tailieunhanh - Báo cáo khoa học: PCR detection of nearly any dengue virus strain using a highly sensitive primer ‘cocktail’

PCR detection of viral pathogens is extremely useful, but suffers from the challenge of detecting the many variant strains of a given virus that arise over time. Here, we report the computational derivation and initial experi-mental testing of a combination of 10 PCR primers to be used in a single high-sensitivity mixed PCR reaction for the detection of dengue virus. | 1FEBS Journal PCR detection of nearly any dengue virus strain using a highly sensitive primer cocktail Charul Gijavanekar1 Maria Anez-Lingerfelt2 Chen Feng3 Catherine Putonti4 5 George E. Fox1 Aniko Sabo6 Yuriy Fofanov1 3 and Richard C. Willson1 7 1 Department of Biology and Biochemistry University of Houston TX USA 2 Department of Chemicaland Biomolecular Engineering University of Houston TX USA 3 Department of Computer Science University of Houston TX USA 4 Department of Biology Loyola University Chicago IL USA 5 Department of Computer Science Loyola University Chicago IL USA 6 Human Genome Sequencing Center Baylor College of Medicine Houston TX USA 7 The Methodist HospitalResearch Institute Houston TX USA Keywords cocktailPCR dengue virus diagnostic PCR primer Correspondence R. C. Willson Department of Chemical and Biomolecular Engineering Department of Biology and Biochemistry University of Houston Houston TX 77204-4004 USA Fax 1 713 743 4323 Tel 1 713 743 4308 E-mail willson@ Present address Scientific and Laboratory Services SW Division PallCorporation Houston TX 77040 USA Received 29 November 2010 revised 2 February 2011 accepted 10 March 2011 doi PCR detection of viral pathogens is extremely useful but suffers from the challenge of detecting the many variant strains of a given virus that arise over time. Here we report the computational derivation and initial experimental testing of a combination of 10 PCR primers to be used in a single high-sensitivity mixed PCR reaction for the detection of dengue virus. Primer sequences were computed such that their probability of mispriming with human DNA is extremely low. A cocktail of 10 primers was shown experimentally to be able to detect cDNA clones representing the four serotypes and dengue virus RNA spiked into total human whole blood RNA. Computationally the primers are predicted to detect 95 of the 1688 dengue strains analyzed with perfect primer match . Allowing up to one