WO2006000140A1

WO2006000140A1 - Primers, probes sequences and methods, useful in the multiple real time fluorescent rt-pcr assay of avian influenza virus subtypes h5, h7 and h9

Info

Publication number: WO2006000140A1
Application number: PCT/CN2005/000667
Authority: WO
Inventors: Jingzhong Lin; Xinglong Xiao; Shengli Liu; Anqing Zhong; Zhifeng Qin; Jianqian Lv; Xianzhong Jin
Original assignee: Taitai Genomics, Inc.; Shenzhen Entry-Exitinspection And Quarantine Bureau Of The People's Republic Of China
Priority date: 2004-06-25
Filing date: 2005-05-13
Publication date: 2006-01-05
Also published as: CN1670221A

Abstract

The present invention relates to primers, probes sequences and methods, useful in the multiple real time fluorescent RT-PCR assay of Avian Influenza virus subtypes H5, H7 and H9; specifically, to primers and probes for RT-PCR amplifing the nucleotide fragments of Avian Influenza virus subtypes H5, H7 and H9 and complementary sequences thereof; as well as to assays for simultaneously type-departing Avian Influenza virus subtypes H5, H7 and H9.

Description

For avian influenza H5, H7 and H9 subtypes

Primers, probe sequences and methods for real-time fluorescent RT-PCR detection

FIELD OF THE INVENTION The present invention relates to RT-PCR amplification primers and probes for highly pathogenic avian influenza H5, H7 and H9 subtype nucleotide fragments, and simultaneous typing assays for avian influenza Ή5, Η7 and Η9 subtypes . BACKGROUND OF THE INVENTION Avian Influenza (AI), also known as European chicken or true chicken, is an acute highly contagious disease caused by influenza A virus. It is an internationally recognized devastating disease that can pass pigs or Other animals are transmitted to humans and therefore have important ecological and public health implications, especially for highly pathogenic avian influenza, which is classified as a Class A infectious disease by the World Organisation for Animal Health. '

Since 1878? After erronci to first reported the avian flu epidemic in Italy, the bird flu virus was isolated in Europe and the Americas, and the avian flu history has long been associated with humans. Avian influenza virus has been found to have 16 serotypes, among which highly pathogenic avian influenza ^: viruses mainly include H5, H7 and H9 subtypes. Short latency, high mortality and sudden death are the main features, which can lead to infection in a short time. The flocks are completely annihilated, and their morbidity and mortality can reach 100%, causing serious economic losses. At present, the avian flu virus has been widely distributed around the world, and each outbreak has caused huge economic losses to the world poultry industry. In 1993, avian flu broke out in Pennsylvania, investing 60 million US dollars to kill 17 million chickens. In 1995, due to bird flu, Mexico eliminated 18 million chickens, blocked 32 million chickens, and 130 million chickens. Emergency vaccination, causing direct economic losses of 1 billion US dollars. In 1997, the Hong Kong SAR government spent 100 million Hong Kong dollars to kill 1.5 million 3⁄4. In 2001, it again spent 80 million Hong Kong dollars to kill 1.2 million chickens. It can be seen that the severe restriction of bird flu affects the development of poultry industry in China and the world, and has caused great blows to the poultry industry. It has become a key target for import and export inspection and quarantine and disease surveillance in various countries.

In recent years, it has been found that avian flu not only seriously harms birds, but also harms mammals, especially those that can infect people and cause death. The avian influenza virus belongs to the RNA virus. Like all RNA viruses, due to the lack of corrective function of RNA polymerase, the genome has a high error rate when it is transcribed, and its genome is segmented, which is prone to rearrangement. Virus has high

Replacement page (Article 26) Degree of variability. The gene fragments encoding the glycoproteins HA and NA in the genome and the gene fragments encoding the non-structural proteins NS 1 and NS2 have large differences among different influenza strains, while other genes are relatively conserved in the avian influenza virus genome. The study found that the virulence of avian influenza virus depends mainly on the interaction between host and virus, and different gene fragments of virus also play different roles in determining the pathogenicity of virus. The main function is to encode HA protein. Gene. By analyzing and comparing the nucleotide sequence and amino acid sequence of a large number of avian influenza viruses, it was found that the amino acid sequence of the HA cleavage site determines the virulence of avian influenza virus. The HA cleavage site of highly pathogenic avian influenza has 6 consecutive sites. Basic amino acids, while low pathogenic avian influenza has 2 basic amino acids.

Despite extensive and in-depth research on avian influenza and the establishment of diagnostic methods, there is currently no method for simultaneous detection of avian influenza H5, H7 and H9 sub-ploughs, and real-time fluorescent PCR technology has great potential for development. High-tech detection technology has been widely used in many fields, so it can be used to solve the problems caused by multi-serotype and high variability of avian influenza virus. Real-time PCR technology was first introduced in 1996. This technology not only achieves a qualitative and quantitative leap in PCR, but also has a more specificity, shorter detection cycle, and efficient PCR than conventional PCR. The characteristics of pollution and high degree of automation have been widely used.

Conventional reverse transcription polymerase chain reaction (RT-PCR) is a technique in which RNA is reverse transcribed into cDNA and then the DNA fragment is rapidly amplified in vitro using DNA polymerase. Fluorescence real-time RT-PCR is based on common RT-PCR. A pair of primers is added to the amplification reaction system, and a specific fluorescent probe is added. The probe is labeled with a fluorescent reporter group at both ends. A fluorescent quenching group of oligonucleotides. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quenching group, so that the fluorescent signal emitted by the fluorescent group of the probe is not detected, and at the 5' end of the Taq enzyme during PCR amplification. Exonuclease activity cleaves the fluorescent probe that specifically binds to the amplified fragment of interest, so that the fluorescent reporter group is released from the reaction system, and the shielding effect of the fluorescence quenching group is removed. At this time, the fluorescence report can be detected. The fluorescence signal of the group, the amount of fluorescence signal is proportional to the amount of amplification product.

Multiple real-time fluorescent PCR is a method of adding multiple pairs of primers and probes in the same reaction system and simultaneously detecting multiple target fragments, which is of great significance in the identification of disease-like diseases and the typing of 'multi-serotype pathogens. Compared with the common PCR method, it has the following advantages: _a . The fluorescent labeling probe is used to further improve the specificity and accuracy of the detection; b. The detection is more sensitive by automatically detecting the change of the fluorescent signal; Perform a fully enclosed detection reaction without post-processing the PCR product to avoid contamination; d. Monitor the results in real time,

Replacement page (Article 26) High detection speed and efficiency; e. Can achieve one tube and multiple inspections, saving time and effort. Because the avian influenza virus has multiple subtypes and the mutation is fast, and the occurrence of the epidemic is complicated, it is urgent to establish a "one-step" typing method for highly pathogenic avian influenza that is sensitive, accurate, reliable, fast and simple. It is used to quickly detect the highly prevalent avian influenza virus subtypes at home and abroad, so as to meet the needs of import and export inspection and quarantine and disease surveillance.

Summary of the invention

• The object of the present invention is to provide primers, probe sequences and methods for multiple real-time fluorescent RT-PCR detection of avian influenza H5, H7 and H9 subtypes.

The object of the present invention can be achieved by the following technical solutions: Primers and fluorescent probes are designed separately by analyzing all reported avian influenza H5, H7 and H9 subtype genomic sequences. Based on the conventional RT-PCR detection technique, a nucleotide probe labeled with two fluorophores is added, and the fluorescent reporter group (R) is labeled at the 5' end of the probe, and the fluorescence quenching group (Q) Marked at the 3' end of the probe, the two constitute an energy transfer structure, that is, the fluorescence emitted by the fluorescent reporter group can be absorbed by the fluorescence quenching group, and when the distance is far, the inhibition is weakened, and the reporter group is weakened. The fluorescence signal is enhanced. During the amplification reaction, the probe hybridizes to the amplified fragment of interest on the template. Since the Taq enzyme has the exonuclease activity at the 5' end to the 3' end, the probe is cleaved during the amplification extension phase, and the fluorescence quenching group is The inhibition of Q) disappeared, and the fluorescent signal of the reporter group was enhanced to detect the H5, H7 and H9 subtypes of avian influenza. The principle of fluorescence RT-PCR detection is shown in Figure 1. Primer and probe design: Multiple pairs of primers and probes were designed by comparing the genomic sequences of all reported avian influenza virus H5, H7 and H9 subtypes, respectively, and selecting non-secondary structure and highly conserved nucleotide segments. The primers are generally about 20 bases in length, and there are no complementary sequences between the primers and the primers, and they should specifically target the avian influenza virus H5, H7 and H9 subtypes, and there is no cross reaction between them, which can be in one reaction tube. It is also used to distinguish between H5, H7 and H9 subtypes.

The primer sequences and probe sequences for nucleotide amplification for detecting the avian influenza H5 subtype include: the upstream primer Η5ρΠ 673 sequence is GACCAGCTACCATGATTGCCA and the complementary sequence is CTGGTCGATGGTACTAACGGT, and the downstream primer H5pr l 600 sequence is GGAGTCAAATTTGGA ATCAATGG and the complementary sequence is The primer pair consisting of CCTCAGTTTAA ACCTTAGTTACC and the upstream primer position of the primer pair extend 10 bases toward the 5' end, and the downstream primer position extends 10 bases toward the 3' end and 10 bases toward the 5' end. The primer sequence and the complementary sequence obtained in the region range; the probe H5pb l 655 sequence is TGCTAGGGAACTCGCCA, the complementary sequence is ACGATCCCTTGAGCGGT, and the 3⁄4 probe extends 10 bases toward the 3' end and 10 base regions toward the 5' end. Probe sequences and complementary sequences obtained in the range.

Primer sequences and probe sequences for nucleotide amplification for detecting avian influenza H7 subtype include -

Replacement page (Article 26) 1. The primer pair consisting of the upstream primer H7pfl292 sequence is CGTGTCCAATTAATGACATTTCC and the complementary sequence is GCACAGGTTAATTACTGTAAAGG, the downstream primer H7prl202 sequence is ATCACAGGCAA ATTGAATCGT and the complementary sequence is TAGTGTCCGTTTAACTTAGCA, and the upstream primer position of the primer pair extends 10 bases toward the 5' end. a primer sequence and a complementary sequence extending 10 bases in the 3' direction and 10 bases in the 5' ^_ end direction; the probe H7pbl247 sequence is TTTGAGCTGATAGACAATGA and the complementary sequence is AAACTCGACTATCTGTTACT, and The probe extends 10 bases in the 3' end direction and a probe sequence and a complementary sequence extending in the range of 10 bases in the 5' end direction.

2. The primer pair consisting of the upstream primer Π7ρΠ664 sequence is GCCATTGCAATGGGCCT and the complementary sequence is CGPTA ACGTTACCCGGA, the downstream primer H7prl736 sequence is AGTAGAAAC 'AAGGGTGTTTTTTCCA and the complementary sequence is TCATCTTTGTTCCC ACAAAAAAGGT, and the upstream primer position of the primer pair extends toward the 5' end. The primer sequence and the complementary sequence obtained by extending the base primer position by 10 bases in the 3' end direction and extending 10 bases in the 5' end direction; the probe H7pbl712 sequence is CGGTGCACTATTTGTATA and the complementary sequence is GCCACGTGATAAACATAT And a probe sequence and a complementary sequence in which the probe extends 10 bases in the 3' end direction and 10 bases in the 5' end direction. Primer sequences and probe sequences for nucleotide amplification for detecting avian influenza H9 subtype include: CCTGTGACACATGCCAAAGA from the upstream primer H9pfl51 sequence and GGACACTGTGT ACGGTTTCT as the complementary sequence, CTTTCGACRATGTAGGACCATTC for the downstream primer H9pr302 sequence and GAAAGCTGYTACATCCTGGTAAG for the complementary sequence The primer pair and the upstream primer position of the primer pair extend 10 bases in the 5' end direction, and the downstream primer position extends 10 bases in the 3' end direction and 10 bases in the 5' end direction. The primer sequence and the complementary sequence; the probe H9pbl75 sequence is CTCCACACAGAGCACAAT and the complementary sequence is GAGGT GTGTCTCGTGTTA, and the probe is extended to 10 bases in the 3' end direction and 10 bases in the 5' end direction. Needle sequence and complementary sequence.

After optimal combination of the above primers and probe sequences, a multiplex real-time fluorescent RT-PCR assay for simultaneous detection of avian influenza H5, H7 and H9 subtypes was established and made for simultaneous detection of avian influenza H5, H7 and H9 subtypes. Multiple real-time fluorescent RT-PCR assay kit.

Avian influenza H5, H7 and H9 subtypes multiplex real-time fluorescence RT-PCR. Detection method using the following

7 3⁄4:

(1) selecting primers and probes as set forth in claims 1-9;

(2) Prepare the template to be tested and extract the genomic RNA of avian influenza virus from various sources:

(3) establishment of the reaction system, a, determine the optimal primer concentration; b, determine the magnesium ion concentration; c,

Replacement page (Article 26) Determine the amount of reverse transcriptase (AMV RnaseXL); d, determine the amount of Taq DNA polymerase (Taq enzyme); e, determine the concentration of dNTPs; f, determine the optimal probe concentration;

(4) selecting the detection channel of the instrument;

(5) On-machine detection. '

Preparation of a template for detection of avian influenza H5, H7 and H9 subtypes in a real-time fluorescent RT-PCR assay using the method QIAamp Viral RNA Mini kit or Trizol nucleic acid extraction reagent extraction method for avian influenza virus from various sources Genomic RNA.

The salient features of the present invention are: Fully utilizing the high-efficiency amplification of PCR technology, the good specificity of nucleic acid hybridization, and the rapid sensitivity of fluorescence detection technology, a high-pathogenic avian influenza H5 and a single detection operation can be performed on one sample. The detection of H7 and H9 subtypes can determine the serotype, and has the advantages of reliable and accurate results, simple operation, time and labor saving, reduced detection cost and improved detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic diagram of real-time fluorescence RT-PCR detection of Taqman probe.

Figure 2 Avian influenza H5, H7 and H9 subtypes of triple real-time fluorescence RT-PCR detection curve Figure 3 Avian influenza H5 and H9 subtype double real-time fluorescence RT-PCR detection curve.

Figure 4 Avian influenza H7 and H9 subtypes of double real-time fluorescence RT-PCR detection curve.

Figure 5 Avian influenza H5 and H7 subtypes of double real-time fluorescence RT-PCR detection curve. The specific method of establishment and optimization of the reaction system: using the inactivated avian influenza virus H5, H7 and H9 subtype strains as samples to be tested, using the QIAamp Viral RNA Mini kit to extract viral genomic RNA, separately stored and stored in a 20 Ό spare.

Establishment and optimization of multiple real-time fluorescence RT-PCR reaction systems for avian influenza H5, H7 and H9 subtypes:

(1) Optimization of primer concentration In the case of other conditions in the reaction system, the primer concentrations of avian influenza H5, H7 and H9 were serially diluted from Ο. ΐμπιοΐ/L to 1.6 mol/L, and combined with each other. The test was carried out, and the final concentration of the optimal primer was determined to be 0.4 inoI/L by analysis and comparison of the test results.

(2) Optimization of magnesium ion concentration The concentration of MgCl _{2 is} increased from 1 mmol/L to 10 mmol/L in 1 mmol/L under the same conditions in the reaction system, and 5 mmol is selected after repeated experiments. /L is the magnesium ion concentration in the kit reaction system.

(3) Optimization of the amount of reverse transcriptase (AMV RnaseXL) After comparing the results of experiments using different concentrations of AMV RNaseXL, 5U was selected as the AMV in the kit reaction system.

Replacement page (Article 26) The amount of RnaseXL.

(4) Optimization of the amount of Taq DNA polymerase (Taq enzyme) By comparing the optimization results of the amount of Taq enzyme (in units of Unit), 5U was selected as the amount of Taq enzyme in the kit reaction system.

(5) Optimization of dNTPs concentration By using different concentrations of dNTPs for detection, 1 mmol/L was selected as the use amount of dNTPs in the kit reaction system after comprehensive evaluation. )

(6) Optimization of probe concentration The probe concentrations of avian influenza H5, H7 and H9 subtypes were from Ο. ι μ mol/L to 0.5 μmol/'L, respectively, under the same conditions in the reaction system. The ratio was measured after serial dilution, and the final concentration of the optimal probe was determined to be 0.2 μmol/L by analysis and comparison of the test results.

The above primers and probes were used to establish the reaction system, and finally the avian influenza H5, H7 and H9 real-time fluorescent RT-PCR reaction systems were determined to be 50 μl. The required components and corresponding concentrations are shown in Table 1.

Table 1 Avian influenza Η5, Η7 and Η9 subtypes of triple real-time fluorescence RT-PCR reaction of various components

Note: 1. When the volume of the multiplex real-time fluorescence RT-PCR reaction is different, each reagent should be adjusted according to the ratio.

2. The instrument used should be adjusted as appropriate.

3. Depending on the source of the test sample, the template addition should be adjusted appropriately. Instrument detection channel selection:

• When performing a three-dimensional real-time fluorescent RT-PCR reaction of avian influenza H5, H7 and H9 subtypes, the collection of the fluorescent signal of the reaction tube in the instrument should be set. The selected fluorescent detection channel is identical to the fluorescent reporter group labeled by the probe. . The specific setting method varies depending on the instrument. Refer to the instrument manual.

Replacement page (Article 26) Example 1

(1) Preparation of the template to be tested

Method 1 Use QI Aamp Viral RNA Mini kit to extract genomic RNA of avian influenza virus from various sources. The specific steps are as follows:

a. Take 560 μl of AVL buffer containing carrier RNA into a 1.5 ml microcentrifuge tube; b. Add 140 μl of plasma, serum, urine sample, cell culture supernatant or swab to PBS wash solution. Oscillating for 15 sec; - c incubation at room temperature (15-25 Ό) for 10 min;

d. instantaneous centrifugation to centrifuge the liquid in the cap to the centrifuge tube;

e. Add 560 μl absolute ethanol, vortex for 15 sec, centrifuge instantaneously, and centrifuge the liquid in the centrifuge tube to the centrifuge tube;

f. Carefully pipet 630 μl of the above liquid into the QIAamp spin column (placed in a 2 ml collection tube). Do not wet the edge of the tube and centrifuge at 8000 rpm for 1 min. Discard the collection tube containing the liquid and place the QIAamp spin column in another 2 ml collection tube;

g. Carefully open the QIAamp spin column cover and repeat step f;

h. Carefully open the QIAamp spin column cover, add 500 μl of AW1 buffer, and centrifuge at 80,00 rpm for 1 min. Place the QIAamp spin column on another clean 2 ml collection tube and discard the collection tube containing the liquid;

i. Carefully open the QIAamp spin column cover, add 500 μl of AW2 buffer, and centrifuge at 1400.0 rpm for 3 min. Place the QIAamp spin column on another clean 2ml collection tube, discard the collection tube containing the liquid, and centrifuge again for 1 min at MOOOrpm;

j. Place the QIAamp spin column in a clean 1.5 ml centrifuge tube, carefully open the lid, add 60 μl AVE elution buffer, incubate for 1 min at room temperature, centrifuge at 8000 rpm for 1 min to obtain viral RNA, 20 °C Store for backup. '

Method 2 Extraction method using Trizol nucleic acid extraction reagent

a. Harvest the allantoic fluid of the avian influenza virus-killed chicken embryo and centrifuge at 12,000 rpm to remove macromolecular impurities. 100 μl of the supernatant was added to a 1.5 ml centrifuge tube, and 300 μM of TrizoL tissue extract was added thereto and shaken well on the shaker. Then centrifuge at 13,000 rpm for 15 min, and transfer the supernatant to a 1.5 ml centrifuge tube;

b. Add 400 μM of pre-cooled isopropanol to the supernatant and shake well on the shaker; c centrifuge at 13000 rpm for 1 min to obtain RNA precipitation;

d. Carefully pour off the supernatant, add 600 μl of 75% ethanol, and wash it by hand upside down several times. (Note: Do not violently shake, prevent RNA breakage and difficult to re-precipitate after RNA precipitation is dissolved); e. Centrifuge at 13000 rpm l Omin, slowly aspirate the supernatant, dry at room temperature for about 25 min, wait until the ethanol is completely

Replacement page (Article 26) Add 25-40μ1 after volatilization

Solubility without RNase (fully boil and mix, 'or repeatedly blow with a gun), store at -20 °C for later use.

(2) Amplification conditions of avian influenza H5, H7 and H9 subtypes of real-time fluorescent RT-PCR reactions were prepared according to Table 1. The PCR tubes were placed in a fluorescent PCR machine and the corresponding fluorescence collection conditions were set. Amplification, the reaction procedure is as follows:

Reverse transcription of RNA was carried out at 50 °C for 30 min, and the reverse transcriptase was inactivated at 95 ° C for 3 min;

Denaturation at 95 °C for 15 sec, 5:> "C 30 sec annealing, 72 °C lmin extension, so repeat 5 cycles for preamplification;

95 °C l Osec denaturation, 60 °C 40 sec annealing, extension, so repeated 40 cycles for the amplification of the target fragment, the test results can be monitored in real time.

(3) Detection results of triple real-time fluorescent RT-PCR of avian influenza H5, H7 and H9 subtypes: Triple real-time fluorescence using specific primers and probes for avian sputum 5, Η7 and Η9 subtypes in a 50μ1 reaction system RT-PCk, a positive sample containing genomic RNA containing the avian influenza virus H5, H7 and H9 subtypes, results in a specific fluorescence curve (Fig. 2). The above test results show that the primers and probes designed by the present invention are specific and work well, and a three-fold real-time fluorescent RT-PCR detection method for avian influenza H5, H7 and H9 subtypes is established.

Example 2

Avian influenza H5 and H9 subtypes dual real-time fluorescence RT-PCR detection:

In a 50μ1 reaction system, double-phase real-time fluorescent RT-PCR was performed using specific primers and probes for avian influenza Η5 and Η9 subtypes to detect positive samples containing genomic RNA of avian influenza virus H5 and H9 subtypes, and the results were obtained. Specific fluorescence curve (Figure 3).

Example 3

Avian influenza H7 and H9 subtypes of double real-time fluorescence RT-PCR detection:

In the 50μ1 reaction system, double-phase real-time fluorescent RT-PCR was performed using specific primers and probes for the influenza Η7 and Η9 subtypes to detect positive samples containing genomic RNA of avian influenza virus H7 and H9 subtypes. Specific fluorescence curve (Figure 4).

Example 4

Avian influenza H5 and H7 subtypes by double real-time fluorescent RT-PCR detection - In a 50μ1 reaction system, double-click real-time fluorescent RT-PCR using specific primers and probes for avian influenza Η5 and Η7 subtypes A positive sample of influenza virus H5 and H7 subtype genomic RNA results in a specific fluorescence curve (Fig. 5).

Example 5

After synthesizing the corresponding primers and probes according to the conventional method, the reagents and positive control nucleotides required for real-time fluorescent RT-PCR were assembled into avian influenza H5, H7 and H9 subtypes.

Replacement page (Article 26) RT-PCR detection kit.

Replacement page (Article 26)

Claims

Claim

A multiplex real-time fluorescent RT-PCR method capable of simultaneously detecting avian influenza H5, H7 and H9 subtypes, characterized in that the method comprises:

(1) By comparing and analyzing the known genomic sequences of avian influenza virus H5, H7 and H9 subtypes, primers and probe sequences capable of detecting the avian influenza virus H5, H7 and H9 subtypes respectively were designed;

(2) using Joe, Rox, Ned, Hex, Tet, Pam or Vic fluorescein to label probe sequences for detection of avian influenza virus H5, H7, H9 subtypes, respectively;

(3) Primers and probes for avian influenza virus 1H5 and H7 or 115 and H9 or H7 and H9, respectively, or for three different subtypes of avian influenza viruses H5, H7 and H9 The primers and probes were placed in the same reaction tube, and a real-time fluorescent RT-PCR reaction was performed using a fluorescent PCR detector.

2. The primer sequence for detecting avian influenza virus H5 subtype according to claim 1, wherein the primer sequence comprises: the upstream primer Hfpfl673 sequence is GACCAGCTACC ATGATTGCCA and the downstream primer Hfpr 1600 sequence is GGAGTCAAATTTGGAATCAATGG. Primer pair; and the complementary sequence of the upstream primer CTGGTCGATGGTACTAACGGT and the complementary sequence of the downstream primer CCTCAGT TTAAACCTTAGTTACC; the upstream primer Hfpf 1673 of the primer pair extends 10 bases toward the 5' end, and the downstream primer H5prl600 extends toward the 3' end 10 The base and the primer sequence and the complementary sequence obtained within a range of 10 bases in the 5' direction.

3. The probe sequence for detecting avian influenza virus H5 subtype according to claim 1, wherein the probe H5pbl655 sequence is TGCTAGGGAACTCGCCA, the complementary sequence is ACGATCCCTTGAGCGGT, and the probe is directed to 3, The probe sequence and its complementary sequence extend in the direction of 10 bases in the end direction and in the range of 10 bases in the 5th direction.

4. The primer sequence for detecting avian influenza virus H7 subtype according to claim 1, wherein the primer sequence comprises: consisting of an upstream primer Η7Ν2ρΠ292 sequence of CGTGTC CAATTAATGACATTTCC and a downstream primer H7N2prl202 sequence of ATCACAGGCAAATTGAA TCGT. Primer pair; and the complement of the upstream primer GCACAGGTTAATTACTGTAAA GG and the complement of the downstream primer TAG TGTCCGTTTAACTTAGCA; the upstream primer Η7Ν2ρΠ292 of the primer pair extends 10 bases toward the 3' end, and the downstream primer H7N2 _P rl202 positions toward the 3' end The primer sequence and the complementary sequence were extended by 10 bases and extended in the range of 10 base regions toward the 5' end.

The probe sequence for detecting avian influenza virus H7 subtype according to claim 1, wherein the probe H7N2pbl247 sequence is TTTGAGCTGATAGACAATGA, complementary sequence

Replacement page (Article 26) It is listed as AAACTCGACTATCTGTTACT, and the probe sequence obtained by extending 10 bases in the 3' end direction and 10 'base regions in the 5' end direction and the complementary sequence thereof.

6. The primer sequence for detecting avian influenza virus H7 subtype according to claim 1, wherein the primer sequence comprises: a primer consisting of an upstream primer H7Nxpfl664 sequence of GCCATTGC AATGGGCCT and a downstream primer H7Nxprl736 sequence of AGTAGAAACAAGGGTGTTTTTTCCA. And the complementary sequence of the upstream primer CGGTAACGTTACCCGGA and the downstream primer TCATCTTTGTTCCCACAAAAAAGGT; the upstream H7Nxpf 1664 position of the primer pair extends 10 bases toward the 3' end, and the downstream primer H7Nxprl736 extends 10 bases toward the 3' end. The primer sequence and the complementary sequence obtained within a range of 10 base regions are extended in the 5' end direction.

7. The probe sequence for detecting avian influenza virus H7 subtype according to claim 1, wherein the probe H7Nxpb 1712 sequence is CGGTGCACTATTTGTATA, the complementary sequence 歹ij is GCCACGTGATAAACATAT, and the probe is 3 The probe sequence obtained by extending 10 bases in the end direction and extending 10 bases in the 5' end direction and its complementary sequence.

8. The primer sequence for detecting avian influenza virus H9 subtype according to claim 1, wherein the primer sequence comprises: from the upstream primer H9pfl51 sequence to CCTGTGACAC ATGCCAAAGA and downstream of the bow I substance H9pr302 sequence 歹 ij a primer pair consisting of CTTTCGACRATGTAGGACCATTC; and a complementary sequence of the upstream primer GGACACTGTGTACGGTTTCT and a complementary sequence of the downstream primer GAAAGCTGYTACATCCTGGTAAG; the upstream primer H9pfl51 of the primer pair extends 10 bases toward the 3' end, and the downstream primer H9pr302 is positioned 3, The primer sequence and the complementary sequence are extended by 10 bases in the direction of 10 bases in the range of 10 bases.

9. The probe sequence for detecting avian influenza virus H9 subtype according to claim 1, wherein the probe H9pb 175 sequence is CTCCACACAGAGCACAAT, the complementary sequence is GAG GTGTGTCTCGTGTTA, and the probe is directed to 3' The probe sequence and the complementary sequence extending within 10 base regions in the end direction and 10 base regions in the 5' end direction.

Replacement page (Article 26)