Detection Of The IpaH Gene Of Shigella Using Molecular Beacon Probe-based Real-time Fluorescent Quantitative Polymerase Chain Reaction

Objective: To detect the ipaH gene of Shigella using molecular beacon probe-based real-time fluorescent quantitative polymerase chain reaction (PCR).Methods: Based on the ipaH gene sequence of strain M32063 of Shigella flexneri recorded in the GenBank, two pairs of primer and one molecular beacon probe were designed. Totally 4 standard strains of Shigella were chosen for the experiment, while Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Streptococcus. were used for negative controls, to perform Shigella ipaH gene-specific PCR amplification. According to the results of amplification as well as the results of the sequence analysis, 1236-1806 nucleotide target sequence of ipaH gene of Shigella flexneri M32063 strain was chosen for specific amplification to extract purified products, followed by their introduction into pGEM-T vector to obtain the recombinant pGEM-T-ipaH clone vector. The bacterial fluid of recombinant pGEM-T-ipaH plasmid was then amplified for the sequence determination in a bio-tech company with subsequent comparison to the nucleotide sequence of ipaH gene of Shigella flexneri M32063 strain recorded in the GenBank. On the other hand, the sterile water was used to dilute the recombinant pGEM-T-ipaH plasmid in ratio of n X 10 to obtain the recombinant pGEM-T-ipaH plasmid in gradient concentrations for PCR and real-time fluorescent quantitative PCR in conditions of different annealing temperatures, different Mg²⁺ concentrations and different probe concentrations to get the final optimized protocols for the real-time fluorescent quantitative PCR. After the following sensitivity analysis and the construction of the positive standard products, the standard curve was established according to the linear relationship between the number of copies and the Ct value. Totally 20 feces samples...