The Establishment Of LAMP To Test The PA And SeV Of Mice And Its Preliminary Application

In life science research, laboratory animals is one of the most basic four support elements, the quality of laboratory animals plays an important role in promoting the repeatability and accuracy in animal experimental science research. Mice is one of the most commonly animal in the modern medical research and life science research, the microbic quality of animals directly affects the accuracy of the experiment.According to GB14922.2-2011, pseudomonas aeruginosa(PA) is one of the must test items in specific pathogen free mice(SPF). Sendai virus(SeV) is one of the must test projects in clean level mice. At present, the methods of detecting pseudomonas aeruginosa and sendai virus in mice included: separation of culture, RT-PCR, Enzyme-linked immunosorbent Assay(ELISA), etc. The traditional cultivation method has high accuracy, but this method is tedious, laborious and time-consuming operation, and the detection rate is low, prone to false negative results. RT-PCR and ELISA detection method have high sensitivity, but equipment demand of the method is higher, and it is easy to produce false positive results.In this paper, Loop-mediated isothermal amplification technology(LAMP) applied six specific primers,which were designed according to the height conservative sequence of pseudomonas aeruginosa and sendai virus genome and PrimerExpler V4 software. The LAMP testing method of mice pseudomonas aeruginosa and sendai virus were established through the optimization of the reaction system conditions. The specificity, stability, repeatability, and sensitivity of the method were verified. They can be used for clinical detection.This study has two main parts.Part1 The establishment of LAMP to test the Pseudomonas aeruginosa in mice and its preliminary applicationObjective: To establish a method for the detection mice’s Pseudomonas aeruginosa by the loop-mediated isothermal amplification(LAMP). Method: Two sets primers, each containing six specific sequences, was designed using V4 PrimerExploer software, according LAMP technology, and the target gene sequence of oprL of mice’s Pseudomonas aeruginosa. Pseudomonas aeruginosa LAMP(PA/LAMP) method was established through optimizing the LAMP reaction system. At the same time, the specificity, stability, repeatability and sensitivity of the method were valided. The initially clinical tastings were performed. Result: The established method of PA/LAMP can detect PA/DNA specifically, with no reactions with other common pathogens. PA/ LAMP can detect different batches at the same time or the same batch at different times of PA/DNA. The detection limit of PA/ LAMP was 0.29 pg. Compared with PCR detection method, it was higher 105 fold. For clinical detection, the positive rate of PA was 27.7%(10/36). All reactions can be completed within 1 h. Visual experiments results can be achieved by adding calcein intuitive. Conclusion: PA/LAMP method has the advantages of specificity, accuracy, sensitivity, rapid and simple. It can be used for the rapid detection of PA in mice.Part2 The establishment of LAMP to test the Sendai virus in mice and its preliminary applicationObjective: To establish a method for the detection mice’s Sendai virus by the loop-mediated isothermal amplification(LAMP). Method: Six specific primers for LAMP were designed with V4 PrimerExploer software, according to the target gene sequence of mice of SeV(gi: 9627219) F protein and LAMP technology. SeV RNA was extracted with virus RNA kit. SeV LAMP(SeV/LAMP) method was established through optimizing the LAMP reaction system. At the same time, the specificity, stability, repeatability and sensitivity of the method were certified. The initially clinical testing was performed. Result: The established method of SeV/LAMP can detect SeV/RNA specifically, with no reactions with other common pathogens; SeV/ LAMP can detect different batches at the same time, or the same batch at different times of SeV. The detection limit of SeV/ LAMP was 0.33 pg. The detection rate of SeV/LAMP was high(respectively 5/30, 2/30) compared with that of ELISA detection method. For clinical detection, the positive rate of SeV was 27.5%(11/40). All reactions can be completed within 1 h. Visual experiments results can be achieved by adding calcein intuitive. Conclusion: SeV/LAMP method has the advantages of specificity, accuracy, sensitivity, rapid and simple. It can be used for the rapid detection of SeV in mice.