PDF(Pubmed)
Abstract：
BACKGROUND: Traditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control.
METHODS: We aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/μl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China.
RESULTS: The established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/μl for S. aureus, 103 copies/μl for S. flexneri, 105 copies/μl for A. caviae, 105 copies/μl for V. vulnificus, 100 copies/μl for S. enterica, 105 copies/μl for P. vulgaris, and 100 copies/μl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species.
CONCLUSIONS: Overall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.
METHODS: We aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/μl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China.
RESULTS: The established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/μl for S. aureus, 103 copies/μl for S. flexneri, 105 copies/μl for A. caviae, 105 copies/μl for V. vulnificus, 100 copies/μl for S. enterica, 105 copies/μl for P. vulgaris, and 100 copies/μl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species.
CONCLUSIONS: Overall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.
摘要：
背景：检测虫媒细菌病原体的传统方法耗时且需要专门的实验室设施，限制其在无法获得此类资源的领域的适用性。因此,快速有效的检测方法已成为疾病预防和控制的迫切需要。
方法：我们比对了7种细菌的核糖体16SrRNA序列（金黄色葡萄球菌，福氏志贺氏菌,鱼气单胞菌,创伤弧菌,肠沙门氏菌,普通变形杆菌,和小肠结肠炎耶尔森氏菌）通过DNASTARLasergene软件。使用DNASTARLasergene和PrimerPremier软件，我们设计了通用引物RLB-F和RLB-R,每种病原体的两种物种特异性探针，和通用探测器（包罗万象）。将7个标准菌株的PCR产物与固定在膜上的特异性寡核苷酸探针杂交以用于特定的实验程序。为了评估PCR-RLB的敏感性，在蒸馏水中将基因组DNA从最初的拷贝数1010连续稀释至100拷贝/μl。这些稀释物用作PCR-RLB敏感性分析的模板。用已建立的PCR-RLB方法对1060只家蝇进行了田间样品中7种蝇传细菌病原体的同时检测,从兰州的各种环境中收集，中国。
结果：已建立的PCR-RLB测定法能够检测金黄色葡萄球菌约103拷贝/μl的细菌菌株，福氏链球菌103拷贝/μl，鱼腥草105拷贝/μl，创伤弧菌105拷贝/μl，肠球菌100拷贝/μl，105个拷贝/μl，和100拷贝/μl的小肠结肠炎。结果表明，与常规PCR相比，已建立的PCR-RLB方法的检出率更高（约100倍）。该方法用于评估兰州各种环境中苍蝇的细菌载体状况。中国。在苍蝇携带的七种细菌病原体中，S、企业（34.57%），S、flexneri（32.1%），肠结肠炎Y（20.37％）被发现是主要物种。
结论：总体而言，这项研究表明，快速有效的PCR-RLB检测技术可以用于监测并因此有效预防和控制昆虫传播疾病的传播。同时,实验结果表明，城市卫生和媒介传播源是病原体传播的重要影响因素。
方法：我们比对了7种细菌的核糖体16SrRNA序列（金黄色葡萄球菌，福氏志贺氏菌,鱼气单胞菌,创伤弧菌,肠沙门氏菌,普通变形杆菌,和小肠结肠炎耶尔森氏菌）通过DNASTARLasergene软件。使用DNASTARLasergene和PrimerPremier软件，我们设计了通用引物RLB-F和RLB-R,每种病原体的两种物种特异性探针，和通用探测器（包罗万象）。将7个标准菌株的PCR产物与固定在膜上的特异性寡核苷酸探针杂交以用于特定的实验程序。为了评估PCR-RLB的敏感性，在蒸馏水中将基因组DNA从最初的拷贝数1010连续稀释至100拷贝/μl。这些稀释物用作PCR-RLB敏感性分析的模板。用已建立的PCR-RLB方法对1060只家蝇进行了田间样品中7种蝇传细菌病原体的同时检测,从兰州的各种环境中收集，中国。
结果：已建立的PCR-RLB测定法能够检测金黄色葡萄球菌约103拷贝/μl的细菌菌株，福氏链球菌103拷贝/μl，鱼腥草105拷贝/μl，创伤弧菌105拷贝/μl，肠球菌100拷贝/μl，105个拷贝/μl，和100拷贝/μl的小肠结肠炎。结果表明，与常规PCR相比，已建立的PCR-RLB方法的检出率更高（约100倍）。该方法用于评估兰州各种环境中苍蝇的细菌载体状况。中国。在苍蝇携带的七种细菌病原体中，S、企业（34.57%），S、flexneri（32.1%），肠结肠炎Y（20.37％）被发现是主要物种。
结论：总体而言，这项研究表明，快速有效的PCR-RLB检测技术可以用于监测并因此有效预防和控制昆虫传播疾病的传播。同时,实验结果表明，城市卫生和媒介传播源是病原体传播的重要影响因素。
