Detection Of Viable Staphylococcus Aureus By Reverse Transcription-PCR

BackgroudStaphylococcus aureus(SA) is an important zoonotic pathogens, which can cause skin and soft tissue infections, food poisoning, traumatic eye and so on.. Bacteria culture is a conventional method of detection of S. aureus at present,and it is reliable, but,the method usually spends over 24h which can not meet rapid detection. Immunological methods include immunofluorescence, radioimmunoassay and enzyme-linked immunosorbent assay. Although these methods have certain specificity and sensitity, it usually requires combination of bacteria culture,and it is difficult to meet the need for clinically rapid detection. Conventional PCR is rapid,specific and highly sensitive. Because DNA degradation of dead bacteria is slow, this method can not distinguish between dead and viable bacteria.For traumatic endophthalmitis, however,it can provide many targeted clinical treatment,if we know whether the bacteria in the samples are live or dead. So,it becomes necessary to develop a new method, which is rapid, sensitive and specific. Especially,the method can distinguish live bacteria. Staphylococcal protein A(SPA),encoded by spa gene, is species-specific and genus-specific. SPA is a superficial protein of the cell wall. Presence of SPA is a characteristic of Staphylococcus aureus. Half-life of prokaryotic's mRNA is very short.Detection of mRNA can distinguish between dead and viable cells.The experiment was done to detect Staphylococcus aureus by RT-PCR with a pair of primers of the spa gene.AimInvestigate some releted questions about viable detection of Staphylococcus aureus by RT-PCR method in order to provide theoretical basis for clinical therapy, to avoid the overuse of antibiotics and reduce the emergence of resistant strains.MethodsAll of bateria,including a standard Staphylococcus awreus(ATCC25923),some clinical strains of Staphylococcus aureus,and some clinical isolates of non-Staphylococcus aureus were verified by conventional phenotypic methods. All the bateria were inoculated in nutrient broth and identified as pure culture.1. One colony-forming unit(CFU) of Staphylococcus aureus (ATCC25923) was dissolved in 2ml nutrient broth and cultured at 35℃for 6-8h. Number of the above baterial broth was counted by plate medium. Meanwhile, the baterial broth was diluted into five concentrations and the dilutions were detected by RT-PCR assay.2. Staphylococcus. aureus(ATCC25923) was killed by different antibotics with different concentrations in vitro envioment. The baterial numbers after effecting certain time were counted by plate medium. The bactericidal concentration-time curves were descriped using the numbers.3. All the strains of Staphylococcus. aureus were detected by two methods to select MRSA.4. The target gene was selected to be used to RT-PCR by comparing RT-PCR with unival PCR using different genes.5. The sensitivity of that Staphylococcus aureus was detected by RT-PCR was measured.6. The specificity of that Staphylococcus aureus was detected by RT-PCR was evaluated.7. The strain of Staphylococcus. aureus(ATCC25923) and a strain of MRSA were distinguished between dead and viable cells by RT-PCR. Results1. Detection of MRS A 12 of 20 strains, accounting for 60%, of Staphylococcus. aureus were MRSA, and 9 of the 12 Staphylococcus. aureus strains'mecA gene were amplified.2. Selection of pairs of primers For the several pairs of primers designed, the pairs of primers of nuc and spa gene could be used to RT-PCR.Contrastly, the pair of primers of spa gene was for follow-up experiments.3. Measuring of sensitivity The lowest concentration of Staphylococcus. aureus detected by RT-PCR was 1.0×104cu/ml,and one single piece of bacterium could be detected by RT-PCR if it was enriched for over 6h.4. Evaluation of specificity All the strains of Staphylococcus. aureus were detected by RT-PCR using the pair of primers primers.There was no crossreaction with E. coli,Pseudomonas aeruginosa,Staphylococcus epidermidis,Pyogenic streptococcus.5. Distinguishment of viable and dead cells mRNA from S. aureus (ATCC25923) became undetectable when dead cells were stored at 4℃temperature for under 2h; for MRSA, mRNA couldn't be detected when cells were inactivited for over 2h.Conclusion1. This pair of primers can be used to detect viable Staphylococcus aureus.2. The method can be used to detect viable MSSA and MRSA,but the mRNA of MRSA can be stored for longer time than MSSA after inactivation by High-pressure steam sterilization.