Microbial Aerosol Monitoring Of The Barrier System And Multiplex PCR Analysis Of Microbe In Specific Pathogen Free Laboratory Animals

To monitor the microbial aerosol of barrier system is an important issue of barrier system environmental monitoring. Pathogenic microbiol aerosol could directly impact the quality of laboratory animals. At present, there were few reports about the monitoring of dynamic microbial aerosol for a barrier system. The evaluation criteria and method for a running dynamic barrier system is also absent as well, which is not good to monitor the contamination sources. The physiological and biochemical tests of bacteria were commonly used to detect the microbe in laboratory animal of barrier system. This kind of test would take longer time and too many steps, and be hard to meet the demand to hugely screening the contamination sources of barrier system. In the present study, the changes of microbial aerosols of the dynamic barrier system and the source of pathogen in microbial aerosols were monitored in order to provide references for the further establishment of microbial aerosol monitoring system of barrier system. The reaction condition of multiplex PCR were optimized in order to established a rapid detection method for barrier system animal susceptible bacteria. Totally2experiments were included.1. Microbial aerosol monitoring of animal barrier systemThe laboratory animal barrier system was chosen to be investigated in this experiment, measuring falling bacteria and aerosol particles larger than or equal0.3um in the different functional area of the barrier system at different time and different working conditions, and analyzing the relevant influenceing factor. The results showed that the falling barrier and aerosol particles is significantly increased following the feed work, and sharply reduced after spray disinfection. Significantly higher at midnight in mouse and rat breeding room, lower at midnight in rabbit breeding room, the amount of falling bacteria significantly increased at working time in the clean corridor and garbage corridor, but at a lower level on weekend. The falling bacteria and aerosol particles larger than or equal0.3um in laboratory animal barrier system is closely related to the animal species, disinfection, personnel and animal feeding. The barrier system is the main place for laboratory animal breeding. The cages, animals and staff are getting in and out of the barrier system every day. The cages could be sterilizinged by high pressure, other things is sprayed or soaked into disinfection to afferent, the air is filtered four times. SPF animals came from qualified isolator. The staff entering the barrier system need to wear aseptic clothing after taking a shower, but it is difficult to accomplish the SPF stage altogether. Detection of the bacteria from the staff, materials, animals and air of barrier system showed that Klebsiella Pneumoniae and Streptococcus Pneumoniae could still be isolated from the staff’s mouth or nasal cavity. Therefore, the staff are possibly the main contamination factor of a barrier system.2. Multiplex pcr for the detection of SPF animal infection microbeThis experiment is intended to establish a rapid detection method for barrier system SPF animal susceptible bacteria (including Staphylococcus Aureus, Klebsiella Pneumoniae, Pseudomonas Aeruginosa and Streptococcus Pneumoniae) with multiplex PCR. Specific primers were designed according to various bacterial virulence genes. The Nuc, Phoe, Opr and PspA is unique for Staphylococcus Aureus, Klebsiella Pneumoniae, Pseudomonas Aeruginosa and Streptococcus Pneumoniae, respectively. To optimize the reaction, the final60.2℃Tm value of the reaction temperature,3.0mM magnesium ion concentration and0.125U/ul Taq enzyme were confirmed as the appropriate condition of four pairs of primers. To diluted the value of the sensitivity of bacterial detection by dilutioning the template genome gradient, the multiplex reaction sensitivity of Staphylococcus Aureus, Klebsiella Pneumoniae, Pseudomonas Aeruginosa and Streptococcus Pneumoniae are22.71,77.67,65.63and45.88pg, respectively. After optimization of the multiplex PCR, it was used to analyze the feces and tracheal samples of infected mice and the target band can be seen clearly, showing that this method can be used to detect susceptible bacteria of SPF animal rapidly.