Development of Dual Fluorescence qPCR for the Detection of Pseudomonas Aeruginosa

Le résumé fourni par la source

Objective: To establish a real-time fluorescence quantitative PCR method for the detection of Pseudomonas aeruginosa (PA), which provides an effective method for the rapid and accurate clinical detection of PA. Methods: Using the principle of fluorescence quantitative PCR technology, the relatively conserved and highly specific DNA sequences of the ETA and OPRL double genes of PA were selected as templates, primers and probes were designed by TaqMan probe method, and standard curves were constructed, so as to establish a dual fluorescence quantitative PCR system for the detection of PA. Results: Orthogonal experiments were used to optimise the reaction system and reaction conditions, and to establish a standard curve for the detection of target genes by dual fluorescence quantitative PCR; the results of specificity experiments showed that except for the two standards that showed positive results, other strains (Staphylococcus aureus, Bacillus thuringiensis, etc.) showed negative results, and the method has good specificity; multiple sets of replicate assays were carried out on the positive standards for 1d and 30d, respectively, and the results showed that the method has good stability. The results showed that the stability of the method was good; the sensitivity experiments showed that the sensitivity of the method for the detection of the two positive QCs could reach 4.68×102copies/ml and 4.54×102copies/ml. Conclusion: Dual real-time fluorescence quantitative PCR for the detection of ETA and OPRL genes can be applied to the rapid detection of PA, which has the advantages of higher sensitivity and better specificity and stability, and has a good application prospect.

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