Rapid Detection Of Shigella Based On IPaH1Gene Liquid Chip Technology

Shigella is the most common pathogens of human bacillarydysentery, a serious threat to human health. Currently Shigella traditionaldetection methods including conventional detection methods (such asisolation culture, biochemical reaction， serotyping), immunologicaldetection methods, molecular biological detection methods. Eachdetection methods have certain limitations. Therefore, the developmentspecific and rapid detection method Shigella in food, drug testing, controlbacillary dysentery pandemic and other aspects is important.Objective: Shigella invasion plasmid antigen H gene (IpaH) for theestablishment of target genes liquid chip detection system to detectShigella in simulated samples.Methods: According to the information Shigella virulence gene andprimer design principles to design highly specific primers, the expressionof ipaH gene levels in Shigella boydii, wild Shigella, Escherichia coli,Enterohemorrhage E. coli (O157:H7), Salmonella enterica subsp,Enterobacter sakazakii, Proteus vulgaris, Bacillus cereus was determinedby PCR, and the optimization of PCR conditions on annealingtemperature and primer concentration was evoluated. A specific probewas designed according amplified IPaH1gene sequence, coupled the probe to microspheres to build liquid chip, With specific binding ofbiotinylated PCR product to create and to optimize the liquid chipdetection condition, and the reproducibility, specificity, sensitivityvalidation, and analog Shigella environmental samples verified to achievethe dual specificity detection on Luminex System.Results: IPaH1gene specific expression were detected inShigella while no expression were detected in Escherichia coli,Enterohemorrhage E. coli (O157:H7), Salmonella enterica subsp,Enterobacter sakazakii, Proteus vulgaris, Bacillus cereus, Enterococcusfaecalis, Staphylococcus aureus, Listeria monocytogenes, Vibrioparahaemolyticus, Clostridium perfringens. PCR conditions wereoptimized, optimal annealing temperature is55.0℃~59.0℃, the finaloptimal concentration of primers is0.1μM, can be directly used in thedetection technology of the liquid chip. The optimized liquid chipdetects，the best conditions denaturation time is2min, hybridizationtemperature54℃, hybridization time is25min, the incubation time is25min. The reproducibility, specificity and sensitivity verification,specificity is100%in a selected limited strain, and the sensitivity ofnucleic acid is10-6ng, the sensitivity of the bacteria is7.8CFU/mL.Simulation validation of Shigella in environmental samples,22samplesof10kinds either positive group or interfere group, the resultingfluorescence signal intensity was significantly higher than the control group. Liquid chip detection accuracy of the analog samples in this studywas100%.