U.S. patent application number 13/103649 was filed with the patent office on 2011-12-22 for rt-late-pcr.
This patent application is currently assigned to BRANDEIS UNIVERSITY. Invention is credited to Cristina Hartshorn, Kenneth E. Pierce, Arthur Henry Reis, JR., John E. Rice, J. Aquiles Sanchez, Lawrence J. Wangh.
Application Number | 20110311971 13/103649 |
Document ID | / |
Family ID | 40472053 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110311971 |
Kind Code |
A1 |
Hartshorn; Cristina ; et
al. |
December 22, 2011 |
RT-LATE-PCR
Abstract
An assay comprising more than one primer pair and more than one
detection probe, a low copy number synthetic amplicon corresponding
to each of the primer pairs. The assay can detect and distinguish
between various sub-types and strains of an influenza virus using
any suitable nucleic acid amplification technique. Related kits and
methods are also described.
Inventors: |
Hartshorn; Cristina;
(Needham, MA) ; Pierce; Kenneth E.; (Natick,
MA) ; Reis, JR.; Arthur Henry; (Arlington, MA)
; Rice; John E.; (Quincy, MA) ; Sanchez; J.
Aquiles; (Framingham, MA) ; Wangh; Lawrence J.;
(Auburndale, MA) |
Assignee: |
BRANDEIS UNIVERSITY
Waltham
MA
|
Family ID: |
40472053 |
Appl. No.: |
13/103649 |
Filed: |
May 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11822536 |
Jul 6, 2007 |
7972786 |
|
|
13103649 |
|
|
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60819000 |
Jul 7, 2006 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12 |
Current CPC
Class: |
C12Q 1/701 20130101 |
Class at
Publication: |
435/6.11 ;
435/6.12 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. (canceled)
2. A reverse transcription-polymerase chain reaction (PCR) method
comprising: a) adding to a reaction vessel a reaction mixture
comprising at least one RNA comprising a target sequence, reverse
transcriptase, DNA amplification reagents that include an excess
primer and a limiting primer for the target sequence, and DNA
polymerase, wherein the concentration of the excess primer is
500-2000 nM and at least five times higher than the concentration
of the limiting primer, and wherein the concentration-adjusted
melting temperature of the limiting primer to the target sequence
is equal to or greater than the concentration-adjusted melting
temperature of the excess primer to the target sequence; b)
incubating the reaction mixture at a first temperature to reverse
transcribe the target sequence from the RNA utilizing the excess
primer and the reverse transcriptase to create a cDNA template for
LATE-PCR amplification, said first temperature being sufficiently
high to disrupt secondary structure of the RNA but sufficiently low
to provide a reverse-transcriptase half-life of at least 2.2
minutes, wherein the effective melting temperature of the excess
primer to the RNA is not lower than 2.degree. C. below said first
temperature; and c) thermally cycling the reaction mixture to
amplify the cDNA template using the excess primer, the limiting
primer and the DNA polymerase to produce both double-stranded
amplification product and single-stranded amplification
product.
3. The method of claim 2 wherein the concentration of the excess
primer is at least ten times higher than the concentration of the
limiting primer.
4. The method of claim 2 wherein the concentration of the excess
primer is at least twenty times higher than the concentration of
the limiting primer.
5. The method of claim 1 wherein the RNA is added to the reaction
mixture as at least one lysed cell or at least one lysed virus.
6. The method of claim 1 wherein the reverse transcriptase and the
DNA polymerase are a single enzyme that has both reverse
transcription activity and DNA polymerase activity.
7. The method of claim 1 wherein the reaction mixture includes a
fluorescently-labeled hybridization probe for detection of the
single-stranded amplification product.
8. The method of claim 1 wherein said at least one RNA comprises at
least two target sequences, and the DNA amplification reagents
include an excess primer and a Limiting primer for each target
sequence.
9. The method of claim 1 wherein said first temperature is in the
range of 50.degree. C. to 60.degree. C.
10. A reaction mixture comprising: (a) at least one RNA, wherein
said RNA comprises a target sequence; (b) reverse transcriptase;
(c) DNA amplification reagents including an excess primer and a
limiting primer for the target sequence, wherein the concentration
of the excess primer is 500-2000 nM and at least five times higher
than the concentration of the limiting primer, and wherein the
concentration-adjusted melting temperature of the limiting primer
to the target sequence is equal to or greater than the
concentration-adjusted melting temperature of the excess primer to
the target sequence; and (d) DNA polymerase.
11. The reaction mixture of claim 10 wherein the concentration of
the excess primer is at least ten times higher than the
concentration of the limiting primer.
12. The reaction mixture of claim 11 wherein the concentration of
the excess primer is at least twenty times higher than the
concentration of the limiting primer.
13. The reaction mixture of claim 10 wherein the RNA is added to
the reaction mixture as at least one lysed cell or at least one
lysed virus.
14. The reaction mixture of claim 10 wherein the reverse
transcriptase and the DNA polymerase are a single enzyme that has
both reverse transcription activity and DNA polymerase
activity.
15. The reaction mixture of claim 10 further comprising a
fluorescently-labeled hybridization probe for detection of the
single-stranded amplification product.
16. The reaction mixture of claim 10 wherein said at least one RNA
comprises at least two target sequences, and the DNA amplification
reagents include an excess primer and a Limiting primer for each
target sequence.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/822,536, filed Jul. 6, 2007, which claims
priority to U.S. Provisional Patent Application Ser. No.
60/819,000, filed Jul. 7, 2006, each of which is hereby
incorporated by reference in their entireties.
BACKGROUND
[0002] Rapid detection and typing of influenza virus and
identification of its various strains is critical to identification
and control of a potential human pandemic. Influenza virus is
composed of eight single-stranded RNA molecules (HA, NA, PB2, PB1,
PA, NS, M, NP) that code for eleven specific proteins. The RNA for
the matrix protein (M) is relatively conserved and is therefore
used to detect and distinguish a Type A virus. M can also be used
to detect and distinguish H5N1.
[0003] The hemagglutinin protein (HA) and neuraminidase protein
(NA) are grouped into 16 and 9 subtypes, respectively, both have
high sequence variability even within subtypes and thus provide an
effective means of monitoring changes that might occur in a virus.
The HA protein protrudes from the surface of the virus and allows
it to attach to a cell to begin the infection cascade. The NA
protein is also located on the surface of the virus and allows the
release of new particles within the infected cell.
[0004] Currently the Eurasian H5N1 virus infects only the lower
lungs in human and is therefore less readily transmitted
human-to-human than annual strains of human influenza that infect
the upper respiratory track. But, mutations within the HA and NA
RNAs are frequent and alter viral infectivity and lethality in
different hosts and their tissues. In addition, gene assortment
among the different viral subtypes is another very worrisome
feature of influenza and could result in recombining RNA sequences
for high infectivity in humans with high lethality.
SUMMARY
[0005] Accordingly, there is a need for an informative influenza
assay that can be performed in the field, i.e., at the point of
care ("POC"). Moreover, in order to save both time and money it
will also be important to make POC assays compatible with more
extensive laboratory analysis, such as sequencing of, for example,
HA and NA. In this way, the evolution of a viral disease and viral
genomics can be analyzed in real-time.
[0006] One embodiment is directed to an assay comprising a
plurality of primer pairs, a plurality of probes, and a low copy
number synthetic amplicon corresponding to each of the plurality of
primer pairs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 illustrates an embodiment of LATE PCR (left) and
provides fluorescence curves produced by LATE PCR.
[0008] FIG. 2 is an agarose gel showing four sets of reactions,
performed in triplicate, each reaction using the same Excess Primer
(thick arrow), plus a different own Limiting Primer (thin arrow).
The melting temperature of the four different Limiting Primers
increases from left to right and the annealing temperature used for
each set of reactions is 2.degree. C. below the melting temperature
of the Limiting Primer.
[0009] FIG. 3 shows agarose gels of identical LATE-PCR without
ELIXIR (left) and with ELIXIR (right). The replicate reactions are
prepared using four different preparations of commercially
available Taq polymerases, both with and without a hot start. The
reactions were incubated at room temperature for 30 minutes before
amplification.
[0010] FIG. 4 is an agarose gel of a pentaplex LATE-PCR without
ELIXIR (left six lanes), monoplex LATE-PCR with ELIXIR (middle five
lanes), and pentaplex with ELIXIR (right six lanes). A molecular
weight ladder is shown in far right lane. In the pentaplex
reactions, all five targets are amplified simultaneously.
[0011] FIG. 5 shows reaction design (left panels) and dF/dT in the
presence of SybrGreen (right panels) where five primer pairs and
(a) one template or (b) one template corresponding to one primer
pair+four low copy number amplicons corresponding to the remaining
four primer pairs are included.
[0012] FIG. 6 shows pyrosequencing of a LATE-PCR monoplex reaction
(left panel) and a multiplex reaction (right panel). The
post-LATE-PCR reactions are split into five aliquots each and
pyrosequencing performed in the presence of a sequencing primer
corresponding to each amplicon.
[0013] FIG. 7 shows an embodiment of a Low-T.sub.m Probe detection
approach.
[0014] FIG. 8 compares amplification and detection of a
high-T.sub.m molecular beacon probe and a low-T.sub.m molecular
beacon probe.
[0015] FIG. 9 compares resolution of single nucleotide polymorphism
in heterozygous CC, heterozygous CT, and homozygous TT using an
anneal down protocol (left) and a melt-up protocol (right).
[0016] FIG. 10. LATE-PCR The captions under the bars indicate the
temperature used for RT prior to LATE-PCR. No RTase (light blue
bar), the inactivated RTase (blue bar), 30 min at 50.degree. C.
(green bar), 30 min at 55.degree. C.; 10 min at 60.degree. C.+20
min at 55.degree. C. (orange bar). LATE-PCR is identical for all
samples.
[0017] FIG. 11. LATE-PCR protocol with 50 nM limiting primer (LP)
and 2 .mu.M RT primer and LATE-PCR excess primer (XP).
[0018] FIG. 12 is a clustal comparison of Influenza Virus M RNA
sequences for H3N2, H5N1, H1N1, and B.
[0019] FIG. 13 is a clustal comparison of Influenza Virus HA RNA
sequences for H3N2, H5N1, H1N1, and B.
[0020] FIG. 14 is a clustal comparison of Influenza Virus NA RNA
sequences for H3N2, H5N1, H1N1, and B.
[0021] FIG. 15 shows a schematic of an embodiment of an assay
[0022] FIG. 16 shows the layout and an possible outcomes for an
exemplary assay.
[0023] FIG. 17 provides primer, probe, and amplicon sequences that
can be used in an embodiment of an influenza virus assay.
DETAILED DESCRIPTION
[0024] All references cited are incorporated herein by
reference.
[0025] An influenza virus assay can detect and distinguish between
various sub-types and strains of an influenza virus using any
suitable nucleic acid amplification technique. This assay can be
performed in a single reaction vessel with all reagents present at
the start of an assay. An assay can use more than one primer pairs
in combination with one or more probes to amplify and detect
specific target nucleic acid sequences of influenza. Using the
information obtained from an amplification reaction it is possible
to distinguish between various sub-types and strains of the an
influenza virus. Specifically, an assay can provide a positive or
negative (yes/no) determination of the likely presence or absence
of influenza virus types A and B, and sub-types H1N1, H3N2, and
H5N1 in a sample. An assay also can be used to monitor for one or
more mutations in an influenza virus strain. Mutations in an
influenza virus, within, for example the HA and NA, can alter viral
infectivity and lethality in different hosts and different
tissues.
[0026] A sample can be any material to be tested, such as, for
example, a biological or environmental sample. Biological samples
can be obtained from any organism. In one embodiment, a sample is
obtained from a mammal, such as a human, or a bird. In one
embodiment, a sample comprises a nasopharyngeal aspirate, blood,
saliva, or any other bodily fluid.
[0027] A nucleic acid amplification method used in an assay can be
a thermal cycling technique, such as a polymerase chain reaction
("PCR") or an isothermal technique. Standard PCR amplification
methods are described in, for example, U.S. Pat. Nos. 4,683,195 and
4,683,202 (both of which are incorporated herein by reference). In
one embodiment, the nucleic acid amplification method is linear
after the exponential ("LATE") PCR ("LATE-PCR"), as described in
U.S. patent application Ser. No. 10/320,893, which is incorporated
by reference. An embodiment of LATE-PCR is illustrated in FIG. 1.
LATE-PCR is an asymmetric PCR method, which uses unequal
concentrations of primers and can yield single-stranded
primer-extension products, or amplicons. LATE-PCR amplifications
and assays typically include at least 30 cycles, at least 60
cycles, or at least 70 cycles. FIG. 2, which shows an agarose gel
showing that amplification product production is less specific when
the melting temperature of the Limiting Primer is below that of the
Excess Primer, demonstrates the specificity advantage that can be
afforded by a properly designed LATE-PCR. An Excess Primer can
misprime at a low annealing temperature, but the reaction can
becomes very specific when the melting temperature of the Limiting
Primer is above the melting temperature of the Excess Primer
(T.sub.mL[0]-T.sub.mX[0].gtoreq.0).
[0028] As used interchangeably herein, the terms "nucleic acid
primer", "primer molecule", "primer", and "oligonucleotide primer"
include short, (for example, between about 16 and about 50 bases)
single-stranded oligonucleotides which, upon hybridization with a
corresponding template nucleic acid molecule, serve as a starting
point for synthesis of the complementary nucleic acid strand by an
appropriate polymerase molecule. Primer molecules can be
complementary to either the sense or the anti-sense strand of a
template nucleic acid molecule. A primer can be composed of
naturally occurring or synthetic oligonucleotides, or a mixture of
the two. If the primers in a pair of PCR primers are used in
unequal concentrations, as is the case in LATE-PCR, primer added at
a lower concentration is a "Limiting Primer", and primer added at a
higher concentration is an "Excess Primer."
[0029] As used herein "amplification target sequence" used
interchangeably with "target sequence" and "target nucleic acid"
refers to a DNA sequence that provides a template for copying by
the steps an amplification technique. An amplification target
sequence can be single-stranded or double-stranded. If the starting
material is RNA, for example messenger RNA, the DNA amplification
target sequence is created by reverse transcription of RNA to
create complementary DNA, and the amplification target sequence is
a cDNA molecule. Thus, in a PCR assay for RNA, a hybridization
probe signals copying of a cDNA amplification target sequence,
indirectly signifying the presence of the RNA whose reverse
transcription produced the cDNA molecules containing the
amplification target sequence. An amplification target sequence
typically is bracketed in length by a pair of primers used to
amplify it. An extension product (or amplicon), whether
double-stranded or, in non-symmetric PCR, single-stranded, is the
exponentially amplified amplicon, bracketed by the primer pair.
[0030] An amplification target sequence can be a single nucleic
acid sequence. In some cases an amplification target sequence will
contain allelic variations or mutations and, thus, will not be a
single sequence, even though amplified by a single primer pair. An
assay for an amplification target sequence containing variations
may use one detection probe for all variations, a single
allele-discriminating probe for one variant, or multiple
allele-discriminating probes, one for each variant.
[0031] Any suitable primer pair can be used, such as, for example,
one of more of the following primer pairs:
TABLE-US-00001 H5 Limiting Primer (reverse complement)
GGATAGACCAGCTACCATGATTGCC, Tm = 66.8 (H5N1), 21.5(H3N2), 30.3
(H1N1), 14.2 (B) Excess Primer-RNA 63.2 linear
GTGGAGTAAAATTGGAATCAATAGG, Tm = 63.6(H5N1), 15.7(H3N2), 53.8(H1N1),
34.6(B) H1 Limiting Primer (reverse complement)
CACCCGTTTCCTATTTCTTTGGCATTATTC, Tm = 22.5(H5N1), 21.6(H3N2),
66.7(H1N1), 30.2(B) Excess Primer, RNA 64.2 linear
CCATGACTCCAATGTGAAG, Tm = 36.1(H5N1), 30.1(H3N2), 63.8(H1N1),
20.3(B) N1 Limiting Primer (reverse complement)
CAGCACCGTCTGGCCAAGAC, Tm = 69.3(H5N1), 69.3(H1N1), 41.0(H3N2),
5.7(B) Excess Primer, RNA 66.3 linear GCAATAACTGATTGGTCAGG, TM =
62.9 (H5N1), 62.7(H1N1), 29.9(H3N2), 40.4(B) H3 Limiting Primer
(reverse complement) CGTTGTATGACCAGAGATCTATTTTAGTGTCCT, Tm =
12.2(H5N1), 67.9(H3N2), 4.6(H1N1), -0.6(B) Excess Primer, RNA 59.7
linear CCATCAGATTGAAAAAGAATTCT, Tm = 29.3(H5N1), 62.7(H3N2),
21.5(H1N1), 22.2(B) B(HA) Limiting Primer (reverse complement)
CAGGAGGTCTATATTTGGTTCCATTGGC, Tm = 14.4(H5N1), 21.0(H3N2),
24.8(H1N1), 67.5(B) Excess Primer, RNA 58.9 linear
CGGTGGATTAAACAAAAGCA, Tm = 12.1(H5N1), 26.0(H3N2), 20.3(H1N1),
62.4(B) B(NA) Limiting Primer (reverse complement)
CCCAATACAGGGGACATCACATTTCTTG, Tm = 10.9(H5N1), 16.2(H1N1),
-4.6(H3N2), 68.9(B) Excess Primer, RNA 69.7 just linear
CATGGGCTGACAGTGAT, Tm = 38.7(H5N1), 43.2(H1N1), 42.5(H3N2), 63.7(B)
M Limiting Primer (reverse complement) GGTGACAGGATTGGTCTTGTCTTTAGC,
Tm = 67.3 (H5N1), 67.3(H3N2), 67.3(H1N1), 14.4(B) Excess Primer
CTAACCGAGGTCGAAAC, Tm = 62.2(H5N1), 62.2(H3N2), 62.2(H1N1), 20.6(B)
N2 Limiting Primer (reverse complement) GATGCAGCTTTTGCCTTCAACAGAG,
Tm = 29.4(H5N1), 16.7(H1N1), 67.4(H3N2), 15.6(B) Excess Primer, RNA
69.4 just before hairpin GGTCCAACCCTAATTCCAA, Tm = 13.6(H5N1),
22.0(H1N1), 63.4(H3N2), 22.7(B) H3 Control Limiting Primer (reverse
complement) CGTTGTATGACCAGAGATCTATTTTAGTGTCCT, Tm = 12.2(H5N1),
67.9(H3N2), 4.6(H1N1), -0.6(B) Excess Primer
CCATCAGATTGAAAAAGAATTCT, Tm = 29.3(H5N1), 62.7(H3N2), 21.5(H1N1),
22.2(B) H5 Delete Region Limiting Primer (reverse complement)
CCTCCCTCTATAAAACCTGCTATAGCTCCAAA, Tm = 69.7(H5N1), 14.3(H3N2),
20.5(H1N1), 6.5(B) Excess Primer CGACTGGGCTCAGAAA, Tm = 62.9(H5N1),
17.4(H3N2), 29.3(H1N1), 17.4(B)
[0032] In one embodiment all of the above primer pairs are
used.
[0033] A target sequence can be present at a starting concentration
of greater than or equal to approximately 1,000,000 copies/sample.
In one embodiment, a target sequence is present at a concentration
of approximately 10 to approximately 1,000,000 copies/sample. In
another embodiment a target sequence is present at a concentration
of less than 10 copies/sample, less than 100 copies/sample, less
than 1,000 copies/sample, less than 10,000 copies/sample, less than
100,000 copies/sample, less than 500,000 copies/sample, or less
than 1,000,000 copies/sample.
[0034] In one embodiment, a target sequence can produce an amplicon
as provided in FIG. 17.
[0035] An assay includes reagents for an amplification reaction,
such as those used in LATE-PCR, a symmetric PCR amplification, or
an isothermal amplification method. For example, the assay mixture
can include each of the four deoxyribonucleotide 5' triphosphates
(dNTPs) at equimolar concentrations, a thermostable polymerase, a
divalent cation, and a buffering agent. An assay mixture can
include additional ingredients, such as, for example, a separate
reverse transcriptase enzyme. Non-natural dNTPs can be used. For
instance, dUTP can be substituted for dTTP and used at three-times
the concentration of the other dNTPs due to the less efficient
incorporation by Taq DNA polymerase.
[0036] An assay also can include reagents that can suppress
mispriming. In one embodiment, a reagent capable of suppressing
mispriming is a single-stranded oligonucleotides capable of forming
a stem-and-loop structure, commonly referred to as a "hairpin"
structure such as those described in, for example, U.S. patent
application Ser. No. 11/252,506, (referred to as an "ELIXIR.TM."),
which is incorporated by reference herein. FIG. 3 demonstrates the
ability of an ELIXIR.TM. to inhibit Taq polymerase, reducing
mispriming and increasing generation of target product. FIG. 4
shows the ability of an ELIXIR.TM. to reduce mispriming in a
multiplex reaction.
[0037] An assay also can include an amplicon corresponding to a
primer pair that is capable of suppressing mispriming. In one
embodiment, one or more copies of an amplicon corresponding to a
primer pair, where there are less targets present than primer
pairs, is (referred to herein as "mono-multiplex"). For example, an
assay can have the capacity of amplify any one of several different
amplicons, but in any particular assay it is possible that either
none or only a few viral target sequences will be present in a
sample. Such reactions mono-multiplex reactions can be difficult to
construct, because they have to suppress mispriming among the
unused primers, while still allowing amplification of the correct
product from any pair of primers. To prevent such mis-priming, it
can be advantageous to add low copy numbers of synthetic amplicons
for each primer pair. In one embodiment, approximately 20 copies of
synthetic amplicons per primer pair can be included in an assay.
FIG. 5 demonstrates how inclusion of low levels of synthetic
amplicons can suppress mispriming in a mono-multiplex reaction. The
difference between the design of unsuppressed and "internally
suppressed monomultiplex" reaction is illustrated in FIG. 5a and
the present and absence of mis-primed products is illustrated in
FIG. 5b.
[0038] In another embodiment, reactions that do not contain
influenza viral sequences have an internal control that rules out
false negatives. This internal control can be observed at a single
wavelength. In one embodiment, the internal control can be observed
at 25.degree. C. and can be generated by amplification of an
internal control target possessing a variant of H3 flanked by the
H3 primers. Detection can be accomplished using any suitable prove,
including, for example, a mismatch-tolerant probe.
[0039] Amplification products can be detected by an end-point
analysis or using a real-time analysis. As used herein, the term
"real time," with respect to an amplification reaction, refers to
the method by which the amplification reaction is detected. In a
"real-time" amplification reaction, accumulation of amplicon or
product is measured during the progression of the reaction, as
opposed to solely after the reaction is complete, the latter being
"end-point" analysis. In one embodiment detection is
quantitative.
[0040] The assays can use any suitable means to detect
amplification, including, but not limited to dyes, such as
intercalating dyes, DNA binding agents, and probe molecules. As
used interchangeably herein, the terms "nucleic acid probe", "probe
molecule", and "oligonucleotide probe" and "hybridization probe"
include defined nucleic acid sequences complementary to a target
nucleic acid sequence to be detected such that the probe will
hybridize to the target. Probes can be detectably labeled, such
that hybridization of a probe to a target sequence can be readily
assessed. A "detectable label" includes moieties that provide a
signal that can be detected and, in some embodiments, quantified.
Such labels are well known to those in the art and include
chemiluminescent, radioactive, metal ion, chemical ligand,
fluorescent, or colored moieties, or enzymatic groups which, upon
incubation with an appropriate substrate, provide a
chemiluminescent, fluorescent, radioactive, electrical, or
colorimetric signal. Methods of detection of such signals are also
well known in the art.
[0041] Probes can be composed of naturally occurring or synthetic
oligonucleotides and include labeled primers. Some hybridization
probes, for example molecular beacon probes, emit an increased
detectable signal upon hybridizing to their complementary sequence
without enzymatic action to hydrolyze the probes to generate a
signal. We refer to such probes as probes that hybridize to their
target and "signal upon hybridization." Other probes, for example
TaqMan.TM. dual fluorescently labeled random coil probes are cut,
or hydrolyzed, during the amplification reaction, and hydrolysis
leads to a detectable signal change. Probes that rely on hydrolysis
as part of signal generation are not probes that "signal upon
hybridization."
[0042] In one embodiment, an assay uses a "molecular beacon probe,"
which is a single-stranded oligonucleotide, typically 25-35
bases-long, in which the bases on the 3' and 5' ends are
complementary. Molecular beacon probes are discussed in, for
example, U.S. Pat. Nos. 5,925,517, 6,037,130, 6,103,476, 6,150,097,
and 6,461,817, and U.S. Patent Appl. Pub. No. 2004/0023269A1, all
of which are incorporated by reference. A molecular beacon probe
can form a hairpin structure at temperatures at and below those
used to anneal the primers to the template (typically below about
60.degree. C.). The double-helical stem of the hairpin brings a
fluorophore attached to one end (often, but not necessarily the '5
end) of a probe in proximity to a quencher attached to the other
end of the probe (typically, but not necessarily, the 3' end). In
the hairpin configuration, probe fluorescence is quenched. If a
probe is heated above the temperature needed to melt the double
stranded stem apart, or a probe is allowed to hybridize to a target
oligonucleotide that is complementary to a sequence within the
single-strand loop of a probe, fluorophore and quencher are
separated, and the resulting conformation shows increased
fluorescence. The strength of a fluorescent signal can increases in
proportion to the amount of a molecular beacon hybridized to an
amplicon. Molecular beacons with different loop sequences can be
conjugated to different fluorophores in order to monitor increases
in amplicons that differ by as little as one base (Tyagi, S. and
Kramer, F. R. (1996) "Molecular Beacons: Probes That Fluoresce Upon
Hybridization," Nat. Biotech. 14:303-308; Tyagi, S. et al., (1998)
"Multicolor Molecular Beacons for Allele Discrimination." Nat.
Biotech. 16: 49-53; Kostrikis, L. G. et al., (1998) "Spectral
Genotyping of Human Alleles," Science 279: 1228-1229).
[0043] Any suitable fluorophore/quencher pair can be used in a
molecular beacon probe. In one embodiment, four probes are used
each with a single fluorophore, wherein the flourophores are texas
red, CY3, CY5, and FAM. Any suitable quencher can be used, such as,
for example, Black Hole.TM. quenchers, dabsyl, and BHQ1. In one
embodiment, an assay include one or more fluorophore/quencher pair,
wherein the pair can be any of texas red/dabsyl, CY5/dabsyl,
FAM/dabsyl, CY5/BHQ1, and CT3/dabsyl. In another embodiment, an
assay uses one or more of the following probes:
TABLE-US-00002 H5 Texas Red-CGCGACTAGGGAACTCGCTCGCG-Dabsyl, Tm =
52.7 (H5N1), 8.0 (H3N2), 24.9(H1N1), 13.0(B) H1
CY3-CGCGGATTGGCTTTTTACTTTCTCACCGCG-Dabsyl, Tm = 7.8(H5N1),
20.1(H3N2), 56.6(H1N1), 12.7(B) N1
FAM-GGCGGATGCTGCTCCCACTACCGCC-Dabsyl, Tm = 56.3(H5N1), 56.3(H1N1),
12.1(H3N2), 25.8(B) H3 CY5-CGCTGAAAGCGTTTCTCGAGGTCCTG-BHQ1, Tm =
9.9(H5N1), 54.5(H3N2), 15.4(H1N1), 9.1(B) B(HA) Beacon Probe 1
Texas Red-GCGAGTTTGCATGTTCTCCTGTCTCGC-Dabsyl, Tm = 19.2(H5N1),
16.2(H3N2), 15.3(H1N1), 52.1(B) Beacon Probe 2
CY5-GCGAGTTTGCATGTTCTCCTGTCTCGC-Dabsyl, Tm = 19.2(H5N1),
16.2(H3N2), 15.3(H1N1), 52.1(B) B(NA) Beacon Probe 1 Texas
Red-GCCGCTCCATTGAAACCATTACGCGGC-Dabsyl, Tm = 26.3(H5N1),
27.9(H1N1), 21.2(H3N2), 53.1(B) Beacon Probe 2 (
CY5-GCCGCTCCATTGAAACCATTACGCGGC-Dabsyl, Tm = 26.3(H5N1),
27.9(H1N1), 21.2(H3N2), 53.1(B) M CY3-GCGCTATAGAGAGAACAGCGC-Dabsyl,
Tm = 33.8 (H5N1), 33.8(H3N2), 33.8(H1N1), 9.6(B) N2
FAM-GGCCGCCTATTACCTCTCGGCC-Dabsyl, Tm = 30.0(H5N1), 27.7(H1N1),
38.9(H3N2), 20.6(B) H3 Control CY5-CGCTGAAAGCGTTTCTCGAGGTCCTG-BHQ1,
Tm = 32.9 vs. modified amplicon sequence CAGGAACTCTAGAAA H5 Delete
Region Fluor-stemTCCTCTCTTTTTTCTTCTTCTCTstem-Dabsyl, Tm =
58.9(H5N1), -3.8(H3N2), 20.4(H1N1), 13.4(B)
[0044] In another embodiment, all of the above molecular beacon
probes are used in an assay. In a further embodiment, an assay can
include nine sequence-specific molecular beacons that are capable
of detecting seven influenza virus targets. In a further
embodiment, three molecular beacons probes, each detectable at a
different wavelength, can form a probe-target hybrid at T.sub.m
45.degree. C. and two additional molecular beacon probes, each
detectable at a different single wavelength, can form a
probe-target hybrid at T.sub.m 30.degree. C. Further, two
additional molecular beacon probes, each detectable a two different
wavelengths, can form a probe-target hybrid at T.sub.m 45.degree.
C. An additional mis-match tolerant probe can also be used to
detect one of the viral targets at 40.degree. C. and a variant of
that sequence present in an internal control at 25.degree. C.
[0045] In one embodiment, an assays can use a "Low-T.sub.m Probe."
A Low-T.sub.m Probe is discussed in U.S. patent application Ser.
No. 10/320,893 and refers to a labeled hybridization probe that
signals upon hybridization to its target, which in a LATE-PCR is
the Excess Primer-Strand generated by extension of the Excess
Primer, and that has a T.sub.m[O].sup.P at least 5.degree. C. below
or at least 10.degree. C. below the T.sub.m[0] of the primer that
hybridizes to and extends along the Excess Primer-Strand, which in
a LATE-PCR is the Limiting Primer. FIG. 7 shows an embodiment of a
Low-T.sub.m Probe detection approach.
[0046] As shown in FIG. 8, Low-T.sub.m Probes can be more specific
over a wider temperature range and can display lower backgrounds.
Low-T.sub.m Probes also show less amplification at higher
concentrations than High-T.sub.m Probes.
[0047] In another embodiment, an assay can use a "Super-Low-T.sub.m
Probe." This probe also is discussed in U.S. patent application
Ser. No. 10/320,893.
[0048] An assay can include more than one probe. In one embodiment,
multiple molecular beacon probes are used. In another embodiment,
the probes are capable of forming probe-target hybrids are more
than one temperature. In a further embodiment, multiple probes can
be used, where a first probe forms a probe-target hybrid at a first
temperature and a second probe forms a probe-target hybrid at a
second temperature. In one embodiment, five molecular beacon probes
can form a probe-target hybrid at a temperature of greater than
45.degree. C. and can be detected at 40.degree. C. and two
molecular beacon probes can form a probe-target hybrid at
30.degree. C. and can be detected at 25.degree. C.
[0049] An assay can also include mismatch tolerant probes, such as,
for example, fluorescent probes. In one embodiment, an assay uses a
mismatch tolerant probes. An assay also can detect probe-target
hybrids as a sample is cooled after PCR amplification ("anneal
down"). This approach can be used in end-point fluorescence
detection. This anneal-down approach can be more sensitive and
provide better resolution than cooling first and then reading
during warm-up (melt-up), because the read-during-cooling approach
can minimize formation of hairpin structures in a target sequence.
FIG. 9 compares resolution of single nucleotide polymorphism in
heterozygous CC, heterozygous CT, and homozygous TT using an anneal
down protocol (left) and a melt-up protocol (right). In one
embodiment, the temperature of an assay reaction is changed from
less than 95.degree. C. to less than 65.degree. C. than 45.degree.
C., to less than 25.degree. C.
[0050] An influenza viral assay (or an assay of any RNA virus) can
involve reverse transcription (RT) as a first step of a detection
reaction. During reverse transcription RNA sequences are converted
to complementary DNA (cDNA), providing a cDNA template for PCR
amplification.
[0051] An approach to RT-PCR is the use of a "One-Step RT-PCR
system." In a system of this type, reagents for both RT and PCR can
be added to a sample in a single mixture and the reaction tube can
be sealed and placed in a thermocycler. The RT and PCR
enzyme-catalyzed reactions are carried out sequentially in the
thermocycler, taking advantage of the different thermostabilities
of the enzymes involved (typically, a reverse transcriptase and a
thermostable DNA polymerase) and by setting an appropriate thermal
profile. An initial incubation at non-denaturing temperature allows
RT to occur first. The temperature then can be raised to initiate
PCR; at this temperature, the reverse transcriptase can be
inactivated, but the DNA polymerase is not. When a "hot start" is
used, DNA polymerase is kept inactive during the RT step by
interaction with a specific antibody. When the temperature is
elevated, the antibody can be denatured and the DNA polymerase
activated. In one embodiment, a multi-functional enzyme, having
both a RTase activity and a DNA polymerase activity, can be used.
In a further embodiment, a multi-functional enzyme having RTase
activity, DNA polymerase activity, and exonuclease activity can be
used, where the exonuclease activity can cleave double-stranded DNA
in TaqMan-type detection method.
[0052] The temperature and duration of RT and PCR steps can be
readily determined by one of skill in the art. In one embodiment,
an RT step can be performed for from less than 2 minutes to more
than 60 minutes at a temperature of from approximately
50-60.degree. C. and a DNA-polymerase step can be performed as a
thermocycle at approximately 95.degree. C. for several cycles as
discussed elsewhere.
[0053] An assay can be performed using any suitable device, such as
a thermal cycler. In one embodiment, an assay is performed using a
portable device, a man-portable device, or a handheld device, such
as, for example, a Bioseeq II. In another embodiment, an assay is
performed using a bench-top device, including, for example, an ABI
Prism 7700 Sequence Detector (Applied Biosystems, Inc., CA)
machine, a Cepheid Smart Cycler, and a Primus PCR thermocycle.
[0054] An assay can be performed in less than or equal to 30
minutes, less than or equal to 20 minutes, less than or equal to 15
minutes, or less than or equal to 10 minutes.
[0055] Assay reagents can be provided as a kit or a consumable. The
reagents can be supplied as a lyophilized preparation. Each reagent
can be supplied separately or as a mixture of one or more reagents.
Reagents also can be supplied on a substrate, such as a bead. A
lyophilized reagent can be stable for more than one year.
[0056] An assay can yield single-stranded products for further use,
for example as starting material for DNA sequencing or as probes in
other reactions, or can be used in other assays. In one embodiment,
single stranded DNA produced by an assay (assay product) can be
sequenced using any suitable sequencing method, such as, for
example, the dideoxy-method or pyrosequencing (Salk et al. (2006)
Anal. Biochem. 353:124, incorporated by reference) by diluting a
fraction of the assay reaction products into a sequencing reaction
mixture. Assay product can be diluted by approximately 1:10,
approximately 1:20, approximately 1:50, approximately 1:100, or
approximately 1:200 or more for use in a sequencing reaction. FIG.
6 shows a LATE-PCR multiplex reaction, in which one sample is split
into five aliquots each spiked with a different sequencing primer,
and sequenced.
[0057] An assay can distinguish Influenza Type B and Type A virus.
In one embodiment, an assay distinguishes Influenza Type B and Type
A virus on the basis of sequences in the HA and NA genes. Within
the Type A viruses an assay can distinguish between subtypes H5
(with or without N1 or N2), H1 (with or without N1 or N2), and H3
(with or without N1 or N2). In one embodiment, an N1 target
sequence used is conserved for the H5 and H1 subtypes and can be
useful for detecting H5N1 and H1N1. In another embodiment, H3N1 can
be determined and such a determination can indicate viral
reassortment. The N2 target sequence used is characteristic of the
H3N2 subtype, thus, detection of H5N2 or H1N2 can indicate viral
reassortment. FIGS. 12-14 shows clustal comparisons of influenza
virus M, HA, and NA proteins for virus H1N1, H5N1, H3N2, and B.
Such analysis is useful in interpreting data obtained from an assay
and from subsequent sequencing of assay products. Using information
obtaining from an assay, it is possible to monitor mutations in a
known virus strain, which allows for detection of and prediction of
changes in virulence and infectivity.
[0058] An exemplary avian influenza assay and possible results of
this exemplary assay are provided in Table I and FIG. 15. FIG. 16
shows a schematic of an embodiment of an assay and FIG. 17 provides
primer, probe sequences, and amplicon sequences that can be used in
an embodiment of an influenza virus assay. In one embodiment, an
assay include all of the primers and probes of FIG. 17 in a
mono-multiplex assay. The features of such a mono-multiplex are
summarized in Table I and the 15 possible outcomes of the reaction
are illustrated 16.
TABLE-US-00003 TABLE I Position Amplicon Target Probe Tm Primers
Sequence Type Color(s) Melting 1 H5 H5 M. Red 45 C. Beacon 2 H1 H1
M. Yellow 45 C. Beacon 3 N1 N1 M. Green 45 C. Beacon 4 H3 H3 EXO-R
Blue 45 C. 6 Type A M gene M gene M. Yellow 30 C. Beacon 7 N2 N2 M.
Green 30 C. Beacon 4/5 Type B HA Type B HA M. Blue only 45 C. only
Beacon Type B HA Type B HA M. Red only 45 C. only Beacon 4/5 Type B
NA Type B NA M Blue only 45 C. only Beacon Type B NA Type B NA M.
Red only 45 C. only Beacon 4/5 Type B HA + Type B HA + M Blue + NA
45 C. NA Beacon Blue Type B HA + Type B HA + M. Red + NA 45 C. NA
Beacon Red 8 H3 int. control mis-matched EX0-R Blue 30 C. H3 H5
int. control no matches no probe H1 int. control no matches no
probe N1 int. control no matches no probe N2 int. control no
matches no probe M int. control no matches no probe Type B HA i.c.
no matches no probe Type B NA i.c. no matches no probe
[0059] The exemplary assay described in this table contains: [0060]
8 pairs of primers [0061] 3 Molecular Beacons with 45.degree. C.
[0062] 2 Molecular Beacons with 30.degree. C. [0063] 2 pairs of
Molecular Beacons both at 45.degree. C. [0064] Total=9 Molecular
Beacons [0065] 1 mismatch-tolerant prove [0066] 1 detectable
internal control [0067] 7 undetected internal controls
Example 1
[0068] Starting with samples of purified RNA, the HA RNA (1770
nucleotides long) and the NA RNA (1400 nucleotides long) are both
be reverse transcribed in toto using random hexamers in a highly
efficient two step RT-procedure. Each reaction also contains low
levels of an M-Gene control DNA. The resulting control and cDNA
molecules are amplified in two parallel multiplex LATE-PCR assays
that each generate six amplicons. The presence of Eurasian H5N1
strain in a sample is established by probing for M, N1 and two
different H5 sequences that are likely to be crucial for
human-to-human transmission and for virulence. Reactions that do
not generate either signal for H5 Eurasian will nevertheless
produce a control DNA signal, proving that they are not false
negatives. Reactions that do signal the presence of the H5 Eurasian
strain from either of two independent probes (one in Multiplex A
and one in Multiplex B) also generates a strong M-gene signal in
both Multiplex A and Multiplex B. However, some samples may
generate a signal for an M protein and only one of two possible HA
signals. This is regarded as an indication of viral evolution. All
samples that generate either one or two HA signals, or an N1 signal
plus an M-gene signal are immediately be processed further for
analysis. The amplicons for the all portions of HA and NA already
are present in the LATE-PCR multiplex reactions. All 10 HA and NA
amplicons are 300-500 by in length and are processed for parallel
pyrosequencing sequencing.
Example 2
[0069] This example is directed to an RT-LATE-PCR assay for the
quantification of Oct4-specific sequences in embryonic mouse cells.
Oct4 is a gene expressed in totipotent and pluripotent cells and,
therefore, preimplantation embryos contain considerable levels of
Oct4 RNA. In addition, each cell contains two copies of the Oct4
gene (Oct4 genomic DNA). In the experiments presented in this
example, Oct4 RNA and DNA are co-purified and co-quantified,
according to a method previously published by this laboratory
(Hartshorn C, Anshelevich A, Wangh L. J. Rapid, single-tube method
for quantitative preparation and analysis of RNA and DNA in samples
as small as one cell. BMC Biotechnol 2005; 5:2). Briefly, single
embryos at the 8-cell stage are transferred to tiny droplets of dry
lysing reagents placed on the lid of PCR tubes. After cell lysis,
which occurs very rapidly, the tube is placed on the lid and
inverted. One-step RT-PCR is performed by adding the reagents in
the same tube already containing the lysed sample. Thus, both RNA
and DNA are present in each sample. LATE-PCR primers are designed
within an exon, also according to our published strategy, so that
amplicons generated by Oct4 cDNA molecules and Oct4 genomic DNA
molecules are identical and detected by the same fluorescent probe,
a sequence-specific molecular beacon conjugated to the TET
fluorescent dye. The final volume of these assays is 50 .mu.l,
according to the instruction of the One-Step RT-PCR kit, but the
volume can be decreased to 25 .mu.l.
[0070] The RT-LATE-PCR reaction is carried out in an ABI Prism 7700
Sequence Detector (Applied Biosystems, CA) and quantification of
"total DNA" (cDNA+genomic DNA) copy numbers for each sample is
achieved by comparison with standard scales prepared with serial
dilutions of commercially available genomic DNA. (Because the Oct4
primers are designed to amplify equally cDNA and genomic DNA,
standard scales of genomic DNA are amplified exactly with the same
efficiency as unknown cDNA samples, ensuring accurate
quantification.) The total number of Oct4 templates in each 8-cell
embryo includes 16 copies of genomic DNA (two copies of the gene
per cell, one on each chromosome 17) while all the other copies are
due to the presence of cDNA and, thus, reflect the Oct4 RNA content
of the embryo.
[0071] Several One-Step RT-PCR kits are tested using this assay and
the efficiencies for Oct4 RNA quantification are compared. FIG. 10
shows the effect of temperature on SuperScript III reverse
transcriptase (Invitrogen).
[0072] The blue and orange bars in the figure are comparable to the
light blue "No RT" bar, indicating that under these temperature
conditions RT does not take place and only genomic Oct4 DNA is
amplified in the samples (16 copies per embryo, as expected). At
55.degree. C. (green bar), however, RT occurs and cDNA is
efficiently generated. Considering that the reverse transcriptase
used for these experiments is active in the 42-60.degree. C. range,
this narrow window of activity is unexpected. To clarify this
point, the thermodynamics of the Oct4 primers during RT is analyzed
and compared to their behavior during PCR.
[0073] Visual OMP 5.0 software (VOMP) is used for this analysis and
the results are summarized in FIG. 11. The two primers used for a
LATE-PCR assay (limiting primer, LP, and excess primer, XP) have
different T.sub.ms and concentrations. In the Oct4 RT-LATE-PCR
assay the most abundant primer (XP) is also the RT primer, being
antisense to Oct4 RNA. As shown in FIG. 11, the effective T.sub.m
of this primer calculated in the presence of double-stranded DNA
during the PCR annealing step (at 55.degree. C.) is of 66.degree.
C., very close to the calculated T.sub.m of 67.degree. C. The
effective T.sub.m of this same primer, however, drops dramatically
to 53.degree. C. in the presence of single-stranded RNA, even if
the temperature of RT is also set at 55.degree. C. This change
results in a much lower percent of primer hybridized during RT than
during the PCR annealing step, although the temperature is the same
during the two steps. Although only 50% of the available primer is
hybridized to target at 55.degree. C., the primer was present at
high concentration (2 .mu.M) which allowed efficient RT. Additional
VOMP analyses show that Oct4 XP's effective T.sub.m during RT does
not change in the 50-60.degree. C. range, which explains why
increasing RT temperature in this case led to RT failure (much less
primer was bound to target at 60.degree. C. than at 55.degree. C.
and, contrary to the manufacturer's indications, the RTase was
completely denatured after the initial 10 minutes at 60.degree. C.,
see next section). On the other hand, the failure of RT at
temperature lower than 55.degree. C. (when, based solely on
T.sub.m, more primer should be hybridized) is probably due to
increased levels of secondary structure of the target RNA
interfering with the ability of the reverse transcriptase to
progress along the template strand.
[0074] These results indicate that primers designed for PCR or
LATE-PCR also should be analyzed in terms of their thermodynamic
modification of a primer's design so that its T.sub.m can meet the
necessary requirements during both RT and PCR. In cases where this
is not possible due to restraints intrinsic to the sequence, a
third primer--designed to hybridize only during the RT step--could
be added to the one-step mixture. In addition, the characteristics
of LATE-PCR are advantageous to promote RT priming in a one-step
assay. In fact, by designating the XP to be also the RT primer we
are able to use higher RT primer concentrations than those used
under standard conditions in RT-PCR assays.
Example 3
[0075] This example demonstrates optimization of RT reaction
parameters.
[0076] Satisfactory RT results are obtained for two different genes
shortening the RT step from 30 to 5 minutes, although a slight loss
of sensitivity is observed. Further reducing RT to 2 or 3 minutes
still yields acceptable results. The reverse transcriptase used was
SensiScript by Qiagen. (Raja et al., 2002. Temperature-controlled
Primer Limit for Multiplexing of Rapid, Quantitative Reverse
Transcription-PCR Assays: Application to Intraoperative Cancer
Diagnostics. Clinical Chemistry 48:8, 1329-1337.)
[0077] RT also performed with SuperScript II (Invitrogen) for 5
minutes. (Raja et al., 2005. Technology for Automated, Rapid, and
Quantitative PCR or Reverse Transcription-PCR Clinical Testing.
Clinical Chemistry 51:5, 882-890.
[0078] RT is successfully carried out for just 1 minute with either
MMLV (Moloney Murine Leukemia Virus RT) or SuperScript III.
(Stanley and Szewczuk, 2005. Multiplexed tandem PCR: gene profiling
from small amounts of RNA using SYBR Green detection. Nucleic Acids
Research 33:20, e180.)
[0079] Based on the above studies, a One-Step RT-PCR assay for
detection of avian flu is designed that will encompass a RT step of
no more than 5 minutes and as low as 1 minute. In doing so, we are
aware that the optimal length of the RT reaction depends on several
factors, including, but not limited to, efficient primer binding
(see Example 2). The intrinsic thermostability of the RT enzyme
also comes into play when choosing the temperature for RT, because
the half-life of any enzyme sharply decreases at increasing
temperatures, although some enzymes are more stable than
others.
[0080] A clear example is provided by the following table posted on
the web by the manufacturer Invitrogen
Summary of RT Half Lives at 50, 55, and 60.degree. C.
TABLE-US-00004 [0081] SuperScript .TM. SuperScript .TM. Temperature
II RT (min) III RT (min) 50.degree. C. 6.1 220 55.degree. C. 2.2 24
60.degree. C. ND 2.5
[0082] From this table it follows that, when working with
SuperScript III at 60.degree. C. or with SuperScript II at
55.degree. C., the optimal RT step duration is no more than 5
minutes in any case, independently from the primer T.sub.ms,
because the enzyme is completely denatured in this period of
time.
[0083] Newer RTases with broader thermostability ranges are
commercially available. For example, StrataScript 5.0 from
Stratatgene, has a half-life of 35 minutes at 55.degree. C. There
is also a number of polymerases commercialized by Roche and derived
from thermophilic bacteria, which have both RTase and DNA
polymerase activity.
[0084] We note that it is important to designing gene-specific RT
primers with T.sub.ms precisely calculated for optimal
hybridization to target at a temperature elevated enough to
minimize the secondary structure of single-stranded, GC-rich RNA
molecules such as those present in viral genomes, but at the same
time allowing a sufficient half-life of the chosen enzyme. The
importance and the subtleties of this approach are not widely
recognized, as shown by the suggestion: "Primers for real-time
RT-PCR should be designed according to standard PCR guidelines"
(Platinum Quantitative RT-PCR ThermoScript One-Step System
instruction sheet, included with product purchased in 2006).
Example 4
[0085] This example demonstrates the use of a Smiths Detection
Bio-Seeq II instrument as a portable, point-of-care assay device.
The Bio-Seeq II used in this example is comprised of four
independently operating thermal cycling units, each encasing a long
thin-walled sample tube having a sample volume of 25 ul. The
primers and probes provided in FIG. 17 are used. Each sample is
viewed using four-color fluorescence optics for dyes that emit at
520 nm, 580 nm, 625 nm, 680 nm. All colors can be viewed
simultaneously without moving parts, a feature of the BioSeeq that
reduces sampling time and lowers the risk of mechanical failure. To
make full use of the broad detection temperature range available in
LATE-PCR each unit can ramp up at 10.degree. C./sec between
25-95.degree. C., and is actively cooled at a rate of at least
2.5.degree. C./sec between 95-25.degree. C. The tolerance for
thermal variance at any chosen hold temperature is .+-.1.degree. C.
The unit is AC or battery powered.
[0086] Each of the LATE-PCR mono-multiplex assays described below
is designed to detect and distinguish any one of 15 possible
outcomes in a single closed-tube. See FIGS. 15 and 16. These assays
are easy to use "in the field" and provide rapid definitive yes/no
answers as to the absence or presence of Influenza Virus sub-types
H1N1, H3N2, B and H5N1. The assays also detect the presence of a
Type B virus, or Type A influenza virus of unknown sub-type and
include internal controls to rule out false negatives.
[0087] Each mono-multiplex reaction includes internal control
target sequences, as shown in FIG. 17, at low copy number
(approximately 20) to insure that all primer pairs are engaged in
amplifying either a viral target sequence or an internal control.
Accordingly, the reaction described below utilizes eight pairs of
primers and eight internal controls.
[0088] Each mono-multiplex reaction is read at end-point by
dropping the temperature to 40.degree. C. and then to 25.degree. C.
Nine sequence-specific molecular beacons are used in this reaction
to detect 7 of the possible viral targets. Three molecular beacons,
each of a single color, form probe-target hybrids at Tm 45.degree.
C. Two additional molecular beacons, each of a single color, form
probe-target hybrids at Tm 30.degree. C. Two additional molecular
beacons, each with two colors, form probe-target hybrids at Tm
45.degree. C.
[0089] One mis-match tolerant probe is used to detect one of the
viral targets at 40.degree. C. and a variant of that sequence
present in an internal control at 25.degree. C. Seven of eight
internal controls go undetected because they possess no targets for
any probe.
[0090] Each mono-multiplex reaction is designed to distinguish
Influenza Type B and Type A viruses on the basis of sequences in
the HA and NA genes. Within the Type A viruses the reaction
distinguishes between subtypes H5 (with or without N1 or N2), H1
(with or without N1 or N2), and H3 (with or without N1 or N2). The
N1 target sequence used is conserved for the H5 and H1 subtypes and
therefore is useful for detecting H5N1 and H1N1. Detection of H3N1
would indicate viral reassortment. The N2 target sequence used is
characteristic of the H3N2 subtype. Therefore detection of H5N2 or
H1N2 would indicate viral reassortment. The mono-multiplex reaction
described here is a one-step RT-LATE-PCR reaction. The chemical
features of this one-step process are described elsewhere.
[0091] One assay provides a reliable means of detecting the
Eurasian subtype of H5. Specimens that test positive for H5
Eurasian in the field are be sent to an analytical laboratory for
complete multiplex analysis and sequencing.
[0092] In a second assay, the H5 amplicon produced in the field
includes the region of the RNA known to code for high pathogenicity
of the Eurasian sub-type. This region is less conserved, but very
important. Under these circumstances the tube that tests positive
in the field for Eurasian H5 is sent to the laboratory for
immediate sequencing of the H5 amplicon. There is no need to
transport the specimen itself or additional amplification
[0093] The next step involves in-depth laboratory analysis of
influenza genes using LATE-PCR multiplexing and nucleic acid
sequencing. Starting with samples of purified RNA, the HA RNA (1770
nucleotides long) and the NA RNA (1400 nucleotides long) are both
be reverse transcribed in toto using random hexamers in a highly
efficient two step RT-procedure. Each reaction also contains low
levels of the same M-Gene control DNA describes for the BioSeeq POC
assays above. The resulting control and cDNA molecules are
amplified in two parallel multiplex LATE-PCR assays that each
generate six amplicons (FIG. 15).
[0094] The possible presence of Eurasian H5N1 strain in a sample
will be established by probing for M, N1 and two different H5
sequences which are likely to be crucial for human-to-human
transmission and for virulence. Reactions that do not generate
either signal for H5 Eurasian still produce a Control DNA signal,
proving that they are not false negatives. Reactions that do signal
the presence of the H5 Eurasian strain from either of two
independent probes (one in Multiplex A and one in Multiplex B) also
generate a strong M-gene signal in both Multiplex A and Multiplex
B. However, some samples may generate a signal for the Matrix
protein and only one of the two possible HA signals. This is
regarded as an indication of viral evolution. All samples that
generate either one or two HA signals, or an N1 signal plus an
M-gene signal will immediately be processed further for analysis.
Amplicons for the all portions of HA and NA are already be present
in the LATE-PCR multiplex reactions. All 10 HA and NA amplicons are
300-500 by in length and are processed for parallel pyrosequenceing
as described above.
Sequence CWU 1
1
59125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ggatagacca gctaccatga ttgcc 25225DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2gtggagtaaa attggaatca atagg 25330DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 3cacccgtttc ctatttcttt
ggcattattc 30419DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 4ccatgactcc aatgtgaag 19520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5cagcaccgtc tggccaagac 20620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6gcaataactg attggtcagg
20733DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7cgttgtatga ccagagatct attttagtgt cct
33823DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8ccatcagatt gaaaaagaat tct 23928DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9caggaggtct atatttggtt ccattggc 281020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10cggtggatta aacaaaagca 201128DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 11cccaatacag gggacatcac
atttcttg 281217DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 12catgggctga cagtgat 171327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13ggtgacagga ttggtcttgt ctttagc 271417DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14ctaaccgagg tcgaaac 171525DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 15gatgcagctt ttgccttcaa cagag
251619DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16ggtccaaccc taattccaa 191732DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17cctccctcta taaaacctgc tatagctcca aa 321816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18cgactgggct cagaaa 161923DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 19cgcgactagg gaactcgctc gcg
232030DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 20cgcggattgg ctttttactt tctcaccgcg
302125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 21ggcggatgct gctcccacta ccgcc 252226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
22cgctgaaagc gtttctcgag gtcctg 262327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
23gcgagtttgc atgttctcct gtctcgc 272427DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
24gccgctccat tgaaaccatt acgcggc 272521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
25gcgctataga gagaacagcg c 212622DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 26ggccgcctat tacctctcgg cc
222715DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 27caggaactct agaaa 152823DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
28tcctctcttt tttcttcttc tct 2329989DNAInfluenza virus 29tagatattga
aagatgagcc ttctaaccga ggtcgaaacg tatgttctct ctatcgttcc 60atcaggcccc
ctcaaagccg aaatcgcgca gagacttgaa gatgtctttg ctgggaaaaa
120cacagatctt gaggctctca tggaatggct aaagacaaga ccaatcctgt
cacctctgac 180taaggggatt ttggggtttg tgttcacgct caccgtgccc
agtgagcgag gactgcagcg 240tagacgcttt gtccaaaatg ccctcaatgg
gaatggggat ccaaataaca tggacaaagc 300agttaaactg tatagaaaac
ttaagaggga gataacattc catggggcca aagaaatagc 360actcagttat
tctgctggtg cacttgccag ttgcatgggc ctcatataca ataggatggg
420ggctgtaacc accgaagtgg catttggcct ggtatgtgca acatgtgaac
agattgctga 480ctcccagcac aggtctcata ggcaaatggt ggcaacaacc
aatccattaa taaaacatga 540gaacaggatg gttttggcca gcactacagc
taaagctatg gagcaaatgg ctggatcaag 600tgagcaggca gcggaggcca
tggagattgc tagtcaggcc aggcaaatgg tgcaggcaat 660gagaaccgtt
gggactcatc ctagctccag tactggtcta agagatgatc ttcttgaaaa
720tttgcagacc tatcagaaac gaatgggggt gcagatgcaa cgattcaagt
gacctgcttg 780ttgttgctgc gagtatcatt gggatcttgc acttgatatt
gtggattctt gatcgtcttt 840ttttcaaatg catctatcga ctcttcaaac
acggcctgaa aagagggcct tctacggaag 900gagtacctga gtctatgagg
gaagaatatc gaaaggaaca gcagaatgct gtggatgctg 960acggcagtca
ttttgtcagc atagagctg 98930985DNAInfluenza virus 30atgagtcttc
taaccgaggt cgaaacgtac gttctctcta tcgtcccgtc aggccccctc 60aaagccgaga
tcgcacagag acttgaaaat gtctttgctg gaaagaatac cgatcttgag
120gctctcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa
ggggatttta 180ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac
tgcagcgtag acgctttgtc 240caaaatgccc ttaatgggaa tggggatcca
aataatatgg acagagcagt taaactgtat 300cgaaagctta agagggagat
aacattccat ggggccaaag aaatagcact cagttattct 360gctggtgcac
ttgccagttg tatgggactc atatacaaca ggatgggggc tgtgaccacc
420gaatcagcat ttggccttat atgcgcaacc tgtgaacaga ttgccgactc
ccagcataag 480tctcataggc aaatggtaac aacaaccaac ccattaataa
gacatgagaa cagaatggtt 540ctggccagca ctacagctaa ggctatggag
caaatggctg gatcgagtga acaagcagct 600gaggccatgg aggttgctag
tcaggccagg cagatggtgc aggcaatgag agccattggg 660actcatccta
gctctagcac tggtctgaaa aatgatctcc ttgaaaattt gcaggcctat
720cagaaacgaa tgggggtgca gatgcaacga ttcaagtgat cctcttgttg
ttgccgcaag 780tataattggg attgtgcacc tgatattgtg gattattgat
cgcctttttt ccaaaagcat 840ttatcgtatc tttaaacacg gtttaaaaag
agggccttct acggaaggag taccagagtc 900tatgagggaa gaatatcgag
aggaacagca gaatgctgtg gatgctgacg atggtcattt 960tgtcagcata
gagctggagt aaaaa 985311039DNAInfluenza virus 31agcaaaagca
ggtagatgtt gaaagatgag tcttctaacc gaggtcgaaa cgtacgttct 60ctctatcatc
ccgtcaggcc ccctcaaagc cgagatcgcg cagaaacttg aagatgtctt
120tgcaggaaag aacaccgatc tcgaggctct catggagtgg ctaaagacaa
gaccaatcct 180gtcacctctg actaaaggga ttttgggatt tgtattcacg
ctcaccgtgc ccagtgagcg 240aggactgcag cgtagacgct ttgtccagaa
tgccctaaat ggaaatggag atccaaataa 300tatggataga gcagtcaagc
tatataagaa gctgaaaaga gaaataacat tccatggggc 360taaggaggtc
gcactcagct actcaaccgg tgcacttgcc agttgcatgg gtctcatata
420caacaggatg ggaacggtga ctacggaagt ggcttttggc ctagtgtgtg
ccacttgtga 480gcagattgca gattcacagc atcggtctca cagacagatg
gcaactacca ccaacccact 540aatcagacat gagaacagaa tggtgctggc
cagcactaca gctaaggcta tggagcagat 600ggcaggatca agtgagcagg
cagcggaagc catggagatc gctaatcagg ctaggcagat 660ggtgcaggca
atgaggacaa ttgggactca tcctaactct agtgctggtc tgagagataa
720tcttcttgaa aatttgcagg cctaccagaa acgaatggga gtgcagatgc
agcgattcaa 780gtgatcctat tgttgttgcc gcaaatatca ttgggatctt
gcacttgata ttgtggattc 840ttgatcgtct tttcttcaaa tgcatttatc
gtcgccttaa atacggtttg aaaagagggc 900ctgctacggc aggggtacct
gagtctatga gggaagagta ccggcaggaa cagcagagtg 960ctgtggatgt
tgacgatggt cattttgtca acatagaatt ggagtaaaaa actaccttgt
1020ttctactaat acggaagac 1039321076DNAInfluenza virus 32atgtcgctgt
ttggagacac aattgcctac ctgctttcat tgacagaaga tggagaaggc 60aaagcagaac
tagcagaaaa attacactgt tggttcggtg ggaaagaatt tgacctagac
120tctgccctgg aatggataaa aaacaaaaga tgcttaactg atatacaaaa
agcactaatt 180ggtgcctcta tctgcttttt aaaacccaaa gaccaggaaa
gaaaaagaag attcatcaca 240gagcctctat caggaatggg aacaacagca
acaaaaaaga aaggcctgat tctagctgag 300agaaaaatga gaagatgtgt
gagctttcat gaagcatttg aaatagcaga aggccatgaa 360agctcagcgc
tactatactg tctcatggtc atgtacctga atcctggaaa ttattcaatg
420caagtaaaac taggaacgct ctgtgctttg tgcgagaaac aagcatcaca
ttcacacagg 480gctcatagca gagcagcgag atcttcagtg cctggagtga
gacgagaaat gcagatggtc 540tcagctatga acacagcaaa aacaatgaat
ggaatgggaa aaggagaaga cgtccaaaaa 600ctggcagaag agctgcaaag
caacattgga gtactgagat ctcttggggc aagtcaaaag 660aatggagaag
gaattgcaaa ggatgtaatg gaagtgctaa agcagagctc tatgggaaat
720tcagctcttg tgaagaaata tctataatgc tcgaaccatt tcagattctt
tcaatttgtt 780cttttatctt atcagctctc catttcatgg cttggacaat
agggcatttg aatcaaataa 840aaagaggagt aaacatgaaa atacgaataa
aaagtccaaa caaagagaca ataaacagag 900aggtatcaat tttgagacac
agttaccaaa aagaaatcca ggccaaagaa acaatgaagg 960aggtactctc
tgacaacatg gaggtattga gtgaccacat aataattgag gggctttctg
1020ccgaagagat aataaaaatg ggtgaaacag ttttggagat agaagaattg cattaa
1076331736DNAInfluenza virus 33tcatctgtca aatggagaaa atagtgcttc
tttttgcaat agtcagtctt gttaaaagtg 60atcagatttg cattggttac catgcaaaca
actcgacaga gcaggttgac acaataatgg 120aaaagaacgt tactgttaca
catgcccaag acatactgga aaagacacac aacgggaagc 180tctgcgatct
agatggagtg aagcctctaa ttttgagaga ttgtagtgta gctggatggc
240tcctcggaaa cccaatgtgt gacgaattca tcaatgtgcc ggaatggtcc
tacatagtgg 300agaaggccaa tccagtcaat gacctctgtt acccagggga
tttcaatgac tatgaagaat 360tgaaacacct attgagcaga ataaaccatt
ttgagaaaat tcagatcatc cccaaaagtt 420cttggtccag tcatgaagcc
tcattagggg tgagctcagc atgtccatac cagagaaagt 480cctccttttt
cagaaatgtg gtatggctta tcaaaaagaa cagtacatac ccaacaataa
540agaggagcta caataatacc aaccaagaag atcttttggt actgtggggg
attcaccatc 600ctaatgatgc ggcagagcag acaaagctct atcaaaaccc
aaccacctat atttccgttg 660ggacatcaac actaaaccag agattggtac
caagaatagc tactagatcc aaagtaaacg 720ggcaaagtgg aaggatggag
ttcttctgga caattttaaa accgaatgat gcaatcaact 780tcgagagtaa
tggaaatttc attgctccag aatatgcata caaaattgtc aagaaagggg
840actcaacaat tatgaaaagt gaattggaat atggtaactg caacaccaag
tgtcaaactc 900caatgggggc gataaactct agtatgccat tccacaatat
acaccctctc accatcgggg 960aatgccccaa atatgtgaaa tcaaacagat
tagtccttgc gactgggctc agaaatagcc 1020ctcaaagaga gagaagaaga
aaaaagagag gattatttgg agctatagca ggttttatag 1080agggaggatg
gcagggaatg gtagatggtt ggtatgggta ccaccatagc aatgagcagg
1140ggagtgggta cgctgcagac aaagaatcca ctcaaaaggc aatagatgga
gtcaccaata 1200aggtcaactc gatcattgac aaaatgaaca ctcagtttga
ggccgttgga agggaattta 1260acaacttaga aaggagaata gagaatttaa
acaagaagat ggaagacggg ttcctagatg 1320tctggactta taatgctgaa
cttctggttc tcatggaaaa tgagagaact ctagactttc 1380atgactcaaa
tgtcaagaac ctttacgaca aggtccgact acagcttagg gataatgcaa
1440aggaactggg taacggttgt ttcgagttct atcataaatg tgataatgaa
tgtatggaaa 1500gtgtaagaaa cggaacgtat gactacccgc agtattcaga
agaagcaaga ctaaaaagag 1560aggaaataag tggagtaaaa ttggaatcaa
taggaattta ccaaatactg tcaatttatt 1620ctacagtggc gagttcccta
gcactggcaa tcatggtagc tggtctatcc ttatggatgt 1680gctccaatgg
gtcgttacaa tgcagaattt gcatttaaat ttgtgagttc agattg
1736341692DNAInfluenza virus 34atgaaagcaa aactactggt cctgttatgt
acatttacag ctacatatgc agacacaata 60tgtataggct accatgccaa caactcaacc
gacactgttg acacagtact tgagaagaat 120gtgacagtga cacactctgt
caacctactt gaggacagtc acaacggaaa actatgtcta 180ctaaaaggaa
tagccccact acaattgggt aattgcagcg ttgccggatg gatcttagga
240aacccagaat gcgaattact gatttccaag gaatcatggt cctacattgt
agaaacacca 300aatcctgaga atggaacatg ttacccaggg tatttcgccg
actatgagga actgagggag 360caattgagtt cagtatcttc atttgagaga
ttcgaaatat tccccaaaga aagctcatgg 420cccaaccaca ccgtaaccgg
agtatcagca tcatgctccc ataatgggaa aagcagtttt 480tacagaaatt
tgctatggct gacggggaag aatggtttgt acccaaacct gagcaagtcc
540tatgtaaaca acaaagagaa agaagtcctt gtactatggg gtgttcatca
cccgcctaac 600ataggggacc aaagggccct ctatcataca gaaaatgctt
atgtctctgt agtgtcttca 660cattatagca gaagattcac cccagaaata
gccaaaagac ccaaagtaag agatcaggaa 720ggaagaatca actactactg
gactctgctg gaacctgggg atacaataat atttgaggca 780aatggaaatc
taatagcgcc atggtatgct tttgcactga gtagaggctt tggatcagga
840atcatcacct caaatgcacc aatggatgaa tgtgatgcga agtgtcaaac
acctcaggga 900gctataaaca gcagtcttcc tttccagaat gtacacccag
tcacaatagg agagtgtcca 960aagtatgtca ggagtgcaaa attaaggatg
gttacaggac taaggaacat cccatccatt 1020caatccagag gtttgtttgg
agccattgcc ggtttcattg aaggggggtg gactggaatg 1080gtagatgggt
ggtatggtta tcatcatcag aatgagcaag gatctggcta tgctgcagat
1140caaaaaagta cacaaaatgc cattaacggg attacaaaca aggtgaattc
tgtaattgag 1200aaaatgaaca ctcaattcac agctgtgggc aaagaattca
acaaattgga aagaaggatg 1260gaaaacttaa ataaaaaagt tgatgatggg
tttctagaca tttggacata taatgcagaa 1320ttgttggttc tactggaaaa
tgaaaggact ttggatttcc atgactccaa tgtgaagaat 1380ctgtatgaga
aagtaaaaag ccaattaaag aataatgcca aagaaatagg aaacgggtgt
1440tttgaattct atcacaagtg taacaatgaa tgcatggaga gtgtgaaaaa
tggaacttat 1500gactatccaa aatattccga agaatcaaag ttaaacaggg
agaaaattga tggagtgaaa 1560ttggaatcaa tgggagtcta tcagattctg
gcgatctact caactgtcgc cagttccctg 1620gttcttttgg tctccctggg
ggcaatcagc ttctggatgt gttccaatgg gtctttgcag 1680tgcagaatat gc
1692351718DNAInfluenza virus 35tattaaccat gaagactatc attgctttga
gctacattct atgtctggtt ttcgctcaaa 60aacttcccgg aaatgacaac agcacggcaa
cgctgtgcct tgggcaccat gcagtaccaa 120acggaacgat agtgaaaaca
atcacgaatg accaaattga agttactaat gctactgagc 180tggttcagag
ttcctcaaca ggtggaatat gcgacagtcc tcatcagatc cttgatggag
240aaaactgcac actaatagat gctctattgg gagaccctca gtgtgatggc
ttccaaaata 300agaaatggga cctttttgtt gaacgcagca aagcctacag
caactgttac ccttatgatg 360tgccggatta tgcctccctt aggtcactag
ttgcctcatc cggcacactg gagtttaaca 420atgaaagctt caattggact
ggagtcactc aaaatggaac aagctctgct tgtaaaagga 480gatctaataa
cagtttcttt agtagattga attggttgac ccacttaaaa ttcaaatacc
540cagcattgaa cgtgactatg ccaaacaatg aaaaatttga caaattgtac
atttgggggg 600ttcaccaccc gggtacggac aatgaccaaa ttagcctata
tgctcaagct tcaggaagaa 660tcacagtctc taccaaaaga agccaacaaa
ctgtaatccc gaatatcgga tctagaccca 720gggtaaggga tatccccagc
agaataagca tctattggac aatagtaaaa ccgggagaca 780tacttttgat
taacagcaca gggaatctaa ttgctcctcg gggttacttc aaaatacgaa
840gtgggaaaag ctcaataatg agatcagatg cacccattgg caaatgcaat
tctgaatgca 900tcactccaaa tggaagcatt cccaatgaca aaccatttca
aaatgtaaac aggatcacat 960atggggcctg tcccagatat gttaagcaaa
acactctgaa attggcaaca gggatgcgaa 1020atgtaccaga gaaacaaact
agaggcatat ttggcgcaat cgcgggtttc atagaaaatg 1080gttgggaggg
aatggtggat ggttggtacg gtttcaggca tcaaaattct gagggaatag
1140gacaagcagc agatctcaaa agcactcaag cagcaatcaa ccaaatcaat
gggaagctga 1200ataggttgat cgggaaaacc aacgagaaat tccatcagat
tgaaaaagaa ttctcagaag 1260tagaagggag aattcaggac ctcgagaaat
atgttgagga cactaaaata gatctctggt 1320catacaacgc ggagcttctt
gttgccctgg agaaccaaca tacaattgat ctaactgact 1380cagaaatgaa
caaactgttt gaaagaacaa agaagcaact gagggaaaat gctgaggata
1440tgggcaatgg ttgtttcaaa atataccaca aatgtgacaa tgcctgcata
gggtcaatca 1500gaaatggaac ttatgaccat gatgtataca gagatgaagc
attaaacaac cggttccaga 1560tcaaaggtgt tgagctgaag tcaggataca
aagattggat cctatggatt tcctttgcca 1620tatcatgttt tttgctttgt
gttgttttgt tggggttcat catgtgggcc tgccaaaaag 1680gcaacattag
gtgcaacatt tgcatttgag tgcattaa 1718361038DNAInfluenza virus
36gatcgaatct gcactgggat aacatcttca aactcacctc atgtggtcaa aacagctact
60caaggggagg tcaatgtgac tggtgtgata ccactgacaa caacaccaac aaaatcttat
120tttgcaaatc tcaaaggaac aaggaccaga gggaaactat gcccagactg
tctcaactgt 180acagatctgg atgtggcctt gggcaggcca atgtgtgtgg
ggaccacacc ttctgcgaaa 240gcttcaatac tccacgacct gttacatccg
ggtgctttcc tataatgcac gacagaacaa 300aaatcgaagt caaggcaact
agccaatctt ctcagaggat atgaaaatat caggttatca 360acccaaaacg
ttatcgatgc agaaaaggca ccaggaggac cctacagact tggaacctca
420ggatcttgcc ctaacgctac cagtaaaagc ggatttttcg caacaatggc
ttgggctgtc 480ccaaaggaca acaacaaaaa tgcaacgaac ccactaacag
tagaagtacc atacatttgt 540acagaagggg aagaccaaat tactgtttgg
gggttccatt cagataacaa aacccaaatg 600aagaacctct atggagactc
aaatcctcaa aagttcacct catctgctaa tggagtaacc 660acacattatg
tttctcagat tggcggcttc ccagatcaaa cagaagacgg aggactacca
720caaagcggca gaattgtcgt tgattacatg atgcaaaaac ctgggaaaac
aggaacaatt 780gtctatcaaa gaggtgtttt gttgcctcaa aaggtgtggt
gcgcgagtgg caggagcaaa 840gtaataaaag ggtccttgcc tttaattggt
gaagcagatt gccttcatga aaaatacggt 900ggattaaaca aaagcaagcc
ttactacaca ggagaacatg caaaagccat aggaaattgc 960ccaatatggg
tgaaaacacc tttgaagctt gccaatggaa ccaaatatag acctcctgca
1020aaactattaa aggaaagg 1038371409DNAInfluenza virus 37atgaatccaa
atcaaaaaat aataaccatt ggatcaatca gtatagcaat cggaataatt 60agtctaatgt
tgcaaatagg aaatattatt tcaatatggg ctagtcactc aatccaaact
120ggaagtcaaa accacactgg agtatgcaac caaagaatca tcacatatga
aaacagcacc 180tgggtgaatc acacatatgt taatattaac aacactaatg
ttgttgctgg aaaggacaaa 240acttcagtga cattggccgg caattcatct
ctttgttcta tcagtggatg ggctatatac 300acaaaagaca acagcataag
aattggctcc aaaggagatg tttttgtcat aagagaacct 360ttcatatcat
gttctcactt ggaatgcaga accttttttc tgacccaagg tgctctatta
420aatgacaaac attcaaatgg gaccgttaag gacagaagtc cttatagggc
cttaatgagc 480tgtcctctag gtgaagctcc gtccccatac aattcaaagt
ttgaatcagt tgcatggtca 540gcaagcgcat gccatgatgg catgggctgg
ttaacaatcg gaatttctgg tccagacaat
600ggagctgtgg ctgtactaaa atacaacggc ataataactg aaaccataaa
aagttggaaa 660aagcgaatat taagaacaca agagtctgaa tgtgtctgtg
tgaacgggtc atgtttcacc 720ataatgaccg atggcccgag taatggggcc
gcctcgtaca aaatcttcaa gatcgaaaag 780gggaaggtta ctaaatcaat
agagttgaat gcacccaatt ttcattatga ggaatgttcc 840tgttacccag
acactggcac agtgatgtgt gtatgcaggg acaactggca tggttcaaat
900cgaccttggg tgtcttttaa tcaaaacctg gattatcaaa taggatacat
ctgcagtggg 960gtgttcggtg acaatccgcg tcccaaagat ggagagggca
gctgtaatcc agtgactgtt 1020gatggagcag acggagtaaa ggggttttca
tacaaatatg gtaatggtgt ttggatagga 1080aggactaaaa gtaacagact
tagaaagggg tttgagatga tttgggatcc taatggatgg 1140acagataccg
acagtgattt ctcagtgaaa caggatgttg tggcaataac tgattggtca
1200gggtacagcg gaagtttcgt tcaacatcct gagttaacag gattggactg
tataagacct 1260tgcttctggg ttgagttagt cagaggactg cctagagaaa
atacaacaat ctggactagt 1320gggagcagca tttctttttg tggcgtaaat
agtgatactg caaactggtc ttggccagac 1380ggtgctgagt tgccattcac
cattgacaa 1409381392DNAInfluenza virus 38ttattggtct cagggagcaa
aagcaggagt tcaaaatgaa tccaaataag aagataataa 60ccatcggatc aatctgtatg
gtaactggaa tggttagctt aatgttacaa attgggaact 120tgatctcaat
atgggtcagt cattcaattc acacagggaa tcaacacaaa gctgaaccaa
180tcagcaatac taattttctt actgagaaag ctgtggcttc agtaaaatta
gcgggcaatt 240catctctttg ccccattaat ggatgggctg tatacagtaa
ggacaacagt ataaggatcg 300gttccaaggg ggatgtgttt gttataagag
agccattcat ctcatgctcc cacttggaat 360gcagaacttt ctttttgact
cagggagcct tgctgaatga caagcactcc aatgggactg 420tcaaagacag
aagccctcac agaacattaa tgagttgtcc tgtgggtgag gctccctccc
480catataactc aaggtttgag tctgttgctt ggtcagcaag tgcttgccat
gatggcacca 540gttggttgac aattggaatt tctggcccag acaatggggc
tgtggctgta ttgaaataca 600atggcataat aacagacact atcaagagtt
ggaggaataa catactgaga actcaagagt 660ctgaatgtgc atgtgtaaat
ggctcttgct ttactgtaat gactgacgga ccaagtaatg 720gtcaggcatc
acataagatc ttcaaaatgg aaaaagggaa agtggttaaa tcagtcgaat
780tggatgctcc caattatcac tatgaggaat gctcctgtta tcctgatgcc
ggcgaaatca 840catgtgtgtg cagggataat tggcatggct caaatcggcc
atgggtatct ttcaatcaaa 900atttggagta tcaaatagga tatatatgca
gtggagtttt tggagacaat ccacgcccca 960atgatggaac aggtagttgt
ggtccggtgt cctctaacgg ggcatatggg gtaaaagggt 1020tttcatttaa
atacggcaat ggtgtctgga tcgggagaac aaaaagcact aattccagga
1080gcggctttga aatgatttgg gatccaaatg ggtggactga aacggacagt
agcttttcag 1140tgaaacaaga tatcgtagca ataactgatt ggtcaggata
tagcgggagt tttgtccagc 1200atccagaact gacaggacta gattgcataa
gaccttgttt ctgggttgag ttgatcagag 1260ggcggcccaa agagagcaca
atttggacta gtgggagcag catatctttt tgtggtgtaa 1320atagtgacac
tgtgggttgg tcttggccag acggtgctga attgccattc accattgaca
1380agtagttgtt ca 1392391410DNAInfluenza virus 39atgaatccaa
atcaaaagat aataacgatt ggctctgttt ctctcaccat ttccacaata 60tgcttcttca
tgcaaattgc catcttgata actactgtaa cattgcattt caagcaatat
120gaattcaact cccccccaaa caaccaagtg atgctgtgtg aaccaacaat
aatagaaaga 180aacataacag agatagtgta tctgaccaac accaccatag
agaaggaaat atgccccaaa 240ctagcagaat acagaaattg gtcaaagccg
caatgtaaca ttacaggatt tgcacctttt 300tctaaggaca attcgattag
gctttccgct ggtggggaca tctgggtgac aagagaacct 360tatgtgtcat
gcgatcctga caagtgttat caatttgccc ttgggcaggg aacaacacta
420aacaacgtgc attcaaatga cacagtacat gataggaccc cttatcggac
cctattgatg 480aatgagttag gtgttccatt tcatctgggg accaagcaag
tgtgcatagc atggtccagc 540tcaagttgtc acgatggaaa agcatggctg
catgtttgtg taacggggga tgataaaaat 600gcaactgcta gcttcattta
caatgggagg cttgtagata gtattgtttc atggtccaaa 660gaaatcctca
ggacccagga gtcagaatgc gtttgtatca atggaacttg tacagtagta
720atgactgatg ggagtgcttc aggaaaagct gatactaaaa tactattcat
tgaggagggg 780aaaatcgttc atactagcac attgtcagga agtgctcagc
atgtcgagga gtgctcctgc 840tatcctcgat atcttggtgt cagatgtgtc
tgcagagaca actggaaagg ctccaatagg 900cccatagtag atataaacat
aaaggattat agcattgttt ccagttatgt gtgctcagga 960cttgttggag
acacacccag aaaaaacgac agctccagca gtagccattg cttggatcct
1020aacaatgaag aaggtggtca tggagtgaaa ggctgggcct ttgatgatgg
aaatgacgtg 1080tggatgggaa gaacgatcag cgagaagtta cgctcaggat
atgaaacctt caaagtcatt 1140gaaggctggt ccaaccctaa ttccaaattg
cagataaata ggcaagtcat agttgacaga 1200ggtaataggt ccggttattc
tggtattttc tctgttgaag gcaaaagctg catcaatcgg 1260tgcttttatg
tggagttgat aaggggaaga aaagaggaaa ctgaagtctt gtggacctca
1320aacagtattg ttgtgttttg tggcacctca ggtacatatg gaacaggctc
atggcctgat 1380ggggcggaca tcaatctcat gcctatataa
1410401463DNAInfluenza virus 40ccaaaatgaa caatgctacc ttcaactata
caaacgttaa ccctatttct cacatcaggg 60ggagtattat tatcactata tgtgtcagct
tcattgtcat acttactata ttcggatata 120ttgctaaaat tcccatcaac
agaaattact gcaccaacaa tgccattgga ttgtgcaaac 180gcatcaaatg
ttcaggctgt gaaccgttct gcaacaaaag gggtgacact tcttctccca
240gaaccggagt ggacataccc gcgtttatct tgcccgggct caacctttca
gaaagcactc 300ctaattagcc ctcatagatt cggagaaacc aaaggaaact
cagctccctt gataataagg 360gaacctttta ttgcttgtgg accaaaggaa
tgcaaacact ttgctctaac ccactatgca 420gcccaaccag ggggatacta
caatggaaca agaggagaca gaaacaagct gaggcatcta 480atttcagtca
aattgggcaa aatcccaaca gtagaaaact ccattttcca catggcagca
540tggagcgggt ccgcatgcca tgatggtaag gaatggacat atatcggagt
tgatggccct 600gacaataatg cattgctcaa aataaaatat ggagaagcat
atactgacac ataccattcc 660tatgcaaaca acatcctaag aacacaagaa
agtgcctgca attgcatcgg gggaaattgt 720tatcttatga taactgatgg
ctcagcttca ggtgttagtg aatgcagatt tcttaagatt 780cgagagggcc
gaataataaa agaaatattt ccaacaggaa gaataaaaca tactgaagaa
840tgcacatgcg gatttgctag caataaaacc atagaatgtg cctgtagaga
taacagttac 900acagcaaaaa gaccctttgt caaattaaac gtggagactg
atacagcaga aataagattg 960atgtgcacag agacttattt ggacaccccc
agaccagatg atggaagcat aacagggcct 1020tgtgaatcta atggggacaa
agggagtgga ggcatcaagg gaggatttgt ccatcaaaga 1080atggcatcca
agattggaag gtggtactct cgaacgatgt ctaaaactaa aaggatgggg
1140atggggctgt atgtcaagta tgatggagac ccatgggctg acagtgatgc
ccttgctttt 1200agtggagtaa tggtttcaat ggaagaacct ggttggtact
cctttggctt cgaaataaaa 1260gacaagaaat gtgatgtccc ctgtattggg
atagagatgg tacatgatgg tggaaaagag 1320acttggcact cagcagctac
agccatttac tgtttaatgg gctcaggaca gctgctgtgg 1380gacactgtca
caggtgttaa tatggctctg taatggagga atggttgagt ctgttctaaa
1440ccctttgttc ctattttgtt tga 146341101DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 41gtggagtaaa attggaatca ataggaattt accaaatact
gtcaatttat tctacagtgg 60cgagttccct agcactggca atcatggtag ctggtctatc
c 1014288DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 42gtggagtaaa attggaatca ataggaattt
accaaatact gtcaatttat tctacagtgc 60actggcaatc atggtagctg gtctatcc
884381DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 43ccatgactcc aatgtgaaga atctgtatga
gaaagtaaaa agccaattaa agaataatgc 60caaagaaata ggaaacgggt g
814461DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 44ccatgactcc aatgtgaaga atctgtataa
agaataatgc caaagaaata ggaaacgggt 60g 6145202DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 45gcaataactg attggtcagg atatagcggg agttttgtcc
agcatccaga actgacagga 60ctagattgca taagaccttg tttctgggtt gagttgatca
gagggcggcc caaagagagc 120acaatttgga ctagtgggag cagcatatct
ttttgtggtg taaatagtga cactgtgggt 180tggtcttggc cagacggtgc tg
20246187DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 46gcaataactg attggtcagg atatagcggg
agttttgtcc agcatccaga actgacagga 60ctagattgca taagaccttg tttctgggtt
gagttgatca gagggcggcc caaagagagc 120acaatttgga catctttttg
tggtgtaaat agtgacactg tgggttggtc ttggccagac 180ggtgctg
1874798DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 47ccatcagatt gaaaaagaat tctcagaagt
agaagggaga attcaggacc tcgagaaata 60tgttgaggac actaaaatag atctctggtc
atacaacg 984883DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 48ccatcagatt gaaaaagaat
tctcagaagt agaagggaga atttatgttg aggacactaa 60aatagatctc tggtcataca
acg 8349122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49cggtggatta aacaaaagca agccttacta
cacaggggaa catgcaaagg ccataggaaa 60ttgcccaata tgggtgaaaa cacccttgaa
gctggccaat ggaaccaaat atagacctcc 120tg 12250105DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 50cggtggatta aacaaaagca agccttacta cggccatagg
aaattgccca atatgggtga 60aaacaccctt gaagctggcc aatggaacca aatatagacc
tcctg 10551119DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 51catgggctga cagtgatgcc
cttgctttta gtggagtaat ggtttcaatg gaagaacctg 60gttggtactc ctttggcttc
gaaataaaag acaagaaatg tgatgtcccc tgtattggg 11952102DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 52catgggctga cagtgatgcc cttgctttta gtggaagaac
ctggttggta ctcctttggc 60ttcgaaataa aagacaagaa atgtgatgtc ccctgtattg
gg 10253152DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 53ctaaccgagg tcgaaacgta cgttctctct
atcatcccgt caggccccct caaagccgag 60atcgcgcaga aacttgaaga tgtctttgca
ggaaagaaca ccgatctcga ggctctcatg 120gagtggctaa agacaagacc
aatcctgtca cc 15254141DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 54ctaaccgagg
tcgaaacgta ccatcccgtc aggccccctc aaagccgaga tcgcgcagaa 60acttgaagat
gtctttgcag gaaagaacac cgatctcgag gctctcatgg agtggctaaa
120gacaagacca atcctgtcac c 14155107DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 55ggtccaaccc taattccaaa ttgcagataa ataggcaagt
catagttgac agaggtaata 60ggtccggtta ttctggtatt ttctctgttg aaggcaaaag
ctgcatc 1075687DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 56ggtccaaccc taattccaaa
ttgcagataa ataggcaagt catagtttta ttctggtatt 60ttctctgttg aaggcaaaag
ctgcatc 875798DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 57ccatcagatt gaaaaagaat
tctcagaagt agaagggaga attcaggaac tctagaaata 60tgttgaggac actaaaatag
atctctggtc atacaacg 985888DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 58cgactgggct
cagaaatagc cctcaaagag agagaagaag aaaaaagaga ggattatttg 60gagctatagc
aggttttata gagggagg 885965DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 59cgactgggct
cagaaatagc cctcaaagag ttatttggag ctatagcagg ttttatagag 60ggagg
65
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