环介导等温扩增(LAMP)技术检测东方蜜蜂微孢子虫Nosema ceranae的研究

Study on detection of Nosema ceranae through Loop-Mediated Isothermal Amplification method

应用环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术建立一种准确、灵敏、快速的蜜蜂微孢子虫检测方法。本研究根据东方蜜蜂微孢子虫Nosema ceranae的依赖DNA的RNA聚合酶Ⅱ大亚基(RPB1)序列,用在线软件Primer Explorer V4.0 online设计4条特异性引物,分别对Mg2+、d NTP、内引物FIP/BIP和甜菜碱浓度及反应温度和时间优化;选择蜜蜂体内常见病原进行该方法的特异性验证,用MseⅠ酶切扩增产物验证其准确性;将N.ceranae的DNA梯度稀释进行灵敏度检测并与PCR比较分析;最后在临床检测中验证该技术的可行性。结果表明,优化的体系可在恒温57℃下完成扩增反应;引物的病原特异性检测仅N.ceranae有梯状条带,MseⅠ酶切产物条带符合理论值;LAMP反应检测的灵敏度较PCR高10倍;能够直接从蜜蜂体内检测出N.ceranae。本研究建立的LAMP检测N.ceranae体系准确、快速、成本低,可为蜜蜂微孢子虫病的检测提供有力的技术支撑。

The objective of this study is to establish a rapid and sensitive method for detecting the Nosema ceranae in epidemiological investigations by the loop-mediated isothermal amplification （LAMP） , and to provide technical supports for monitoring and prevention of N. ceranae in honeybee. Four specific primers were designed based on the particular sequence of DNA-dependent RNA polymerase II largest subunit （RPB1 ） of the N. ceranae using the soft of PrimerExplorer V4. 0 online. The concentration of Mg2＋ , dNTP, inner FIP/BIP, betaine and reaction times and reaction empretures were optimized. The specificity of LAMP was tested by using genomic DNA of the common pathogens that infected honybee. In order to verify accuration, the LAMP amplified products were digested with Mse I restriction enzyme. Moreover, the sensitivity of LAMP was compared with normal PCR by employing ten-fold serially diluted DNA templates of N. ceranae. Finally, the practical reliability was achieved in clinical trials. The results showed that ： The amplification was successfully performed under isothermal condition at 57 °C . The templates of common pathogens were prepared and tested through this method, however, the unique laddrdder-like bands can only amplified from N. ceranae DNA template. Several bands of enzyme digested with Mse I are consistent with theory. Sensitivity of the LAMP assay was 10-fold higher than the conventional PCR method, which was capable of detecting N. ceranae directly from the infected bee. Therefore, the LAMP for identifying N. ceranae described here was proved to be precise, fast as well as cost-saving and could be applied to other associated research domain.