CN110643738A - Primer combination, kit and PSR method for detecting H1N1 influenza A virus - Google Patents
Primer combination, kit and PSR method for detecting H1N1 influenza A virus Download PDFInfo
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Abstract
The invention discloses a primer combination, a kit and a PSR (phosphoenolpyruvate carboxylase) method for detecting H1N1 influenza A virus, belonging to the technical field of biological detection. The invention uses the primer combination to carry out PSR reaction on a sample to be detected, detects the PSR reaction product and determines whether the sample to be detected contains the influenza A H1N1 virus. The method of the invention has simple operation: the reaction system is only required to be put into a constant-temperature water bath kettle at the temperature of 64-67 ℃ to complete the reaction; and the result judgment is simple and convenient: the result can be judged by naked eyes, or by a fluorescence amplification detector or a turbidimeter; rapid and efficient amplification; the specificity is strong: the primer combination can only amplify the RNA of HA gene of influenza A H1N1 virus.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer combination, a kit and a PSR method for detecting H1N1 influenza A virus.
Background
Influenza (influenza), the most common epidemic disease, is very easy to cause various complications and complications, and seriously threatens the life and property safety of people. The disease is acute viral respiratory infectious disease caused by influenza virus (inflenzavirus), is national legal report type C infectious disease, has high infectivity and rapid transmission, can cause outbreak in multiple regions and epidemic in the global scope in a short time, is infectious disease which is mainly detected and prevented all over the world, and is one of infectious diseases which are not effectively controlled by human beings so far.
Influenza viruses, which are representative species of the Orthomyxoviridae family (Orthomyxoviridae), are polymorphic, having a spherical, filamentous, and rod-like structure with an envelope, matrix protein, and core moiety in sequence from outside to inside. Among them, Influenza virus directed only to human is a causative agent of Influenza (flu), and is classified into Influenza a virus (Influenza a virus), Influenza B virus (Influenza B virus), and Influenza c virus (Influenza cv virus) according to difference in Nucleoprotein (NP) and M protein antigenicity, and there is no cross immunity between the three types. Influenza a viruses often cause pandemics, even concurrent outbreaks internationally; influenza b viruses generally cause epidemics in a small range, regionally; the primary infectious subject of influenza c virus is infants and young children, and typically only causes the common type of cold. The animal influenza virus can cause infection and disease attack of various animals such as birds, pigs, horses, bats and the like, and is a pathogen of animal epidemic diseases such as bird flu, swine flu, horse flu and the like.
Influenza a is the most common influenza virus and the most serious pathogen of all influenza viruses, and is variable and infectious. Influenza a viruses have various subtypes with the development of time, are originally called as avian influenza, can infect human beings after the variation of virus genes, have high infectivity to human beings, and once cause pandemics worldwide for many times.
Influenza a viruses are subdivided into many subtypes according to the difference in antigenicity between the external glycoproteins Hemagglutinin (HA) and Neuraminidase (NA), wherein H represents hemagglutinin and is divided into 17 subtypes (H1-H17); n represents neuraminidase, and there are 10 subtypes (N1 to N10), which are designated as SIV subtypes according to their combinations. Influenza b and c viruses currently have no subtype detected. Influenza a viruses have undergone several major variations over the last century, with each new subtype appearing with a larger pandemic. From studies of various subtypes of influenza a viruses, it was found that human can be directly infected with: types a H1N1, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, and H10N 8. The H1, H5 and H7 subtypes are highly pathogenic, and H1N1, H5N1 and H7N9 are worthy of intensive research.
Influenza A H1N1 is a viral infectious disease of respiratory tract, and its etiology is influenza A H1N1 virus, which is named influenza A H1N1 virus because its internal antigens (including inner membrane (M) protein and Nuclear (NP) protein) are classified as type A and its surface antigens Hemagglutinin (HA) and Neuraminidase (NA) are both type 1. HA is the main antigen, and the encoded HA protein HAs immunogenicity, induces hemagglutination inhibition antibodies, HAs the characteristics of hemagglutination inhibition and virus infectivity neutralization, and can enable a human to generate protective antibodies. Neuraminidase (NA) is another major surface antigen belonging to class ii glycoproteins, NA which is associated with the specificity and virulence of the virus, has critical biological functions affecting the release of influenza virions and preventing aggregation of virions, and is one of the major surface antigens of influenza virus.
Influenza a H1N1 is a relatively common viral respiratory virus, and belongs to the group of orthomyxoviridae (0 rtmoylovidae) and Influenza a (Influenza virus a), and its genetic material is RNA, and has a single negative strand structure, a genome of about 13.6kb, and is composed of 8 independent fragments with different sizes, each encoding 10 different proteins: PA (RNA polymerase subunit PA), HA, NA, PBI (RNA polymerase subunit PB1), PB2(RNA polymerase subunit PB2), NP (nucleoprotein), M (matrix protein), NS (non-structural protein, encoded by the same RNA fragment), these proteins are closely related to the pathogenic capability and host specificity of the virus. The typical virus particle is spirally symmetrical, the inside is nucleocapsid, the particle is spherical, the diameter of the particle is 80 nm-120 nm, and the particle is provided with a capsule membrane. The envelope HAs a number of protuberant glycoproteins (also called spikes) arranged in radial arrays, hemagglutinin HA, neuraminidase NA and matrix protein M2. The influenza A H1N1 virus particle has a capsule membrane, so the influenza A H1N1 virus particle is sensitive to organic solvents such as ether, acetone, chloroform and the like; the product can be inactivated at 56 ℃ for 30min, and is sensitive to heat sources; although it is also sensitive to UV light, it is often possible to inactivate the viruses by UV light and to compensate for each other by recombination to produce infectious virions.
The pathogenicity and the fatality rate of influenza caused by the influenza A H1H1 virus are extremely high, and the pathogenicity and the fatality rate pose a serious threat to the safety of human life and property. Influenza patients and asymptomatic infected persons are the main source of infection, and contact with the patient's respiratory secretions and body fluids can also cause infection. After infection, the virus is rapidly propagated in a large amount in respiratory tracts, even finally leads to the degeneration and necrosis of epithelial cells of each bronchus, bronchiole and alveolus, and pathological sections are observed by a microscope and contain a large amount of neutrophilic granulocytes and monocytes.
After a person is infected with influenza A H1N1, the incubation period is 1-7 days, mostly 1-3 days, and potential infectivity exists in the incubation period. Therefore, it is very important to identify and take effective preventive and control measures in time to prevent the influenza A H1N1 from developing into severe cases in the early stage of the influenza A, and to reduce the mortality.
The influenza A H1N1 is an acute viral respiratory infectious disease, the treatment of which is not slow, and in fact, no treatment method with strong operability and no adverse reaction is invented in the current modern scientific research. Influenza is difficult to control so far, and the main reason is the continuous variation of virus surface antigens, especially the variation of antigenicity of surface glycoproteins hemagglutinin and neuraminidase. The outbreak range and the outbreak degree of the influenza virus are closely related to the antigenic variation and the recombination frequency of the influenza virus, and mainly comprise two types, namely antigen drift caused by point mutation of surface proteins HA and NA and antigen transfer caused by reassortment of 8 gene segments. Antigen drift of HA and NA genes can excite the antigenicity of the virus to change, so that the antibody generated by the influenza virus can not prevent the invasion of new variant virus; genetic structural changes have led to strains that can be transmitted from animals to other organisms, even humans, and this is "antigen transfer" which has 3 pathways and often causes pandemics. Vaccines are the most critical medical action during influenza epidemics that can effectively reduce the number of infected individuals and reduce the mortality.
The gene recombination is a more effective way for the influenza virus to escape the immune pressure of the organism, and the novel influenza A H1N1 has strong gene recombination capability. The process of producing a new strain, which is recombinant, after two different types of viruses have infected the same cell at the same time. The reasons for recombination are: the genome of the influenza A H1N1 virus contains 8 RNA segments which are independent of each other, and provides favorable conditions for the RNA segments from each virus strain to be combined and recombined sufficiently, thereby assembling a new virus particle. When detecting the variation and recombination of virus, the change of surface protein (HA and NA) should be studied, and the evolution of internal factors should not be ignored. Meanwhile, because the initial symptoms are not obviously different from the daily cold, whether the human is infected by the influenza A H1N1 virus or not can not be judged from clinical signs, and laboratory reagents and equipment are required to be used for detection. Therefore, it is an urgent problem to be solved by those skilled in the art to provide a PSR method capable of rapidly detecting and diagnosing H1N1 influenza a virus on site.
Disclosure of Invention
In view of the above, the invention provides a primer combination, a kit and a PSR method for detecting H1N1 influenza A virus, which are simple to operate, rapid and efficient in amplification and strong in specificity.
In order to achieve the purpose, the invention adopts the following technical scheme:
a primer combination for detecting H1N1 influenza A virus has the following specific primer sequences:
6-FP：5’-CCTGTACGACGGCAATGTGGAACAGTGTCATCATTTGAAAGGTTT-3’；SEQ ID NO.21；
6-BP：5’-AAGGTGTAACGGCAGCATGTCCGAATTTCCTTTTTTAACTAGCCAT-3’；SEQ ID NO.22；
6-LF：5’-ACTTGTCTTGGGGAATATCTC-3’；SEQ ID NO.23；
6-LB：5’-ATGCTGGAGCAAAAAGCT-3’；SEQ ID NO.24。
further, a kit comprising the above primer combination.
Further, the kit also comprises 2XPSR reaction buffer, a double-indication system and a positive control.
Further, the 2XPSR reaction buffer contained the following components: pH8.8Tris-HCl, 20 mmol; KCl, 50 mmol; MgSO (MgSO)4，8mmol；(NH4)2SO4，10mmol；Tween20，0.1％；Betaine，0.8mol。
Further, the dual indicator system comprises a color developing liquid and/or a fluorescent dye; the color development liquid comprises 0.08mM cresol red and 0.02mM phenol red; the fluorescent dye is SYBR Green I or EveGreen.
Further, the positive control is influenza a H1N1 virus.
Further, a PSR method for detecting H1N1 influenza a virus, comprising the steps of: and carrying out PSR reaction on the sample to be detected by using the primer combination, detecting a PSR reaction product, and determining whether the sample to be detected contains the influenza A H1N1 virus.
Further, the PSR reaction system is as follows: mu.L of template RNA, 2.4. mu.L of primer mixture, 12.5. mu.L of 2XPSR reaction buffer, 3.5. mu.L of dNTPs, 1.0. mu.L of Bst DNA polymerase, and 1.0. mu.L of AMV reverse transcriptase are made up to 25. mu.L with double distilled water;
the primer mixed solution comprises 0.8 mu L of each of 50 mu mol/L FP and BP and 0.4 mu L of each of 50 mu mol/L LF and LB; the molar ratio of the main primers FP and BP to the primers LF and LB can be 2-4: 1.
The final concentration of the dNTPs is 1.4 mmol/L/seed;
the final concentrations of Bst DNA polymerase and AMV reverse transcriptase were 8U.
Further, the reaction condition of the PSR reaction is 64-67 ℃ and 30-60 min.
Further, the method for determining whether the sample to be detected contains the influenza A H1N1 virus by detecting the PSR reaction product comprises the following steps: if the amplification can be carried out, the sample to be detected contains H1N1 influenza A virus; if the amplification cannot be carried out, the sample to be tested does not contain the influenza A H1N1 virus.
Further, the method for determining whether the sample to be detected contains the influenza A H1N1 virus by detecting the PSR reaction product is a, b or c:
a. turbidity method: and detecting the turbidity change curve of the PSR reaction product of the sample to be detected by a turbidity meter, if the turbidity change curve of the PSR reaction product of the sample to be detected is in an ascending state, determining that the sample to be detected is the influenza A H1N1 virus, and if the turbidity change curve of the PSR reaction product of the sample to be detected is not in the ascending state, determining that the sample to be detected is not the influenza A H1N1 virus.
Mixing the materials in the PSR reaction system in a reaction tube of 0.2ml, and performing reaction in a LA-320c real-time turbidimeter under the following conditions: 60min at 65 ℃; along with the reaction, the positive reaction tube becomes turbid due to the generated white precipitate by-products, and the turbidity meter can detect the change of the light absorption value at 650nm, so that the reaction can be monitored in real time, the turbidity of the reaction tube is measured every 6s, and a reaction curve is generated; negative controls were set for each reaction with double distilled water.
The reactions that occur during the PSR reaction are as follows:
(DNA)n-1+dNTP→(DNA)n+P2O7 4-
P2O7 4-+2Mg2+-→Mg2P2O7↓ (magnesium pyrophosphate, white precipitate)
b. A color development method: adding a color development liquid into the PSR reaction, observing the color of the PSR reaction product of the sample to be detected, if the PSR reaction product of the sample to be detected is yellow, the sample to be detected is influenza A H1N1 virus, and if the PSR reaction product of the sample to be detected is red, the sample to be detected is not influenza A H1N1 virus.
When the deoxyribonucleotide molecule is combined with a newly synthesized DNA double strand under the action of DNA polymerase, a hydrogen ion is generated at the same time, and the hydrogen ion is gradually increased along with the PSR reaction, so that the pH value of the reaction system is reduced, and the color development is realized through the change of the pH value. A positive reaction occurs and yellow color is displayed because the concentration of hydrogen ions in the reaction solution is increased; the pH at which the negative control occurred was unchanged, and the solution color remained red.
c. Fluorescence method: adding a fluorescent dye into the PSR reaction, and carrying out amplification reaction in a fluorescence quantitative instrument: if an S-type amplification curve appears in the reaction stage and the dissolution curve is positive by a single peak, the reaction is positive; no amplification curve exists in the reaction stage, and no peak appears in the dissolution curve, which is negative; and (4) result judgment standard: under the premise that the positive control generates positive reaction and the negative control generates negative reaction, the sample to be detected generates an S-shaped amplification curve in the reaction stage, the dissolution curve is a single peak and is close to the position of the positive control single peak (the Tm change range is within 1-3 ℃), the sample to be detected is judged to be positive, and otherwise, the sample to be detected is negative.
According to the technical scheme, compared with the prior art, the invention discloses a primer combination and a PSR method for detecting H1N1 influenza A virus, which are simple to operate: the reaction system is only required to be put into a constant-temperature water bath kettle at the temperature of 64-67 ℃ to complete the reaction; the result identification is simple and convenient: the result of color development can be observed by naked eyes, or the result can be judged by a fluorescence detector and a turbidity meter; rapid and efficient amplification; the specificity is strong: the primer combination can only amplify the RNA of HA gene of influenza A H1N1 virus.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a PSR reaction curve diagram of 6 sets of primers for HA gene of influenza A virus H1N1 according to the present invention;
FIG. 2 is a graph showing the reaction curves of 8 PSR temperature gradients of HN6 primer of the present invention;
FIG. 3 is a PSR turbidity assay result of an HA gene PSR specificity test of the influenza A H1N1 virus of the present invention;
wherein each reference numeral represents:
1, influenza a H1N1 virus; 2, negative control; 3, H2N3 influenza a virus; 4, influenza a H5N1 virus; influenza a H7N9 virus; adenovirus type 6, 14; 7, 7-type adenovirus; adenovirus type 8, 55; 9, human parainfluenza virus; 10, respiratory syncytial virus; 11, bordetella pertussis; 12, mycobacterium tuberculosis; 13, staphylococcus aureus; 14, shigella flexneri; 15, staphylococcus epidermidis; 16, enterococcus faecalis;
FIG. 4 is a diagram showing PSR specificity experiment-PSR color development method results of HA gene of influenza A H1N1 virus of the present invention;
FIG. 5 is a PSR fluorescence detection result of specific experiment of influenza A H1N1 HA gene PSR;
wherein each reference numeral represents:
1. influenza a H1N1 virus; 2. negative control; 3. an adenovirus; 4. human parainfluenza virus; 5. respiratory syncytial virus; 6. tubercle bacillus; 7. (ii) bordetella pertussis; 8. staphylococcus aureus bacteria;
FIG. 6 is a diagram showing the sensitivity of the PSR method for detecting H1N1 influenza A virus-the PSR turbidimetry detection result;
FIG. 7 is a graph showing the sensitivity of the PSR method of the present invention to H1N1 influenza A virus-PSR coloration;
FIG. 8 is a graph showing the sensitivity of the PCR method of the present invention for detecting H1N1 influenza A virus;
wherein each reference numeral in fig. 7-8 represents:
1. 1000000 copies/. mu.l, 2, 100000 copies/. mu.l, 3, 10000 copies/. mu.l, 4, 1000 copies/. mu.l, 5, 100 copies/. mu.l, 6, 10 copies/. mu.l, 7, 1 copies/. mu.l, 8, 0 copies/. mu.l, 9, negative control.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Preparation of 2xPSR reaction buffer (2 × RM):
(1) preparation of 20 × RM pre: 2.64g of ammonium sulfate, 1.49g of potassium chloride, 3.95g of magnesium sulfate heptahydrate and 2.0ml of Tween 20 are fully dissolved in a 100ml volumetric flask, the pH value is adjusted to 8.0 by using 1% KOH solution, finally the volume is fixed to 100ml, and the mixture is stored at 4 ℃.
(2) Preparation of betaine solution (0.375 g/ml): taking 7.5g betaine in a 20ml volumetric flask, fully dissolving and fixing the volume to 20ml, and storing at 4 ℃.
(3) Preparation of 2 × RM: the reaction buffer solution of PSR should avoid repeated freeze thawing, so it is generally prepared into 2ml small system; mu.l of 20 XMM pre was pipetted into a 2ml centrifuge tube, 53.33. mu.l of Tris-HCl solution (1.5M, pH8.8), 1.0ml of betaine solution (0.375g/ml) and 746.67. mu.l of deionized water were added to make up 2ml, and the tube was stored at-20 ℃.
Color development liquid: 0.0765g of cresol red (Sigma, USA) and 0.0177g of phenol red (Sigma, USA) are weighed and dissolved in 100ml of pure water, and the molar concentrations of the cresol red and the phenol red are 2.0mM and 0.5mM respectively; when in use, 1. mu.l of the color developing solution is taken in a 25. mu.l PSR reaction system.
Example 1 determination of optimal primers
6 sets of primers were designed based on the gene sequence of the specific gene HA gene (GenBank accession number FJ966974.1) of influenza A H1N1 virus, see Table 1 for details.
TABLE 1 influenza A H1N1 HA Gene PSR primer sequences
And (3) carrying out PSR reaction by respectively using the 6 sets of primers, detecting a PSR reaction product, and determining whether a sample to be detected contains the influenza A H1N1 virus.
The PSR reaction system is as follows: mu.L of template RNA, 12.5. mu.L of 2XPSR reaction buffer, 2.4. mu.L of primer mix (0.8. mu.L each of FP and BP at 50. mu. mol/L, 0.4. mu.L each of LF and LB at 50. mu. mol/L), 3.5. mu.L of dNTPs, 1.0. mu.L of Bst DNA polymerase, and 25. mu.L of double distilled water.
The reaction conditions were 65 ℃ for 60min, and the results are shown in FIG. 1. As can be seen from the figure, the 6 th primer HN6 has the PSR reaction firstly, the reaction time and the amplification amount are better than those of other primers, and the primer combination is selected as the optimal primer combination.
Example 2 determination of optimum reaction temperature
According to the optimal PSR primers selected previously, reactions were carried out at 8 different temperatures ranging from 61 ℃ to 68 ℃ for 60min, and the results are shown in FIG. 2. As can be seen from the figure, the reaction does not have a great difference in the peak-off time between 61 ℃ and 67 ℃, but the curve at 65 ℃ has a large ascending gradient and high amplification efficiency, and is selected as the optimum temperature.
Example 3 specificity test (turbidity method and color development method)
Double distilled water was used as a negative control, and set 6 primer HN6 was used to amplify the RNA of HA gene of influenza A H1N1 virus and 14 pathogen (see Table 2) nucleic acids were used as controls. As can be seen from FIG. 3, the 6 th primer HN6 only amplified RNA of influenza A H1N1 HA gene, and could generate precipitation due to increased turbidity of PSR reaction, while the other control group did not amplify, indicating that the primer is specific to influenza A H1N1 HA gene; as can be seen in FIG. 4, only the influenza A H1N1 virus changed color. To exclude interference from sample bacterial nucleic acids, several bacterial nucleic acids were selected as controls to check for primer specificity.
TABLE 2 viruses and strains used and sources thereof
Example 4 specificity assay (fluorescence method)
Respectively detecting influenza A H1N1 virus, negative control (double distilled water), adenovirus, human parainfluenza virus, respiratory syncytial virus, tubercle bacillus, Bordetella pertussis and Staphylococcus aureus; the results of the fluorescence method are shown in FIG. 5, the influenza A H1N1 virus shows a typical S-shaped amplification curve, the CT value is 15.7, and the virus is judged to be positive; the other samples have no amplification curve and are judged to be negative.
Example 5PSR vs PCR sensitivity
To compare the PSR method with the detection sensitivity of the gold standard PCR method, RNA of H1N1 influenza A virus of a test subject was extracted, quantified and diluted with a 10-fold gradient of RNA to ensure final concentrations of 1000000 copies/. mu.l, 100000 copies/. mu.l, 10000 copies/. mu.l, 1000 copies/. mu.l, 100 copies/. mu.l, 10 copies/. mu.l, 1 copy/. mu.l and 0 copies/. mu.l. The PSR reaction was performed, and the PCR method was used as a reference to determine and evaluate the PSR detection sensitivity. 3-FP and 3-BP were used as PCR primers, the template RNA was identical to that used in the PSR method, and the total reaction volume was 25. mu.l.
The PCR product was electrophoresed at 120V for 35min on a 1% EB-containing agarose gel and examined under a gel imaging system. The reaction system and reaction conditions are shown in tables 3 and 4.
TABLE 3 PCR reaction System
TABLE 4 PCR reaction conditions
10-fold gradient dilution of RNA templates of influenza A H1N1 virus were subjected to PSR reaction and PCR reaction, respectively, and the results of the reactions were compared in sensitivity, as shown in FIGS. 6 to 8. The detection results of the two interpretation methods of the turbidity method detection and the PSR color development method are the same, the PSR detection result is that the lowest detection concentration is 100 copies/mu l, the PCR detection result is an amplification band of 184bp, and the lowest detection concentration is 100 copies/mu l, and the result shows that the sensitivity of the PSR detection method for the influenza A H1N1 is consistent with that of the common PCR detection.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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cgagtcatga ttgggccatg acagtgtcat catttgaaag gttt 44
<210> 18
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 18
agtaccgggt tagtactgag cgaatttcct tttttaacta gccat 45
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 19
acttgtcttg gggaatatct c 21
<210> 20
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 20
<210> 21
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 21
cctgtacgac ggcaatgtgg aacagtgtca tcatttgaaa ggttt 45
<210> 22
<211> 46
<212> DNA
<213> Artificial Sequence
<400> 22
aaggtgtaac ggcagcatgt ccgaatttcc ttttttaact agccat 46
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 23
acttgtcttg gggaatatct c 21
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 24
Claims (10)
1. A primer combination for detecting H1N1 influenza A virus is characterized in that the specific primer sequence is as follows:
6-FP：5’-CCTGTACGACGGCAATGTGGAACAGTGTCATCATTTGAAAGGTTT-3’；SEQ ID NO.21；
6-BP：5’-AAGGTGTAACGGCAGCATGTCCGAATTTCCTTTTTTAACTAGCCAT-3’；SEQ ID NO.22；
6-LF：5’-ACTTGTCTTGGGGAATATCTC-3’；SEQ ID NO.23；
6-LB：5’-ATGCTGGAGCAAAAAGCT-3’；SEQ ID NO.24。
2. a kit comprising the primer combination of claim 1.
3. The kit of claim 2, further comprising a 2x PSR reaction buffer, a dual indicator system, and a positive control.
4. The kit according to claim 3, wherein the 2XPSR reaction buffer comprises the following components: Tris-HCl (pH8.8), 20 mmol; KCl, 50 mmol; MgSO (MgSO)4，8mmol；(NH4)2SO4，10mmol；Tween20，0.1％；Betaine，0.8mol。
5. The kit according to claim 3, characterized in that said dual indicator system comprises a chromogenic solution and/or a fluorescent dye; the color development liquid comprises 0.08mM cresol red and 0.02mM phenol red; the fluorescent dye is SYBR Green I or EveGreen.
6. A PSR method for detecting H1N1 influenza a virus, comprising the steps of: performing a PSR reaction on a sample to be tested by using the primer combination of claim 1, detecting a PSR reaction product, and determining whether the sample to be tested contains the influenza A virus H1N 1.
7. A PSR method for detecting H1N1 influenza A virus according to claim 6, wherein the PSR reaction system is as follows: mu.L of template RNA, 2.4. mu.L of primer mixture, 12.5. mu.L of 2XPSR reaction buffer, 3.5. mu.L of dNTPs, 1.0. mu.L of LBstDNA polymerase and 1.0. mu.L of AMV reverse transcriptase, and making up to 25. mu.L with double distilled water;
the primer mixed solution comprises 0.8 mu L of each of 50 mu mol/L FP and BP and 0.4 mu L of each of 50 mu mol/L LF and LB;
the final concentration of the dNTPs is 1.4 mmol/L/seed;
the final concentration of the BstDNA polymerase and AMV reverse transcriptase was 8U.
8. The PSR method for detecting the influenza A H1N1 virus according to claim 6, wherein the PSR reaction is carried out at 64-67 ℃ for 30-60 min.
9. The PSR method for detecting H1N1 influenza A virus according to claim 6, wherein the PSR reaction product is detected by the method for determining whether the sample to be tested contains H1N1 influenza A virus: if the amplification can be carried out, the sample to be detected contains H1N1 influenza A virus; if the amplification cannot be carried out, the sample to be tested does not contain the influenza A H1N1 virus.
10. The PSR method for detecting H1N1 influenza A virus according to claim 6, wherein the method for detecting the PSR reaction product to determine whether the sample to be tested contains H1N1 influenza A virus is a, b or c:
a. turbidity method: detecting a turbidity change curve of the PSR reaction product of the sample to be detected by a turbidity meter, wherein if the turbidity change curve of the PSR reaction product of the sample to be detected is in an ascending state, the sample to be detected is the influenza A H1N1 virus, and if the turbidity change curve of the PSR reaction product of the sample to be detected is not in the ascending state, the sample to be detected is not the influenza A H1N1 virus;
b. a color development method: adding a color development liquid into the PSR reaction, observing the color of the PSR reaction product of the sample to be detected, if the PSR reaction product of the sample to be detected is yellow, the sample to be detected is influenza A H1N1 virus, and if the PSR reaction product of the sample to be detected is red, the sample to be detected is not influenza A H1N1 virus;
c. fluorescence method: adding a fluorescent dye into the PSR reaction, and carrying out amplification reaction in a fluorescence quantitative instrument: if an S-type amplification curve appears in the reaction stage and the dissolution curve is positive by a single peak, the reaction is positive; no amplification curve exists in the reaction stage, and no peak appears in the dissolution curve, which is negative; and (4) result judgment standard: and on the premise that the positive control generates a positive reaction and the negative control generates a negative reaction, the sample to be detected generates an S-shaped amplification curve in the reaction stage, the dissolution curve is a single peak and is close to the position of the positive control single peak, the sample to be detected is judged to be positive, and otherwise, the sample to be detected is negative.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
Non-Patent Citations (2)
| Title |
|---|
| W. LIU等: "Polymerase Spiral Reaction (PSR)：A novel isothermal nucleic acid", 《SCIENTIFIC REPORTS》 * |
| 马文等: "聚合酶螺旋反应快速检测甲型H1N1流感病毒", 《军事医学》 * |
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