CN108977585B - Subtype typing detection method for influenza A virus - Google Patents
Subtype typing detection method for influenza A virus Download PDFInfo
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Abstract
The invention discloses an influenza A virus subtype typing detection method, which comprises the following steps: step one, designing and synthesizing a primer; step two, preparing carboxyl magnetic beads connected with forward primers; step three, extracting influenza A virus RNA; step four, RT-PCR; step five, magnetic adsorption and washing; and step six, detection. The subtype typing detection method for the influenza A virus can be used for typing the influenza A virus subtype with high flux, rapidness, sensitivity and specificity, and has good application value.
Description
Technical Field
The invention belongs to the field of biotechnology detection, and particularly relates to an influenza A virus subtype typing detection method.
Background
Influenza a viruses are common influenza viruses and are very susceptible to variation, and thus there are many influenza a virus subtypes. These subtypes are known as "avian influenza". Influenza a has high human pathogenicity, and has caused a worldwide pandemic many times. Among influenza a viruses, avian influenza virus subtypes that have been found to be able to directly infect humans are: types a H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, and H10N 8. Among them, subtypes H1, H5, and H7 are highly pathogenic, and H1N1, H5N1, and H7N9 are particularly interesting. Therefore, it is very necessary to detect influenza a virus subtypes H1N1, H5N1, and H7N 9. Currently there is no good detection method for these subtypes.
Disclosure of Invention
The invention aims to provide a method for quickly and sensitively detecting influenza A virus subtypes.
The specific scheme is as follows: a subtype typing detection method of influenza A virus comprises the following steps:
step one, designing and synthesizing a primer: according to influenza A virusThe specific gene sequence of the type, and respectively designs a corresponding forward primer and a corresponding reverse primer; the 5' end of the forward primer is connected with-NH2-(CH2)n-TmObtaining a magnetic bead connection forward primer, wherein n is 4-8, and m is 14-18; carrying out fluorescence labeling on the 5 'end band of the reverse primer to obtain the reverse primer with the 5' end band of the fluorescence labeling, and selecting different fluorescence labels aiming at different influenza A virus subtypes; synthesizing forward primers of all subtypes of the influenza A virus, connecting the forward primers with magnetic beads and reverse primers with fluorescence labels at 5' ends;
step two, preparing carboxyl magnetic beads connected with forward primers: connecting the magnetic beads of each subtype of the influenza A virus obtained in the step one with a forward primer respectively to be connected with carboxyl magnetic beads, so as to obtain the carboxyl magnetic beads of each subtype of the influenza A virus, which are connected with the magnetic beads and connected with the forward primer;
step three, extracting influenza A virus RNA;
step four, RT-PCR: taking the forward primer of each subtype of the influenza A virus obtained in the step one, the reverse primer with the fluorescent label at the 5' end and the carboxyl magnetic bead connected with the magnetic bead and connected with the forward primer of each subtype of the influenza A virus obtained in the step two as primers, and taking the influenza A virus RNA obtained in the step three as a template to carry out RT-PCR reaction;
step five, magnetic adsorption and washing: after the RT-PCR reaction is finished, the magnetic beads are adsorbed and gathered under the magnetic action, washing the magnetic beads by using a washing solution, and removing fluorescent labels which are not connected with the magnetic beads;
step six, detection: and D, detecting the fluorescent labeling signals on the magnetic beads obtained in the fifth step by using a microplate reader or a flow cytometer so as to determine the subtype of the influenza A virus.
Further, a positive control template aiming at each subtype of the influenza A virus is added in the fourth step; the positive control template is a plasmid with a DNA fragment amplified by RT-PCR in the fourth step.
Further, influenza a virus subtypes include influenza a virus subtype H1N1, influenza a virus subtype H5N1, and influenza a virus subtype H7N 9.
Further, the sequence of the H1N1 subtype forward primer is shown in SEQ ID NO. 7; the sequence of the H5N1 subtype forward primer is shown in SEQ ID NO. 8; the sequence of the H7N9 subtype forward primer is shown in SEQ ID NO. 9; the sequence of the reverse primer with the fluorescent label at the 5' end of the H1N1 subtype is shown as SEQ ID NO. 4; the sequence of the reverse primer with the fluorescent label at the 5' end of the H5N1 subtype is shown as SEQ ID NO. 5; the sequence of the reverse primer with the fluorescent label at the 5' end of the H7N9 subtype is shown as SEQ ID NO. 6.
Further, the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of subtype H1N1, the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of subtype H5N1 and the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of subtype H7N9 are CY5, VIC and FAM, respectively; or the fluorescent marker of the reverse primer with the fluorescent marker at the 5 ' end of the H1N1 subtype, the fluorescent marker of the reverse primer with the fluorescent marker at the 5 ' end of the H5N1 subtype and the fluorescent marker of the reverse primer with the fluorescent marker at the 5 ' end of the H7N9 subtype are respectively VIC, FAM and CY 5; or the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of the H1N1 subtype, the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of the H5N1 subtype and the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of the H7N9 subtype are FAM, CY5 and VIC respectively.
Further, n is 6 and m is 16.
Further, the H1N1 subtype positive control template is a plasmid with a DNA sequence shown as SEQ ID NO. 10; the H5N1 subtype positive control template is a plasmid with a DNA sequence shown as SEQ ID NO. 11; the H7N9 subtype positive control template is a plasmid with a DNA sequence shown in SEQ ID NO. 12.
Further, a negative control template is added in the fourth step; the negative control template was RNase-free water.
Further, the fifth step is specifically: after RT-PCR reaction is finished, standing the PCR tube on a first electromagnetic chuck, switching on a power supply of the first electromagnetic chuck to ensure that magnetic beads are attached to the bottom of the PCR tube under the magnetic action, and sucking away liquid by using a pipette; adding a washing solution, placing a second electromagnetic chuck at the top of the PCR tube, and switching on and off power supplies of the first electromagnetic chuck and the second electromagnetic chuck alternately, so that magnetic beads soaked in the washing solution move back and forth at the bottom and the top of the PCR tube to achieve the purpose of washing; finally, turning off the power supply of the second electromagnetic chuck, turning on the power supply of the first electromagnetic chuck, standing, and then sucking away liquid by using a liquid transfer gun; the wash was RNase-free water.
Furthermore, in the fourth step, the ratio of the amount of the substance of the reverse primer with the fluorescent label at the 5' end of each subtype of the influenza A virus to the amount of the substance of the forward primer of each subtype of the influenza A virus is 10-20: 1. Because the quantity of the reverse primer with the fluorescence mark at the 5 'end is 10 to 20 times of that of the forward primer, the reverse primer with the fluorescence mark at the 5' end which is not matched with the forward primer for RT-PCR amplification can be matched with the forward primer connected on the carboxyl magnetic beads for RT-PCR amplification, so that a target gene is connected to the carboxyl magnetic beads, and the RT-PCR amplification is prevented from only occurring between the reverse primer and the forward primer.
The subtype typing detection method for the influenza A virus can detect an influenza A virus sample in a high-throughput, rapid, sensitive and specific manner, carries out nucleic acid typing on the influenza A viruses with high pathogenicity, namely H1N1 subtypes, H5N1 subtypes and H7N9 subtypes, and has important application value.
The primer is connected on the magnetic bead and is more favorable to improving RT-PCR efficiency than fixing at the container bottom, because for fixed primer, the magnetic bead is in the container bottom under the magnetic field effect that first electromagnetic chuck produced when circular telegram only, in case the power of disconnection first electromagnetic chuck, and the magnetic field disappears, and the magnetic bead can be very easy again leave the container bottom, consequently, the primer on the magnetic bead can be better mix with other reagents.
Because the viruses of different subtypes correspond to different fluorescent labels and the generated RT-PCR product is combined on the magnetic bead, the RT-PCR product combined on the magnetic bead can be separated from the fluorescent label primer without RT-PCR reaction through the magnetic adsorption effect, the influence of other fluorescent signals is avoided, and the detection result can be read by adopting an enzyme labeling instrument. No laboratory is required to be equipped with real-time quantitative RT-PCR related equipment and design related probes.
When the device is used, the second electromagnetic chuck and the first electromagnetic chuck can be switched on and off alternately, so that the magnetic beads move back and forth between the bottoms of the micropores and the tops of the micropores, and the purposes of uniform mixing and full washing are achieved.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a schematic diagram of a PCR tube array;
FIG. 2 is a graph showing the results of fluorescence detection in example 1.
Detailed Description
The present invention is further illustrated by the following specific examples in conjunction with the attached drawings, but it should be understood that these examples are only illustrative and not limiting of the scope of the invention. The carboxyl magnetic beads are commercially available carboxyl magnetic beads, such as those available from Zhongkoraiming (Beijing) science and technology, Inc. in the examples described below. RT-PCR reagents are commercially available RT-PCR reagents, which in the examples below are from the Tiangen One-step RT-PCR Kit (QIAGEN One step RT-PCR Kit). The reagents used in the following examples are commercially available unless otherwise specified. The main apparatus is as follows: Bio-Rad _ Mycycler PCR instrument, Bio-Rad iMark microplate reader.
Example 1
1. Design and Synthesis of primers
According to the HA gene sequences of influenza A viruses H1N1, H5N1 and H7N9 in a Genebank database, a corresponding core primer, a reverse primer (referred to as a reverse primer in Table 1 for short) with a fluorescence label at the 5' end and a forward primer are designed, and the sequences are shown in Table 1.
TABLE 1 primer sequences
The 5' end of the H1N1 reverse primer is connected with a fluorescent label CY5 to form an H1N1 reverse primer with a fluorescent label CY 5; connecting a fluorescent label VIC to the 5' end of the H5N1 reverse primer to form a H5N1 reverse primer with the fluorescent label VIC; and connecting the fluorescence labeled FAM to the 5' end of the H7N9 reverse primer to form a H7N9 reverse primer of which the fluorescence is labeled FAM.
In this example the 5' linker molecule is-NH2-(CH2)6-. Therefore, the magnetic bead connection forward primers corresponding to H1N1, H5N1 and H7N9 are respectively 5' -NH2-(CH2)6-TTTTTTTTTTTTTTTTCCACAACGGAAAACTATGT-3’，5’-NH2-(CH2)6-TTTTTTTTTTTTTTTTCATGTCCATACCATGGGAG-3 'and 5' -NH2-(CH2)6-TTTTTTTTTTTTTTTTGAGGCAATGCAAAATAGAATACAGAT-3'. The above primers were synthesized by Shanghai Biotechnology engineering services, Inc.
After adding a specific linker sequence to the gene sequences amplified by the forward primer and the reverse primer of the HA genes of 3 subtypes in Table 1, the number of bases was 80-160bp (as shown in Table 2), which was synthesized and ligated to pGEM-T vector plasmid by Shanghai Bioengineering services, Inc., and the plasmid of each subtype was quantified to 1. mu.g/ml.
TABLE 2 plasmid inserted Gene sequences of the subtypes
2. Preparation of magnetic beads with coupled magnetic beads to Forward primer
Preparing a connecting liquid: 0.1M 2-morpholinoethanesulfonic acid, 0.25% (v/v) Tween-20, 10mM carbodiimide hydrochloride, adjusted to pH 5 with hydrochloric acid and/or sodium hydroxide.
Preparing a purification buffer solution: 10mM PBS pH7.4, 0.25% (v/v) Tween-20.
Preparing magnetic bead preservation solution: 20 nmol/. mu.l Tris base, 20 mmol/. mu.l magnesium chloride, 50 mmol/. mu.l potassium chloride, 0.25% (v/v) Tween-20, 0.5% (m/m) BSA.
For example, the preparation of magnetic beads with H1N1 attached to them and the carboxyl beads with the forward primer attached to them: taking 1mg of magnetic beads with 200nm particle size and modified carboxyl on the surface, and respectively cleaning the magnetic beads by using 200 mu l of 0.01M sodium hydroxide solution and deionized water; then 1nmol of magnetic beads of H1N1 was added to the beads to attach the forward primer (linker molecule-NH)2-(CH2)6Core primer sequence shown as SEQ ID NO. 1) and 100. mu.l of the ligation solution, incubating at room temperature for 5h, and gently shaking with a mixer; then separating the carboxyl magnetic beads connected with the magnetic beads and the forward primer by using a magnetic separator, and washing for 2 times by using a purification buffer solution; finally, the washed carboxyl magnetic beads connected with the magnetic beads and the forward primer are stored in 100 mu l of magnetic bead storage solution at 4 ℃ for standby.
Similarly, a carboxyl-based magnetic bead with a H5N 1-linked magnetic bead-linked forward primer was prepared, except that the H1N 1-linked magnetic bead-linked forward primer was replaced with a H5N 1-linked magnetic bead-linked forward primer (the linker molecule was-NH)2-(CH2)6The sequence of the core primer is shown in SEQ ID NO. 2).
Similarly, a carboxyl-based magnetic bead with a H7N 9-linked magnetic bead-linked forward primer was prepared, except that the H1N 1-linked magnetic bead-linked forward primer was replaced with a H7N 9-linked magnetic bead-linked forward primer (the linker molecule was-NH)2-(CH2)6The sequence of the core primer is shown in SEQ ID NO. 3).
3. Influenza a virus RNA extraction
Mu.l of influenza A virus sample solution was taken, 500. mu.l of the lysate was added thereto, and 50. mu.l of viral RNA was extracted according to the RNeasy Mini Kit (QIAGEN, cat # 74104).
4、RT-PCR
(1) System configuration: the reaction solution was used with QIAGEN One step RT-PCR Kit (cat # 210212). The system configuration is specifically shown in table 3.
TABLE 3 RT-PCR reaction System configuration
| Components | Volume (μ l) |
| 5 XQIAGEN One step RT- |
10 |
| |
2 |
| QIAGEN One step RT- |
2 |
| RNase inhibitors | 0.2 |
| Carboxylic magnetic bead # connected with magnetic bead connected with forward primer | 10(μg) |
| Reverse primer with fluorescence mark at 5' |
1 |
| |
1 |
| |
10 |
| RNase-free water | To 50 |
The carboxyl magnetic beads with magnetic beads connected with the forward primer are a mixture of carboxyl magnetic beads with magnetic beads connected with H1N1 connected with the forward primer, carboxyl magnetic beads with magnetic beads connected with H5N1 connected with the forward primer and carboxyl magnetic beads with magnetic beads connected with H7N9 connected with the forward primer in equal proportion (m/m).
The reverse primer with the fluorescent label at the 5' end is a mixture of H1N1 reverse primer with the fluorescent label of CY5, H5N1 reverse primer with the fluorescent label of VIC and H7N9 reverse primer with the fluorescent label of FAM in equal proportion (ratio of the amount of substances).
The forward primer is a mixture of H1N1 forward primer, H5N1 forward primer and H7N9 forward primer in equal proportion (amount of substances).
The # template was as follows according to loading into different microwells: viral RNA extract, p-H1N1, p-H5N1, p-H7N9, RNase-free water (as negative control), 10-fold dilution of p-H1N1, 100-fold dilution of p-H1N1, 10-fold dilution of p-H5N1, 100-fold dilution of p-H5N1, 10-fold dilution of p-H7N9, and 100-fold dilution of p-H7N 9. The loading position of each template on the PCR tube array (8X 12 array) in this example is shown in FIG. 1:
p-H1N 1: a1, B12, G11, H2 and E7;
p-H5N 1: a2, B11, G12, H1 and D7;
p-H7N 9: b2, a11, H12, G1, and D6;
negative control: b1, a12, H11, G2, and E6;
10-fold dilution of p-H1N 1: b3, B4 and B5;
100-fold dilution of p-H1N 1: c3, C4, and C5;
10-fold dilution of p-H5N 1: d3, D4 and D5;
100-fold dilution of p-H5N 1: e3, E4 and E5;
10-fold dilution of p-H7N 9: f3, F4, and F5;
100-fold dilution of p-H7N 9: g3, G4 and G5;
viral RNA extract: the remaining micropores.
(2) Reaction procedure
The reaction mixture was added to a PCR tube according to the system shown in Table 3, and placed in a PCR apparatus, and RT-PCR reaction was started according to the above reaction procedure.
5. Magnetic adsorption and washing
After the reaction is finished, taking the PCR tube out of the PCR instrument, and opening the PCR tube cover; then installing a first electromagnetic chuck at the bottom of the PCR tube, connecting a power supply of the first electromagnetic chuck, and adsorbing magnetic beads at the bottom of the PCR tube; the liquid in the PCR tube was then carefully pipetted away without aspirating the beads.
Adding RNase-free water into the PCR tube to wash and remove residual reverse primer which is not combined with the magnetic beads and provided with fluorescent labels at the 5' end. The specific mode is as follows:
the second electromagnetic chuck is arranged on the upper surface of the PCR tube cover, and the power supply of the first electromagnetic chuck is turned off while the power supply of the second electromagnetic chuck is turned on, so that the magnetic beads at the bottom of the PCR tube move towards the direction of the PCR tube cover. Then the power supply of the second electromagnetic chuck is turned off and simultaneously the power supply of the first electromagnetic chuck is turned on, so that the magnetic beads move from the top to the bottom of the PCR tube. After reciprocating for several times, the state of the power supply of the first electromagnetic chuck is started while the power supply of the second electromagnetic chuck is stopped, so that the magnetic beads are adsorbed at the bottom of the PCR tube. The liquid in the PCR tube was then pipetted away, leaving the magnetic beads.
6. Detection of
And (3) placing the PCR tube into an enzyme-labeling instrument or a flow cytometer, and detecting by adopting the excitation wavelength and the detection wavelength corresponding to CY5, VIC and FAM. The excitation wavelength 620-650nm of CY5, and the detection wavelength 675-730 nm; the excitation wavelength of VIC is 500-535nm, and the detection wavelength is 560-580 nm; the excitation wavelength of FAM is 450-490nm, and the detection wavelength is 515-530 nm.
Fig. 2 shows a schematic diagram of the detection results. Corresponding fluorescent markers can be detected for 5 repeats of 3 groups of positive controls (p-H1N1, p-H5N1 and p-H7N9), which indicates that the detection has good repeatability and strong specificity; no fluorescent marker was detected for 5 replicates of the negative control, demonstrating that the test results were acceptable; the 3 groups of positive controls diluted by 100 times can still detect corresponding fluorescent labels, which indicates that the sensitivity is high and the specificity is strong. CY5 fluorescent markers are detected by B7 and C9, and the subtype of the influenza A virus at the positions of B7 and C9 is proved to be H1N 1; VIC fluorescent markers were detected by F8, F9 and H10, demonstrating that the influenza A virus subtype at positions F8, F9 and H10 is H5N 1; FAM fluorescent markers were detected by D9 and E10, demonstrating that the influenza A subtype at positions D9 and E10 was H7N 9. The remaining microwells to which the influenza A RNA extract was added did not detect fluorescent markers, indicating that their subtypes are not among H1N1, H5N1, and H7N 9.
Therefore, the subtype typing detection method for the influenza A virus can quickly and simply distinguish 3 subtypes of the influenza A virus, and has the advantages of good repeatability, high sensitivity and strong specificity. For specificity, more virus types can be further adopted for verification, and the invention only verifies the specificity among H1N1, H5N1 and H7N 9. Therefore, the high specificity in the present invention means only high specificity among H1N1, H5N1 and H7N 9.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
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Claims (2)
1. A subtype typing detection method for influenza A viruses is characterized by comprising the following steps:
step one, designing and synthesizing a primer: respectively designing a corresponding forward primer and a reverse primer according to specific gene sequences of all subtypes of the influenza A virus; in the forward directionThe 5' end of the substance is linked to-NH2-(CH2)n-TmObtaining magnetic bead ligation forward primer, n =6, m = 16; carrying out fluorescence labeling on the 5 'end band of the reverse primer to obtain a reverse primer with the 5' end band of the fluorescence labeling, and selecting different fluorescence labels aiming at different influenza A virus subtypes; synthesizing the forward primer of each subtype of the influenza A virus, the magnetic bead connecting forward primer and the reverse primer with the 5' end provided with the fluorescent label;
step two, preparing carboxyl magnetic beads connected with forward primers: connecting the magnetic beads of each subtype of the influenza A virus obtained in the step one with a forward primer respectively to be connected with carboxyl magnetic beads to obtain the carboxyl magnetic beads of each subtype of the influenza A virus, which are connected with the magnetic beads and the forward primer;
step three, extracting influenza A virus RNA;
step four, RT-PCR: performing RT-PCR reaction by using the forward primer of each subtype of the influenza A virus obtained in the step one, the reverse primer with the fluorescent label at the 5' end and the carboxyl magnetic beads connected with the magnetic beads and connected with the forward primer of each subtype of the influenza A virus obtained in the step two as primers and the influenza A virus RNA obtained in the step three as a template; in the fourth step, the ratio of the amount of substances of the reverse primer with the fluorescence label at the 5' end of each subtype of the influenza A virus to the amount of substances of the forward primer of each subtype of the influenza A virus is 10-20: 1;
step five, magnetic adsorption and washing: after the RT-PCR reaction is finished, the magnetic beads are adsorbed and gathered under the magnetic action, washing the magnetic beads by using a washing solution, and removing fluorescent labels which are not connected with the magnetic beads;
step six, detection: detecting the fluorescent labeling signals on the magnetic beads obtained in the fifth step by adopting an enzyme labeling instrument or a flow cytometer so as to determine the subtype of the influenza A virus;
positive control templates aiming at all subtypes of the influenza A virus are added in the fourth step; the positive control template is a plasmid with the RT-PCR amplified DNA fragment in the fourth step;
the influenza a virus subtypes include influenza a virus subtype H1N1, influenza a virus subtype H5N1 and influenza a virus subtype H7N 9; the sequence of the H1N1 subtype forward primer is shown in SEQ ID NO. 7; the sequence of the H5N1 subtype forward primer is shown in SEQ ID NO. 8; the sequence of the H7N9 subtype forward primer is shown in SEQ ID NO. 9; the sequence of the reverse primer with the fluorescent label at the 5' end of the H1N1 subtype is shown as SEQ ID NO. 4; the sequence of the reverse primer with the fluorescent label at the 5' end of the H5N1 subtype is shown as SEQ ID NO. 5; the sequence of the reverse primer with the fluorescent label at the 5' end of the H7N9 subtype is shown as SEQ ID NO. 6;
the H1N1 subtype positive control template is a plasmid with a DNA sequence shown as SEQ ID NO. 10; the H5N1 subtype positive control template is a plasmid with a DNA sequence shown as SEQ ID NO. 11; the H7N9 subtype positive control template is a plasmid with a DNA sequence shown as SEQ ID NO. 12; a negative control template is added in the fourth step; the negative control template is water without RNase;
the fifth step is specifically as follows: after RT-PCR reaction is finished, standing a PCR tube on a first electromagnetic chuck, switching on a power supply of the first electromagnetic chuck to ensure that magnetic beads are attached to the bottom of the PCR tube under the magnetic action, and sucking away liquid by using a pipette; adding the washing solution, placing a second electromagnetic chuck at the top of the PCR tube, and switching on and off power supplies of the first electromagnetic chuck and the second electromagnetic chuck alternately, so that magnetic beads soaked in the washing solution move back and forth at the bottom and the top of the PCR tube to achieve the purpose of washing; finally, turning off the power supply of the second electromagnetic chuck, turning on the power supply of the first electromagnetic chuck, standing, and sucking away liquid by using a liquid transfer gun; the wash solution is RNase-free water;
the influenza a virus subtype typing detection method is not used for disease diagnosis or treatment.
2. The method for subtype typing detection of influenza A virus according to claim 1, wherein the fluorescent label of the reverse primer carrying a fluorescent label at the 5 ' end of subtype H1N1, the fluorescent label of the reverse primer carrying a fluorescent label at the 5 ' end of subtype H5N1 and the fluorescent label of the reverse primer carrying a fluorescent label at the 5 ' end of subtype H7N9 are CY5, VIC and FAM, respectively; or the fluorescent marker of the reverse primer with the fluorescent marker at the 5 ' end of the H1N1 subtype, the fluorescent marker of the reverse primer with the fluorescent marker at the 5 ' end of the H5N1 subtype and the fluorescent marker of the reverse primer with the fluorescent marker at the 5 ' end of the H7N9 subtype are respectively VIC, FAM and CY 5; or the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of the H1N1 subtype, the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of the H5N1 subtype and the fluorescent label of the reverse primer with the fluorescent label at the 5 ' end of the H7N9 subtype are FAM, CY5 and VIC respectively.
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