CN114427009A - Multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus - Google Patents
Multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to a multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus. The method comprises the steps of mixing a respiratory tract sample with immunomagnetic beads respectively coupled with influenza A virus antibodies, influenza B virus antibodies and respiratory syncytial virus antibodies, mixing and incubating at a certain temperature, separating immunomagnetic bead-virus complexes by using a magnetic tool, re-suspending the complexes in a salt ion buffer solution, heating and cracking the re-suspended immunomagnetic bead-virus complexes, separating the magnetic beads by using the magnetic tool, and directly detecting a cracked product (liquid part) by using a fluorescent PCR detection reagent.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus.
Background
Currently, methods for influenza virus and respiratory syncytial virus applied in clinical examination mainly include: a method for separately detecting a virus nucleic acid, such as an influenza A virus nucleic acid detection reagent (fluorescence PCR method), a respiratory syncytial virus nucleic acid detection reagent (fluorescence PCR method); a plurality of virus nucleic acid combined detection methods, such as influenza A virus/respiratory syncytial virus nucleic acid combined detection reagent (fluorescence PCR method) and the like. However, the currently applied fluorescence PCR detection method, as an enzymatic reaction, still has the disadvantages of tedious sample treatment, high requirement on nucleic acid purity, long PCR detection time, high automation cost and the like, and the existing detection method can not meet the requirement on sensitivity in clinical examinations such as blood screening and the like; the above disadvantages cause that the current fluorescent PCR detection method for influenza virus and respiratory syncytial virus is limited in application in clinical examination, and especially cannot meet the requirements of outpatient and emergency treatment.
Disclosure of Invention
The invention aims to provide a multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus, which aims to overcome the defects of complex sample treatment, high requirement on nucleic acid purity, long PCR detection time, high automation cost and the like in the prior detection technology.
Based on the purpose, the invention adopts the following technical scheme:
a multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus specifically comprises the following steps:
s1, preparation of immunomagnetic beads: respectively coupling three antibodies of influenza A, influenza B and respiratory syncytial virus to carboxyl modified superparamagnetic nano-beads to obtain immunomagnetic beads of the three coupled virus antibodies,
s2, enrichment of viruses: mixing and incubating sample preserving fluid with positive influenza A virus or positive influenza B virus or positive respiratory syncytial virus with immunomagnetic beads of three kinds of coupled virus antibodies;
s3, separating the immunomagnetic bead-virus complex by using a magnetic tool, and resuspending the immunomagnetic bead-virus complex in TE buffer solution for heating and cracking;
s4, separating the magnetic beads by using a magnetic tool to obtain enriched and concentrated viruses, and directly detecting liquid in the cleavage product by using a multiplex fluorescent PCR reagent, wherein the fluorescent PCR reagent comprises the following primer probes (table 1):
TABLE 1
The main process of the detection method of the invention is as follows: mixing a respiratory tract sample with immunomagnetic beads respectively coupled with an influenza A virus antibody, an influenza B virus antibody and a respiratory syncytial virus antibody, mixing and incubating at a certain temperature, separating an immunomagnetic bead-virus compound by using a magnetic tool, re-suspending the compound in a salt ion buffer solution, heating and cracking the re-suspended immunomagnetic bead-virus compound, separating the magnetic beads by using the magnetic tool, and directly detecting a cracking product (liquid part) by using a fluorescent PCR detection reagent. By using a multiplex fluorescence PCR technology, 13 primer probes are added into the same detection hole, and the detection hole comprises 3 fluorescence channels, so that multiple subtypes of influenza A viruses (novel influenza A H1N1 virus (2009), seasonal influenza A H1N1 virus, H3N2, H5N2 and H7N9), influenza B viruses (Yamagata type and Victoria type) and respiratory syncytial viruses (A type and B type) can be detected. The influenza A, the influenza B and the respiratory syncytial virus can be distinguished during detection, so that the accurate judgment of the disease condition is facilitated, and the early diagnosis and treatment of diseases are assisted.
The fluorescent PCR detection reagent contains a PCR reaction buffer solution with inhibition resistance and anti-interference and DNA polymerase for rapid amplification, and can effectively amplify and detect samples containing various interference substances.
Preferably, the three mixed antibodies of influenza a, influenza b and respiratory syncytial virus are monoclonal virus surface antibodies.
Preferably, the specific method for enriching the virus in step S2 is: sample 200. mu.L of the storage solution was mixed with 5. mu.L each of three kinds of immunomagnetic beads conjugated with virus antibodies, and the mixture was mixed at room temperature (25-37 ℃) for 6-15 minutes.
Preferably, the thermal cracking in step S3 is: heating at 85-95 deg.C for 5-15 min to crack virus and release nucleic acid.
Preferably, the fluorescent PCR reagent further comprises primer probes of the following internal standards:
| primer and method for producing the same | Sequence (5 '-3') | Serial number |
| IC F | CATCGCTGGTAACACCACC | SEQ ID No:14 |
| IC R | AGTCCCTATGACAGAGAGAGAAG | SEQ ID No:15 |
| IC P | CY5-TCACTGGGCAGCAGCATGTGGCACC-BHQ2 | SEQ ID No:16 |
Preferably, the fluorescent PCR reagent (15 μ L) of step S4 comprises the following components:
| composition of | Serial number | Concentration of mother liquor | The dosage per person (mu L) |
| IFA F | SEQ ID No:1 | 100uM | 0.12 |
| IFA F1 | SEQ ID No:2 | 100uM | 0.12 |
| IFA R | SEQ ID No:3 | 100uM | 0.12 |
| IFA P | SEQ ID No:4 | 100uM | 0.045 |
| IFB F | SEQ ID No:5 | 100uM | 0.12 |
| IFB R | SEQ ID No:6 | 100uM | 0.12 |
| IFB P | SEQ ID No:7 | 100uM | 0.045 |
| RSVA F | SEQ ID No:8 | 100uM | 0.10 |
| RSVA R | SEQ ID No:9 | 100uM | 0.10 |
| RSVA P | SEQ ID No:10 | 100uM | 0.08 |
| RSVB F | SEQ ID No:11 | 100uM | 0.10 |
| RSVB R | SEQ ID No:12 | 100uM | 0.10 |
| RSVB P | SEQ ID No:13 | 100uM | 0.06 |
| IC F | SEQ ID No:14 | 100uM | 0.015 |
| IC R | SEQ ID No:15 | 100uM | 0.015 |
| IC P | SEQ ID No:16 | 100uM | 0.015 |
| Purified water | / | 8.125 | |
| Total up to | 9.4 |
Preferably, the PCR amplification reaction conditions are as follows: maintaining at 52 deg.C for 15 min; maintaining at 95 deg.C for 1 min; (95 ℃ for 5 s; 58 ℃ for 30s and fluorescence collected) 45 cycles.
Preferably, the diameter of the superparamagnetic nano bead is 200nm to 350nm, and the surface of the superparamagnetic nano bead is provided with carboxyl (-COOH) groups.
Preferably, the preparation of the immunomagnetic beads specifically comprises the following steps: connecting the monoclonal surface antibody to the surface of the superparamagnetic nano-bead with carboxyl (-COOH) on the surface by using an EDC activation method, and blocking the magnetic bead coupled with the antibody by using BSA.
Preferably, the air-drying preservation method of the immunomagnetic beads comprises the following steps: placing the immunomagnetic beads in an oven, adjusting the temperature to 30-37 ℃, starting ventilation, standing for more than 10 hours, taking out, and sealing and storing at 2-8 ℃.
Preferably, the virus antibody coupled to the immunomagnetic beads is a nail type influenza virus Neuraminidase (NA) antibody, an influenza B virus Neuraminidase (NA) antibody, a respiratory syncytial virus F protein antibody, and is a monoclonal antibody or a polyclonal antibody.
Compared with the prior nucleic acid combined fluorescence PCR detection technology for the influenza A/B virus and the respiratory syncytial virus, the invention has the beneficial effects that: the sample is easy to process, the detection sensitivity is greatly improved due to enrichment and concentration, the specificity is strong through two measures of immune recognition and gene recognition, the fluorescent PCR detection time is short due to the use of a rapid PCR reagent, and the like.
Drawings
FIG. 1 is a schematic diagram of the nucleic acid-based fluorescence PCR detection process for influenza A/B virus and respiratory syncytial virus of the present invention;
FIG. 2 is an amplification curve diagram of the fluorescence PCR detection of the positive samples of influenza A virus, influenza B virus or respiratory syncytial virus after enrichment, wherein a is the positive sample of influenza A virus, b is the positive sample of influenza B virus, and c is the positive sample of respiratory syncytial virus;
FIG. 3 is an amplification curve diagram for direct nucleic acid extraction (influenza A/B and respiratory syncytial virus nucleic acid (fluorescence PCR) assay) detection of influenza A virus, influenza B virus or respiratory syncytial virus positive samples, wherein a, influenza A virus positive samples, b, influenza B virus positive samples, c, respiratory syncytial virus positive samples;
FIG. 4 is a direct fluorescence PCR detection amplification curve diagram of an influenza A virus, influenza B virus or respiratory syncytial virus positive sample stock solution, wherein a is an influenza A virus positive sample, b is an influenza B virus positive sample, and c is a respiratory syncytial virus positive sample;
FIG. 5 is a fluorescent PCR detection amplification curve diagram of a sample supernatant fluid of a positive sample of influenza A virus, influenza B virus or respiratory syncytial virus after virus immunization enrichment, wherein a is the positive sample of influenza A virus, b is the positive sample of influenza B virus, and c is the positive sample of respiratory syncytial virus.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
The reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1 development of detection reagents for influenza A virus, influenza B virus and respiratory syncytial virus
First, design of primer Probe
According to the literature report, the corresponding conserved regions of influenza A virus, influenza B virus and respiratory syncytial virus are selected as the target genes of the kit, and 2 pairs of primers and corresponding probes are respectively designed for each virus. And according to the experimental result, comprehensively considering the aspects of detection rate, amplification linearity, amplification efficiency and the like, and selecting a primer combination for each virus. The primer pairs and probe sequences for each finally selected virus are shown in Table 2.
TABLE 2
The design of the internal standard primer probe of the invention is as follows:
in the invention, human genes are introduced as internal standards, the inhibition of the internal standard genes on the target gene detection and the intensity of internal standard gene amplification signals are comprehensively considered, and finally selected internal standard primers and probe sequences are shown in Table 3 through the contrast screening of different internal standard primer probes.
Primer and probe sequences of internal standard in Table 3
Secondly, establishing and optimizing a reaction system
In order to improve the reliability and the precision of the experiment to the utmost extent and obtain the experiment result with high positive coincidence rate, the invention optimizes the main components of a gene amplification system, particularly enzyme mixed liquor and PCR reaction buffer solution, and specifically comprises the following steps:
the enzyme mixture consists of:
the composition of the amplification reaction solution 1 was:
optimization of primer probe concentration in the amplification reaction solution 2: under the condition that other components in the reaction system are unchanged, the concentration of the primer is subjected to gradient test, the concentration of the probe is subjected to gradient test, and after multiple comparison tests, the finally determined concentration of the primer and the concentration of the probe are shown in table 4.
TABLE 4 composition of amplification reaction solution 2
After the amplification reaction conditions are optimized, the method is determined as follows:
maintaining at 52 deg.C for 15 min; maintaining at 95 deg.C for 1 min; (95 ℃ for 5 s; 58 ℃ for 30s and fluorescence collected) 45 cycles.
Example 2 detection of influenza A, B or respiratory syncytial Virus Positive samples
A multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus, a flow chart is shown in figure 1, and the specific process is as follows:
method and device
1. Preparing nucleic acid combined detection reagent (a non-extraction fluorescence PCR method) for influenza A/B virus and respiratory syncytial virus.
(1) Preparation of PCR reaction solution
| Name of raw materials | Per volume (μ L) |
| Enzyme mixture | 1.6 |
| Amplification reaction solution 1 | 4 |
| |
9.4 |
| Sample(s) | 5 |
(2) Subpackaging PCR reaction solution
The PCR reaction solution was dispensed into a PCR tube at a volume of 15. mu.L/reaction.
(3) Preparation of immunomagnetic beads: respectively coupling three antibodies of influenza A, influenza B and respiratory syncytial virus to carboxyl modified superparamagnetic nano beads to obtain immunomagnetic beads of the three coupled virus antibodies. The diameter of the super-cis nanometer magnetic bead is 200nm-350 nm.
Connecting the monoclonal surface antibody to the surface of a super-cis nano magnetic bead with carboxyl (-COOH) on the surface by using an EDC activation method, and blocking the magnetic bead coupled with the antibody by using BSA.
The air-drying preservation method of the immunomagnetic beads comprises the following steps: placing the immunomagnetic beads in an oven, adjusting the temperature to 30-37 ℃, starting ventilation, standing for more than 10 hours, taking out, and sealing and storing at 2-8 ℃.
2. Sample processing
Taking 3 clinical respiratory tract samples (a sample plate 1 is positive to influenza A virus, a sample 2 is positive to influenza B virus, and a sample 3 is positive to respiratory syncytial virus), and carrying out the following three groups of treatment on each sample in parallel;
(1) treatment group-Immunocontraction group
Respectively adding 5 mu L of immune magnetic beads coupled with influenza A virus antibodies, influenza B virus antibodies and respiratory syncytial virus antibodies into a centrifugal tube, then adding 200 mu L of samples, mixing the samples with immune magnetic beads, and gently blowing and sucking the samples for 5-6 times by using a pipette;
incubation for 10 minutes at room temperature;
performing magnetic separation by using a magnetic frame, discarding the supernatant, and adding 25 mu L of TE into the rest magnetic beads;
heating at 90 deg.C for 5 min. Performing magnetic separation by using a magnetic frame;
taking 5 mu L of supernatant, adding the supernatant into a PCR tube, and preparing for detection on a machine;
(2) control group 1-extraction test group
Adding 200 mu L of sample into a nucleic acid extraction reagent for nucleic acid extraction, adding 5 mu L of extracted nucleic acid into a PCR tube, and preparing for detection on a computer;
(3) control group 2-stock solution test group
After the samples are mixed evenly, directly sampling 5 mu L of the sample, adding the sample into a PCR tube, and preparing for on-machine detection;
(4) control group 3-supernatant test group
Taking the supernatant obtained after the magnetic separation of the immune enrichment group in the step (1) as a sample, heating the sample at 90 ℃ for 5 minutes, taking 5 mu L of the supernatant, adding the supernatant into a PCR tube, and preparing for detection on a computer.
3. Detection on machine
And (3) putting the PCR tube with the sample added into a fluorescence PCR instrument for fluorescence PCR amplification, wherein the detection procedure comprises the following steps: maintaining at 52 deg.C for 15 min; maintaining at 95 deg.C for 1 min; (95 ℃ for 5 s; 58 ℃ for 30s and fluorescence collected) for 45 cycles.
Second, results and analysis
After the operation of the equipment detection program is finished, observing an amplification curve graph, and reading a Ct value;
the amplification curve of the fluorescence PCR detection of the enriched positive sample of the influenza A virus, the influenza B virus or the respiratory syncytial virus without nucleic acid purification is shown in figure 2, and the amplification curves of a control group 1 (not enriched), a control group 2 and a control group 3 are respectively shown in figure 3, figure 4 and figure 5. The detection results (fluorescence curve Ct values) of the treatment groups are shown in Table 5.
TABLE 5
Description of the drawings: FAM: respiratory syncytial virus; and (3) VIC: b, virus of influenza; ROX: influenza A virus.
As can be seen from fig. 2, fig. 3, fig. 4 and fig. 5, and table 5, the detection efficiency (higher Ct value) of the detection control group after the immune enrichment treatment (the present invention) is higher than that of the detection control group after the conventional extraction, and the detection efficiency is also significantly superior to that of the direct amplification; and the comparison with the detection result of the supernatant liquid shows that the immune enrichment of the invention has better virus capture capacity.
Example 3
A primer probe combination for detecting influenza A, influenza B and respiratory syncytial virus consists of primer probes shown in a table 2 and internal standard primer probes shown in a table 3. The combination is used for detecting positive samples of influenza A virus, influenza B virus or respiratory syncytial virus.
Example 4
A detection kit for influenza A, influenza B and respiratory syncytial virus comprises a fluorescent PCR reagent for multiplex fluorescent PCR detection, wherein the fluorescent PCR reagent contains the primer probe combination described in the embodiment 3, and contains DNA polymerase, reverse transcriptase, magnesium ions, dNTP, surfactant and the like.
PCR reagent composition (15 μ L):
the composition of the amplification reaction solution 2 is shown in Table 4.
When the kit is used, 5 mu L of sample is added, and the reaction system is 20 mu L.
The embodiments in the present specification are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same or similar parts in the embodiments are referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points can be referred to the description of the method part.
The multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus provided by the invention is described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall into the protection scope of the claims of the present invention.
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<120> multiple enrichment detection method for influenza A, influenza B and respiratory syncytial virus
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<400> 2
catggartgg ctaaaga 17
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence (IFA R)
<400> 3
ggcattytgg acaaakcgt 19
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (IFA P)
<400> 4
cagtcctcgc tcactgggca c 21
<210> 5
<211> 19
<212> DNA
<213> Artificial sequence (IFB F)
<400> 5
gagcgtyttr atgaaggac 19
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (IFB R)
<400> 6
<210> 7
<211> 26
<212> DNA
<213> Artificial sequence (IFB P)
<400> 7
ccaattcgag cagctgaaac tgcggt 26
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence (RSVA F)
<400> 8
<210> 9
<211> 18
<212> DNA
<213> Artificial sequence (RSVA R)
<400> 9
<210> 10
<211> 26
<212> DNA
<213> Artificial sequence (RSVA P)
<400> 10
ccatcctctg tcrgttcttg ttaagc 26
<210> 11
<211> 23
<212> DNA
<213> Artificial sequence (RSVB F)
<400> 11
aagcaagtat gtaagagaaa gat 23
<210> 12
<211> 22
<212> DNA
<213> Artificial sequence (RSVB R)
<400> 12
aatgtccatt gtgaacataa ta 22
<210> 13
<211> 26
<212> DNA
<213> Artificial sequence (RSVB P)
<400> 13
tccaatatag taggagtaac atcrcc 26
<210> 14
<211> 19
<212> DNA
<213> Artificial sequence (IC F)
<400> 14
catcgctggt aacaccacc 19
<210> 15
<211> 23
<212> DNA
<213> Artificial sequence (IC R)
<400> 15
agtccctatg acagagagag aag 23
<210> 16
<211> 25
<212> DNA
<213> Artificial sequence (IC P)
<400> 16
tcactgggca gcagcatgtg gcacc 25
Claims (10)
1. A multiple enrichment detection method for non-diagnosis or treatment purposes of influenza A, influenza B and respiratory syncytial virus is characterized by comprising the following steps:
s1, preparation of immunomagnetic beads: respectively coupling three antibodies of influenza A, influenza B and respiratory syncytial virus to carboxyl modified superparamagnetic nano-beads to obtain immunomagnetic beads of the three coupled virus antibodies,
s2, enrichment of viruses: mixing and incubating sample preserving fluid with positive influenza A virus or positive influenza B virus or positive respiratory syncytial virus with immunomagnetic beads of three coupling virus antibodies;
s3, separating the immunomagnetic bead-virus complex by using a magnetic tool, and resuspending the immunomagnetic bead-virus complex in TE buffer solution for heating and cracking;
s4, separating the magnetic beads by using a magnetic tool to obtain enriched and concentrated viruses, and directly detecting liquid in the cleavage product by using a multiple fluorescent PCR reagent, wherein the fluorescent PCR reagent comprises the following primer probes:
2. the multiplex enrichment detection method of claim 1, wherein the virus antibody coupled to the immunomagnetic beads is a influenza A Neuraminidase (NA) antibody, an influenza B Neuraminidase (NA) antibody, a respiratory syncytial virus F protein antibody, a monoclonal antibody or a polyclonal antibody.
3. The multiplex enrichment detection method according to claim 1, wherein the enrichment of the virus in step S2 is specifically performed by: sampling 200 mu L of the preservation solution, mixing the preservation solution with 5 mu L of each immunomagnetic bead of three kinds of coupled virus antibodies, and uniformly mixing the mixture for 6 to 15 minutes at room temperature (25 to 37 ℃);
the thermal cracking in step S3 is: heating at 85-95 deg.C for 5-15 min to crack virus and release nucleic acid.
4. The multiplex enrichment detection method of claim 1, wherein the fluorescent PCR reagents further comprise primer probes for the following internal standards:
5. the multiplex enrichment detection method of claim 1, wherein the fluorescent PCR reagent (15 μ L) of step S4 comprises the following components:
。
6. The multiplex enrichment detection method of claim 1, wherein the PCR amplification reaction conditions are: maintaining at 52 deg.C for 15 min; maintaining at 95 deg.C for 1 min; (95 ℃ for 5 s; 58 ℃ for 30s and fluorescence collected) 45 cycles.
7. The multiplex enrichment detection method of claim 1, wherein the immunomagnetic beads are specifically prepared by: connecting the monoclonal surface antibody to the surface of the superparamagnetic nano-bead with carboxyl (-COOH) on the surface by using an EDC activation method, and blocking the magnetic bead coupled with the antibody by using BSA.
8. The multiplex enrichment detection method according to claim 1, wherein the air-dried preservation method of the immunomagnetic beads comprises: placing the immunomagnetic beads in an oven, adjusting the temperature to 30-37 ℃, starting ventilation, standing for more than 10 hours, taking out, and sealing and storing at 2-8 ℃.
9. A primer probe combination for detecting influenza a, influenza b and respiratory syncytial virus, the combination comprising the following primer probes:
and primer probes of the following internal standards:
。
10. A kit for detecting influenza A, influenza B and respiratory syncytial virus, comprising a fluorescent PCR reagent for multiplex fluorescent PCR detection, wherein the fluorescent PCR reagent contains the primer probe combination of claim 9.
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