Primer Design.ppt

Development of a Real Time RT-PCR Assay for Neuraminidase Subtyping of Avian Influenza Virus Yanyan Huang Shandong Academy of Agricultural Sciences , Mazhar Khan and Ion Mandoiu University of Connecticut Primer Design Introduction Pool Design Experimental Validation AIV NA Subtyping Assay Discussion Conclusions Primer Pool N1 N2 N3 N4 N5 N6 N7 N8 N9 A 2,6,7 - + - - - + + - - B 4,5,7,8 - - - + + - + + - C 3,5,9 - - + - + - - - + D 1,4,6,9 + - - + - + - - + Position of PCR products in the NA segment We selected for each NA subtype one pair of primers For each pair, detection limits were determined using RRT-PCR on serially diluted RNA standards 10 potential primer dimers were identified using autodimer The ILP formulation had 85 variables pool candidates and 45 constraints that was solved to optimality in a fraction of a second using CPLEX; the four resulting pools are shown below Experiments show that RRT-PCR with primer pools can detect and differentiate NA RNA of all nine subtypes extracted from AIV-infected allantoid fluids. Amplification and dissociation properties of N4 RNA in primer pool tests are shown below; full results will be presented in [Huang et al 2010]. In this study, 9 pairs of neuraminidase NA subtype-specific primers were designed and successfully used in real time RT-PCR with four primer-pool reactions to differentiate 9 NA subtypes of AIV The RRT-PCR assays are sensitive and can detect in vitro transcribed RNA of different NA subtypes ranging from 176 to 4000 copies per reaction, or 2-30fg of AIV RNA The assays also possess good specificity. There was no cross reaction between RNA of different NA subtypes in RRT-PCR with each subtype-specific primers, and no amplification was displayed for RNA of IBV, IBDV, NDV This study validated further the powerful function of Primer Hunter for the design of subtyping primers and also introduced a sensitive and specific method for NA subtyping of AIV The quadruplicate primer pool test de