| Staphylococcus aureus ( S.aureus. SA ) is a major bacterial pathogen that is responsible for a broad and divergent range of human and animal infections.A -strain of SA named SAm0109 was isolated from laboratory mice which were killed by SA infection in Jiangsu province in September of 2001. The bacteria produced golden-yellow pigment, # -hemolytic action and the power of fermenting mannitol, sucrose and coagulase test was positive. The strain of SA can cause mice dead.To study the mechanisms of pathogenicity of disease due to SA, experimental mice were used for this purpose. Experimental strains of SA were SA1800 and SAm0109. After ineoulating with the bacteria, the clinical symptoms and pathogenic changes of experimental mice were examined. Two strains of SA could cause disease in experimental mice. Obvious invasion of the bacteria was found. It demonstrates that the LD50 of SA1800 to experimental mice is 10-4.1 / 0.2 ml. and the LD50 of SA.m0109 to experimental mice is 10-5.1/ 0. 2 ml. It was a good animal model for studying the pathological mechanisms of SA.Aceording to the sequence of the nuc gene, which encodes the thermostable nuclease (thermonuclease. TNase ) of SA, a pair of oligonucleotide primers which are special to the sequence of nuc gene were designed and synthesized, and were used to amplif} a sequence of nuc gene by the polymerase chain reaction ( PCR ). DNA extracted from SA was amplified and DNA from Staphylococcus epidermidis,Streptococcus pyogenes, Escherichia coli was used for control. The PCR product was detected by agarose gel electrophoresis analysis. The SA revealed positive results indicated by the presence of a 676 bp specific fragment on gel. while non-SA gave negative results demonstrating that the primers should be specific for SA tested. The PCR would be able to detect as little as 3 pg SA-DNA as determined by amplification of serially diluted SA-DNA. The whole process of DNA extraction, amplification and eiectrophoresis could be finished within 4 h. 240 clinical samples taken randomly from laboratory rats and mice were submitted to detect by PCR and culture with a positive detection rate of 2.9 % ( 7 / 240 ) and an agreement of 99.6 %. The results showed the PCR assay developed was a rapid, sensitive and specific method for detecting SA. It provides a useful tool for direct detect clinical specimens, and should be suitable for detecting SA in laboratory animals.Using the genome DNA of the clinically isolated strain of SAm0109 as template, the fragment flanking nuc gene was amplified by PCR. The expected size fragment was obtained and was cloned into the plasmid pGEM-T. The recombinant plasmid of pGEM-NUC was identified by restriction enzyme analysis. PCR and sequencing, which proved completely its validity.To establish a sensitive dot blotting for detecting SA, a DNA fragment of 676 bps of the nuc gene was applified from SA genome DNA by PCR and labelled with horse-radish peroxidase as the probe. The purified genomic DNAs of SA1800. SAm0109 and Staphylococcus epidermidis. Streptococcus pyogenes, Escherichia coli were blotted onto the Hybond N+ nylon membrane, fixed using UV irradiation. hybridized under high stringency conditions, and detected using the Enhanced Chemiluminesence kit ( ECL ). The probe reacted positively with the SA genomic DNAs, but not with that of the non-SA with a sensitivity of 1 pg. 322 clinical samples taken randomly from specific pathogen free ( SPF ) rats and mice were submitted to detect by the dot blotting and previously established PCR and culture with a positive detection rate of 3.1 % ( 10 / 322 ) and an agreement of 100 %. These data demonstrated that the established dot blotting was a quick, reliable, sensitive and specific method for detecting SA in laboratory rats and mice. |
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