CN109722492B - Method for detecting H5 and H7N9 subtype highly pathogenic avian influenza virus and H9 subtype avian influenza virus - Google Patents
Method for detecting H5 and H7N9 subtype highly pathogenic avian influenza virus and H9 subtype avian influenza virus Download PDFInfo
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Abstract
The invention provides a rapid detection method for simultaneously detecting and distinguishing H5 and H7N9 subtype highly pathogenic avian influenza viruses and H9 subtype avian influenza viruses, which is characterized in that a primer and a probe are designed in a conserved region by analyzing nucleotide sequences of HA genes of the H5 subtype highly pathogenic avian influenza viruses, the H7N9 subtype highly pathogenic avian influenza viruses and the H9 subtype avian influenza viruses which are separated in recent years, and a real-time fluorescent quantitative RT-PCR nucleic acid rapid detection method for the H5 and H7N9 subtype highly pathogenic avian influenza viruses and the H9 subtype avian influenza viruses is established. The H5 and H7N9 subtype highly pathogenic avian influenza virus and the H9 subtype avian influenza virus real-time fluorescence RT-PCR method provided by the invention has the advantages of high sensitivity, short detection time, no need of open links such as electrophoresis and the like, detection of three subtype avian influenza viruses in one closed reaction tube can be completed by only one fluorescence PCR instrument, and a reaction curve can be checked in real time in the detection process to rapidly judge the result.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a method for simultaneously and rapidly detecting and distinguishing H5 and H7N9 subtype highly pathogenic avian influenza viruses and H9 subtype avian influenza viruses.
Background
Avian influenza viruses can be classified into highly pathogenic and low pathogenic avian influenza viruses according to differences in pathogenicity to poultry. Among them, highly pathogenic avian influenza viruses include partial strains of H5 and H7 subtypes. The H9 subtype avian influenza virus belongs to a low pathogenic strain.
However, it was found during the cultivation that immunization with vaccines accelerates the virus variation, so that the vaccine must be updated faster. With the evolution of viruses, H5 subtype avian influenza viruses are rapidly mutated all the time; from the first N28, re-1 to Re-4, re-5, to Re-6, re-8, to the latest Re-11, re-12. Viral variation is due to variation of nucleotides in the hemagglutinin gene (HA). Currently, there are 10 branches of avian influenza virus subtype H5 worldwide. The branches existing or popular in China comprise 4 branches including the 0 th branch, the 2.3.4.4 branch, the 2.3.2.1 branch and the 7 th branch, and are all highly pathogenic viruses. The HA nucleotide homology of the strains circulating in China with the virus A/goose/Guangdong/1/1996 (H5N 1) originally isolated in 1996 is only 90.6% (Re-11) and 91.2% (Re-12). The nucleotide homology of HA of 2.3.4.4 branch and 2.3.2.1 branch represented by Re-11 and Re-12 vaccine strains is only 88.5%. The rapid variation of HA nucleotide increases the difficulty of the detection method using nucleotide as target gene, which results in the decrease of specificity and sensitivity of the detection method using HA gene as target gene, and results in false negative.
After the H7N9 virus is mutated into a highly pathogenic strain, huge economic losses are caused to the poultry industry. The infection symptoms are very similar to those of the H5 subtype highly pathogenic avian influenza virus, and are difficult to distinguish clinically. Although the H9 subtype avian influenza virus belongs to a low pathogenic strain and does not directly kill poultry when being singly infected, the avian influenza virus can be mixed with other pathogens to be infected, so that the death rate of infected poultry can be increased. Therefore, the detection and the differentiation of the H5 and H7N9 subtype highly pathogenic avian influenza virus and the H9 subtype avian influenza virus have important significance for the diagnosis, the prevention and the treatment of diseases.
Disclosure of Invention
The invention provides a rapid detection method for simultaneously detecting and distinguishing H5 and H7N9 subtype highly pathogenic avian influenza viruses and H9 subtype avian influenza viruses, which is characterized in that a primer and a probe are designed in a conserved region by analyzing nucleotide sequences of HA genes of the H5 subtype highly pathogenic avian influenza viruses, the H7N9 subtype highly pathogenic avian influenza viruses and the H9 subtype avian influenza viruses which are separated in recent years, and a real-time fluorescent quantitative RT-PCR nucleic acid rapid detection method for the H5 and H7N9 subtype highly pathogenic avian influenza viruses and the H9 subtype avian influenza viruses is established.
The invention firstly provides a primer group and a probe for detecting H5 subtype highly pathogenic avian influenza virus, and the sequence information is as follows:
wherein the sequence of the upstream primer is ATGGAAAARAACGTYACTGT (SEQ ID NO: 1),
the sequence of the downstream primer is AGCCATCCWGCYACACTRCAAT (SEQ ID NO: 2);
the probe sequence is AAGACATACTGGARAAGACACAYAAYGGGA (SEQ ID NO: 3),
wherein, the 5 'end of the probe is marked with FAM, and the 3' end is marked with BHQ1;
secondly, the invention provides a primer group and a probe for detecting H7N9 subtype highly pathogenic avian influenza virus, and the sequence information is as follows:
wherein the sequence of the upstream primer is AGTCTKCTGCTKGCWACAGGRATG (SEQ ID NO: 4),
the sequence of the downstream primer is CTTCCCATCCATTTTCAATGAAAC (SEQ ID NO: 5);
the probe sequence is as follows:
CGGAMTRMDACCAAAGRGAAAACGGAMTRMDAGAGGCCT(SEQ ID NO：6)，
wherein the 5 'end of the probe is marked with Cy5, and the 3' end of the probe is marked with BHQ2;
the invention further provides a primer group and a probe for detecting the H9 subtype avian influenza virus, and the sequence information of the primer group and the probe is as follows:
wherein the sequence of the upstream primer is AACYTACAAAATCCTCA (SEQ ID NO: 7),
the sequence of the downstream primer is AATGTTGCAYCTGCAAGA (SEQ ID NO: 8);
the probe sequence is as follows: GCAATGGGGTTYGCTGCCTTYTTRTTCTGG (SEQ ID NO: 9),
wherein the 5 'end of the probe is marked with Hex, and the 3' end of the probe is marked with BHQ1;
the primer group and the probe are used for preparing a kit for detecting H5 and H7N9 subtype highly pathogenic avian influenza viruses and H9 subtype avian influenza viruses by using a real-time fluorescent quantitative RT-PCR method;
in another aspect, the present invention provides a method for detecting highly pathogenic H5 and H7N9 subtype avian influenza viruses and H9 subtype avian influenza viruses, comprising the steps of:
1) Preparing a reaction system:
dNTPs and Mg are added into a polymerase chain reaction tube in sequence 2+ 25. Mu.l of 2 XqRT-PCR reaction buffer, 2.0. Mu.l of upstream primer at a concentration of 10. Mu. Mol/L, 2.0. Mu.l of downstream primer at a concentration of 10. Mu. Mol/L, and,1.5 mul of probe with the concentration of 10 mul mol/L, 2.5 mul of enzyme mixture and 6.0 mul of virus nucleic acid sample to be detected;
2) The reaction steps are as follows:
sealing the reaction system prepared in the step 1), and placing the reaction system on a fluorescent quantitative PCR instrument for reaction. Reaction conditions are as follows: the first stage, reverse transcription is carried out at 50 ℃/10min; second stage, pre-denaturation at 95 ℃/2min; the third stage, 95 ℃/10s,60 ℃/30s,40 cycles; collecting fluorescence during each cycle of annealing extension during the third phase;
3) And (4) detecting a result:
if the FAM channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, the H5 subtype highly pathogenic avian influenza virus is judged to be positive;
if the Cy5 channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, the subtype H7N9 highly pathogenic avian influenza virus is judged to be positive.
And if the Hex channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, judging that the H9 subtype avian influenza virus is positive.
The H5 and H7N9 subtype highly pathogenic avian influenza virus and the H9 subtype avian influenza virus real-time fluorescence RT-PCR method provided by the invention have the advantages of high sensitivity, short detection time, no need of open links such as electrophoresis (easy to cause environmental pollution), capability of completing detection of the three subtype avian influenza viruses in one closed reaction tube by only one fluorescence PCR instrument, capability of viewing a reaction curve in real time in the detection process and capability of quickly judging the result. The virus isolation method as the standard of the avian influenza diagnosis gold needs more equipment, longer time and higher hardware requirements (highly pathogenic avian influenza virus needs to be completed in a biosafety third-level laboratory).
Drawings
FIG. 1 is a diagram showing the sequence alignment of HA of the avian influenza virus subtype H5,
FIG. 2 is a diagram of an HA sequence alignment of H7N9 subtype avian influenza virus,
FIG. 3 is a diagram showing the sequence alignment of HA of the H9 subtype avian influenza virus,
FIG. 4 shows the verification of primer probe in triple real-time fluorescent quantitative RT-PCR method,
FIG. 5 is a diagram of the sensitivity test of the real-time fluorescent quantitative RT-PCR method established by the present invention.
Detailed Description
The present invention will be described in detail below by way of examples.
Example 1: determination of conserved sequence region and screening of primer probe
The applicant researches HA nucleotide sequences of different NA subtypes and different branches of H5 subtype influenza viruses by a molecular biological method (table 1), and compares the nucleotide sequences with HA sequences of H5 strains which are separated and stored in laboratories where the applicant is located to determine conserved regions (figure 1). The sequence alignment shows that the HA sequence of the H5 subtype influenza virus is in a nt106-nt174 region, and the sequence of the region is as follows:
ATGGAAAARAACGTYACTGTWACRCATGCYMAAGACATACTGGARAAGACACAYAAYGGGARGCTYTG；
the nt210-nt242 region with the sequence of
ATTGYAGTGTRGCWGGATGGCTHCTYGGRAAYCC is more conservative; wherein R = a or G, Y = C or T, M = or C, W = a or T.
Table 1: h5 virus strain information table used for designing primers and probes in the invention
According to the design principle of a fluorescent quantitative RT-PCR primer probe, a plurality of pairs of primers and probes are designed in different conserved regions for screening, and the optimal primer group and probe for detecting the H5 subtype avian influenza virus are finally determined according to the detected sensitivity, and the sequence information is as follows:
wherein the sequence of the upstream primer is ATGGAAAARAACGTYACTGT (SEQ ID NO: 1),
the sequence of the downstream primer is AGCCATCCWGCYACACTRCAAT (SEQ ID NO: 2);
the sequence of the probe is AAGACATACTGGARAAGACACAYAAYGGGA (SEQ ID NO: 3), wherein the 5 'end of the probe is marked with FAM, and the 3' end of the probe is marked with BHQ1;
the applicant researches HA nucleotide sequences of virulent strains and attenuated strains of the H7N9 subtype avian influenza virus (table 1) by a molecular biological method, and compares the nucleotide sequences with HA sequences of H7N9 strains which are separated and stored in a laboratory where the applicant is located to determine a conserved region (figure 2). The sequence alignment shows that the HA sequence of the H7N9 subtype avian influenza virus strong and weak virus is in the region from nt907 to nt956, and the sequence of the region is as follows:
TTTCAGAACATWGAYAGCAGRRCARTTGGAAAATGYCCRAGATAKGTTAA；
nt964-nt995 in the sequence
AGTCTKCTGCTKGCWACAGGRATGAAGAATGT；
nt1028 to nt1076 region of sequence
GAGGCCTRTTTGGTGCTATAGCDGGTTTCATTGAAAATGGATGGGAAGG is more conservative; wherein R = a or G, Y = C or T, M = a or C, W = a or T, K = G or T.
Comparing HA sequences of a high-pathogenicity strain and a low-pathogenicity strain of the H7N9 subtype avian influenza virus, and displaying that the high-pathogenicity strain is mainly formed by inserting 12 basic groups in an nt1016-nt1027 region, and the sequence is AACGGAMTRMDA; wherein R = a or G, M = a or C, D = G, a or T.
Table 2: information table of H7N9 virus strain used for designing primers and probes in the invention
According to the design principle of a fluorescent quantitative RT-PCR primer probe, a plurality of pairs of primers and probes are designed in different conserved regions for screening, and the optimal primer group and probe for detecting the H7N9 subtype highly pathogenic avian influenza virus are finally determined according to the detected sensitivity, and the sequence information is as follows:
wherein the sequence of the H7 upstream primer is AGTCTKCTGCTKGCWACAGGRATG (SEQ ID NO: 4),
the sequence of the H7 downstream primer is CTTCCCATCCATTTTCAATGAAAC (SEQ ID NO: 5);
the probe sequence is
CGGAMTRMDACCAAAGRGAAAACGGAMTRMDAGAGGCCT (SEQ ID NO: 6), wherein the 5 'end of the probe is labeled with Cy5 and the 3' end is labeled with BHQ2;
the applicant researches the HA nucleotide sequence of the H9 subtype influenza virus by a molecular biological method (Table 3), and compares the HA nucleotide sequence with the HA sequence of the H9 strain which is separated and stored in a laboratory where the applicant is positioned to determine the conserved region (figure 3). The sequence alignment shows that the HA sequence of the H9 subtype influenza virus is in a nt120-nt147 region, and the sequence of the region is as follows:
CCTGTGACACATGYAAAGAA；
nt157-nt185 region, sequence AATGGRATGCTRTGTGCAAC;
nt1249-nt1292 region with the sequence
ATGATCAAYRATAARRTTGATGAYCAAATYCARGAYATATGGGC；
nt1541-nt1681 region of sequence
TCAARCTGGRATCTGARRRAACYTACAAAATCCTCAYMATTTATTCGACTGYYGCCTCATCYCTTGYDATTGCAATGGGGTTYGCTGCCTTYTTRTTCTGGGCCAKGTMYMYYGSRTCTTGCAGRTGCAACATTWGTATAT is more conservative; wherein R = a or G, Y = C or T, M = or C, W = a or T, S = G or C, D = G, a or T.
Table 3: h9 virus strain information table used for designing primers and probes in the invention
According to the design principle of a fluorescent quantitative RT-PCR primer probe, a plurality of pairs of primers and probes are designed in different conserved regions for screening, and the optimal primer group and probe for detecting the H9 subtype avian influenza virus are finally determined according to the detected sensitivity, and the sequence information is as follows:
wherein the sequence of the upstream primer is AACYTACAAAATCCTCA (SEQ ID NO. 7),
the sequence of the downstream primer is AATGTTGCAYCTGCAAGA (SEQ ID NO. 8);
the sequence of the probe is GCAATGGGGTTYGCTGCCTTYTTRTTCTGG (SEQ ID NO. 9), wherein the 5 'end of the probe is marked with Hex, and the 3' end of the probe is marked with BHQ1.
In order to verify the designed real-time fluorescent RT-PCR primer probes, three pairs of primer probes are configured in one reaction tube, H5 and H7N9 subtype highly pathogenic avian influenza viruses and H9 subtype avian influenza viruses stored in a laboratory are selected for fluorescent RT-PCR detection, and as a result, in the triple real-time fluorescent RT-PCR method, the respective primer probes can only specifically detect the corresponding viruses without cross amplification (figure 4).
Example 2: establishing a detection method
The detection reaction system and reaction conditions are established, and 25 mul of 2 XTRT-PCR reaction buffer solution (containing dNTPs and Mg) is added into the PCR tube 2+ ) 2.0 μ L (concentration is 10 μmol/L) of the upstream primer of the first step, 2.0 μ L (concentration is 10 μmol/L) of the downstream primer of the first step, 1.5 μ L (concentration is 10 μmol/L) of the probe of the first step, 2.5 μ L of the enzyme mixture (reverse transcriptase, RNase inhibitor, taq enzyme with 5'→ 3' exo activity), and 6.0 μ L of the viral nucleic acid to be detected (extracted from clinical samples or other samples by using a nucleic acid extraction kit); and then sealing the reaction system, and placing the reaction system on a fluorescent quantitative PCR instrument for reaction. Reaction conditions are as follows: the first stage, reverse transcription at 50 deg.C/10 min; second stage, pre-denaturation at 95 ℃/2min; the third stage, 95 ℃/10s,60 ℃/30s,40 cycles; fluorescence was collected during the third stage for each cycle of annealing extension. If the S-type fluorescence curve appears and the Ct value is less than or equal to 38, the result is judged to be positive, otherwise, the result is judged to be negative.
The real-time fluorescent quantitative RT-PCR technology is used for quickly detecting nucleic acid of H5 and H7N9 subtype highly pathogenic avian influenza virus and H9 subtype avian influenza virus, and comprises the following steps:
first step (and primer): according to the nucleic acid sequences (namely SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 in the sequence table) specified by the invention, the upstream and downstream primers and probes required by the amplification reaction are artificially synthesized;
the second step (configuration of reaction system): 25 μ l of 2 xqRT-PCR buffer (containing dNTPs and Mg) was added to the PCR tube in sequence 2+ ) 2.0 μ L (concentration is 10 μmol/L) of the upstream primer of the first step, 2.0 μ L (concentration is 10 μmol/L) of the downstream primer of the first step, 1.5 μ L (concentration is 10 μmol/L) of the probe of the first step, 2.5 μ L of the enzyme mixture (reverse transcriptase, RNase inhibitor, taq enzyme with 5'→ 3' exo activity), and 6.0 μ L of the viral nucleic acid to be detected (extracted from clinical samples or other samples by using a nucleic acid extraction kit);
the third step (reaction): and (3) sealing the reaction system prepared in the second step, and then placing the reaction system on a fluorescent quantitative PCR instrument for reaction. Reaction conditions are as follows: the first stage, reverse transcription is carried out at 50 ℃/10min; second stage, pre-denaturation at 95 ℃/2min; the third stage, 95 ℃/10s,60 ℃/30s,40 cycles; fluorescence was collected during the third stage for each cycle of annealing extension.
Fourth step (result detection): if the FAM channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, the H5 subtype highly pathogenic avian influenza virus is judged to be positive; if the Cy5 channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, the subtype H7N9 highly pathogenic avian influenza virus is judged to be positive; and if the Hex channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, judging that the H9 subtype avian influenza virus is positive.
Example 3: effect detection of primers and probes
1. The primer probe and the method which are screened and established are adopted to carry out sensitivity test on H5 and H7N9 subtype highly pathogenic avian influenza virus and H9 subtype avian influenza virus, and the method comprises the following steps:
the first step is as follows: extracting virus RNA, and determining the content of the virus RNA by using a trace nucleic acid analyzer. Diluting RNA by 10 times, taking 6.0 mu l of diluted RNA template, and adding the diluted RNA template into 44.0 mu l of qRT-PCR premix;
the second step is that: detecting by adopting the established real-time fluorescent quantitative RT-PCR method to determine the sensitivity of the kit;
the result shows that the real-time fluorescent quantitative RT-PCR method established by the invention can detect the H5 virus RNA template of 0.1fg, can detect the H7N9 virulent RNA template of 0.004fg and can detect the H9 virus RNA template of 0.01fg (figure 5).
2. The specificity test is carried out on the real-time fluorescent quantitative RT-PCR detection method of the H5 and H7N9 subtype highly pathogenic avian influenza virus and the H9 subtype avian influenza virus, and the method comprises the following steps:
the first step is as follows: the H1-H16 subtype avian influenza virus stored in the selected room comprises an H1N1 subtype, an H2N2 subtype, an H3N2 subtype, an H4N4 subtype, an H5N1 subtype, an H5N2 subtype, an H5N3 subtype, an H5N6 subtype, an H5N8 subtype, an H6N8 subtype, an H7N9 subtype, an H8N6 subtype, an H9N2 subtype, an H10N8 subtype, an H11N2 subtype, an H12N2 subtype, an H13N1 subtype, an H14N4 subtype, an H15N1 subtype and an H16N1 subtype. The H5 subtype avian influenza virus comprises strains of 4 branches including the 0 th branch, the 2.3.4.4 branch, the 2.3.2.1 branch and the 7.2 th branch; the subtype H7N9 includes H7N9 highly pathogenic strains and H7N9 low pathogenic strains. Meanwhile, other avian viruses including newcastle disease virus, avian infectious bronchitis virus and infectious bursal disease virus are selected. Extracting RNA as a template;
the second step is that: detecting by adopting an established real-time fluorescent quantitative RT-PCR method to determine the specificity of the kit;
the third step: according to the real-time fluorescent quantitative RT-PCR method established by the invention, the H5 primer probe group and the H5 subtype avian influenza virus which can detect 4 branches including the 0 th branch, the 2.3.4.4 branch, the 2.3.2.1 branch and the 7.2 th branch have no detection on other subtypes of avian influenza virus and other avian viruses; the H7N9 primer probe group can detect H7N9 highly pathogenic strains, and other subtype avian influenza viruses (including H7N9 low pathogenic strains) and other avian viruses are not detected; the H9 primer probe set can detect all H9N2 subtype avian influenza viruses, and has no detection on other subtypes of avian influenza viruses and other avian viruses (Table 4). The results show that the sensitivity of the technique is 100.0% and the specificity is 100.0%.
Table 4: the invention establishes a real-time fluorescent quantitative RT-PCR method specificity test result table
Example 4: and (5) application detection of clinical samples.
100 parts of clinical suspected H5 and H7N9 subtype highly pathogenic avian influenza and H9 subtype avian influenza onset poultry samples are collected in 2014-2018, wherein the clinical suspected H5 and H7N9 subtype highly pathogenic avian influenza onset poultry samples mainly comprise tissues such as lung, liver, spleen, trachea, rectum and the like. Taking a small amount of tissue sample, shearing, adding 5-10 times volume of PBS buffer solution, and homogenizing or vibrating and crushing. After freezing and thawing for 3 times, centrifuging for 5 minutes at 10000rpm, and taking the supernatant for later use. RNA is directly extracted from part of the supernatant, and the real-time fluorescence RT-PCR nucleic acid rapid detection method for the H5 and H7N9 subtype highly pathogenic avian influenza virus and the H9 subtype avian influenza virus is used for detection; another part of the supernatant was inoculated into 10-day-old chick embryos, virus isolation was performed, and the symbol rate of the two methods was compared.
The results show that: 100 parts of clinical pathogenic tissue samples are detected, the H5 and H7N9 subtype highly pathogenic avian influenza virus and the H9 subtype avian influenza virus real-time fluorescence RT-PCR nucleic acid rapid detection method are used for detection, 56 parts of samples are positive by the H5 virus, 15 parts of samples are positive by the H7N9 strong virus, and 18 parts of samples are positive by the H9 virus. The virus separation result also shows that 89 parts of the virus separation result are positive, and the sequencing result shows that the RT-PCR method is consistent with the virus separation result and is in a corresponding relationship, and the neutralization rate is 100%. The results show that the sensitivity of the technique is 100.0% and the specificity is 100.0%.
Sequence listing
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Claims (9)
1. A primer group and a probe for detecting H5 subtype highly pathogenic avian influenza virus, H7N9 subtype highly pathogenic avian influenza virus and H9 subtype avian influenza virus are characterized in that the primer group and the probe comprise:
1) The upstream primer for detecting the H5 subtype highly pathogenic avian influenza virus has a sequence of SEQ ID NO:1,
the sequence of the downstream primer for detecting the H5 subtype highly pathogenic avian influenza virus is SEQ ID NO:2;
the sequence of the probe for detecting the H5 subtype highly pathogenic avian influenza virus is SEQ ID NO:3;
2) The upstream primer for detecting the H7N9 subtype highly pathogenic avian influenza virus has a sequence of SEQ ID NO:4,
the sequence of the downstream primer for detecting the H7N9 subtype highly pathogenic avian influenza virus is SEQ ID NO:5;
the sequence of the probe for detecting the H7N9 subtype highly pathogenic avian influenza virus is SEQ ID NO:6;
3) The upstream primer for detecting H9 subtype avian influenza virus has a sequence of SEQ ID NO:7,
the downstream primer for detecting the H9 subtype avian influenza virus has a sequence of SEQ ID NO:8;
the probe for detecting H9 subtype avian influenza virus has a sequence of SEQ ID NO:9;
the 5 'end and the 3' end of the probe for detecting the H5 subtype highly pathogenic avian influenza virus, the probe for detecting the H7N9 subtype highly pathogenic avian influenza virus and the probe for detecting the H9 subtype avian influenza virus are respectively marked by different fluorescein.
2. The primer set and the probe for detecting the highly pathogenic avian influenza virus of the H5 subtype according to claim 1, wherein the 5 'end of the probe is labeled with FAM and the 3' end of the probe is labeled with BHQ1.
3. The primer set and the probe for detecting the highly pathogenic avian influenza virus subtype H7N9 according to claim 1, wherein the 5 'end of the probe is labeled with Cy5, and the 3' end of the probe is labeled with BHQ2.
4. The primer set and the probe for detecting the H9 subtype avian influenza virus according to claim 1, wherein the 5 'end of the probe is marked with Hex, and the 3' end of the probe is marked with BHQ1.
5. Use of the primer set and the probe according to claim 1 for the preparation of a kit.
6. A fluorescence quantitative RT-PCR detection kit, which is characterized by comprising the primer group and the probe of claim 1.
7. The primer set and the probe as claimed in claim 1 are applied to detection of highly pathogenic H5 and H7N9 subtype avian influenza virus and H9 subtype avian influenza virus for non-disease diagnosis and treatment purposes.
8. A method for detecting highly pathogenic avian influenza virus of H5 and H7N9 subtypes and avian influenza virus of H9 subtype for the purpose of non-disease diagnosis and treatment by using the primer set and probe of claim 1, comprising the steps of:
1) Preparing a reaction system: adding 25 mul of 2 xqRT-PCR reaction buffer solution containing dNTPs and Mg < 2+ >, 2.0 mul of upstream primer with the concentration of 10 mul mol/L, 2.0 mul of downstream primer with the concentration of 10 mul mol/L, 1.5 mul of probe with the concentration of 10 mul mol/L, 2.5 mul of enzyme mixture and 6.0 mul of virus nucleic acid sample to be detected into a polymerase chain reaction tube in sequence; the upstream primer, the downstream primer and the probe are the primer group and the probe for detecting the H5 subtype highly pathogenic avian influenza virus, the H7N9 subtype highly pathogenic avian influenza virus and the H9 subtype avian influenza virus in claim 1;
2) The reaction steps are as follows:
sealing the reaction system prepared in the step 1), and then placing the reaction system on a fluorescent quantitative PCR instrument for reaction; reaction conditions are as follows: the first stage, reverse transcription is carried out at 50 ℃/10min; second stage, pre-denaturation at 95 ℃/2min; the third stage, 95 ℃/10s,60 ℃/30s,40 cycles; collecting fluorescence during each cycle of annealing extension in the third stage;
3) And (4) detecting a result:
and judging the virus type of the detected sample according to the fluorescent development results of different probes.
9. The method of claim 8, wherein the results, one of the criteria for determining, are as follows:
if the FAM channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, the FAM channel is positive for the H5 subtype highly pathogenic avian influenza virus;
if the Cy5 channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, the subtype H7N9 highly pathogenic avian influenza virus is positive;
if the Hex channel has an S-type fluorescence curve and the Ct value is less than or equal to 38, the H9 subtype avian influenza virus is positive.
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