Development And Application Of LAMP For Detection Anaplasma Phagocytophilum And Anaplasma Bovis

Anaplasmosis is caused by members of Anaplasma that are spread by ticks and then parasite in the blood cells of sheep, cattle, deer and other animals, even human, leading to chronic or acute infectious diseases in animals and human being. This disease results in symptoms such as weight loss, decreased milk production, abortion, severely can lead to death, causing serious economic losses. The known Anaplasma pathogenes include A. marginale, A. centrale, A. phagocytophilum, A. ovis, A. bovis, and A. platys. The establishment of efficient, rapid and accurate detection method is particularly urgent to diagnose the disease and then to prevent and control it. In our study, a loop-mediated isothermal amplification (LAMP) method with high specificity and high sensitivity was established for detecting A.phagocytophilum and A. bovis.The positive sheep blood DNA samples preserved in our laboratory were used to establish LAMP method to detect A. phagocytophilum. Firstly,4 specific primers complementary to the 16S rRNA gene of A. phagocytophilum were designed using Primer Explorer V4(http://primerexplorer. jp/elamp4.0.0/index.html)to do preliminary experiment and then the the reaction condition was optimized. The results showed that the most optimal reaction temperature and time were 65℃ and 60 min, respectively, and in 25μL reaction mixture, the most optimal concentrations of MgSO4、dNTP Mixture、Betaine and Bst DNA polymerase were 8 mM、1.4 mM、 0.6 mM and 8U/25μL, respectively. No cross-reactions were observed with A. bovis, A. ovis, Theileria luwenshuni, Babesia motasi and the Schistosoma japonicum. What’s more, the LAMP assay could be able to detect as few as 1 copy of 16S rRNA gene in 1μL DNA. After that, the practicality of the LAMP was assessed by testing 94 samples and comparing with the results of nest-PCR, the detection rates was 56.38% versus 12.77%. The results demonstrated that the LAMP assay was significantly more sensitive than the nest-PCR. In addition, the reaction results of A. phagocytophilum can be easily decided rapidly by adding SYBR Green I. At last,10 positive samples and 10 negative samples determined by nest-PCR were picked out to be used to compare the sensitivity of our method and two other reported LAMP methods by Lei and Lee, the detection rates was 45%,10% and 0, respectively, the results showed that the LAMP method we developed was more sensitive than the two others. Our results indicated that the LAMP method was a useful approach for detecting A. phagocytophilum infections.As the same, species-specific LAMP primers were designed based on 16S rRNA gene sequences of A. bovis in NCBI. After optimization of reaction temperature and reaction condition, the results showed that the most optimal reaction temperature and time were 62℃ and 60 min, and in 25 μL reaction mixture, the most optimal concentrations of MgSO4、dNTP Mixture、Betaine and Bst DNA polymerase were 8mM、1.0 mM、0.6 mM and 8U/25μL, respectively. Moreover, no cross-reactions were observed with A. phagocytophilum, A. ovis, T. luwenshuni, B. motasi and the S. japonicum. The LAMP assay could be able to detect as few as 1 copy of 16S rRNA gene in 1μL DNA. The practicality of the LAMP was assessed by testing 120 samples and comparing with the results of nest-PCR, the detection rates was 67.50% versus 37.83%. The results demonstrated that the LAMP was significantly more sensitive than the nest-PCR. What’s more, the reaction results of A. bovis can be easily decided by adding SYBR Green I. There is no report about the LAMP assay for detecting A. bovis in the world now. Our results stated clearly that the LAMP assay was a useful method for detecting A. bovis infections.In this study, we developed the LAMP for detecting the A. phagocytophilum and A. bovis, has good sensitivity and specificity, and are better than the existing detection methods. This will provide a new technical method for diagnosing A. phagocytophilum and A. bovis.