Methods For Nucleic Acid Detection Of Methicillin-resistant Staphylococcus Aureus(MRSA)

Pathogenic bacteria have developed resistant to antibiotics which was widely used during therapy. Related diseases and infections are difficult to be cured because antibiotics cannot suppress drug-resistant pathogens. Currently, drug-resistant pathogen have become a worldwide problem and threat for human health. Obviously, it is especially important to develop rapid and accurate detection method for drug-resistant pathogens in clinical samples, food samples and environmental samples. The detection results have vital roles in subsequent treatment and disposal options. This study focused on rapid detection of Methicillin-Resistant Staphylococcus aureus( MRSA) by molecular analysis. First, we studied the different peptidoglycan hydrolases used to extract nucleic acids of S.aureus(including MRSA). Cell walls of S. aureus(including MRSA) are difficult to be destroyed due to compact peptidoglycan layers. Therefore, we mainly studied whether a new phage lysin could replace existing commercial peptidoglycan hydrolases(Lysostaphin and Achromopeptidase) to extract nucleic acids of S.aureus(including MRSA). Experimental result showed that the lysin was superior to the existing commercial hydrolytic enzymes in extracting the nucleic acids from S.aureus(including MRSA). Second, we studied the magnetic beads labeled antibody capturing S.aureus(including MRSA) combined with the above-mentioned phage lysin was applied to detection of MRSA in complex samples, and the results showed the LOD of MRSA detection could reach to ~10 2 CFU/ml. Finally, droplet digital PCR was utilized to detect MRSA in the mixed Staphylococci. In this section, different extraction methods of bacterial nucleic acids and duplex PCR strategies were evaluated to eliminate the interference of Methicillin-Resistant Coagulase-Negative Staphylococci(MR-Co NS) in detection of MRSA. It was found that dual amplification of mec A and SCCmec-orf X junction via dd PCR was able to detect MRSA from the mixed Staphylococci directly. Meanwhile, this method can directly figure out the number of MRSA in samples via absolute quantification.