Antibacterial activity of crude protein and development of species-specific molecular marker for fungus Ophiocordyceps sobolifera

Abstract

The medicinal mushroom Ophiocordyceps sobolifera has been used in traditional Chinese medicine; however, the proven medical benefits of the crude protein are limited. Therefore, in this study, forty-two isolates of O. sobolifera were identified using their internal transcribed spacer (ITS) sequences. Then, the antibacterial activities of the crude proteins were evaluated against strains of methicillin-resistant and -susceptible Staphylococcus aureus and Bacillus cereus using agar well diffusion technique. The results revealed that the crude protein had a good inhibitory effect against S. aureus and B. cereus. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the crude proteins ranged from 0.156–10.00 mg/mL and 0.156–10.00 mg/mL, respectively. Among them, the Cod-NB1302 strain had the broadest inhibitory activity against the tested bacterial pathogens with MIC values of 0.625–1.25 mg/mL and MBC values of 0.625–1.25 mg/mL. Then, the species-specific molecular marker derived from random amplified polymorphic DNA (RAPD) was developed for the identification of O. sobolifera. The results indicated that the OPT13 arbitrary primer was able to generate two species-specific DNA fragments from O. sobolifera. Specific fragments were then cloned, sequenced, and used to design the species-specific primers. Only one species-specific primer set, the SpsoL_f01/SpsoL_r01 primer pair, produced a prominent amplicon of 118 bps in O. sobolifera under stringent conditions without non-specific detection after being applied to other species, with the exception of O. longissima. Moreover, the detection limit of this primer was 5.84 ng of genomic DNA, and this primer set could be applied in all strains of O. sobolifera.