期刊
JOURNAL OF VIROLOGICAL METHODS
卷 222, 期 -, 页码 182-191出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2015.06.014
关键词
DNA array; High-throughput sequencing; Virus identification; Porcine reproductive and respiratory; syndrome virus; PRRSV; Propidium monoazide; PMA; Ethidium bromide monoazide; EMA
资金
- Canadian Swine Health Board (CSHB)
- Natural Sciences and Engineering Research Council of Canada (NSERC)
Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HIS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HIS to identify viruses. (C) 2015 Elsevier B.V. All rights reserved.
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