CAJ | 학술논문

建立了一种基于环介导等温核酸扩增技术（LAMP）的松材线虫高灵敏可视化闭管检测方法。针对松材线虫核糖体 DNA 的序列保守区域设计 LAMP 引物，通过优化 LAMP 体系中的 Mg2+、甜菜碱浓度和反应温度等因素，建立了环介导等温扩增法；并结合蜡封反应管对产物进行检测，检测结果可直接通过肉眼观察 SYBR Green I 荧光显色进行判定。结果表明，本方法可检测到低至10拷贝/管的松材线虫核酸片段，可对单条线虫进行检测，并且具有很高的特异性，能区分检测松材线虫与拟松材线虫。由于整个反应恒温进行，无需热循环仪；闭管检测极大地降低了扩增产物交叉污染的风险；检测速度快，整个检测过程只需40 min，为松材线虫的现场快速筛检提供了一种简便、高灵敏、高特异的工具。
건립료일충기우배개도등온핵산확증기술（LAMP）적송재선충고령민가시화폐관검측방법。침대송재선충핵당체 DNA 적서렬보수구역설계 LAMP 인물，통과우화 LAMP 체계중적 Mg2+、첨채감농도화반응온도등인소，건립료배개도등온확증법；병결합사봉반응관대산물진행검측，검측결과가직접통과육안관찰 SYBR Green I 형광현색진행판정。결과표명，본방법가검측도저지10고패/관적송재선충핵산편단，가대단조선충진행검측，병차구유흔고적특이성，능구분검측송재선충여의송재선충。유우정개반응항온진행，무수열순배의；폐관검측겁대지강저료확증산물교차오염적풍험；검측속도쾌，정개검측과정지수40 min，위송재선충적현장쾌속사검제공료일충간편、고령민、고특이적공구。
A visualized closed-tube loop-mediated isothermal amplification(LAMP)assay with high sensitivity meth-od to detect pine wood nematode(Bursaphelenchus xylophilus)was established. LAMP primers were designed aiming at the conservative domain of ribosomal DNA sequence of B. xylophilus through an optimization of LAMP reaction con-ditions such as concentration of Mg2 + ,betaine,and reaction temperature etc and established the LAMP method. The results of determination was observed through naked eyes by judging color changes of fluorescent coloration SYBR Green I of the product sealed with wax at the bottom of the detection tube. The results indicated that the sensitivity of the proposed method was as low as 10 copies of genomic DNA fragment of B. xylophilus,and it was sensitive enough to detect a single nematode. The method had a very high specificity,and could easily distinguish B. xylophilus from B. mucronatus. Because the whole reaction was carried out in constant temperature,no thermal cycler was needed. The closed-tube LAMP reaction greatly decreased the risk of cross-contamination from amplified products. The assay was very fast,it needed only 40 minutes for the whole detection process. Therefore,the established method provided a simple,highly sensitive and highly specific tool for rapid screening of pine wood nematode on the site.