CN114959121A - Primers and probes for detecting influenza virus subtypes H5, H7 and H9, and application, reagent and digital PCR kit thereof - Google Patents

Abstract

The invention provides a primer and a probe for detecting influenza virus H5, H7 and H9 subtypes and its application, reagent and digital PCR kit, and relates to the field of biotechnology. The primers and probes for detecting influenza virus H5, H7 and H9 subtypes provided by the present invention, wherein the primers have the nucleic acid sequences shown in SEQ ID NO.1-6, and the probes have the nucleic acid sequences shown in SEQ ID NO.7-9 The primers and probes have strong specificity and high sensitivity, and can be used to prepare products for detecting influenza virus H5, H7, and H9 subtypes, and realize the detection of influenza virus H5, H7, and H9 subtypes.

Description

Primers and probes for detection of influenza virus H5, H7, H9 subtypes and their applications, reagents and digital PCR kits

technical field

The present invention relates to the field of biotechnology, in particular to a primer and probe for detecting influenza virus H5, H7 and H9 subtypes and its application, reagent and digital PCR kit.

Background technique

Influenza virus belongs to the Orthomyxoviridae family in virus taxonomy. It is mainly transmitted through droplets in the air, contact between susceptible and infected persons, or contact with contaminated items. Generally, the autumn and winter seasons are the high incidence period. Influenza viruses include human influenza viruses and animal influenza viruses. Human influenza viruses are divided into three types: A (A), B (B), and C (C), which are the pathogens of influenza. Among them, influenza A virus is prone to mutation in antigenicity and can be further divided into subtypes such as H1N1, H5N1, and H7N9, among which H and N represent the two surface glycoproteins of influenza virus, respectively. So far, the avian influenza virus subtypes that can directly infect humans are: H5N1, H7N1, H7N2, H7N3, H7N7, H9N2 and H7N9 subtypes. According to the different pathogenicity and virulence of avian influenza virus, avian influenza can be divided into highly pathogenic avian influenza, low pathogenic avian influenza and non-pathogenic avian influenza. The H5N1 subtype and the H7N9 subtype of highly pathogenic avian influenza are of particular concern, which not only caused human casualties, but also severely damaged the poultry industry.

The existing influenza virus detection methods include molecular diagnosis and immunodiagnosis. Molecular diagnostic techniques include reverse transcription-polymerase chain reaction (RT-PCR), loop-mediated isothermal nucleic acid amplification (LAMP), nucleic acid molecular hybridization, digital PCR, gene chips, etc. Immunodiagnostic techniques include enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay, chemiluminescence, and colloidal gold.

Digital PCR has higher sensitivity and accuracy than traditional RT-PCR, and can detect samples with a minimum single copy number. By randomly assigning nucleic acids to a large number of reaction units for amplification reaction, after the amplification is completed, the fluorescence signal of each reaction unit is collected, and finally the original concentration or content of the sample is calculated by direct counting or Poisson distribution formula. technology. However, there is still a lack of a kit that can simultaneously detect influenza virus H5, H7, and H9 subtypes and is suitable for droplet digital PCR.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide primers and probes for detecting influenza virus H5, H7, and H9 subtypes, so as to solve at least one of the above problems.

The second object of the present invention is to provide the application of the above primers and probes in the preparation of influenza virus H5, H7 and H9 subtype detection products.

The third object of the present invention is to provide a reagent.

The fourth object of the present invention is to provide a digital PCR kit.

In a first aspect, the present invention provides a primer and probe for detecting influenza virus H5, H7, and H9 subtypes, and the primers include a first primer pair, a second primer pair, and a third primer pair;

The first primer pair has the nucleic acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2; the second primer pair has the nucleic acid sequences shown in SEQ ID NO.3 and SEQ ID NO.4; The third primer pair has the nucleic acid sequences shown in SEQ ID NO.5 and SEQ ID NO.6;

The probe includes a first probe, a second probe and a third probe;

The first probe has the nucleic acid sequence shown in SEQ ID NO.7; the second probe has the nucleic acid sequence shown in SEQ ID NO.8; the third probe has the nucleic acid sequence shown in SEQ ID NO.9 Nucleic acid sequences shown.

As a further technical solution, the 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a fluorescence quenching group.

As a further technical solution, the fluorescent reporter group includes FAM, CY5 or HEX;

Preferably, the fluorescence quenching group includes TAMRA, BHQ1 or BHQ2.

In the second aspect, the present invention provides the application of the above primers and probes in the preparation of influenza virus H5, H7, H9 subtype detection products;

Preferably, the detection product includes a digital PCR kit.

In a third aspect, the present invention provides a reagent, including the primers and probes.

In a fourth aspect, the present invention provides a digital PCR kit, including the primers and probes and a digital PCR reaction master mix.

As a further technical solution, the digital PCR reaction master mix includes: Buffer, Mg ²⁺ , dNTPs, hot-start Taq enzyme and reverse transcriptase.

As a further technical solution, a positive control substance is also included;

The positive controls include H5 subtype H gene fragments, H7 subtype H gene fragments and H9 subtype H gene fragments.

As a further technical solution, the positive control substance is a recombinant plasmid containing H5 subtype H gene fragment, H7 subtype H gene fragment and H9 subtype H gene fragment.

As a further technical solution, a negative control substance is also included;

The negative control substance is nuclease-free water.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides primers and probes for detecting influenza virus H5, H7 and H9 subtypes, wherein the primers have nucleic acid sequences as shown in SEQ ID NO.1-6, and the probes have nucleic acid sequences as shown in SEQ ID NO.7-9 The nucleic acid sequence, the primers and probes have strong specificity and high sensitivity, and can be used to prepare products for detecting influenza virus H5, H7, and H9 subtypes, and realize the detection of influenza virus H5, H7, and H9 subtypes.

The digital PCR kit provided by the invention adopts the method of digital PCR to detect the target nucleic acid, and its detection sensitivity is obviously better than that of fluorescent quantitative PCR or colloidal gold, and can realize the detection of the amount of a single copy template.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is H5 subtype primer probe screening;

Fig. 2 is H7 subtype primer probe screening;

Fig. 3 is H9 subtype primer probe screening;

Fig. 4 is the triple real-time fluorescence quantitative PCR amplification curve diagram of influenza virus H5, H7, H9 subtype;

Fig. 5 is the schematic diagram of the experimental result of the specificity of the primer probe of influenza virus H5 subtype;

Fig. 6 is the schematic diagram of the experimental result of the specificity of the primer probe of influenza virus H7 subtype;

Fig. 7 is the schematic diagram of the experimental result of the specificity of the primer probe of influenza virus H9 subtype;

Figure 8 is a schematic diagram of the experimental results of the sensitivity of the influenza virus H5 subtype primer probe;

Figure 9 is a schematic diagram of the experimental results of the sensitivity of the influenza virus H7 subtype primer probe;

FIG. 10 is a schematic diagram of the experimental results of the sensitivity of the primer probe of influenza virus H9 subtype.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. If no specific conditions are specified, follow the general conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

In a first aspect, the present invention provides a primer and probe for detecting influenza virus H5, H7, and H9 subtypes, and the primers include a first primer pair, a second primer pair, and a third primer pair;

The first primer pair has the nucleic acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2; the second primer pair has the nucleic acid sequences shown in SEQ ID NO.3 and SEQ ID NO.4; The third primer pair has the nucleic acid sequences shown in SEQ ID NO.5 and SEQ ID NO.6;

The probe includes a first probe, a second probe and a third probe;

The first probe has the nucleic acid sequence shown in SEQ ID NO.7; the second probe has the nucleic acid sequence shown in SEQ ID NO.8; the third probe has the nucleic acid sequence shown in SEQ ID NO.9 Nucleic acid sequences shown.

In some preferred embodiments, a fluorescent reporter group is attached to the 5' end of the probe, and a fluorescence quenching group is attached to the 3' end.

In some preferred embodiments, the fluorescent reporter groups include but are not limited to FAM, CY5 or HEX; the fluorescence quenching groups include but are not limited to TAMRA, BHQ1 or BHQ2; Other fluorescent reporter groups and fluorescence quenchers.

The primers and probes for detecting influenza virus H5, H7 and H9 subtypes provided by the present invention have strong specificity and high sensitivity, and can be used to prepare products for detecting influenza virus H5, H7 and H9 subtypes, and realize the detection of influenza virus H5, H7 and H9 subtypes. Detection of H7 and H9 subtypes.

In the second aspect, the present invention provides the application of the above primers and probes in the preparation of influenza virus H5, H7, H9 subtype detection products;

Preferably, the detection product includes a digital PCR kit.

The detection primers and probes provided by the present invention have strong specificity and high sensitivity, and therefore, can be used to prepare products for detecting influenza virus H5, H7, and H9 subtypes, thereby realizing the detection of influenza virus H5, H7, and H9 subtypes. .

In a third aspect, the present invention provides a reagent, including the primers and probes.

The specific types of reagents in the present invention are not limited, and the above-mentioned primers and probes may be included, which can be used for influenza virus H5, H7, and H9 subtypes. Since the above-mentioned reagents contain the above-mentioned primers and probes, they have the advantages of strong specificity and high sensitivity.

In a fourth aspect, the present invention provides a digital PCR kit, including the primers and probes and a digital PCR reaction master mix.

In some preferred embodiments, the digital PCR reaction master mix includes: Buffer, Mg ²⁺ , dNTPs, hot-start Taq enzyme and reverse transcriptase.

The digital PCR kit provided by the present invention adopts the method of droplet digital PCR, which can realize the accurate detection of H5/H7/H9 subtype virus samples, so as to achieve the purpose of early differential diagnosis and epidemic monitoring of influenza virus, so as to provide Influenza virus diagnosis provides a new method.

In some preferred embodiments, a positive control substance is also included, which is used to monitor the homogeneity of the test samples and ensure the comparability of the test results.

In the present invention, the positive controls include H5 subtype H gene fragments, H7 subtype H gene fragments and H9 subtype H gene fragments.

In some preferred embodiments, the positive control substance is a recombinant plasmid containing H5 subtype H gene fragment, H7 subtype H gene fragment and H9 subtype H gene fragment.

In some preferred embodiments, a negative control is also included to monitor whether the detection reagent is contaminated.

In the present invention, the negative control substance is nuclease-free water.

The present invention is further described below through specific examples and comparative examples, however, it should be understood that these examples are only used for more detailed description, and should not be construed to limit the present invention in any form.

Example 1

A primer and probe for detecting influenza virus H5, H7, H9 subtypes, comprising the first primer pair H5-F, H5-R; the second primer pair H7-F, H7-R; the third primer pair H9-F , H9-R; the first probe H5-P; the second probe H7-P; the third probe H9-P, the sequences of primers and probes are shown in Table 1.

Table 1

H5-F GAATCAATAGGAACTTACCAAATACTG (SEQ ID NO. 1) H5-R CTGCATTGTAACGACCCATTG (SEQ ID NO. 2) H5-P TTTATTCAACAGTGGCGAGTTCCC (SEQ ID NO. 7) H7-F CTAATTGATGGTTGGTATGGTTTC (SEQ ID NO. 3) H7-R AATTGCCGATTGAGTGCTTTT (SEQ ID NO. 4) H7-P CAGAATGCACAGGGAGAGGGAACTGCT (SEQ ID NO. 8) H9-F CTGGAATCTGAAGRRACTTACAAA (SEQ ID NO. 5) H9-R AGAACAARAAGGCAGCAAAACC (SEQ ID NO. 6) H9-P ACHATTTATTCGACTGTYGCCTC (SEQ ID NO. 9)

Comparative Example 1-1 to Comparative Example 1-3

Comparative Examples 1-1 Comparative Examples 1-3 provide a primer and a probe for detecting influenza virus H5, respectively, and the sequences of the primers and probes are shown in Table 2.

Table 2

Comparative Example 2-1 to Comparative Example 2-3

Comparative Examples 2-1 to 2-3 respectively provide a primer and a probe for detecting influenza virus H7, and the sequences of the primers and probes are shown in Table 3.

table 3

Comparative Example 3-1 to Comparative Example 3-3

Comparative Examples 3-1 to 3-3 respectively provide a primer and a probe for detecting influenza virus H9, and the sequences of the primers and probes are shown in Table 4.

Table 4

Example 2

A kit comprising the primers and probes of Example 1, a reaction master mix, a positive control substance and a negative control substance. The reaction master mix is: qScript XLT One-Step RT-qPCR ToughMix (QuantaBio).

Positive control substance: recombinant plasmid containing H5 subtype H gene fragment, H7 subtype H gene fragment, and H9 subtype H gene fragment.

Negative control: nuclease-free water.

Test Example 1 Primer Probe Screening

1. Amplification method

Viral nucleic acid extraction was carried out by magnetic bead method, and the nucleic acid was extracted from influenza virus samples using the viral nucleic acid extraction kit produced by Hangzhou Bioer Technology Co., Ltd. Prepare a digital PCR reaction system as shown in Table 5, with a total volume of 25ul.

table 5

digital PCR reaction

Droplet Generation:

Add 25 μL of the reaction system to the droplet reaction chip, close the lid, and transfer it to the Naica automated droplet chip digital PCR instrument. At the same time, turn on the pressure pump and adjust the value to about 1.28, and increase the pressure to atmospheric pressure + 1000 mbar to generate droplets.

PCR amplification:

After the droplets are generated, the PCR amplification program is entered, as shown in Table 6.

Table 6

After the amplification, take out the chip and put it into the droplet reader for data reading.

2. Primer Probe Screening

The nucleic acids of influenza virus H5, H7, and H9 subtypes were extracted respectively. For influenza virus H5 subtype nucleic acid, the first primer pair and the first probe in Example 1, and the samples of Comparative Examples 1-1 to 1-3 were used respectively. The primer probes were amplified according to the above-mentioned amplification method, and the results are shown in Figure 1 . (The curves in Figure 1 are the amplification curves of Example 1, Comparative Example 1-1, Comparative Example 1-2, and Comparative Example 1-3 in order from left to right).

For influenza virus H7 subtype nucleic acid, the second primer pair and the second probe in Example 1, and the primer probes of Comparative Examples 2-1 to 2-3 were amplified according to the above-mentioned amplification method, and the results were as follows: shown in Figure 2. (The curves of FIG. 2 are the amplification curves of Example 1, Comparative Example 2-1, Comparative Example 2-2, and Comparative Example 2-3 in order from left to right).

For influenza virus H9 subtype nucleic acid, the third primer pair and the third probe in Example 1, and the primer probes of Comparative Examples 3-1 to 3-3 were amplified according to the above-mentioned amplification method, and the results were as follows: shown in Figure 3. (The curves in FIG. 3 are the amplification curves of Example 1, Comparative Example 3-1, Comparative Example 3-2, and Comparative Example 3-3 in order from left to right).

Prepare the mixed nucleic acid of influenza virus H5, H7, H9 subtype, for the mixed nucleic acid of influenza virus H5, H7, H9 subtype, adopt the primers and probes provided in Example 1 to amplify according to the above-mentioned amplification method, and the mixed nucleic acid concentration Dilute 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , and 10 ^-5 in turn, and the results are shown in Figure 4 .

It can be seen from the figure that the amplification curves of H5, H7, and H9 subtype primers and probes combined do not affect each other, and the fluorescence intensity is high, and the curve is S-shaped, indicating that the selected primer pairs and probes are very good.

Test Example 2 Digital PCR Specificity Experiment

Select influenza A/H1N1 influenza virus, A/H3N2 influenza virus, A/H5N1 influenza virus, A/H7N9 influenza virus, and A/H9N2 influenza virus that have homology to the nucleic acid sequence of influenza virus and cause the same or similar clinical symptoms. Influenza B virus, novel coronavirus, positive control and negative control are set at the same time. The primers and probes provided in Example 1 were used for detection respectively, and the results were shown in Figure 5, Figure 6 and Figure 7 (in Figure 5, the abscissas represent: 1.H5N1, 2.H1N1, 3.H3N2, 4.5. H7N9, 5H9N2, 6. Influenza B virus, 7 new coronaviruses, 8 negative controls; in Figure 6, the abscissas represent: 1.H7N9, 2.H1N1, 3.H3N2, 4.H5N1, 5H9N2, 6.B Type influenza virus, 7 novel coronavirus, 8 negative control; in Figure 7, the abscissas represent: 1.H9N2, 2.H1N1, 3.H3N2, 4.H5N1, 5H7N9, 6. Type B influenza virus, 7 novel coronavirus virus, 8 negative control), as can be seen from the figure, only H5, H7, H9 can be detected, and other various templates are negative, indicating that the detection method of the present invention does not cross-react with other viruses, and has strong specificity sex.

Test Example 3 Digital PCR Sensitivity Experiment

The positive samples of influenza virus H5, H7, and H9 subtypes were used for 4-fold gradient dilution successively, and the dilution was 4096 times. As shown in Figure 8, Figure 9 and Figure 10 (in Figure 8, the abscissas represent: 1. H5N1 nucleic acid, 2. H5N1 nucleic acid diluted 4 times, 3. H5N1 nucleic acid diluted 16 times, 4. H5N1 nucleic acid diluted 64 times, 5. H5N1 nucleic acid diluted 256 times, 6. H5N1 nucleic acid diluted 1024 times, 7. H5N1 nucleic acid diluted 4096 times; in Figure 9, the abscissas represent: 1. H7N9 nucleic acid, 2. H7N9 nucleic acid diluted 4 times, 3. H7N9 nucleic acid Dilute 16 times, 4.H7N9 nucleic acid is diluted 64 times, 5.H7N9 nucleic acid is diluted 256 times, 6.H7N9 nucleic acid is diluted 1024 times, and 7.H7N9 nucleic acid is diluted 4096 times; in Figure 10, the abscissas represent: 1.H9N2 nucleic acid, 2. H9N2 nucleic acid diluted 4 times, 3. H9N2 nucleic acid diluted 16 times, 4. H9N2 nucleic acid diluted 64 times, 5. H9N2 nucleic acid diluted 256 times, 6. H9N2 nucleic acid diluted 1024 times, 7. H9N2 nucleic acid diluted 4096 times). The results show that the H5 subtype of this system can produce stable amplification at a template amount of 1 copy (No. 6 in FIG. 8 ). The H7 subtype produced stable amplification at a template amount of 2 copies (No. 6 in Figure 9). The H9 subtype can produce stable amplification at a template amount of 1 copy (No. 6 in Figure 10).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

SEQUENCE LISTING

<110> Hangzhou Bioer Technology Co., Ltd.

<120> Primers and probes for detection of influenza virus H5, H7, H9 subtypes and their applications, reagents and digital PCR reagents

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<170> PatentIn version 3.5

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Claims (10)

1. a primer and a probe for detecting influenza virus H5, H7, H9 hypotype, it is characterized in that, described primer comprises the first primer pair, the second primer pair and the 3rd primer pair; The first primer pair has the nucleic acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2; the second primer pair has the nucleic acid sequences shown in SEQ ID NO.3 and SEQ ID NO.4; The third primer pair has the nucleic acid sequences shown in SEQ ID NO.5 and SEQ ID NO.6; The probe includes a first probe, a second probe and a third probe; The first probe has a nucleic acid sequence as shown in SEQ ID NO.7; the second probe has a nucleic acid sequence as shown in SEQ ID NO.8; the third probe has a nucleic acid sequence as shown in SEQ ID NO.8. The nucleic acid sequence shown in 9. 2. The primer and probe according to claim 1, wherein the 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a fluorescence quenching group. 3. primer and probe according to claim 2, is characterized in that, described fluorescent reporter group comprises FAM, CY5 or HEX; Preferably, the fluorescence quenching group includes TAMRA, BHQ1 or BHQ2. 4. the application of the primer described in any one of claim 1-3 and probe in the preparation of influenza virus H5, H7, H9 subtype detection product; Preferably, the detection product includes a digital PCR kit. 5. A reagent, characterized in that it comprises the primer and probe according to any one of claims 1-3. 6. A digital PCR kit, characterized in that it comprises the primers and probes described in any one of claims 1-3 and a digital PCR reaction premix. 7 . The digital PCR kit according to claim 6 , wherein the digital PCR reaction premix solution comprises: Buffer, Mg ²⁺ , dNTPs, hot-start Taq enzyme and reverse transcriptase. 8 . 8. digital PCR kit according to claim 6, is characterized in that, also comprises positive control substance; The positive controls include H5 subtype H gene fragments, H7 subtype H gene fragments and H9 subtype H gene fragments. 9 . The digital PCR kit according to claim 8 , wherein the positive control substance is a recombinant plasmid containing H5 subtype H gene fragment, H7 subtype H gene fragment and H9 subtype H gene fragment. 10 . 10. The digital PCR kit according to claim 6, further comprising a negative control substance; The negative control substance is nuclease-free water.

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