7种细菌性虫媒病病原体的反向线状印迹杂交检测技术的建立  

Establishment of reverse line blot hybridization for detection of seven bacterial insect-borne disease pathogens

作　　者：李雅琪 叶得河[1] 马永华 LI Yaqi;YE Dehe;MA Yonghua(College of Animal Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《甘肃农业大学学报》2024年第3期31-39,共9页Journal of Gansu Agricultural University

基　　金：甘肃农业大学学科建设专项基金项目(GUA-XKJS-2018-073);甘肃农业大学青年导师基金项目(GAU-QDFC-2020-11);甘肃农业大学人才专项经费项目(2017RCZX-11);甘肃省科技厅重点研发项目(18YF1FA080);甘肃省科技厅国际合作项目(144WCGA169);国家自然科学基金项目(32160843);甘肃省现代丝路寒旱农业科技支撑项目(GSLK-2021-18);甘肃省自然科学基金项目(18JR3RA167)。

摘　　要：【目的】建立快速高效的细菌性虫媒病的检测方法。【方法】运用反向线状印迹杂交技术(reverse line blot,RLB),通过对金黄色葡萄球菌(Staphylococcus aureus)、福氏志贺菌(Shigella flexneri)、豚鼠气单胞菌(Aeromonas caviae)、创伤弧菌(Vibrio vulnificus)、肠沙门氏菌肠亚种鼠伤寒血清型(Salmonella enterica subsp.enterica serovar Typhimurium)、普通变形杆菌(Proteus vulgaris)、小肠结肠炎耶尔森菌(Yersinia enterocolitica)7种细菌的核糖体16S RNA进行序列比对,利用DNAStar和Primer premier软件设计出了用于PCR-RLB杂交反应的基因组DNA样品进行PCR扩增的通用引物(RLB-F,RLB-R),和特异性探针以及通用探针(Catch-all),通用下游引物(RLB-R)5'端用生物素标记和探针5'端连接氨基团(NH_(2)^(+))。【结果】成功建立高效、快速、特异且能同时检测7种细菌PCR-RLB的方法,并确定了通用引物最佳浓度为25μmol/L,金黄色葡萄球菌和福氏志贺菌探针的最佳浓度分别为50μmol/L,其他5种病原探针的最佳浓度为100μmol/L。使用PCR-RLB检测方法,可对上述7种细菌的检测敏感性可达到10^(-6)~10^(-11) ng/μL,远远高于PCR的10^(-3)~10^(-8) ng/μL。【结论】首次同时对7种细菌进行检测,并确定了不同引物及探针的最佳浓度。该方法不仅提高了检测效率,同时还提高了检测的灵敏度,为开发新的检测手段提供了理论依据。【Objective】 The study aimed to establish a rapid and efficient method for the detection of bacterial insect-borne diseases.【Method】 Reverse line blot(RLB) was used to detect Staphylococcus aureus,Shigella flexneri,Aeromonas caviae,Vibrio vulnificus,Salmonella enterica subsp.enterica serovar Typhimurium,Proteus vulgaris,and Yersinia enterocolitica.The 16sRNA sequences were compared,and universal primers(RLB-F,RLB-R) for the PCR-RLB hybridization reaction were designed using DNA Star and Primer premier software.Specificity probes,universal probes(Catch-all),universal downstream primers(RLB-R),and biotin-labeled 5' end probes linked with an amino group(NH_(2)^(+)) were developed.【Result】A highly efficient and rapid assay with a 25 μmol/L concentration was successfully developed for the simultaneous detection of the seven targeted bacteria.The concentrations for Staphylococcus aureus and Shigella flexneri probes were 50 μmol/L,while the other five pathogen probes had concentrations of 100 μmol/L.The sensitivity of PCR-RLB was 10^(-6) to 10^(-11) ng/μL,significantly higher than that of PCR(10^(-3) to 10^(-8) ng/μL).【Conclusion】 This study presented the simultaneous detection of seven different bacteria for the first time using RLB.Optimal concentrations for primers and probes were determined.The developed method not only improves detection efficiency but also enhances detection sensitivity.These findings provided a theoretical basis for the development of new detection methods for bacterial insect-borne diseases.

关 键 词：细菌性虫媒病 反向线状印迹杂交技术 PCR扩增 

分 类 号：S855[农业科学—临床兽医学]