| Honeybees play a pivotal role in both agriculture development and natural ecosystems. However, the Nosemosis caused by the pathogen of Nosema ceranae has decreased the ability of pollination, even destroyed the whole honeybees population. It is a great significance for apiculture to establish a simple, direct, quick method to detect Nosemosis.Loop-mediated isothermal amplification(LAMP) is a rapid, simple and sensitive isothermal amplification tool, which is consist of two pairs of specific primers targeting the six regions and a strand displacement DNA polymerase.To identify N. ceranae with LAMP, 6 specific primers were designed based on eight different segments of 16 S rDNA of N. ceranae which isolated from Youyang Chongqing. Bst DNA polymerase with strand displacement activity was used at a constant temperature 60℃ for 60 minutes. We established a rapid, specific and sensitive LAMP system for detecting the N. ceranae, which can be applied to simple places without special instrument. The LAMP assay has high sensitivity and high specificity compared with standard PCR. It is also provided a powerful tool for the detection and prevention of N. ceranae in apiculture.The results are as follows:1. Design of primers and optimization of reaction conditions of LAMPN. ceranae which isolated from Youyang Chongqing used as subject for the loop-mediated isothermal amplification(LAMP) with specific primers targeting 16 S rDNA,ptp2,swp12. After the experiment of three groups of primers, 16 S rDNA was selected as the detection target.Then the 16 S rDNA segment was amplified from DNA of N. ceranae and then the recombinant plasmid pMD19-T-Nc 16 S rDNA was constructed.In order to establish the best reaction conditions, the recombinant plasmid pMD19-T-Nc 16 S rDNA as the template, the temperature, time,the concentration of the inner primers and the dosage of Bst DNA polymerase were optomized.The optimized condition of LAMP assay is that the concentration of inner primers are 5 μmol/L and the dosage of polymerase was 800 U,reaction at 60 ℃ for one hour.2. The specificity and sensitivity of LAMP method for the detection of N. ceranaeIn order to detect the specificity of LAMP method for the detection of N. ceranae,we used the bee’s common pathogen Ascosphaera apis and Chinese sacbrood virus as the control of N. ceranae.we found that only the genomic DNA of N. ceranae had the corresponding amplified bands, suggesting that the detection system could specifically detect microsporidium.In order to determine the sensitivity of LAMP to detect the N. Ceranae,the recombinant plasmid pMD19-T-Nc 16 S rDNA and the DNA of N. ceranae was subjected to LAMP and PCR testing, respectively.For the recombinant plasmid,LAMP has ability to detect as low as 1.735×10-2ng /μL comparing PCR which can detect1.735×10-1ng /μL.And LAMP has ability to detect as low as 1.475×10-3ng/μL DNA comparing PCR which can detect 1.475×10-1 ng/μL DNA, suggesting LAMP has higher sensitivity than PCR.3. Detection of midgut infected with N. ceranae with LAMP methodWe extracted genomic DNA of uninfected normal and infected with different concentrations of N. ceranae midgut,and determined by LAMP and PCR.The statistics of ten visual field of the number of N. ceranae spores observed under microscope,11 spores were observed in the visual fields of 105/mL while spores were not observed in the visual fields of 104/mL and 103/mL.The results of detefting genomic DNA of midgut show that LAMP has ability to detect as low as 1.2×104/mL,while only the positive plasmid was amplified by PCR method.So for infected N. ceranae midgut, the detection sensitivity of the LAMP assay is much higher than conventional PCR sensitivity.In summary, was studied the application of LAMP method in N. ceranae,it shows that the 16 S rDNA could be used as a target to detect N. ceranae by LAMP method.And our studies lay the foundation for establishing LAMP combined with a lateral flow strip in future. |
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