Development of microarray gene chip for detection of pathogenic bacteria in port and waterway

ObjectiveGene chip to detect pathogenic bacteria in port and waterway was developed in order to lay a foundation for gene chip detection technology targeting port and waterway. MethodThe genomic DNA of the target strains were extracted by conventional methods. 16S rDNA universal primer and conserved region primers of gyrB gene were used for PCR amplification. Oligonucleotide probe designed on target fragments obtained by amplification was carried out using AlleleID 6.0 and Array Designer 4.25. Through PCR screening and validation, amino-modified target probes were spotted on aldehyde slide via microarrayer. Chip hybridization conditions were optimized, and was applied to detection of water and sediment samples from port and waterway. In this way, detection effects of microarray gene chip were validated.ResultThe optimized chip hybridization conditions were: probe concentration 10 μmol/L, ultraviolet crosslinking time 2.0 h and hybridization temperature 65 ℃. It increased sensitivity of gene chip detection, and was of high speed, high throughput and accuracy compared with traditional detection method. The microarray gene chip could detect seven pathogenic strains from port and waterway: Vibrio cholerae, Enterobacter cloacae, V. alginolyticus, V. harveyi, V. parahemolyticus, Aeromonas hydrophila and V. vulnificus. No specific hybridization was detected. ConclusionMicro-array gene chip detection technology targeting pathogenic bacteria in port and waterway is highly specific, sensitive, rapid and convenient. It can be used for marine environmental monitoring and marine products quality and safety detection in port and their waterway and surrounding areas.