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 DesignIntroduction Pool Design

Experimental ValidationAIV 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 described decreases the amount of reactions needed to differentiate NA subtypes from 9 to 4. This reduces costs and labor, and could also improve sensitivity of detection when the quantity of viral RNA is limited

References

• Y. Bao, P. Bolotov, D. Dernovoy, B. Kiryutin, L. Zaslavsky, T. Tatusova, J. Ostell, and D. Lipman. The influenza virus resource at the national center for biotechnology information. J. Virol., 82(2), pp. 596-601, 2008

• J. Duitama, D.M. Kumar, E. Hemphill, M. Kahn, I.I. Mandoiu, and C.E. Nelson. PrimerHunter: A Primer Design Tool for PCR-Based Virus Subtype Identification, Nucleic Acids Research 37, pp. 2483-2492, 2009

• Y. Huang, M. Kahn, and I.I. Mandoiu. Manuscript in preparation

• S. Rash and D. Gusfield. String barcoding: Uncovering optimal virus signatures, Proc. 6th Annual International Conference on Computational Biology, pp. 254-261, 2002

• D. Suarez, A. Das, and E. Ellis. Review of rapid molecular diagnostic tools for avian influenza. Avian Diseases, 51, pp. 201-208, 2007

Subtype TargetsNon-

TargetsAvg. %

DissimilarityForward Primers

Reverse Primers

PrimerPairs

N1 110 578 8.8 97 71 553

N2 241 447 11.7 77 44 234

N3 65 623 8.8 45 61 113

N4 15 673 7.1 370 360 9665

N5 32 656 6.4 355 353 8380

N6 77 611 10.3 29 43 7

N7 22 666 7.2 97 103 480

N8 84 604 6.7 140 211 1785

N9 42 646 6.6 292 305 6310

PrimerHunter Parameters & Results

• PrimerHunter was run with default parameters: primer length 20-25, amplicon length 75-200, GC content 25%-75%, Max mononucleotide repeat 5, matching mask M = 11, no required 3’ GC clamp, primer concentration 0.8μM, salt concentration 50mM, Tmin_target =Tmax_nontarget = 40oC

• Between 7 and 9,665 pairs of primers were found by PrimerHunter for each subtype, full results are available at http://dna.engr.uconn.edu/software/PrimerHunter/

Sequence Data

• Complete Avian Influenza NA sequences from North America available in NCBI flu database [Bao et al. 2008] as of March 2008

• Phylogenetic analysis detected a N1 sequence mislabeled as N4

• For each subtype Ni, we used all available Ni sequences as targets, and all sequences with different subtype as non-targets

• To reduce the number of reactions needed to identify the subtype of a sample we perform RRT-PCR reactions with pools of primer pairs.

• Pools must be designed so that the result of these reactions uniquely identifies the subtype present in the sample. As noted by [Rash and Gusfield 2002], this is equivalent to ensuring that for every pair of subtypes there is a pool that results in a positive signal for one but not for the other.

• Additional constraints: Each subtype results in positive amplification for at least one pool

Bounded pool size (no more than m pairs in a pool) No primer dimers (set of pairs of subtypes whose primers are

predicted to form dimers is denoted by D; we used the autodimer tool at the National Institute of Standards and Technology with a total score threshold of 4 to predict primer dimers)

• Optimization objective: Minimize the number of pools Subject to this, minimize total size of pools

• Notations N = number of subtypes (N=9 for NA subtyping) P = set of of candidate pools, i.e., subsets of size at most m

of {1,…,N} that do not contain any pair in D xp = 0/1 variable set to 1 if pool p is selected and to 0

otherwise M = constant larger than N|P|:

• In [Duitama et al. 2009] we presented an open source software tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. We have also confirmed the sensitivity and specificity of PrimerHunter's primers for hemagglutinin (HA) subtyping of Avian influenza virus using individual real time PCR (R-PCR) reactions testing the presence of each subtype.

• To increase sensitivity of detection when the quantity of viral RNA is limited, we propose using PCR reactions with pools of subtype-specific primer pairs designed so that each subtype yields a unique amplification pattern.

• In this poster we describe an integer linear program (ILP) for designing the minimum number of uniquely decodable pools subject to primer non-dimerization constraints. We also present results of real time RT-PCR reactions demonstrating the sensitivity of the assay for NA subtyping of AIV.

• Avian influenza virus (AIV) belongs to the influenza type A genus of the Orthomyxoviridae family of RNA viruses. It is a highly mutable virus, with the Haemagglutinin (HA) and the Neuraminidase (NA) genes being the most variable. To date, 16 HA and 9 NA subtypes have been identified.

•C.W.Lee and Y.M. Saif. Avian influenza virus. Comparative Immunology, Microbiology & Infectious Diseases, 32:301-310,

2009

• Rapid AIV subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks, and global-scale surveillance of highly pathogenic subtypes.

• Polymerase Chain Reaction (PCR) has become the method of choice for virus subtype identification due to its high sensitivity and specificity, fast response time, and affordable cost [Suarez et al. 2007].