The Establishment Of A Multiplex PCR For Distinguishing Four Microbes In Pyometra And The Tentative Observation Of The Antibacterial Effect Of Lysozyme

Pyometra, also known as cystic endometritis-pyometra complex, is an endometritiscaused by bacteria after a course of cystic endometrial hyperplasia (CEH) which is usuallyresulted from the abnormality of canine endocrine and hormone receptors in the uterus. It is acommon reproductive system disease in aged bitches and secondary bacterial infection is asignificant factor. To this day, a variety of bacteria have been isolated from uterus contents ofbitches with pyometra, including Streptococcus, Salmonella, Staphylococcus aureus,Escherichia coli, etc. The development of a multiplex PCR assay, which could be used todistinguish those microbes, is important for the future research on pyometra with molecularbiology techniques.On the basis of gene sequences of Streptococcus EF-Tu (Elongation Factor Tu),Salmonella invA (invasion protein A), Staphylococcus aureus nuc (thermostable nuclease) andEscherichia coli phoA (Alkaline phosphatase),4pairs of primers were designed andsynthesized. DNA fragments of166bp,284bp,406bp,555bp were amplified. After theoptimization of multiplex PCR conditions, the results indicated that the optimal annealingtemperature was at54℃; the optimal primer concentration were100nmol/L,300nmol/L,400nmol/L,60nmol/L respectively; the optimal concentration of Mg2+was1.5mmol/L. Thesensitivity of this multiplex PCR was also determined, the lowest DNA detection limits were16.5pg/μl,3.11pg/μl,21.0pg/μl and3.52pg/μl respectively. Using this multiplex PCR assay,we detected9pyometra samples. The detection rate of Streptococcus was88.9percent,Salmonella55.6percent, Staphylococcus aureus11.1percent, Escherichia coli33.3percent;and mixed infections accounted for two-thirds of all collected cases. The results showed thatthis established multiplex PCR was a simple, fast, high sensitive method, which could providenot only a new thinking to the future microbiological research of pyometra, but also sometechnical supports for exploring the microbes and the pathogenetic regularity of this canine disease.Antibacterial liquid medium with lysozyme was applied to tentatively observe theantibacterial activity of lysozyme on those four microbes; its concentration was changed inthe range of19.5μg/ml to10.0mg/ml by10-fold concentration gradient. Results demonstratedthat lysozyme had best inhibitory effect on Staphylococcus aureus and worst on Salmonella.The minimum inhibitory concentration of lysozyme against each bacteria was respectivelybetween0.313mg/ml to0.625mg/ml,2.50mg/ml to5.00mg/ml,0.0781mg/ml to0.156mg/ml,and0.625mg/ml to1.25mg/ml.