vibriosis and the on-site detection of VV infection/contamination of aquatic products.
作者
罗元丰
林子琴
王玲梦
董晶晶
袁褀恒
楼永良
赵玲玲
肖星星
LUO Yuan-feng;LIN Zi-qin;WANG Ling-meng;DONG Jing-jing;YUAN Qi-heng;LOU Yong-liang;ZHAO Ling-ling;XIAO Xing-xing(Wenzhou Key Laboratory of Sanitary Microbiology,School of Laboratory Medicine and Life Science,Wenzhou Medical University,Wenzhou,Zhejiang,325035,China)
出处
《动物医学进展》
北大核心
2023年第7期55-61,共7页
Progress In Veterinary Medicine
基金
国家级大学生创新训练项目(202010343025)
温州市基础性科技项目(Y20210109)
国家自然科学基金项目(82002117)。
