ppt - University of Connecticut

Report
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
• 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
•C.W.Lee and Y.M. Saif. Avian influenza virus. Comparative
subtypes have been
Immunology, Microbiology & Infectious Diseases, 32:301-310,
2009
identified.
• 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].
• 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.
Pool Design
• 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:
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
 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)
PrimerHunter Parameters & Results
• Optimization objective:
• 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/
Subtype
Targets
NonTargets
Avg. %
Dissimilarity
Forward
Primers
Reverse
Primers
Primer
Pairs
N1
N2
N3
N4
N5
N6
N7
N8
N9
110
241
65
15
32
77
22
84
42
578
447
623
673
656
611
666
604
646
8.8
11.7
8.8
7.1
6.4
10.3
7.2
6.7
6.6
97
77
45
370
355
29
97
140
292
71
44
61
360
353
43
103
211
305
553
234
113
9665
8380
7
480
1785
6310
Experimental Validation
AIV NA Subtyping Assay
• 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
Position of PCR products in the NA segment
• 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
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)
+
-
-
+
-
+
-
-
+
• 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].
 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|:
Discussion & Conclusions
• 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 RRTPCR 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

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