A simple RT-multiplex PCR method for diagnosis of L-A and M totiviruses in Saccharomyces cerevisiae

Author:

Quintero Juan1,Jimenez Juan1,Garzón Andrés1ORCID

Affiliation:

1. Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/CSIC. Carretera de Utrera Km 1, Sevilla. Spain

Abstract

Killer yeasts and their toxins have many potential applications in environmental, medical and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait is usually performed phenotypically, on the basis of their toxin production and immunity. In this study, we describe a simple and specific RT-multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated to the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primers addition to the RT reaction. Using a mixture of specific primers at the PCR step, this RT-multiplex PCR protocol provides accurate diagnosis of both L-A and M totivirus in all its known variants L-A-1/M1, L-A-2/M2, L-A-28/M28 and L-A-lus/Mlus to be found in infected killer yeasts. By means of this method, expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified, but importantly, asymptomatic L-A-2/M2 infected cells, as well as unexpected L-A-lus/M2 totiviral associations, were also found. Importance The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, development of simple methods for their detection significantly facilitates their study. The simplified RT-multiplex PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depends on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provides valuable insights into L-A/M viral associations and their coadaptional events in nature.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

Cited by 3 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3