title: [Investigation of RNA viral genome amplification by multiple displacement amplification technique].
abstract: In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.
authors.affiliation: Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China. bh_pz@sina.com
authors.forename: Zheng
authors.initials: Z
authors.lastname: Pang
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Li
authors.forename: Chuan
authors.initials: C
authors.lastname: Li
authors.forename: Mi-Fang
authors.initials: MF
authors.lastname: Liang
authors.forename: De-Xin
authors.initials: DX
authors.lastname: Li
mesh_terms: D002044:Bunyaviridae Infections / Q000175:diagnosis* / Q000821:virology; D002460:Cell Line; D011088:DNA Ligases / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral / Q000235:genetics*; D006801:Humans; D016856:Phlebovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases