| The major pathogens of a respiratory infectious disease in rabbits are Bordetella bronchiseptica and Pasteurella multocida. Conventional diagnosis method was difficult to diagnosis quickly and accurately, moreover misdiagnosis would delay the treatment of the disease due to the co-infection of these two pathogens as well as common characteristics in colonial morphology, clinical symptoms and pathological changes epidemiology. Therefore, to establish a simple, rapid, sensitive and specificity detection method is particularly important. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that relies on a strand displacement DNA polymerase, efficient, fast, specific amplification of the target gene in Isothermal conditions, no requirement of the template heat denaturation, long time temperature cycle and cumbersome electrophoresis process. The methodology with LAMP is a new way for the rapid pathogens diagnosis clinically.Four LAMP primers were designed by Primer Explorer software, Version4, based on16S rRNA gene and Kmtl gene. The set of LAMP reaction system conditions were optimized including temperature, time, dNTPs concentration, Mg2+concentration, etc. In this study8bacterial strains were detected by LAMP in order to evaluate the specificity of primers. The result showed only the target strain was positive while other7strains were negative. It was demonstrated that LAMP has good specificity in present study.The limitation of detecting Bb and Pm by LAMP and PCR were compared in the study. The templates of Bb and Pm were prepared by Qiagen kit. Result showed that limitation of detecting Bb and Pm by LAMP were1.65CFU/mL and1.29CFU/mL respectively, while the traditional PCR assay were165CFU/mL and129CFU/mL. It showed that LAMP was100times higher sensitive than ordinary PCR.In this study, the optimized LAMP detection method were used to test the stability and repeatablity of15Bb positive samples and10Pm positive samples, all the test results were positive, indicating that the method has good stability and reproducibility.The three genomic DNA extract methods were compared in this study. The result showed LAMP had no strict requirement on the DNA extract method. Thus the boiling method was viable, the time and cost would be greatly reduced by LAMP from present results.In this study totally54Bb samples and46Pm samples were analyzed by LAMP and conventional PCR methods. The results showed that the positive rate, sensitivity, specificity and according rate of Bb by LAMP was77.78%,100%,85.71%, and96.3%respectively. The positive rate, sensitivity, specificity and according rate of Pm by LAMP was47.73%,100%,88.46%and93.18%respectively.LAMP reaction was very quick, finishing in70min. The detection process would be more quickly and efficiently from the easily judging according to the changing color results adding1μL SYBR GREEN I (1000x) after the termination of reaction. Consistent results of restriction endonuclease fragments verified the correctness of the LAMP method. Moreover, LAMP reaction was easily to operate only needing a simple thermostatic water bath without the expensive PCR instrument, thus it deserves for further application in the grassroots level. |
PDF Full Text Request