Establishment And Preliminary Application Of Multiplex TaqMan Fluorescent Quantitative PCR For Respiratory Bacterial Pathogens In Rabbits

In the process of rabbit breeding,the respiratory diseases is the second most common diaease with high morbidity after intestinal diseases in rabbits.With the continuous development of the scale of rabbit farming in China,respiratory infectious diseases have brought huge losses to rabbit breeding industry.Among them,Pasteurella multocida is one of the main pathogenic bacteria of respiratory diseases in rabbits,which causes rhinitis,pneumonia,sepsis and other clinical symptoms,and often leads to acute outbreak of respiratory disease due in intensive culture,causing a large number of deaths.Bordetella bronchiseptica is often co-infected with Pasteurella multocida,which mainly occurred in young rabbits.It also can be co-infected with parasites and spread widely in rabbits populations.Pseudomonas aeruginosa and Klebsiella pneumonia are the other two common pathogens of respiratory diseases in rabbits,and pulmonary hemorrhage and suppuration are common symptoms in infected rabbits after necropsy.The clinical symptoms and pathological changes of the four pathogens are similar,and showing mixed infection in clinical detection.At the same time,it should be noted that the infection of Pseudomonas aeruginosa and Klebsiella pneumonia in humans are also very common at home and abroad.Therefore,it is important to establish a rapid,accurate and sensitive method for the diagnosis of common respiratory bacterial pathogens in rabbits.1.Establishment of quadruple TaqMan fluorescent quantitative PCRIn order to establish a more rapid and effective detection method for pathogenic bacteria detecyin of respiratory diseases in rabbits,four pairs of specific primers and four TaqMan probes were designed,which were referring to the conserved sequences of kmt gene of Pasteurella multocida,fla gene of Bordetella bronchiseptica,gyr A gene of Pseudomonas aeruginosa and pho E gene of Klebsiella pneumoniae in Gen Bank.The specificity,sensitivity and repeatability of the method were tested.The results showed that the standard curve of this method had a good linear relationship while the standard plasmid concentration is from 2.35×10~6to 2.35 copies/μL.Amplification signals with high specificity were showed in positive templates,and no cross reaction with other different bacterial and viral genomes.The lower limit of copy number detected by TaqMan multiplex fluorescent quantitative PCR was 2.25 copies/μL,the lowest sensitivity was10～100 times higher than conventional PCR,and the coefficient of variation of repeatability test was less than 2%.2.Preliminary application of quadruple TaqMan fluorescent quantitative PCRUsing this method,153 clinical samples in rabbit farms from Shandong,Jiangsu,Gansu,Heilongjiang,Fujian and Sichuan Chongqing Region were detected.86 Pasteurella multocida,39 Bordetella bronchiseptica,24 Pseudomonas aeruginosa,67 Klebsiella pneumoniae and 64 mixed were identified in samples,which was 90.84%consistent with conventiona PCR result and 100%consistent with the sequence results.It can analyze the proportion of bacteria in the mixed infected samples by cycle threshold,which can identidy the leading parhogens.In conclusion,the TaqMan quadruple fluorescent quantitative PCR to detect four different respiratory bacterial pathogens quantitatively was successfully established with high specificity and sensitivity,laying a foundation for clinical medication and providing a more accurate method for the diagnosis and epidemiological investigation of respiratory diseases in rabbits.