Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species, with Pleurotus spp. as an Example

Abstract

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.

Degradation of Oxo-Biodegradable Plastic by Pleurotus ostreatus

“…The alignment of this sequence with other sequences found in the database showed a 92% similarity with the sequence EF514247.1, described as Pleurotus ostreatus strain CGMCC 5.37 [22]. Thus, the sequencing confirmed the previous morphologic classification of the isolate PLO6 as the species P. ostreatus .…”

Leucocalocybe, a new genus for Tricholoma ongolicum (Agaricales, Basidiomycota)

“…Primers LROR/LR5 (Michot et al, 1984) were used for the amplification of LSU (D1 and D2) of nrDNA. Reaction mixtures followed those in Yang et al (2007). The thermal cycling conditions consisted of an initial denaturation at 95°C for 5 min; followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 35 s, extension at 72°C for 1 min; and a final extension at 72°C for 10 min.…”

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

“…Current methods include macroscopic and microscopic visualization, presumptive identification using Marquis', Ehrlich's, and Froehde's reagents, thin layer chromatography, gas chromatography‐mass spectrometry (GC‐MS), and high‐performance liquid chromatography . Mushroom DNA has been differentiated by restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) techniques . While both of these DNA methods utilize the polymerase chain reaction (PCR), they also require post‐PCR gel electrophoresis to analyze and visualize the PCR results.…”