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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species with Pleurotus spp. as an Example
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Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species with Pleurotus spp. as an Example

机译：限制性内切核酸酶的自动筛选用于菌种侧耳属菌种的PCR限制性片段长度多态性鉴定。举个例子

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摘要

A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.
机译：设计了一种基于PCR的内部转录间隔区（ITS）核糖体DNA序列的PCR限制性片段长度多态性（RFLP）分析的分子方法，以快速鉴定真菌物种，以侧耳属属为例。基于对侧耳属ITS序列进行系统发育分析的结果，开发了PCR-RFLP核酸内切酶自动筛选（PRE Auto）程序来筛选限制性核酸内切酶，以区分不同物种的多个序列。 PRE Auto程序会分析核酸内切酶识别位点，并根据物种识别来计算导入到程序中的序列中片段的大小。通过计算序列组的平均系数和组内序列的平均系数对每个限制性核酸内切酶进行评分，然后根据评分系统的结果显示所选限制性内切酶的虚拟电泳图谱，以显示快速确定候选核酸内切酶。共使用代表151个ITS序列的85个单倍型进行分析，并筛选了2,992个限制性核酸内切酶以寻找用于鉴定物种的候选者。该方法已通过对代表12种平菇的28个样品的实验进行了验证。限制性内切酶的消化结果显示，除四个错误识别的样品外，PRE Auto程序预期的DNA片段模式相同。来自14个样品的ITS序列（本研究中获得了9个序列），包括4个最初被错误识别的样品，通过PCR-RFLP分析证实了物种的同一性。本文开发的方法可用于鉴定其他活微生物的种类。

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