The Research Of RT-PCR Detection And Transmission Of DsRNA Virus In Lentinus Edodes

The based on the traditional dsRNA extraction technology, the little amount of dsRNA extraction technology and the plant nucleic acid extraction kit, this study established a method of cellulose-column centrifugal extraction for virus dsRNA of Lentinus edodes, which could stability extract dsRNA from0.3g mushrooms mycelia in2.5h. The compared with the traditional dsRNA extraction technology and the little amount of dsRNA extraction technology, this method have the advantages of that a small sample, a simple of the operation, rapid, efficient and big extractions. As the same time, the dsRNA can satisfy the subsequent gene cloning experiments of molecules.The based on RT-PCR detection of the dsRNA virus from the fungi and plants, this research developed and optimized the system of RT-PCR detection with the principle of economy, high efficiency and stability for dsRNA virus from Lentinus edodes. A high number of amplification products and good reproducibility was achieved using a20μl of RT response consisting of100ng the total RNA,0.25mmol/L dNTPs,0.15U mol/L reverse primer,2.5U/μl M-MLV and the heat preservation before RT reaction should be95℃in above and a30μl of PCR response consisting of2μl cDNA template,1.5mmol/L Mg2+,0.08mmol/L dNTPs,0.16μmol/L forward and reverse primer,0.07U/μl Taq polymerase.This research discussed the mode of transmission for dsRNA virus in Lentinus edodes according to the researches of dsRNA virus from some other fungi. The test results showed that the dsRNA virus either could be spread between strains, which might be affected by the specific fusion and different genotypes between strains, or could be spread through spores, which was the account for100%in the detection single spore strains of this study.