林木外生菌根真菌彩色豆马勃巢式PCR检测

为探讨林木外生菌根真菌的分子检测方法,利用真菌通用引物ITS1-F/ITS4-B扩增了外生菌根真菌彩色豆马勃的核糖体DNA内转录间隔区并进行了序列测定.通过序列比较,设计了一对彩色豆马勃特异性引物PtF/PtR.利用该对特异性引物与ITS1-F/ITS4-B组合进行巢式PCR,能从供试的彩色豆马勃10个菌株中特异性地扩增出1条347 bp的条带,而供试的其他6个参比菌株未出现扩增产物.经分析,该巢式PCR检测技术的灵敏度可达到10 fg的DNA,是常规PCR检测的1 000倍.利用该技术从马尾松苗木菌根中检测到目的外生菌根真菌.这表明采用本研究设计的特异性引物,利用巢式PCR技术可以灵敏、准确地从林木外生菌根中检测出彩色豆马勃.

Detection of Forest Ectomycorrhizal Fungi-Pisolithus tinctorius by Nested PCR

For investigating a suitable molecular approach to detect ectomycorrhizal fungi(EMF),the internal transcribed spacer(ITS) region of the EMF Pisolithus tinctorius was amplified with universal primers ITS1-F/ITS4-B and sequenced.Based on the conserved sequence determined by alignment using Clustal W,a pair of specific primers named PtF/PtR were designed by Primer premier 5.0.The specificity of the primer pair was then analyzed in 10 P.tinctorius strains together with 6 reference strains,and a 347 bp product was amplified from all P.tinctorius strains but there was no product from the 6 reference strains.A nested PCR procedure using ITS1-F/ITS4-B as the first-round primers followed by PtF/PtR increased detection sensitivity 1 000-fold to 10 fg DNA template.The target EMF from the mycorrhiza of Pinus massoniana potted seedlings inoculated with P.tinctorius was detected by this technique.The results indicated that the nested PCR assay could be used as a sensitive and accurate way to detect forest EMF P.tinctorius.