Simultaneous detection of seven bacterial pathogens transmitted by flies using the reverse line blot hybridization assay.

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 10 to 10 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 10 copies/μl for S. aureus, 10 copies/μl for S. flexneri, 10 copies/μl for A. caviae, 10 copies/μl for V. vulnificus, 10 copies/μl for S. enterica, 10 copies/μl for P. vulgaris, and 10 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.