CAJ | 학술논문

454焦磷酸测序已成为真菌群落结构研究的常用手段，文中对454焦磷酸测序的Barcoded PCR扩增中的模板质量浓度、Mg2＋和引物浓度等反应条件进行了优化，并对扩增所得产物的特异性进行了鉴定；同时就不同Taq酶对测序结果的影响进行了讨论。结果表明：DNA模板经适度稀释能显著提高反应效率；Mg2＋及引物浓度对扩增反应效率有影响，建议上下游引物浓度均为0．1μmol· L-1，Mg2＋浓度可不做调整；使用特异性好、活性较强的Taq酶对保证扩增产物的真菌特异性至关重要，建议选用热启动Taq酶。
454초린산측서이성위진균군락결구연구적상용수단，문중대454초린산측서적Barcoded PCR확증중적모판질량농도、Mg2＋화인물농도등반응조건진행료우화，병대확증소득산물적특이성진행료감정；동시취불동Taq매대측서결과적영향진행료토론。결과표명：DNA모판경괄도희석능현저제고반응효솔；Mg2＋급인물농도대확증반응효솔유영향，건의상하유인물농도균위0．1μmol· L-1，Mg2＋농도가불주조정；사용특이성호、활성교강적Taq매대보증확증산물적진균특이성지관중요，건의선용열계동Taq매。
454 pyrosequencing became a common method in analyzing fungal community structure.We explored the optimal con-tents of template DNA, Mg2+and primer in Barcoded PCR, identified the specificity of PCR products and discussed the se-lection of Taq polymerase.PCR reaction efficiency can be significantly improved after DNA template being diluted.Con-tents of Mg2+and primer can affect amplification efficiency, and we recommended 0.1μmol· L-1 of primer.Adjusting Mg2+content is not essential.The specificity and the activity of Taq polymerase are important, therefore, the hot-strrat Taq polymerase is recommended .