Using Junction Probe Technology For Rapid Detection Of Staphylococcus Aureus

Purpose:1. To establish an isothermal nucleic acid detection method, Junction Probe (JP), based on restriction endonuclease, suitable for both research and clinic laboratory.2. To develop a rapid method to detect Staphyloccocus aureus using JP technology.Method:1. Three pairs of probes were designed and tested. By using the probes with highest reactivity and reaction reproducibility, the molar ratio of the probes was optimized, the detection specificity was investigated and the double-stranded targets were tested.2. Based on the biological nature of Staphyloccocus aureus, the bacterial strains from hospitals were grown on LB agar and the genomic DNA were obtained by the purification of alkali treatment method.3. Single strand DNA were obtained by combining PCR amplification and magnetic beads extraction, and Staphyloccocus aureus was detected using JP technology.Discussion:1.The feasibility of the method to detect Staphyloccocus aureus was realized by detecting the artificial single-strand DNA target. The PA1-PB1probe pair was screened to give the fastest reaction rate and best reproducibility. The experiments showed that the detection limit of this system is4nM of temple DNA, and it can distinguish Staphylococcus aureus from Staphylococcus epidermidis, which contains SNP in the target sequence. The experiments also showed that the systems can not detect double stranded DNA, so single stranded target must be prepared while detecting clinical samples.2.The cultivation results of Staphylococcus aureus showed that the bacteria does not need stringent growing conditions, and LB medium is good enough, which satisfied our subsequent experimental requirement.3.This method can quickly and effectively detect Staphylococcus aureus, and has great advantages over traditional biochemical identification methods. The system has a great S/N ratio, resulting from both of the PCR and JP signal amplification in the detection of single-strand DNA samples. With the two-stage examination, the positive ratio is up to97.25%by our PCR-JP method, which is more specific than PCR technology alone.