苇状羊茅内生真菌与多年生黑麦草内生真菌实时荧光PCR检测研究

苇状羊茅内生真菌Neotyphodium coenophialum和多年生黑麦草内生真菌N．lolii对美国、新西兰等国家的畜牧业曾经造成过巨大损失。以N．coenophialum和N．lolii及其近似种N．huerfanum、N．chisosum、N．aotearoae、N．sp．共6种18个菌株,以及苇状羊茅和多年生黑麦草8个品种种子为供试材料,根据Tub-2基因设计了通用Taqman探针及引物,根据NC25基因设计了N．coenophialum和N．lolii的特异Taqman探针及共用引物,通过通用探针的单色荧光PCR和特异探针的双色荧光PCR,建立了N．coenophialum和N．lolii的菌丝及单粒种子稳定可靠、特异性强的荧光PCR检测方法,检测灵敏度达到单粒种子,使检测时间由至少一个月缩短至7～8个小时。

Detection of Neotyphodium coenophialum and N.Iolii basedon real-time fluorescence PCR

In some countries such as USA and New Zealand,livestock production has suffered great losses from the infection of Neotyphodium coenophialum and N.lolii.Based on eighteen strains belonging to six species of Neotyphodium and the seeds of eight varieties of Festuca arundinacea and Lolium perenne,the universal Taqman probe and its primer of all the tested strains according to Tub-2 gene,and the special Taqman probes(nc-probe for N.coenophialum and nl-probe for N.lolii)and their common primer according to NC25 gene have been designed according to seqences from GenBank and sequencing.Using single-colored real-time fluorescence PCR with universal probe and the double-colored real-time fluorescence PCR with the special probes,the stable, credible and strongly special detection method for mycelium and single seed has been successfully established. The sensitivity of detection is greatly high and is able to detect the mycelia of N.coenophialum and N.lolii in a single grass seed,and detection duration is shortened from at least one month to 7~8 hours.