Dipartimento Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy.
Dipartimento Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy.
Int J Food Microbiol. 2015 May 4;200:47-51. doi: 10.1016/j.ijfoodmicro.2015.01.020. Epub 2015 Feb 7.
Torulaspora delbrueckii yeast strains are being increasingly applied at the industrial level, such as in the winemaking process, and so their identification and characterisation require effective, fast, accurate, reproducible and reliable approaches. Therefore, the development of typing techniques that allow discrimination at the strain level will provide an essential tool for those working with T. delbrueckii strains. Here, 28 T. delbrueckii strains from various substrates were characterised using different PCR-fingerprinting molecular methods: random amplified polymorphic DNA with polymerase chain reaction (RAPD-PCR), minisatellites SED1, AGA1, DAN4 and the newly designed T. delbrueckii (Td)PIR, and microsatellites (GAC)5 and (GTG)5. The aim was to determine and compare the efficacies, reproducibilities and discriminating powers of these molecular methods. RAPD-PCR using the M13 primers and the newly designed TdPIR3 minisatellite primer pair provided discrimination of the greatest number of T. delbrueckii strains. TdPIR3 clustered the 28 strains into 16 different groups with an efficiency of 100%, while M13 clustered the strains into 17 different groups, although with a lower efficiency of 89%. Moreover, the TdPIR3 primers showed reproducible profiles when the stringency of the PCR protocol was varied, which highlighted the great robustness of this technique. In contrast, variation of the stringency of the M13 PCR protocol resulted in modification of the amplified profiles, which suggested low reproducibility of this technique.
毕赤酵母菌株在工业领域的应用越来越广泛,例如在酿酒过程中,因此需要有效的、快速的、准确的、可重复的和可靠的方法来对其进行鉴定和特性描述。因此,开发能够在菌株水平上进行区分的分型技术将为研究毕赤酵母菌株的人员提供重要工具。本研究使用不同的 PCR 指纹图谱分子方法,包括随机扩增多态性 DNA 聚合酶链反应(RAPD-PCR)、微卫星 SED1、AGA1、DAN4 和新设计的毕赤酵母(Td)PIR,以及微卫星(GAC)5 和(GTG)5,对来自不同基质的 28 株毕赤酵母菌株进行了特征描述。目的是确定和比较这些分子方法的功效、可重复性和区分能力。使用 M13 引物的 RAPD-PCR 和新设计的 TdPIR3 微卫星引物对提供了对最多数量的毕赤酵母菌株的区分。TdPIR3 将 28 株菌株聚类为 16 个不同的组,效率为 100%,而 M13 将菌株聚类为 17 个不同的组,尽管效率较低,为 89%。此外,当改变 PCR 方案的严格性时,TdPIR3 引物显示出可重复的图谱,这突出了该技术的强大稳健性。相比之下,改变 M13 PCR 方案的严格性会导致扩增图谱的改变,这表明该技术的可重复性较低。