U.S. patent application number 12/573855 was filed with the patent office on 2010-05-06 for kit for detecting non-pathogenic or pathogenic influenza a subtype h5 virus.
This patent application is currently assigned to Hai Kang Life Corporation Limited. Invention is credited to Lung-Sang Ko, Lok-Ting Lau, Ka-Lun So, Albert Cheung-Hoi YU.
Application Number | 20100112548 12/573855 |
Document ID | / |
Family ID | 4578275 |
Filed Date | 2010-05-06 |
United States Patent
Application |
20100112548 |
Kind Code |
A1 |
YU; Albert Cheung-Hoi ; et
al. |
May 6, 2010 |
KIT FOR DETECTING NON-PATHOGENIC OR PATHOGENIC INFLUENZA A SUBTYPE
H5 VIRUS
Abstract
Current methods for detecting influenza A subtype H5 virus, for
example cell culture, haemagglutination-inhibition, fluorescent
antibody and enzyme immunoassay, and reverse transcriptase
polymerase chain reaction (RT-PCR) may have the disadvantages of
low sensitivity and low specificity. Furthermore, such methods are
relatively difficult to use, and may not be suitable for routine
detection on a daily basis. The kit for detecting H5 virus of this
invention may provide a user-friendly alternative that is
relatively more sensitive and specific to H5 virus. The detection
kit utilizes two specially designed primers A and B for the
replication of H5 virus, and a specific capture probe for
immobilizing the amplified viral RNA. An additional primer C is
also designed for the detection of pathogenic H5 virus. The
detection of H5 virus by the detection kit may be accomplished
within one day if desired.
Inventors: |
YU; Albert Cheung-Hoi; (Pok
Fu Lam, HK) ; So; Ka-Lun; (Clear Water Bay, HK)
; Ko; Lung-Sang; (Kowloon, HK) ; Lau;
Lok-Ting; (Tung-Chung, HK) |
Correspondence
Address: |
HESLIN ROTHENBERG FARLEY & MESITI PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
US
|
Assignee: |
Hai Kang Life Corporation
Limited
Kowloon
HK
|
Family ID: |
4578275 |
Appl. No.: |
12/573855 |
Filed: |
October 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10398207 |
Apr 7, 2003 |
7611837 |
|
|
PCT/CN01/01458 |
Sep 27, 2001 |
|
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12573855 |
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Current U.S.
Class: |
435/5 ;
536/23.1 |
Current CPC
Class: |
C12Q 1/701 20130101;
Y02P 20/582 20151101 |
Class at
Publication: |
435/5 ;
536/23.1 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C07H 21/04 20060101 C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 5, 2000 |
CN |
00106310.0 |
Claims
1-40. (canceled)
41. A purified and isolated DNA molecule or the complementary DNA
molecule comprising essentially of any one of the DNA sequences set
forth in SEQ ID NOS. 1 to 14.
42-55. (canceled)
56. A purified and isolated DNA molecule or the complementary DNA
molecule including: (i) a first DNA sequence for binding to at
least a portion of RNA sequence of haemagglutinin gene-containing
influenza A subtype H5 virus; and (ii) a second DNA sequence
encoding a promoter DNA sequence of a RNA polymerase; such that the
purified and isolated DNA molecule extends in the presence of an
enzyme and DNA nucleotides to generate a DNA sequence including:
(a) a DNA sequence complementary to at least a portion of the RNA
sequence of H5 virus; and (b) a DNA sequence encoding the promoter
DNA sequence of a RNA polymerase when the first purified and
isolated DNA molecule binds to at least a portion of the RNA
sequence of H5 virus; wherein the purified and isolated DNA
molecule or the complementary DNA molecule consists of at least one
of the DNA sequences set forth in SEQ ID NOs. 1 to 4 and 6 to
14.
57. The molecule as claimed in claim 56, wherein the RNA polymerase
is bacteriophage T7 RNA polymerase, and the second DNA sequence
encodes the DNA sequence sets forth in SEQ ID NO.4.
58. A purified and isolated DNA molecule or the complementary DNA
molecule including: (i) a first DNA sequence encoding at least a
portion of RNA sequence of haemagglutinin gene-containing influenza
A subtype H5 virus; and (ii) a second DNA sequence encoding a DNA
sequence for binding to a detection molecule; such that the
purified and isolated DNA molecule extends in the presence of an
enzyme and DNA nucleotides to generate a DNA sequence including:
(a) a third DNA sequence encoding at least a portion of the RNA
sequence of the H5 virus; and (b) a fourth DNA sequence encoding
the DNA sequence for binding a detection molecule when the purified
and isolated DNA molecule binds to a DNA molecule including a DNA
sequence complementary to at least a portion of the H5 virus;
wherein the first DNA sequence encodes at least one of the DNA
sequences set forth in SEQ ID NOs. 5, 6, 7, 9, 10 and 11 which are
bindable at regions between 914 to 940, 866 to 961, 846 to 981,
1017 to 1042, 970 to 1063 and 950 to 1083, of the haemagglutinin
gene, respectively.
59. The molecule as claimed in claim 58, wherein the second DNA
sequence encodes the DNA sequence set forth in SEQ ID NO. 8.
60. A purified and isolated DNA molecule according to claim 41, or
the complementary DNA molecule thereof, wherein said DNA molecule
encodes at least one of the DNA sequences set forth in SEQ ID NOs.
5, 6, 7, 9, 10 and 11 which are bindable at regions between 914 to
940, 866 to 961, 846 to 981, 1017 to 1042, 970 to 1063 and 950 to
1083, of the haemagglutinin gene, respectively.
61. A purified and isolated DNA molecule or the complementary DNA
molecule comprising: (i) a DNA sequence encoding at least a portion
of RNA sequence of haemagglutinin gene-containing influenza A
subtype H5 virus for binding to a target molecule, wherein the
target molecule includes a nucleic acid sequence complementary to
at least a portion of the H5 virus; and (ii) an immobilizer; such
that the target molecule is immobilized when bound to the second
purified and isolated DNA molecule; wherein the first DNA sequence
encodes at least one of the DNA sequences set forth in SEQ ID NOs.
12, 13 and 14.
62. A purified and isolated DNA molecule or the complementary DNA
molecule thereof comprising: (i) a first DNA sequence encoding at
least a portion of RNA sequence of haemagglutinin gene-containing
influenza A subtype H5 virus for binding to a target molecule,
wherein the target molecule includes a nucleic acid sequence
complementary to at least a portion of the RNA sequence of
influenza A subtype H5 virus; and (ii) a signal generator; such
that a signal is generated from the target molecule when the target
molecule is bound to the purified and isolated DNA molecule;
wherein said signal generator is selected from the group consisting
of ruthenium-bipyridine complex [Ru(bpy).sub.3].sup.2+, radioactive
.sup.32P, chemiluminescent fluorescent, luciferin, luciferase,
enzymatic alkaline phosphatase, enzymatic horseradish peroxidase
and eletrodemiluminescence molecules.
63. A purified and isolated DNA molecule according to claim 41, or
the complementary DNA molecule thereof, wherein said DNA molecule
is capable of binding at regions between 1107 to 1132, 1060 to 1140
and 1040 to 1160, 914 to 940, 866 to 961, 846 to 981, 1017 to 1042,
970 to 1063 and 950 to 1083 of haemagglutinin gene of influenza A
subtype H5 virus, chosen from DNA sequences of SEQ ID NOs. 1 to 3,
5 to 7 and 9 to 11, respectively.
64. (canceled)
65. A purified and isolated DNA molecule that binds to a region of
the haemagglutinin gene of influenza A subtype H5 virus, or a
purified and isolated complement of said DNA molecule, wherein said
isolated DNA molecule consists of a nucleotide sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10,
and SEQ ID NO:11, and wherein said region of the haemagglutinin
gene is selected from the group consisting of nucleotides
1107-1132, nucleotides 1060-1140, nucleotides 1040-1160,
nucleotides 914-940, nucleotides 866-961, nucleotides 846-981,
nucleotides 1017-1042, nucleotides 970-1063, and nucleotides
950-1083 of the haemagglutinin gene.
66. (canceled)
67. A kit for detecting influenza A subtype H5 virus in a
biological sample, said kit comprising: (i) an isolating agent for
isolating RNA molecules of the H5 virus from the biological sample
and producing isolated RNA molecules therefrom; (ii) a nucleic acid
amplifying agent for amplifying the isolated RNA molecules of the
H5 virus and producing amplified target molecules for detection,
wherein the nucleic acid amplifying agent includes: (a) a first
purified and isolated DNA molecule consisting of: (1) a first DNA
sequence consisting of any one of the DNA sequences of SEQ ID NO.
1, SEQ ID NO. 2 and SEQ ID NO. 3; and (2) a second DNA sequence
encoding a promoter DNA sequence of an RNA polymerase; and (b) a
second purified and isolated DNA molecule consisting of: (1) a
third DNA sequence encoding at least a portion of the RNA sequence
of the H5 virus; and (2) a fourth DNA sequence encoding a fifth DNA
sequence for binding to a detection molecule; and (iii) a nucleic
acid detecting agent for detecting the target molecule; wherein the
nucleic acid detecting agent includes the detection molecule.
68. The kit for detecting H5 virus as claimed in claim 67, wherein
the kit further comprises a lysis agent for stabilizing nucleic
acids in the biological sample.
69. The kit for detecting H5 virus as claimed in claim 67, further
comprising an adsorbent for adsorbing nucleic acids.
70. The kit for detecting H5 virus as claimed in claim 67, wherein
the target molecule is a RNA molecule.
71. The kit for detecting H5 virus as claimed in claim 67, wherein
the RNA polymerase is bacteriophage T7 RNA polymerase.
72. The kit for detecting H5 virus as claimed in claim 67, wherein
the DNA sequence encoding the promoter DNA sequence of the RNA
polymerase is set forth in SEQ ID NO. 4.
73. The kit for detecting H5 virus as claimed in claim 67, further
comprising a reverse transcriptase.
74. The kit for detecting H5 virus as claimed in claim 67, further
comprising a RNaseH.
75. The kit for detecting H5 virus as claimed in claim 67, wherein
the second purified and isolated DNA molecule or the fifth DNA
sequence includes the DNA sequence set forth in SEQ ID NO. 8.
76. The kit for detecting H5 virus as claimed in claim 67, further
comprising a detection probe for generating a signal.
77. The kit for detecting H5 virus as claimed in claim 67, further
comprising an immobilizer whereby the target molecule can be
immobilized when bound to the second purified and isolated DNA
molecule.
Description
FIELD OF THE INVENTION
[0001] This invention relates to apparatus for detecting influenza
A subtype H5 virus.
BACKGROUND OF THE INVENTION
[0002] Avian influenza (Influenza A) viruses infect a variety of
animals, including humans, pigs, horses, sea mammals, and birds.
Recent phylogenetic studies of Influenza A viruses have revealed
species-specific lineages of viral genes and have demonstrated that
the prevalence of interspecies transmission depends on the animal
species. They have also revealed that aquatic birds are the source
of all influenza viruses in other species.
[0003] The emergence of a "new" Influenza A virus in humans is
possible. Serological and virological evidence suggests that since
1889 there have been six instances of the introduction of an
influenza virus with an HA subtype that had been absent from human
population for some time. Three human subtypes of HA have appeared
cyclically--subtype H2 in 1889, H3 in 1900, H1 in 1918, H2 again in
1957, H3 again in 1968, and H1 again in 1977. The first human
infection with avian influenza A subtype H5N1 was reported in 1997,
which resulted in the death of a 3-year-old boy. This first report
leads to the need for the routine screening for H5 virus in
animals, particularly chicken, in stopping the spread of the
viruses.
[0004] Many methods for viral identification are currently being
used, including cell culture, haemagglutination-inhibition,
fluorescent antibody and enzyme immunoassay, and reverse
transcriptase polymerase chain reaction (RT-PCR). However, these
methods all share the same problems--they have relatively low
sensitivity and low specificity. Furthermore, the detection time
may be too long for routine detection purposes, and such methods
are relatively difficult to be utilized.
[0005] The current methodologies applied for detecting influenza A
subtype H5 virus includes immunodiagnostic assay and virus culture.
Examples of immunodiagnostic essay include haemagglutinin
inhibition (HI) assay and immuno assay. However, immunodiagnostic
assay may have the disadvantage of low sensitivity. Furthermore, as
the target of immunodiagnostic assay is usually a specific protein,
the underlying genetic nature of a target may not be obtained
directly. In addition, the initial derivation of antibodies is
ultimately dependent upon the antigenicity of the protein analysis
in the immune host animal and therefore, cross-reactivity may
occur.
[0006] Although virus culture is an accurate and low cost detection
method, it is relatively labour intensive and requires a lot of
space for incubation. The culturing process may be slow and cannot
meet the demand of daily inspection. In addition, virus culture can
not provide the detection results directly and has to reply upon
further confirmation by other detection methods, which may be very
expensive.
OBJECT FOR THE INVENTION
[0007] Therefore, it is an object of this invention to design a
user-friendly diagnostic kit for detecting H5 virus such that the
sensitivity and specificity may be improved.
[0008] Another object of this invention to design a kit for
detecting influenza A subtype H5 virus such that the detection time
and the overall costs for detection may be reduced.
[0009] It is yet another object of this invention to design a kit
for detecting influenza A subtype H5 virus such that the
pathogenicity of the H5 virus may be detected directly.
[0010] As a minimum, it is an object of the present invention to
provide the public with a useful choice.
SUMMARY OF THE INVENTION
[0011] Accordingly, this invention provides a kit for detecting
non-pathogenic or pathogenic influenza A subtype H5 virus in a
biological sample including: [0012] an isolating agent for
isolating the RNA molecules of H5 virus from the biological sample;
[0013] a nucleic acid replicating agent for replicating a target
molecule, wherein the target molecule includes: [0014] a nucleic
acid sequence complementary to at least a portion of the RNA
sequence Of H5 virus; and [0015] a nucleic acid sequence for
binding to a detection molecule; [0016] a nucleic acid detecting
agent for detecting the target molecule, wherein the nucleic acid
detecting agent includes the detection molecule.
[0017] It is another aspect of this invention to provide a purified
and isolated DNA molecule or the complementary DNA molecule
including: [0018] a first DNA sequence for binding to at least a
portion of the RNA sequence of influenza A subtype H5 virus; and
[0019] a second DNA sequence encoding a promoter DNA sequence of a
RNA polymerase such that the purified and isolated DNA molecule
extends in the presence of an enzyme and DNA nucleotides to
generate a DNA sequence including: [0020] a DNA sequence
complementary to at least a portion of the RNA sequence of H5
virus; and [0021] a DNA sequence encoding the promoter DNA sequence
of a RNA polymerase
[0022] when the first purified and isolated DNA molecule binds to
at least a is portion of the RNA sequence of H5 virus.
[0023] It is yet another aspect of this invention to provide a
purified and isolated DNA molecule or the complementary DNA
molecule including: [0024] a first DNA sequence encoding at least a
portion of the RNA sequence of non-pathogenic or pathogenic
influenza A subtype H5 virus; and [0025] a second DNA sequence
encoding a DNA sequence for binding to a detection molecule such
that the purified and isolated DNA molecule extends in the presence
of an enzyme and DNA nucleotides to generate a DNA sequence
including: [0026] a DNA sequence encoding at least a portion of the
RNA sequence of non-pathogenic or pathogenic influenza A subtype H5
virus; and [0027] a DNA sequence encoding the DNA sequence for
binding a detection molecule
[0028] when the purified and isolated DNA molecule binds to a DNA
molecule including a DNA sequence complementary to at least a
portion of the RNA sequence of non-pathogenic or pathogenic H5
virus.
[0029] This invention also provides a purified and isolated DNA
molecule or the complementary DNA molecule consisting of a first
DNA sequence encoding either one of the DNA sequences set forth in
SEQ ID No.5, 6, 7, 9, 10, or 11.
[0030] It is yet another aspect of this invention to provide a
purified and isolated DNA molecule or the complementary DNA
molecule including: [0031] a first DNA sequence encoding at least a
portion of the RNA sequence of influenza A subtype H5 virus for
binding to a target molecule, wherein the target molecule includes
a nucleic acid sequence complementary to at least a portion of the
RNA sequence of influenza A subtype H5 virus; and [0032] an
immoblizer
[0033] such that the target molecule is immoblized when bound to
the purified and isolated DNA molecule.
[0034] It is another aspect of this invention to provide a purified
and isolated DNA molecule or the complementary DNA molecule
including: [0035] a first DNA sequence encoding at least a portion
of the RNA sequence of influenza A subtype H5 virus for "binding to
a target molecule, wherein the target molecule includes a nucleic
acid sequence complementary to at least a portion of the RNA
sequence of influenza A subtype H5 virus; and [0036] a signal
generator
[0037] such that a signal is generated from the target molecule
when the target molecule is bound to the purified and isolated DNA
molecule.
[0038] This invention also provides the use of a first and a second
purified and isolated DNA molecules in the manufacture of a kit for
the detection of influenza A subtype H5 virus in a biological
sample, wherein: [0039] the RNA molecule of H5 virus is isolated
from the biological sample by an isolating agent; [0040] a target
molecule is replicated by a nucleic acid replicating agent
including the first and the second purified and isolated DNA
molecules, wherein the target molecule includes: [0041] a nucleic
acid sequence complementary to at least a portion of the RNA
sequence of H5 virus; and [0042] a nucleic acid sequence for
binding to a detection molecule; [0043] the target molecule is
detected by a nucleic acid detecting agent, wherein the nucleic
acid detecting agent includes the detection molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] A preferred embodiment of this invention will now be
described with reference to the following figures:
[0045] FIG. 1 shows the flow chart of the overall procedures of the
detection of influenza A subtype H5 virus by the kit of this
invention.
[0046] FIG. 2 shows the detailed procedures for the detection of
influenza A subtype H5 virus by the detection kit of this
invention.
[0047] FIG. 3 shows the isolation of the viral RNA molecules from
the biological sample.
[0048] FIG. 4 shows the amplification of a portion of the influenza
A subtype H5 viral RNA molecule by two DNA molecules, primers A and
B.
[0049] FIG. 5 shows the immobilization of the amplified RNA
molecule while bound to a detection probe.
[0050] FIGS. 6-19 show the nucleic acid sequences SEQ ID Nos.1-14
for this invention, respectively, which are used in the DNA
molecules of the detection kit for amplification and detection
purposes.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0051] Preferred embodiments of this invention are now described
with reference to the figures. List 1 is a part list so that the
reference numerals in the figures may be easily referred to.
[0052] The concentration of influenza A subtype H5 in a biological
sample, for example chicken blood, may be very low such that
detection of the presence of H5 viral RNA may not be performed on
the biological sample directly. In order to increase the number of
the viral RNA molecules to a sufficient amount for the detection
purpose, a suitable amplification technology is required. Nucleic
acid sequence-based amplification (NASBA) is known to be a flexible
technology with particular use for the amplification of RNA. The
amplified RNA molecules may then be detected by suitable
technology. NASBA is a rapid, highly sensitive and highly specific
method for the detection of influenza virus subtype H5. Results can
be obtained in as little as one day. In addition, it can
discriminate between pathogenic and non-pathogenic H5 strains
directly.
[0053] Influenza virus contains its genetic material in the form of
a single strand of viral ribonucleic acid (RNA). Influenza A
subtype H5 viral RNA contains the genes necessary for its
reproduction and one of the essential genes is called
haemagglutinin. This gene is approximately 1756 nucleotides in
length, and the nucleotides are numbered from the 5' end of the
molecule.
[0054] FIG. 1 shows the overall procedures for the detection of H5
virus by the detection kit. As shown in FIG. 1, the target H5 viral
nucleic acid molecule, which is in the form of a single strand of
RNA molecule, is firstly extracted from a biological sample. The
compatible biological sample types may include blood, serum/plasma,
peripheral blood mononuclear cells/peripheral blood lymphocytes
(PBMC/PBL), sputum, urine, faeces, throat swabs, dermal lesion
swabs, cerebrospinal fluids, cervical smears, pus samples, food
matrices, and tissues from various parts of the body including
brain, spleen, and liver. Other samples that have not been listed
may also be applicable. The nucleic acid extraction process of the
detection kit of this invention is accomplished by an isolating
agent.
[0055] After the target H5 viral RNA molecule is extracted from the
biological sample, the amount of RNA molecules in the sample may
not be sufficient to be detected. Therefore, a portion of the H5
viral RNA molecule is replicated to a target nucleic acid molecule
by an appropriate amplification technique, for example, NASBA. The
target nucleic acid molecules may then be detected by suitable
methods.
[0056] After the overall procedures of the detection kit of the
invention described, the details of each procedure will now be
discussed.
[0057] The H5 viral RNA molecule may be isolated from the
biological sample by applying a suitable isolating agent to the
biological sample. Preferably, a lysis agent may be applied before
the isolating agent. The lysis agent, for example, a lysis buffer,
is responsible for dissolving the proteins and lipids, and
denaturing the proteins in the biological sample such that these
materials may be removed from the sample more easily. Furthermore,
the lysis agent may also serve as a buffer for stablizing the RNA
molecule for long term storage purposes. As shown in FIG. 2, the
RNA molecule may be stable in the lysis buffer for up to 48 hours
at room temperature and may be stored indefinitely at -70.degree.
C. The advantages for doing so is that it may not be necessary to
perform the analysis at the sampling site, which may not be
suitable for carrying out such processes.
[0058] An example of suitable lysis buffer may include 5M guanidine
thiocyanate and Tris/HCl. The lysis buffer forms no invention of
the detection kit and the compositions suitable for its purpose are
well known to the art. Therefore, the detailed composition of the
lysis buffer will not be discussed here. Lysis agents having
different compositions that can still achieve the purposes of
dissolving proteins and lipids, denaturing proteins, and stablizing
the RNA molecules may be utilized in the detection kit of this
invention.
[0059] After the lysis agent has been applied to the biological
sample, the next step is the isolation of the nucleic acid
molecules from the sample through the use of an isolating agent.
FIG. 3 describes the overall isolation procedure in the detection
kit. After the lysis agent is applied to the biological sample,
nucleic acids (10) together with other unwanted components are in
the form of a solution. An adsorbent, for example silica (12), may
then be added into the solution to adsorb the nucleic acids (10),
resulting in a nucleic acids/silica mixture (14). After that,
proteins and lipid and other unwanted materials in the solution may
be washed away by suitable eluents, for example, 5M guanidine
thiocyanate and Tris/HCl solution, Tris/HCL solution, 70% ethanol,
or acetone, or their combinations. After the mixture (14) is washed
with sufficient amount of eluents, the nucleic acids (10) in the
silica (12) may then be isolated by centrifugation.
[0060] After the nucleic acids (12) contained in the sample are
isolated, an amplification agent may then be applied to the mixture
of nucleic acid such that the H5 viral RNA molecule is replicated
for detection purposes, for example NASBA technique. Three purified
and isolated DNA molecules are designed for the amplification
purpose, which are termed primers A to C.
[0061] FIG. 4 shows a schematic diagram for the amplification of
the H5 viral RNA by NASBA in this invention. As shown in the
figure, the amplification process is initiated by the annealing of
primer A (22) to the target H5 viral RNA (20), which is a
single-stranded RNA molecule. The primer A (22) is designed such
that it is capable of binding to the targeted RNA molecule, and
further includes a DNA sequence encoding the promoter for a RNA
polymerase, preferably bacteriophage T7 RNA polymerase. The precise
location of binding depends upon the strain of virus examined. The
binding site may change after a certain period of time. The
important technical feature of Primer A is that it remains capable
of binding to a portion of H5 virus.
[0062] Accordingly, primer A (22) includes a binding sequence
encoding a DNA sequence complementary to at least a portion of the
H5 viral RNA (20). For the purpose of this invention, it is found
that the region suitable for binding in the H5 viral RNA (20) is a
region between nucleotides 1107 to 1132 of the haemagglutinin gene
of H5 virus, which is found to contain the least number of
nucleotides for the binding function. Therefore, the binding
sequence of Primer A preferably includes a DNA sequence that is
complementary to region between nucleotide 1107 to 1132 of the
haemagglutinin gene of H5 virus, which is set forth in SEQ ID No. 1
in FIG. 6. It should be noted that SEQ ID No. 1 is formally written
in the 5'-3' direction. As a result, the orientation of binding
with respect to the viral gene is from "back" to "front".
[0063] As an alternative, nucleotides 1060 to 1140 (SEQ ID No.2,
FIG. 7) or nucleotides 1040 to 1160 (SEQ ID No.3, FIG. 8) of the
haemagglutinin gene of H5 virus may be used for the binding purpose
in primer A (22).
[0064] For the amplification purpose, which will be described in
more detail in the specification, primer A (22) further includes a
DNA sequence encoding a promoter of a RNA polymerase, for example
bacteriophage T7 RNA polymerase. A suitable promoter DNA sequence
is set forth in SEQ ID No.4 (FIG. 9). The promoter sequence is
preferably attached to the 5' end of the binding sequence, such
that the binding sequence may extend at the 3' end when Primer A
binds to H5 viral RNA. If other RNA polymerase is utilized, the
promoter sequence will have to be changed accordingly.
[0065] After the primer A binds to the H5 viral RNA, the primer A
is extended through the action of a suitable reverse transcriptase,
for example Avian Myoblastosis Virus-Reverse Transcriptase (AMV-RT)
in the presence of suitable nucleotides at the 3' end of Primer A.
Therefore, an extended Primer A (24) including the following
sequences is resulted: [0066] (a) a DNA sequence that is
complementary to a portion of H5 viral RNA; and [0067] (b) a DNA
sequence encoding a promoter for a RNA polymerase.
[0068] The H5 RNA portion of the resulting DNA:RNA hybrid (26) is
eliminated through the action of RNase H. This allows for the
primer B (28) to anneal to the extended Primer A (24) at a position
that is upstream from the primer A (22) annealing site. Therefore,
in order for the primer B (28) to bind to the extended Primer A
(24), primer B (28) includes a first binding DNA sequence encoding
a portion of the H5 viral haemagglutinin gene sequence. Preferably,
this first DNA sequence of primer B (28) encodes nucleotides 914 to
940 of the haemagglutinin gene of H5 virus (SEQID No.5, FIG. 10).
As an alternative, nucleotides 866 to 961 (SEQID No.6, FIG. 11) or
nucleotides 846 to 981 (SEQID No.7, FIG. 12) of H5 viral
haemagglutinin gene may be utilized.
[0069] To achieve the detection purpose, primer B (28) may further
include a second DNA sequence that is complementary to the nucleic
acid sequence of a detection molecule. If the detection molecule is
designed in a way such that it includes a DNA sequence encoding a
portion of the RNA sequence of H5 virus such that it may bind to
the amplified RNA molecules, it may not be necessary for primer B
to include the second DNA sequence. In this case, primer B may
consist of merely the first DNA sequence.
[0070] As an alternative, a second DNA sequence encoding the DNA
sequence set forth in SEQ ID No.8 (FIG. 13) is included in Primer
B. The second DNA sequence is preferably attached to the 5' end of
the binding sequence of Primer B. SEQ ID No. 8 is subjected to
change if other detection nucleic acid sequences are used.
[0071] After the annealing of primer B (28) to the extended Primer
A (24), primer B (28) extends down through the T7 RNA ploymerase
promoter at the end of the extended primer A (24) through the
action of AMV-RT. As a result, a double-stranded DNA copy (30) of
the original H5 viral RNA target sequence is produced, encoding an
intact T7 RNA polymerase promoter at one end and a portion of H5
viral RNA sequence at the other end. This promoter is then
recognized by the 17 RNA polymerase, resulting in the production of
large amount of target RNA molecules (32) that include a RNA
sequence complementary to a portion of the original H5 viral RNA
sequence.
[0072] Primer B may be used to determine non-pathogenic strains of
influenza A subtype H5 virus. For the detection of the pathogenic
H5 virus, a primer C is used in place of primer B. Again primer C
is a purified and isolated DNA molecule including the following DNA
sequences: [0073] a first DNA sequence encoding at least a portion
of the RNA sequence of pathogenic H5 virus; and [0074] a second DNA
sequence encoding a DNA sequence complementary to a detection DNA
sequence. As in the case of Primer B, this second DNA sequence may
be a purely optional component.
[0075] The function and working of Primer C is the same as Primer
B, except that the target is pathogenic H5 virus.
[0076] Preferably, the first DNA sequence of primer C encodes
nucleotide 1017 to 1042 of the haemagglutinin gene of H5 virus (SEQ
ID No.9, FIG. 14). As an alternative, nucleotide 970 to 1063 (SEQ
ID No.10, FIG. 15) or nucleotide 950 to 1083 (SEQ ID No.11, FIG.
16) of H5 viral haemagglutinin gene may be utilized.
[0077] It is found that Primer C does not replicate the target H5
viral RNA efficiently from freshly isolated nucleic acids from the
samples with Primer A (22). Therefore, it is preferred that Primer
C is applied to amplified RNA from samples testing positive for H5
virus using Primers A (22) and B (28).
[0078] The product of the amplification process is a large quantity
of target RNA molecules (32) each containing the following RNA
sequences: [0079] (a) a RNA sequence complementary to a portion of
the original H5 viral RNA (pathogenic or non-pathogenic); and
[0080] (b) a RNA sequence complementary to the nucleic acid
sequence of a detection molecule, if Primer B or C includes the
corresponding second DNA sequence.
[0081] The target RNA molecule (32) of this particular embodiment
further includes a RNA sequence encoding the promoter for T7 RNA
polymerase, which is automatically included during the
amplification process by the T7 RNA polymerase. However, this
segment of RNA sequence may have no function in the detection
step.
[0082] The detection of the target RNA molecule (32) is illustrated
in FIG. 5. The target RNA molecule (32) may be detected by binding
to the detection molecule that is capable of generating a signal,
for example the detection probe (40). The signal may be generated
from a signal generator (41) that is attached to the detection
probe (40). In this particular preferred embodiment as shown in
FIG. 5, the signal generator (41) is a ruthenium-bipyridine complex
[Ru(bpy).sub.3].sup.2+. As an alternative, the signal generator
(41) may be radioactive (e.g. .sup.32P), chemiluminescent (e.g.
luciferin/luciferase), fluorescent (e.g. fluorescein), enzymatic
(e.g. alkaline phosphatase, horseradish peroxidase), or other
electrochemiluminescence molecules.
[0083] If primers B or C includes the corresponding second DNA
sequence that is complementary to a detection DNA sequence, the
target RNA molecule (32) includes a RNA sequence complementary to
the nucleic acid sequence of the detection molecule. The advantage
of utilizing such a design is that commercially available detection
molecules may be used.
[0084] If primers B or C consists of merely the first DNA sequence
that encodes a portion of the H5 viral haemagglutinin gene
sequence, a new detection molecule is required. In this case, the
detection molecule may include: [0085] a nucleic acid sequence
encoding a portion of H5 viral RNA sequence that is complementary
to that encoded in the target RNA molecule (32); and [0086] a
signal generator.
[0087] The target RNA molecule (32) is contained in a mixture
together with other undesired components including the unamplified
nucleic acids contained in the original sample, the primers A, B,
and C, the unreacted nucleotides, and most importantly, the unbound
detection molecules. Therefore, the target RNA molecule (32) may be
immoblized by a capture molecule, for example the capture probe
(42), such that the undesired components may be washed away. The
capture probe (42) is capable of binding to the target RNA molecule
(32). This may be achieved by including a nucleic acid sequence
encoding a portion of H5 viral RNA sequence that is complementary
to that encoded in the target RNA molecule (32). The capture probe
(42) is further attached to an immobilizer (44), which may
immobilize the target RNA molecule (32) so that other undesired
components may be washed away. The immobilizer (44) as shown in
FIG. 5 is a magnetic particle that may be attracted to a working
electrode. Other immobilizers may also be utilized, for example, a
piece of polymer with a number of capture probes (42) attached
on.
[0088] The sequence of the detection probe may be complementary to
any region of the amplified RNA product whose ends are defined by
primers A and B or by primers A and C. However, the detection probe
sequence cannot overlap that of the capture probe, as this would
affect the interaction of the amplified RNA with the capture probe
and vice versa.
[0089] As shown in FIG. 5, the target RNA molecule may be
immobilized together with the detection probe (40). Therefore,
there may be no restriction on the timing of the addition of the
capture probe (42) into the mixture. The capture probe (42) may be
added after or before the addition of the detection probe (40), as
long as the capture probe is added before the washing step.
[0090] As the nucleic acid sequences of Primers A, B, and C, and
the capture probe are now known, the synthesis of the corresponding
complementary DNA molecules will be apparent to one skilled in the
art. Such complementary DNA molecules may be used as templates in
the synthesis of Primers A, B, and C, and the capture probe.
[0091] The present invention is now illustrated by the following
non-limiting examples. It should be noted that various changes and
modifications can be applied to the following example and processes
without departing from the scope of this invention. Therefore, it
should be noted that the following example should be interpreted as
illustrative only and not limiting in any sense.
Example
[0092] The detailed components of the detection kit of this example
are listed as follows:
A. Lysis Buffer
[0093] 50.times.0.9 ml Lysis buffer (5M guanidine thiocyanate,
Triton X-100, Tris/HCl)
B. Nucleic Acid Isolation Components
[0093] [0094] 5.times.22 ml Wash Buffer (5M guanidine thiocyanate,
Tris/HCl) [0095] 5.times.0.8 ml Silica (Hydrochloric acid-activated
silicon dioxide particles) [0096] 5.times.1.5 ml Elution buffer
(Tris/HCl)
C. Nucleic Acid Amplification Components
[0096] [0097] 5.times.60 .mu.l Enzyme solution (Avian Myoblastosis
Virus-Reverse Transcriptase (AMV-RT), RNase-H, T7 RNA polymerase
stabilized with bovine serum albumin) [0098] 5.times.10 mg Reagent
spheres (lyophilised spheres with nucleotides, dithiothreitol and
MgCl.sub.2). Contained in a foil pack with silica gel desiccant
[0099] 1.times.0.6 ml Reagent sphere diluent (Tris-HCl, 45% DMSO)
[0100] 1.times.1.6 ml KCl solution [0101] 1.times.70 .mu.l
H5-primer mixture
D. Nucleic Acid Detection Components
[0101] [0102] 1.times.0.9 ml Generic ECL detection probe
(Ruthenium-labelled DNA oligonucleotide Preservative: 5 g/L
2-chloroacetamide) [0103] 1.times.0.7 ml H5-capture probe
(Biotinylated-oligonucleotide Preservative: 5 g/L
2-chloroacetamide) [0104] 2.times.1.7 ml Instrument Reference
Solution (Ruthenium-labelled paramagnetic beads) The materials as
listed above are intended to be used for 50 test reactions.
[0105] Readily available materials not included in the detection
kit that will be used in the test reactions are listed as
follows:
TABLE-US-00001 Material Recommended Source 70% (v/v) Ethanol
(prepared from 96- Merck 1.00983 100% (v/v) ethanol, ACS quality);
use nuclease-free water for dilution Acetone, analytical grade
SIGMA A4206 Diethylpyrocarbonate for the preparation SIGMA D5758 of
RNase-free water
Preparation of Reagents
A. Lysis Buffer
[0106] Pre-warm Lysis buffer for 30 min at 37.degree. C. before
starting the release procedure. [0107] Mix the Lysis buffer vial
every 10 min during the incubation to ensure that any crystals have
fully dissolved. [0108] Allow Lysis buffer to cool to room
temperature. [0109] Protect Lysis buffer from excessive heat or
light.
B. Nucleic Acid Isolation Reagents
[0109] [0110] Bring all reagents to room temperature before use.
[0111] Re-use of reagents: If less than 10 samples are being
analysed, the remainder of the isolation reagents may be stored at
-20.degree. C. for up to two weeks.
1. Wash Buffer
[0111] [0112] Pre-warm Wash buffer for 30 min at 37.degree. C.
before starting the isolation procedure. [0113] Mix the Wash buffer
vial every 10-min during the incubation to ensure that any crystals
have fully dissolved. [0114] Allow Wash buffer to cool to room
temperature. [0115] Protect Wash buffer from excessive heat or
light.
C. Nucleic Acid Amplification Reagents
[0115] [0116] Bring all reagents to room temperature before use.
[0117] Re-use of reagents: The reconstituted Reagent spheres and
the unused Enzyme solution can be re-used within two weeks provided
they have been stored at -70.degree. C. Re-use of all other
amplification reagents is possible if the unused portions have been
stored at -20.degree. C.
1. Preparation of Reagent Spheres/KCl Solution
[0117] [0118] Add 80 .mu.l Reagent sphere diluent to the
lyophilised Reagent spheres and immediately vortex well. DO NOT
centrifuge. [0119] Add 30 .mu.l of KCl solution to the diluted
spheres and vortex.
2. Preparation of the Target RNA-Specific Primer Solution
[0119] [0120] Transfer 110 .mu.l of the Reagent sphere/KCl solution
into a fresh test tube and add [0121] 10 .mu.l of the H5-primer
mixture. Mix well by vortexing. DO NOT centrifuge.
3. Enzyme Solution
[0121] [0122] Thaw the Enzyme solution at room temperature and mix
gently by flicking the tube with fingers. DO NOT vortex any
solution containing enzymes. Centrifuge tube contents before
use.
D. Nucleic Acid Detection Reagents
[0122] [0123] Re-use of detection reagents is possible if the
unused reagents have been stored at 2-8.degree. C.
1. Capture and Detection Probe
[0123] [0124] Detection of specific RNA amplicons is carried out
with the generic detection probe in the kit in combination with an
H5-capture probe previously coupled to paramagnetic beads.
2. H5 RNA Hybridisation Solution
[0124] [0125] Vortex H5-capture beads until an opaque solution has
formed. [0126] For N H5 RNA-specific reactions: add (N+2).times.10
.mu.l H5 RNA-specific capture beads to a fresh test tube add
(N+2).times.10 .mu.l generic ECL probe [0127] Vortex hybridisation
solution before use. RNA Amplification in vitro
A. Nucleic Acid Release and Isolation
[0127] [0128] 1. Pre-warm Lysis buffer tubes for 30 min and vortex
regularly before starting nucleic acid release. [0129] 2.
Centrifuge Lysis buffer tubes for 30 sec at 10,000.times.g. [0130]
3. Add 100 .mu.l target RNA to Lysis buffer tubes and vortex.
[0131] In Lysis buffer, specimens can be stored: [0132]
indefinitely at -70.degree. C. [0133] up to 14 days at 2-8.degree.
C. [0134] up to 48 hours at 25.degree. C. [0135] 4. Vortex the
Silica suspension and add 50 .mu.l to each sample RNA/Lysis buffer
tube. [0136] 5. Incubate RNA/Lysis buffer/Silica tubes for 10 min
at room temperature (vortex tubes every 2 min to prevent silica
from settling to the bottom). [0137] 6. Centrifuge RNA/Lysis
buffer/Silica tubes for 30 sec at 10,000.times.g. [0138] 7.
Carefully remove the supernatant (do not disturb the pellets) and
add 1 ml Wash buffer to each tube. [0139] 8. Vortex tubes until the
pellets have resuspended completely. [0140] 9. Centrifuge tubes for
30 sec at 10,000.times.g. [0141] 10. Repeat steps (7) to (9) [0142]
Once with Wash buffer [0143] Twice with 70% ethanol [0144] Once
with acetone. [0145] 11. After the final wash step, carefully
remove any residual acetone with a 100 .mu.l pipette. [0146] 12.
Dry the silica pellets in open test tubes for 10 min at 56.degree.
C. on a heating block. [0147] 13.When dry, add 50 .mu.l Elution
buffer to each test tube. [0148] 14.Vortex tubes until the pellets
have resuspended completely. [0149] 15. Incubate the resuspended
silica for 10 min at 56.degree. C. to elute the nucleic acid
(vortex the test tubes after 5 min). [0150] 16. Centrifuge tubes
for 2 min at 10,000.times.g. [0151] 17. Transfer 5 .mu.l of each of
the nucleic acid supernatants to a fresh tube and begin the
amplification reaction within 1 hr.
B. Nucleic Acid Amplification
[0151] [0152] 1. For each H5 RNA reaction, pipette 5 .mu.l of the
nucleic acid extract into a fresh test tube. [0153] 2. Add 10 .mu.l
of the H5 RNA-specific amplification solution. The amplification
solution contains Primers A and B for the detection of
non-pathogenic H5 virus. [0154] 3. Incubate tubes for 5 min at
65.degree. C. in a heating block. [0155] 4. Cool tubes for 5 min at
41.degree. C. in heating block. [0156] 5. Add 5 .mu.l of Enzyme
solution and mix well by flicking the test tube with finger. [0157]
6. Immediately return test tubes to 41.degree. C. for 10 min.
[0158] 7. Briefly centrifuge the tubes and incubate them for 90 min
at 41.degree. C. in a water bath. [0159] 8. Detection of the
amplification products may now be performed. As an alternative, the
amplification products may be stored at -20.degree. C. for up to 1
month. [0160] 9. If the detection result for the presence of
non-pathogenic H5 virus is positive, an amplification solution
containing Primers A and C for the detection of pathogenic 115
virus may now be applied onto the amplification products by
repeating steps 2-8.
C. Nucleic Acid Detection
[0160] [0161] 1. Vortex the hybridisation solutions until opaque.
Add 20 .mu.l of target RNA hybridisation to each of the
hybridisation tubes. [0162] 2. For the amplification reactions:
[0163] Add 5 .mu.l H5 RNA amplification reaction. [0164] Cover the
hybridisation tubes with adhesive tape. [0165] Mix the
hybridisation tubes until an opaque solution forms. [0166] 3. Use
adhesive tape to cover the hybridisation tubes. This is to prevent
evaporation and contamination. [0167] 4. Incubate hybridisation
tubes for 30 min at 41.degree. C. [0168] 5. Add 300 .mu.l assay
buffer to each hybridisation tube.
[0169] The samples are now ready to be detected for the presence of
H5 virus by a suitable detection equipment. The detection in this
example is performed on a suitable system equipped with a
photomultiplier tube.
[0170] The results of H5 virus detection are listed in the
following tables. The results are confirmed by DNA sequencing using
a Perkin Elmer ABI 310 Genetic Analyzer.
TABLE-US-00002 TABLE 1 Results of the detection for non-pathogenic
H5 virus by the detection kit using Primers A and B H5 Subtype
Detection Case/sample no. Detectable Count Result Classification
258/97 4229 Positive 977/97-2 6961 Positive 1000/97 33835 Positive
1258/97-2 2500 Positive 1258/97-3 2400 Positive 1258/97-4 10494
Positive 1258/97-5 3089 Positive 1258/97-9 4883 Positive 1258/97-10
Protocol optimization 437/99-4 5165 Positive 437/99-6 22200
Positive 437/99-8 5142 Positive 437/99-10 511 Positive Negative
control 1 1 Negative Negative control 2 1 Negative Negative control
3 35 Negative
TABLE-US-00003 TABLE 2 Results of the detection for pathogenic H5
virus by the detection kit using Primers A and C H5 Pathogenicity
Detection Case/sample no. Detectable Count (.times.10.sup.3) Result
Classification 258/97 11800 Positive 977/97-2 5300 Positive 1000/97
23100 Positive 1258/97-2 61800 Positive 1258/97-3 85100 Positive
1258/97-4 68400 Positive 1258/97-5 15800 Positive 1258/97-9 5400
Positive 1258/97-10 Protocol optimization 437/99-4 48400 Positive
437/99-6 27100 Positive 437/99-8 21100 Positive 437/99-10 11600
Positive Negative control 1 1 Negative Negative control 2 3
Negative Negative control 3 2 Negative
[0171] As shown in the above example, it can be realized that the
detection kit may be used conveniently in various testing sites
including farms. Furthermore, the detection kit is relatively
easier to use than existing methods, and may be able to provide the
detection results in a shorter time--the detection results may be
available within one day if desired. As it is a RNA-based detection
system, the specificity and the sensitivity may be enhanced--the
detection kit is specific to H5 virus, and the concentration of the
H5 virus in the sample may no longer be important as the virus will
be replicated to a target molecule for detection.
[0172] It will be apparent to one skilled in the art that the
primers, detection probe, and capture probe may be also useful in
the form of RNA molecules. DNA molecules are preferred due to
stability reason.
[0173] Although the preferred embodiment of this invention has been
described in previous paragraphs, it should be apparent to one
skilled in the art that modifications and alternative editions of
this invention are possible, and such modifications and editions
are still within the scope of this invention, which is set forth in
the following claims. In addition, the embodiments of this
invention shall not be interpreted restrictively by the examples or
figures only.
TABLE-US-00004 List 1 Reference No. Description 10 nucleic acids in
sample 12 silica 14 nucleic acids/silica mixture 20 H5 viral RNA 22
Primer A 24 Extended Primer A 26 Extended Primer A(DNA): H5 viral
RNA hybrid 28 Primer B 30 double-stranded DNA copy of H5 virus 32
target RNA molecule 40 detection probe 41 signal generator 42
capture probe 44 immobilizer
Sequence CWU 1
1
14125DNAInfluenza A Subtype H5 Virus 1tcccctgctc attgctatgg tggta
25281DNAInfluenza A Subtype H5 Virus 2gtatccactc ccctgctcat
tgctatggtg gtacccatac caaccatcta ccatgccctg 60ccatcctccc tctataaaac
c 813120DNAInfluenza A Subtype H5 Virus 3gtggattctt tgtctgcagc
gtatccactc ccctgctcat tgctatggtg gtacccatac 60caaccatcta ccatgccctg
ccatcctccc tctataaaac tgctatagct ccaaatagtc 120431DNAInfluenza A
Subtype H5 Virus 4aattctaata cgactcacta tagggagaag g
31527DNAInfluenza A Subtype H5 Virus 5tgccattcca caacatacac cccctca
27696DNAInfluenza A Subtype H5 Virus 6actgcaacac caagtgtcaa
actccaatgg gggcgataaa ctctagtatg ccattccaca 60acatacaccc cctcaccatc
ggggaatgcc ccaaat 967136DNAInfluenza A Subtype H5 Virus 7aagtgaattg
gaatatggta actgcaacac caagtgtcaa actccaatgg gggcgataaa 60ctctagtatg
ccattccaca acatacaccc cctcaccatc ggggaatgcc ccaaatatgt
120gaaatcaaac agatta 136820DNAInfluenza A Subtype H5 Virus
8gatgcaaggt cgcatatgag 20925DNAInfluenza A Subtype H5 Virus
9gagagaagaa gaaaaaagag aggac 251094DNAInfluenza A Subtype H5 Virus
10tcaaacagat tagttcttgc gactggactc agaaataccc ctcaaaggga gagaagaaga
60aaaaagagag gactatttgg agctatagca ggtt 9411134DNAInfluenza A
Subtype H5 Virus 11aatgccccaa atatgtgaaa tcaaacagat tagttcttgc
gactggactc agaaataccc 60ctcaaaggga gagaagaaga aaaaagagag gactatttgg
agctatagca ggttttatag 120agggaggatg gcag 1341222DNAInfluenza A
Subtype H5 Virus 12ctatttggag ctatagcagg tt 221391DNAInfluenza A
Subtype H5 Virus 13ggactcagaa atacccctca aagggagaga agaagaaaaa
agagaggact atttggagct 60atagcaggtt ttatagaggg aggatggcag g
9114131DNAInfluenza A Subtype H5 Virus 14acagattagt tcttgcgact
ggactcagaa atacccctca aagggagaga agaagaaaaa 60agagaggact atttggagct
atagcaggtt ttatagaggg aggatggcag ggcatggtag 120atggttggta t 131
* * * * *