Study And Utility Of Multiplex PCR In Rapid Detection Of Methicillin-Resistant Staphylococcus

Objective To detect methicillin-resistant staphylococcus aureus(MRSA) and methicillin-resistant coagulase-negative staphylococcus (MRCNS) by multiplex PCR. And to Investigate the Epidemiology circumstance of methicillin-resistant staphylococcus(MSR) in the region of Qingdao.Methods We detected staphylococcus 262 species by microscan bacteria appraiser, staphylococcus aureus (SA)86 species and coagulase-negative staphylococcus 176 species, we amplified the DNA fragment by multiplex PCR utilizing universal 16S rRNA of staphylococcus primers, mecA gene of methicillin-resistant staphylococcus(MRS) primers and femA gene of staphylococcus aureus primers . The results were compared with Agar Dilution. And proceed to statistics the analysis to the Epidemiology circumstance of Qingdao region MRS.Results The specific genetic amplification fragments of 16S rRNA of staphylococcus, mecA gene and femA gene is 1088bp, 165bp and 314bp, respectively. Amplified, ethidium bromide-stained DNA fragments were visualized after electrophoresis on agarose gel.We found that 262 species presenced 1088bp belts;among the 86 SA species found to presence 314bp belts, 67 species also presenced 165bp belts;among the 176 CNS species, 139 species presenced 165bp belts.We considered Agar Dilution as gold criteria and determined MRS by mecA positive.AII the culture-positive samples were also detected by PCR(n=204), but, among the 58 species found to be MRS negative by microbiological criteria, 2 species were scored as postive.These data yielded mecA gene PCR assay' s sencitivity , specificity, negative predictive values and positive predictive values were 100%, 96.6%, 100% and 99.0%, respectively. We determined MRSA by mecA positive and femA positive.AII the culture-positive samples were also detected by PCR(n=66), but, among the 196 species found to be MRSA negative by microbiological criteria, 1 species were scored as postive.These data yielded mecA gene combining with femA gene PCR assay' s sencitivity , specificity, negative predictive values and positive predictive values were 100%, 99.5%, 100% and 98.5%, respectively.The Epidemiology result of Qingdao region MRS:The staphylococcus tolerance rate was 78.6%, among them coagulase-negative staphylococcus tolerance rate was 79.0%, tolerance rate as 78.0% in the staphylococcus aureus.Conclusion The result revealed that the method of detecting mecA gene by multiplex PCR could sensitively, rapidly and specifically detect methicillin-resistant staphylococcus.the united detection of mecA gene and femA gene could effectively detect MRSA. The MRS has become the increasingly serious problem in staphylococcus infection.