Luchi N, Capretti P, Vettraino A M, Vannini A, Pinzani P, Pazzagli M
Dipartimento di Biotecnologie Agrarie, Sezione di Patologia vegetale, Università degli Studi di Firenze, Piazzale delle Cascine, 28 I-50144 Florence, Italy.
Lett Appl Microbiol. 2006 Jul;43(1):33-8. doi: 10.1111/j.1472-765X.2006.01920.x.
To develop a quantitative real-time PCR (Rt PCR) assay for the early detection of Biscogniauxia nummularia, a xylariaceous fungus that causes strip-canker and wood decay on European beech (Fagus sylvatica L.).
The molecular assay was based on TaqMan chemistry using species-specific primers and a fluorogenic probe designed on the ITS1 sequence of rRNA gene clusters. The specificity of the oligonucleotides and the probe were tested using the DNA of B. nummularia isolates from different geographic areas, of phylogenetically related species, and of some fungi commonly colonizing European beech bark and wood. A total of 31 symptomless and symptomatic shoots of European beech were collected from three forest sites in the Apennine Mountains of Italy. The percentage of positive detections of B. nummularia with the TaqMan assay was 78.6%, compared with only 14.3% of positive isolations on growth media for two sites.
In shoots, the quantitative Rt PCR assay detected down to 8.0-fg fungal DNA per microgram of total DNA extracted.
The assay developed in quantitative Rt PCR, by using TaqMan chemistry, revealed a rapid and sensitive method useful for the early detection of B. nummularia in symptomless European beech twigs.
开发一种定量实时PCR（Rt PCR）检测方法，用于早期检测引起欧洲山毛榉（Fagus sylvatica L.）条溃疡和木材腐朽的炭角菌科真菌——钱币状炭团菌。
该分子检测方法基于TaqMan化学法，使用物种特异性引物和根据rRNA基因簇的ITS1序列设计的荧光探针。使用来自不同地理区域的钱币状炭团菌分离株、系统发育相关物种以及一些常见于欧洲山毛榉树皮和木材上的真菌的DNA，对寡核苷酸和探针的特异性进行了测试。从意大利亚平宁山脉的三个森林地点采集了总共31个欧洲山毛榉无症状和有症状的嫩枝。TaqMan检测法检测到钱币状炭团菌的阳性率为78.6%，而在两个地点的生长培养基上阳性分离率仅为14.3%。
在嫩枝中，定量Rt PCR检测法能检测到每微克总DNA中低至8.0 fg的真菌DNA。
通过TaqMan化学法开发的定量Rt PCR检测法，揭示了一种快速且灵敏的方法，可用于早期检测无症状欧洲山毛榉嫩枝中的钱币状炭团菌。
Zotero 插件