The Establishment And Preliminary Applications Of Multiple PCR For Detecting Staphylococcus Aureus, Pseudomonas Aeruginosa And Mycoplasma Pulmonis In Laboratory Animals

Objective:Laboratory animals are the basis for life science research,and they are closely linked to the health of animal practitioners and animal operators.Therefore,the microbiological quality control of laboratory animals has been incorporated into the national standards.Mycoplasma pulmonis,Pseudomonas aeruginosa,and Staphylococcus aureus are common susceptible pathogens of laboratory animals,even in rats,mice,guinea pigs,and hamsters rabbits which infection rate can reach a few percent,or even tens of percent.So the above pathogens are required to exclude in the microbiological testing of laboratory animal.Currently Staphylococcus aureus,Pseudomonas aeruginosa and Mycoplasma pulmonis are mainly detected by isolated and purified culture,biochemical tests or other methods which are time-consuming and require experienced technical staff to identify objective indicators and subjective judgments.Such as S aureus are often 48 h from bacteria isolated to issue test reports,Pseudomonas aeruginosa can take up to 72 h,and Mycoplasma pulmonis detection is required 21 days to complete.The PCR(Polymerase Chain Reaction)technology for its simple,rapid,and accurate has been widely used in medical diagnosis and other fields.In theory,it also can be applied to the pathogen detection of laboratory animals.But ordinary PCR method can only detect one pathogen one time.It can not meet the actual needs of single-tube detection of multiple pathogens,but can not meet the large sample,rapid and accurate detection.Different sizes of nucleic acid fragments can be amplified using multiplex PCR in the same reaction tube system.If multiplex PCR for detecting microbes are applicated in laboratory animal,it can simultaneously detect many pathogens and it is suitable for a large number of conventionalmicrobial detection and identification of laboratory animal.It has a high sensitivity,high efficiency,high specificity and low cost and other advantages.The present study is aimed to bulide a multiplex PCR for detecting three kinds of experimental animal pathogens(Staphylococcus aureus,Pseudomonas aeruginosa and Mycoplasma pulmonis)and test the operability and accuracy of the method.Methods:1.Staphylococcus aureus standard strain was incubated in SP agar.After culturing 24 h,it was transferred to the blood agar medium to isolate and purify.Coagulase tests and mannitol fermentation tests was carried out using microscope.The standard strain of Pseudomonas aeruginosa was inoculated into NAC liquid medium and then transferred to NAC solid medium to isolate and purify.Biochemical identification.Oxidase test and 42?growth test was operated.The Mycoplasma pulmonis was cultured in a liquid medium for a week and then observed.2.Specific primers of Mycoplasma pulmonis,Staphylococcus aureus,and Pseudomonas aeruginosa were designed by retrospective research.The specific of primers for each pathogen were analyzed by Blast.PCR amplification products were sequenced.and verify correctness by bioinformatic analysis.3.We optimized PCR Reaction System and detected the specificity and sensitivity of PCR method.The template DNA was diluted to a series of 1:10,and respectively diluted DNA was used to amplify as a template.Colonies were nourished in ordinary nutrient broth.and expanded in 37 ? thermostatic shaker.The colonies(diluted by 10-fold gradient.)was inoculated to a solid medium,then extract nucleic acid was amplified by PCR.Other non-target bacterias were amplified for specific experiments.4.The DNA of Mycoplasma pulmonis,Staphylococcus aureus and Pseudomonas aeruginosa were amplified simultaneously in the same reaction system.And bacterial universal primer was added to the reaction system as an internal quality control.Multiplex PCR was establish to detecte three kinds ofpathogens by adjusting and optimizing the reaction system.Rat and mice were a total of 45.Nucleic acids collected from animal dung and trachea and amplified to dectect three pathogens by multiplex PCR.While routine testing were carried out to inspect the practicality and accuracy of multiplex PCR method.Results:1.Yellow colonies of Staphylococcus aureus was formed on the SP solid medium.Transfer it to the blood agar,forming hoary and ?-hemolytic ring around the colonies.Bacteria were Gram-positive cocci,arranged in grape-like clusters.Mannitol fermentation test and coagulase test was positive.Pseudomonas aeruginosa colonies were flat,uneven and spreading with serrated edge on NAC agar plates.Most colonies could produce a green pigment in the colony as well as the surrounding medium.The bacteria was Gram-negative bacilli,of different lengths.Whether negative or positive,the result of biochemical identification was correct.Oxidase test and 42 ?growth test were positive.The liquid culture of Mycoplasma pulmonis had shown a color change from red to yellow.2.We have received three kinds of pathogens specific primers,which was verified by Blast comparison.The PCR product sizes of Staphylococcus aureus,Pseudomonas aeruginosa and Mycoplasma pulmonis were respectively 153 bp,375bp and 266 bp.The similarities of its sequencing results corresponding to Genbank sequence were respectively 97.00%,98.90%and 99.59%.3.Specificity testing of PCR: The specific result was that except the standard strains,other species were amplified negative.Sensitivity testing of PCR: the minimum detectable amount of nucleic acid in DNA were 1pg and10 pg for Staphylococcus aureus and Pseudomonas aeruginosa,separately.The minimal limited colonies were 2 CFU and 14 CFU of Staphylococcus aureus and Pseudomonas aeruginosa,separately.The minimum detectable amount of nucleic acid in DNA was 1pg for Mycoplasma pulmonis.4.A multiplex PCR amplification systems(containing Staphylococcusaureus,Mycoplasma pulmonis,Pseudomonas aeruginosa and bacterial common gene)were established.Agarose gel electrophoresis showed four bands: 153 bp,266 bp,375 bp and 520 bp,respectively.The colonies of laboratory animal were tested by PCR.The results were consistent with microbiological testing.The results of animal faeces was discrepant compared with conventional culture identification methods.Conclusions: We developed a specific,sensitive and reproducible multiplex PCR detection system by Staphylococcus aureus,Pseudomonas aeruginosa and Mycoplasma pulmonis.This method was consistent with the conventional detection methods in experimental animals microbiological testing,but more rapid,simple,and accurate.Preliminary validated the feasibility and practicality of this detection method,laid its foundation for future use in routine testing of laboratory animal microorganisms.