Establishment And Preliminary Applications Of Multiplex PCR For Detecting Pathogens In Laboratory Animals

Objective:Laboratory animals is the basics and conditions of biological science research.As replacer of mankind to bear medicine safety and therapeutic effect,the quality of laboratory animals directly affects the accuracy of scientific experiments in a broad range of fields.Along with the continuous improvement of the scientific research in domestic,the microorganism quality control of laboratory animals becomes much stricter.Staphylococcus aureus,Pseudomonas aeruginosa,Pasteurella multocida,Bordetella bronchiseptica,Mycoplasma pneumoniae,Clostridium piliformis,Klebsiella pneumoniae are not only the seven pathogens which needs to be eliminated in microorganism quality control of laboratory animals in domestic but also the pathogens to which laboratory animals are commonly susceptible.Currently,pathogens identification of laboratory animals depends on methods such as isolation and purification culture,colonial morphology,microscopic observation and biochemical test,which takes long time and high expense and relies on experienced analyst to avoid possible missing detection and misjudgment.PCR(Polymerase Chain Reaction)has already been widely applied to medical and health industry due to its advantages of simple,rapid and accurate.However,microorganism quality control of experimental animals has neither introduced PCR analysis method even nor multiplex PCR,which is far from satisfactory of identifying varieties of pathogens or large-scale samples simply,rapidly and accurately.Multiplex PCR can amplify the different fragments in the same tube and reaction system,which is simple,convenient and efficient.The application of multiplex PCR to pathogenic microorganism detection in laboratory animals can realize detection of multiplex pathogens simultaneously,which is suitable for detection and identification of large number of specimens and ofadvantages of high sensitivity,high efficiency,high specificity and low cost.Even though applied widely in other industries,multiplex PCR has less been applied for detecting pathogens in laboratory animals and researches in relation to this are limited in number.This study is aimed at establishing multiplex PCR detection method to seven laboratory animal pathogens,S.aureus,P.aeruginosa,P.multocida,B.bronchiseptica,M.pneumoniae,Clostridium piliformis,and K.pneumoniae,and comparing it with traditional microbiological detection methods or serological methods to validate the feasibility and accuracy of the multiplex PCR detection method.Method:1 Standard strains culture and DNA extraction: inoculate S.aureus standard strain on SP agar,P.aeruginosa standard strain on NAC solid agar,K.pneumoniae on DHL agar,and culture at 37 ? for 18-24 h;inoculate P.multocida and B.bronchiseptica on blood agar respectively and culture at37? for 24-48h;inoculate M.pneumoniae into special M.pneumoniae fluid medium for seven gradients and collect after one week.Extract DNA with the TIANamp DNA Kit according to the manufacturer's instructions.DNA of clostridium piliformis was kindly given by the National Institutes for Food and Drug Control and Institute of Laboratory Animal Sciences.2 Primers design: design the specific primers of S.aureus,P.aeruginosa,K.pneumoniae,P.multocida,B.bronchiseptica,M.pneumoniae and clostridium piliformis based on literatures and Primer 5.0 software.Analyze the specificity of primer by Blast.Amplify with PCR and detect the sequence to identify.3 Specificity and sensitivity test: to verify the specificity of PCR method,try to amplify nucleic acid fragments of nontarget strains.Assess the sensitivity of PCR method by testing template DNA of gradient dilution by 10 times.4 Establishing and application of multiplex PCR methods for four groups of pathogens:4.1 Amplify three bacteria,S.aureus,P.aeruginosa and K.pneumoniae,ina reaction system simultaneously.Meanwhile,add a pair of universal primer16 S rRNA as control to optimize the multiplex reaction system and amplification conditions,and to verify the specificity and sensitivity of the multiplex system.Apply this multiplex reaction system to detect artificially infected positive samples and specimens of 76 cases of mice and rats.At the same time,analyze all specimens with traditional method.4.2 Amplify three pathogens,P.multocida,B.bronchiseptica and M.pneumoniae,in a reaction system simultaneously to verify the specificity and sensitivity of the multiplex system and to establish a multiple reaction system.Apply this multiplex reaction system to detect artificially infected tracheal secretions sample,swab specimens and specimens of 182 cases of experimental animals.Meanwhile,analyze all specimens with traditional culture method or serology method.4.3 Amplify five pathogens,S.aureus,P.aeruginosa,P.multocida,B.bronchiseptica and M.pneumoniae,in a reaction system simultaneously to optimize multiplex reaction system and amplification conditions and verify the specificity and sensitivity of this system,to establish a multiplex reaction system.Apply this multiplex reaction system to detect artificially infected specimens of excrement,tracheal secretion and cotton swab,and specimens of182 cases of laboratory animals.Meanwhile,analyze all specimens with traditional culture method or serology method.4.4 Amplify three bacteria,S.aureus,P.aeruginosa and clostridium piliformis,in a reaction system simultaneously to verify the specificity and sensitivity of this system,to establish a multiplex reaction system.Apply this multiple reaction system to detect excrement specimens of 112 cases of experimental animals.Meanwhile,analyze all specimens with traditional culture method or serology method.Results:1 S.aureus,P.aeruginosa,K.pneumoniae,P.multocida and B.bronchiseptica grew well on agar plates;color of fluid medium for M.pneumoniae incubation changed from red to yellow.2 10 specific primers were obtained and their specificity were verified through the Blast.The size of primer amplification product were respectively153 bp(S.aureus),600 bp(P.aeruginosa),368 bp(K.pneumoniae),520 bp(universal primers),356 bp(P.multocida),237 bp(B.bronchiseptica),266 bp(M.pneumoniae),639 bp(clostridium piliformis),and 197 bp(P.aeruginosa).The sequence analysis results of amplification product corresponded with the sequences in Genebank.3 Specificity and sensitivity test of PCR: Specificity of PCR: the amplification in standard strains trials showed positive,while the amplification in other strains trails showed negative.Sensitivity of PCR: the limit of detection(LOD)for S.aureus DNA was 1pg;the LOD for P.aeruginosa was 10pg;the LOD for K.pneumoniae was 10 pg,the LOD for P.multocida was 1pg,the LOD for B.bronchiseptica was 10pg;the LOD for M.pneumoniae was 1pg;the LOD for clostridium piliformis was 100 pg,equals to 600 copies/?L.4 Multiplex PCR methods for four groups of pathogens:4.1 Multiplex PCR detection method for S.aureus(153bp),P.aeruginosa(600bp),K.pneumoniae(368bp)and universal 16 S rRNA(520bp)was established.Multiplex PCR was applied to analyze artificial infection samples and all results were positive.Analysis results of 76 specimens of feces of rats and mice with multiplex PCR showed that one specimen of mouse feces was P.aeruginosa positive,while the others were negative.The results of multiplex PCR method corresponded with those of traditional culture method.4.2 Multiplex PCR detection method for P.multocida(356bp),B.bronchiseptica(237bp)and M.pneumoniae(266bp)was established.Multiplex PCR was applied to analyze artificial infection samples,and all results were positive.Analysis results of 182 cases of cotton swabs of rats,mice and rabbits by multiplex PCR showed that 19 cases of rabbits swab were P.multocida positive;9 cases of rats swab were M.pneumoniae positive;others were negative.The results of P.multocida and B.bronchiseptica by traditional culture method were negative.There were 9 positive results of M.pneumoniaeby serology method.4.3 Multiplex PCR detection method for S.aureus(153bp),P.aeruginosa(600bp),P.multocida(356bp),B.bronchiseptica(237bp)and M.pneumonia(266bp)was established.Multiplex PCR was applied to analyze artificially infected samples of feces,tracheal secretions and nasopharyngeal cotton swabs and all results were positive.The results of 182 case of feces and cotton swabs of rats and mice by multiplex PCR showed one P.aeruginosa positive,while others were negative.The results of multiplex PCR method corresponded with those of traditional culture method and serology method:The results of P.multocida and B.bronchiseptica by culture were negative,the results of M.pneumoniae by serology were negative.4.4 Multiplex PCR detection method for S.aureus(153bp),P.aeruginosa(197bp),clostridium piliformis(639bp)was established.Multiplex PCR was applied to analyze 112 samples of feces of rats,mice and rabbits.All results were negative.The results of detecting S.aureus and P.aeruginosa by culture were negative and the results of detecting clostridium piliformis by serology were negative.Conclusions:Based on the characteristics of pathogens and methods to obtain specimens,aiming at seven pathogens,S.aureus,P.aeruginosa,P.multocida,B.bronchiseptica,M.pneumoniae,clostridium piliformis,and K.pneumoniae,multiplex PCR methods for four groups of pathogens were established.All methods showed high sensitivity and specificity,which is able to detect all artificial infection samples.The results showed that multiplex PCR has high practicability and accuracy.The results are consistent with those obtained by traditional culture method or serology method.There are several specimens,which are reported as negative by traditional culture method or serology method,detected by multiplex PCR as positive results.The established methods will lay foundation for multiplex PCR application for detecting microorganism in laboratory animals.