CN108192996A - A kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings combine and its application - Google Patents
A kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings combine and its application Download PDFInfo
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Abstract
The invention belongs to molecular Biological Detection fields, are related to a kind of multiple RT RPA primers for influenza A virus detection and H1 and H3 partings and combine and its apply.The invention discloses a kind of multiple RT RPA primers for influenza A virus detection and H1 and H3 partings to combine, RT RPA primers including being used to detect influenza A virus Matrix genes, H1 hypotype HA genes and H3 hypotype HA genes, sequence is successively as shown in SEQ ID NO.1 to NO.6.The multiple RT RPA primers combination can be used for detecting influenza A virus and identifying H1 and H3 hypotypes.Present invention system for the first time using multiple RT RPA methods come detect influenza A virus and and H1 and H3 hypotypes are identified, take short, high sensitivity, high specificity, can fast and effeciently be used for the detection of influenza A virus and H1 and H3 partings.
Description
Technical field
The invention belongs to technical field of molecular biological detection, are related to influenza A virus detection and its Subtype side
Method, and in particular to (reverse transcription recombinates enzymatic polymerization to a kind of multiple RT-RPA for influenza A virus detection and H1 and H3 partings
Enzymatic amplification technology) primer combines and its application.
Background technology
Influenza virus (Influenza virus) is the causal organism of common respiratory tract infectious disease, is had high
Incidence and lethality because of the features such as its infectiousness is strong, incubation period is short, pathogenic strong, spread speed is fast, greatly threaten people
Class health and lives.According to incompletely statistics, adult's infection rate of the annual seasonal current Influenza Virus in the whole world is 5~10%, infant
It is 20~30%, causes 25 to 500,000 people dead.On taxology, influenza virus is broadly divided into first, second and the third three classes, three
Orthomyxoviridae family (Orthomyxoviridae) is belonged to, is 8 segments, sub-thread minus-stranded rna virus, wherein due to Flu-A disease
Malicious (Influenza A virus) variability is strong, so as to repeatedly cause to be very popular.According to influenza A virus surface glycoprotein,
Hemagglutinin (Hemagglutinin, HA) and neuraminidase (Neuraminidase, NA), can be divided into a variety of hypotypes,
Middle seasonal current Influenza Virus H1N1 and H3N2 are most commonly seen.
Influenza a virus infection without clinical symptoms, can cause fever, cough, sore-throat, headache etc. to be faced sometimes sometimes
Bed symptom, it is similar to symptom caused by the pathogen infections such as adenovirus, Respiratory Syncytial Virus(RSV), therefore, it is difficult to according to clinical symptoms
Infected by influenza infection carries out precise Identification.Research shows that compared with test in laboratory, the positive prediction of clinical symptoms and sign
It is 18~87% to be worth, and is had greatly uncertain.The common test in laboratory method of influenza A virus mainly has separation to train
Foster, serology and Protocols in Molecular Biology etc.；Influenza virus is separately cultured using chicken embryo or dog kidney passage cell, needs 2~5
It time belongs to hysteresis quality detection, for medical treatment patient, can not provide and timely and effectively diagnose.Clinically, it is main to utilize
Influenza virus quick diagnosis technology detects antigen or neuraminidase activity, although the technology can be detected quickly, sensitivity compared with
It is low, and cannot identify infection hypotype.Detection technique based on nucleic acid is now widely used for detection influenza A virus, so
And most methods are based on PCR technologies, although round pcr can realize more target amplifications and parting detection, this method
Testing cost and technology are more demanding, easy to pollute, the scene of can not carrying out or immediately detection.Therefore, be badly in need of one kind can immediately, it is simple
Just, accurate nucleic acid detection method, detection and Subtype for influenza A virus.
Recombinase polymeric enzymatic amplification technology (Recombinase polymerase amplification, RPA) is a kind of
Nucleic acid isothermal amplification technology forms nucleic acid-protein compound by recombinase and oligonucleotides and carrys out scan templates double-stranded DNA, seeks
Look for homologous sequence, then carry out chain exchange, using single strand binding protein stablize single stranded DNA, prevent oligonucleotides due to migration and
Discharge, and then utilize archaeal dna polymerase synthetic DNA.It is reacted different from PCR, RPA does not need to nucleic acid denaturation, and optimal reaction temperature is
37~41 DEG C, it can also carry out at normal temperatures, without special installation, greatly accelerate reaction speed, it can be by single point in 10min
Son is expanded to 1012.In addition, RPA and reverse transcription reaction (reverse transcription, RT) are combined, it can be to RNA templates
It is expanded.In addition, RPA only needs pair of primers that amplification can be completed, available for multiple reaction system, while multiple targets are expanded
Gene.At present, RPA technologies have been used for the quick detection of a variety of causal organisms；However multiple RPA technologies are applied to Flu-A
Viral diagnosis and parting, then have not been reported.Therefore, based on influenza A genes sequence analysis, specific nucleic acid can be designed
Primer to build the multiple RT-RPA amplification systems of influenza A virus, realizes that quickly detection and H1 and H3 divide influenza A virus
Type instructs patient's rational use of medicines.
Invention content
In view of the deficiencies of the prior art, and in order to improve clinical influenza A virus detection and the technical merit of parting, this
Invention provides a kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings and combines and its apply, this
Be for the first time using multiple RT-RPA methods detection influenza A virus and and H1 and H3 hypotypes are identified, take it is short, sensitive
Degree is high, high specificity, can be efficiently used for the detection of influenza A virus and H1 and H3 partings.
Using bioinformatics technique, according to discovery time and separately point taxonomic revision influenza A genes group sequence
Row, aligned sequences so as to obtain consensus sequence, on this basis, further compare all sequences, obtain influenza A virus
Gene conserved region is separately designed for influenza A virus stromatin (Matrix) gene (for differentiating influenza A virus)
With the RT-RPA amplimers of HA genes (be used for influenza A virus parting), and optimizational primer combination and reaction system are established
Influenza A virus detection and the multiple RT-RPA amplification methods and kit of parting.
For achieving the above object, the present invention adopts the following technical scheme that：
A kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings combine, and are detected including being used for
The RT-RPA primers of influenza A virus Matrix genes, forward primer sequence is as shown in SEQ ID NO.1, reverse primer sequence
Row are as shown in SEQ ID NO.2；For detecting the RT-RPA primers of influenza A virus H1 hypotype HA genes, forward primer sequence
Row are as shown in SEQ ID NO.3, and reverse primer sequences are as shown in SEQ ID NO.4；For detecting influenza A virus H3 hypotypes
The RT-RPA primers of HA genes, forward primer sequence is as shown in SEQ ID NO.5, reverse primer sequences such as SEQ ID NO.6 institutes
Show.
Further, each primer concentration of the multiple RT-RPA primers combination is 10 μM；Influenza A virus
The Product Sequence that Matrix genes, H1 hypotypes and H3 hypotype HA genes are expanded through RT-RPA is respectively such as SEQ ID NO.7, SEQ
Shown in ID NO.8 and SEQ ID NO.9.
The inspection combined containing a kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings
Test agent box, the kit is detected for influenza A virus and H1 and H3 partings, and kit includes described in claim 1
Multiple RT-RPA primers combination.The kit also comprises the following components：Buffer solution, enzyme reaction solution, without RNase water, reverse transcription
Enzyme, RNase inhibitor, magnesium acetate solution, negative control and positive control；The positive control is 1.0 × 105 copies/
The external synthesizing single-stranded RNA standard items of μ L；The negative control is normal person's throat swab of inactivation.
The application that a kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings combine, profit
Influenza A virus is detected with the multiple RT-RPA primers combination and H1 and H3 hypotypes are identified.
A kind of multiple RT-RPA methods for being used for influenza A virus detection and H1 and H3 partings, include the following steps：
(1) sample to be tested total serum IgE is extracted；
(2) multiple RT-RPA primers combination described in claim 1 is designed and synthesized, to extract the total serum IgE of sample to be tested
For template, multiple RT-RPA amplifications are carried out；After RT-RPA amplifications, influenza A virus Matrix genes, H1 hypotypes and H3 hypotypes
Product Sequence of the HA genes through amplification is respectively as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9；
After (3) 42 DEG C of water-bath 4min, mixing, 42 DEG C of reaction 30min；It is detected again through agarose gel electrophoresis；According to electrophoresis
As a result judged：If there is the band of 279bp sizes, contain influenza A virus in sample to be tested；If occur 279 simultaneously
Two bands of bp and 216bp sizes then contain influenza A virus H1 hypotypes in sample to be tested；If occur 279bp and 394 simultaneously
Two bands of bp sizes then contain influenza A virus H3 hypotypes in sample to be tested；If occur simultaneously 279bp, 216bp and
Tri- bands of 394bp then contain influenza A virus H1 and H3 hypotype simultaneously in sample to be tested；If there are not 279bp sizes
Band, and there is the band of 216bp or/and 394bp, then need sequence verification again.
1. design of primers.
(1) it is a pair of for expanding the RT-PRA primers of influenza A virus Matrix genes, as influenza A virus
Detection primer, sense primer is as shown in SEQ ID NO.1, shown in downstream primer SEQ ID NO.2.
Influenza A virus Matrix gene RT-RPA detection primers (influenza A virus detection primer)：
MF146：GGCTCTCATGGAATGGCTAAAGACAAGAC (SEQ ID NO.1),
MR425：TTGTATATGAGGCCCATGCAACTGGCAAGTG(SEQ ID NO.2)；
(2) it is a pair of for detecting the RT-RPA primers of influenza A virus H1 type HA genes, as influenza A virus H1
The detection primer of type, sense primer is as shown in SEQ ID NO.3, shown in downstream primer SEQ ID NO.4.
Influenza A virus H1 type RT-RPA amplimers (H1 serotype specific primers)：
H1F1325：TGTTGGTTCTATTGGAAAATGAAAGAACTTT (SEQ ID NO.3),
H1R1540：TGTTTAATTTTGCTTCCTCTGAGTATTTTGG(SEQ ID NO.4)；
(3) it is a pair of for detecting the RT-RPA primers of influenza A virus H3 type HA genes, as influenza A virus H3
Type detection primer, sense primer is as shown in SEQ ID NO.5, shown in downstream primer SEQ ID NO.6.
Influenza A virus H3 type RT-RPA amplimers (H3 serotype specific primers)：
H3F763：GAATAAGCATCTATTGGACAATAGTAAAAC(SEQ ID NO.5)
H3R1156：CCCTCAGAATTTTGATGCCTGAAACCGTACCA(SEQ ID NO.6).
2. multiple RT-RPA methods.
The present invention also provides a kind of for influenza A virus detection and the multiple RT-RPA methods of H1 and H3 type partings.
(1) multiple RT-RPA amplimers group, the primer sets include following primer pair：
Influenza A virus Matrix gene RT-RPA amplimers are to (influenza A virus detection primer)：
MF146：GGCTCTCATGGAATGGCTAAAGACAAGAC (SEQ ID NO.1),
MR425：TTGTATATGAGGCCCATGCAACTGGCAAGTG(SEQ ID NO.2)；
Influenza A virus H1 type RT-RPA amplimers (H1 serotype specific primers)：
H1F1325：TGTTGGTTCTATTGGAAAATGAAAGAACTTT (SEQ ID NO.3),
H1R1540：TGTTTAATTTTGCTTCCTCTGAGTATTTTGG(SEQ ID NO.4)；
Influenza A virus H3 type RT-RPA amplimers (H3 serotype specific primers)：
H3F763：GAATAAGCATCTATTGGACAATAGTAAAAC (SEQ ID NO.5),
H3R1156：CCCTCAGAATTTTGATGCCTGAAACCGTACCA(SEQ ID NO.6).
Sense primer and downstream primer in above-mentioned primer pair are 10 μM；A type stream is expanded using multiple RT-RPA methods
The Product Sequence of Influenza Virus Matrix genes, H1 type HA genes and H3 type HA genes is respectively SEQ ID NO.7, SEQ ID
Shown in NO.8 and SEQ ID NO.9.
(2) standard items and other reactants.Influenza A virus Matrix gene RNAs standard items, H1 type HA gene RNAs
Standard items, H3 type HA gene RNAs standard items, reaction buffer inhibit without RNase water, enzyme reaction solution, reverse transcriptase, RNase
Agent, magnesium acetate (MgAc), positive control and negative control.
It is as follows for building the primer sequence of each RNA standard items of influenza A virus：
A, for building the primer of the RNA standard items of influenza A virus Matrix genes：
MF102：GATCACTAATACGACTCACTATAGGGCAGAGACTTGAAGATGTCTT (SEQ ID NO.10),
MR502：TGCTGGGAGTCAGCAATCTG(SEQ ID NO.11)；
B, for building the primer of the RNA standard items of influenza A virus H1 type HA genes：
H1F1083：GATCACTAATACGACTCACTATAGGGGGTAGATGGATGGTACGGTT (SEQ ID NO.12),
H1R1612：CGACAGTTGAATAGATCGCC(SEQ ID NO.13)；
C, for building the primer of the RNA standard items of influenza A virus H3 type HA genes：
H3F376：GATCACTAATACGACTCACTATAGGGTTATGCCTCCCTTAGGTCAC (SEQ ID NO.14),
H3R1156：CCCTCAGAATTTTGATGCCTG(SEQ ID NO.15).
Using in-vitro transcription technology, it is respectively synthesized influenza A virus Matrix gene RNAs standard items, H1 type HA genes
RNA standard items and H3 type HA gene RNA standard items, structure and optimization for multiple RT-RPA reaction systems.
RNA standard items (influenza A virus Matrix gene RNAs standard items, H1 types HA gene RNAs standard items and H3 types
HA gene RNAs standard items), using influenza A virus Matrix genes, H1 types and H3 type HA genes as target gene, using with
The primer (SEQ ID NO.10 to SEQ ID NO.15) of t7 rna polymerase promoter sequence is synthesized double using RT-PCR technology
Chain DNA, then using the synthesizing single-stranded RNA of t7 rna polymerase, single stranded RNA sequence is followed successively by SEQ ID NO.16, SEQ ID
NO.17 and SEQ ID NO.18 (being the RNA standard items of Matrix genes, H1 type HA genes and H3 type HA genes).
The no RNase water is stayed overnight for 37 DEG C, then high steam using 0.5% pyrocarbonic acid diethyl ester processing deionized water
Sterilizing.
The negative control is normal person's oropharyngeal swab specimen of inactivation.
The positive control is a concentration of 1 × 105Copies/ μ L single stranded RNA standard items.
Described reaction buffer (10mM Tris (pH 8.0), 5%DMSO, 5%PEG-1000,4mM DTT, 0.5mM
EDTA, 0.1mg/mL BSA, 2mM dATP, 200mM dNTPs), enzyme reaction solution (30ng/ μ L Bsu, 250ng/ml gp32,
140ng/ml UvsX, 22ng/ml UvsY), reverse transcriptase and RNase inhibitor be commercially available in market.
(3) RT-RPA reaction systems.
A kind of detection influenza A virus and the multiple RT-RPA methods amplification system of parting include：Every 50 μ L amplification systems
In the 29.5 μ L containing buffer solution, 6 μ L of primer sets, 0.5 μ L, RNase inhibitor of reverse transcriptase 0.5 μ L, no RNase water 8.0 μ L are to be measured
Sample rna extracting solution is 3.0 μ L, and reaction starts 2.5 μ L of liquid.Reaction condition is：42 DEG C of 4min of water-bath, 42 DEG C of 30min after mixing.
Electrophoretic analysis is carried out using 1.5% Ago-Gel, detects influenza A virus and analysis infection (H1 in medical specimen
And H3) hypotype.
(4) methodology validation.
The advantages and positive effects of the present invention are as follows：
(1) single tube Multiple detection：The present invention establishes the multiple RT-RPA inspections of influenza A virus and H1 and H3 hypotypes for the first time
Survey method can once realize the detection of three target genes, so as to be directly realized by the detection of influenza A virus and identification H1
With H3 hypotypes, testing cost is reduced, detection time is saved, can quickly detect immediately；
(2) high sensitivity：The present invention detects influenza A virus and H1 and H3 partings using multiple RT-RPA methods, for
Matrix genes, H1 and H3 type HA genes are minimum to can detect 104Copies/ reacts；
(3) specificity is high：In the present invention, using bioinformatics method, carried out according to the discovery time of virus and place
Classification, then obtains its conserved sequence, and then on this basis, compares analysis, designs primer, coverage height and specificity
By force；
(4) without especial equipment requirements：The reaction condition of the method applied in the present invention (multiple RT-RPA) is isothermy
(37-42 DEG C), it is easy to use without special heating equipment.
In conclusion the multiple RT-RPA methods for being used for influenza A virus detection and its subtype typing of the present invention,
Specific high, applicability is wide, saves detection time, and easy to operate, can be effectively applied to the inspection of clinical medicine sample
It surveys.
Description of the drawings
Fig. 1 is using primer specifics of the subtypes of influenza A virus H1N1 and H3N2 as the multiple RT-RPA methods of template
Property detection electrophoretogram；Wherein, Lane M are DL2000DNA Marker；Lane 1-7：H1N1 positive samples, H3N2 are positive
Sample, H1N1 and H3N2 mixing positive sample, influenza B virus positive sample, Respiratory Syncytial Virus(RSV) A positive samples, breathing
Road syncytial virus B positive samples, negative control；Primer is MF146/MR425, H1F1325/H1R1540 and H3F763/
H3R1156。
Fig. 2 is the electrophoretogram for verifying multiple RT-RPA methods detection sensitivity；Wherein, Lane M are DL2000 DNA
Marker；Lane 1-7 are that the multiple RT-RPA of single stranded RNA template of gradient dilution expands electrophoresis result；A is mono- for Matrix and H1
Chain RNA template mixture gradient dilution liquid；B is Matrix and H3 single stranded RNA template mixture gradient dilution liquid；C is
Matrix, H1 and H3 single stranded RNA template mixture gradient dilution liquid.
Fig. 3 is the electrophoretogram of 19 parts of collect specimen testing results；Wherein, Lane M are DL2000DNA Marker；Lane
1-19 is oropharyngeal swab specimen testing result；A is the testing result using multiple RT-RPA methods；B and C is respectively Matrix genes
With the RT-Nested PCR method testing result of H1 type HA genes.
Specific embodiment
Technical solutions according to the invention are further described in detail below by specific embodiment, but it is necessary to
It points out that following embodiment is served only for the description to invention content, does not form limiting the scope of the invention.
All solvents and reagent that the present embodiment uses are that commercially available finished product, primer and probe give birth to work biotechnology for Shanghai
Co., Ltd (Shanghai) synthesizes；Viral RNA extracts kit (Code No.DP315-R) is purchased from Tiangeng biochemical technology Co., Ltd
(Beijing)；TwistAmp Basic Kit are purchased from TwistDx companies (Britain)；Reverse transcriptase AMVReverse
Transcriptase (Code No.M5108) and RNase inhibitor (Code No.N2111) are (beautiful purchased from Promega companies
State, WI, Madison).
1. the preparation of standard items
(1) nucleic acid extraction
Reference virus RNA extracts kits (Tiangeng is biochemical, Code No.DP315-R) specification, using the side of column chromatography
RNA in method extraction oropharyngeal swab specimen, it is specific as follows：1.0mL oropharyngeal swab specimens are taken, 12000r/min centrifugation 20min are sucked
After 860 μ L supernatants, 560 μ L Carrier RNA working solutions are added in, be vortexed concussion 15s, is then placed at room temperature for 15 min, of short duration
After centrifugation, 560 μ L absolute ethyl alcohols are added in, 15s is shaken, is transferred in adsorption column (RNase-Free adsorption column CR2), 8000rpm
1min abandons waste liquid, and adsorption column is put back in collecting pipe, is separately added into solution GD and RW and is cleaned, be eventually adding 60 μ L without
RNase water elutions.
(2) RT-PCR is expanded
Reverse transcription (RT) reaction system (20 μ L)：In the PCR pipe of 0.2 μ L, it is separately added into 5 μ L RNA, 1.0 μ L
Random Primer(0.5μg/μL)、MgCl2(25mM)4μL、Reverse Transcription 10×Buffer 2μL、
dNTP Mixture (10mM)2μL、AMV Reverse Transcriptase 0.25μL、RNase Inhibitor 0.25μ
L supplies no RNase water to 20 μ L.Reaction condition：20 DEG C of 10min, 42 DEG C of 30min.
PCR amplification system (25 μ L)：2 × PCR Mix, 12.5 μ L, sense primer (10 μM) and downstream primer (10 μM) are each
0.5 2 μ L of μ L, cDNA, supply no RNase water to 25 μ L.Primer pair (MF102/MR502, H1F1083/ is respectively adopted
H1R1612 and H3F376/H3R1156) carry out PCR amplification.Reaction condition：95℃5min；95℃30s, 50℃30s,72℃
30s；95℃30s,55℃30s,72℃30s；72℃10min.PCR product is subjected to 1.2% agarose gel electrophoresis analysis, is cut
Glue recycles, and is sequenced.
(3) synthesis of single stranded RNA
In the PCR pipe of 0.2mL, 10 × Transcription Buffer 2 μ L, ATP (50mM) 2 μ L are sequentially added,
GTP (50mM) 2 μ L, CTP (50mM) 2 μ L, UTP (50mM) 2 μ L, RNase Inhibitor (40 U/ μ L) 0.5 μ L, T7RNA
2 μ L of Polymerase (50U/ μ L), 5 μ L purified pcr products, 42 DEG C of reaction 2h.After room temperature cooling, RNase free are added in
DNase I (5U/ μ L) 2 μ L, 37 DEG C of 2h.Finally, purifying obtains single stranded RNA, and RNA concentration is measured using micro-spectrophotometer.
2. the design and screening of primer
The influenza A genes sequence delivered in GeneBank databases is very numerous, and variability is big, to improve
The applicability and specificity of primer, by the gene order in database according to delivering the time and classifying with finding, utilization is soft
Part DNAMAN carries out sequence alignment, obtains the homogeneity sequence of different regions and time of occurrence, is then compared again
It is right, obtain gene conserved region.In addition, for RPA primers, since recombinase is combined with primer, so as to be carried out to target zones
Amplification, for primer length generally in 30~35nt, longer primer, which is easy to cause mispairing and forms secondary structure, causes amplification to imitate
Rate is relatively low.Therefore, the present embodiment devises multipair primer, and carries out primer screening using substance RT-RPA methods, after screening
RPA primers it is as shown in table 1.
RPA primers after the screening of table 1
3. the foundation of multiple RT-RPA amplification systems
Using the above-mentioned RPA primers screened, the single stranded RNA (1 × 10 synthesized with in-vitro transcription5Copies/ μ L) it is mould
Plate carries out the optimization of multiple RT-RPA amplification systems.RT-RPA amplification systems (50 μ L)：It is sequentially added in 0.2mL centrifuge tubes
10 μM of 6 μ L of primer are (specific as follows：0.75 1.0 1.5 1.5 μ L of μ L, H1R1540 of μ L, H1F1325 of μ L, MR425 of MF146,
0.5 0.75 μ L of μ L, H3R1156 of H3F763), 29.5 μ L of buffer solution, template ribonucleic acid is 3 0.5 0.5 μ of μ L, RNase of μ L, AMV
L supplies no RNase water to 47.5 μ L, after mixing, adds in MgAc (280mM) 2.5 μ L.Centrifuge tube is placed in 42 DEG C of incubation 4min
Afterwards, mixing then reacts 30min for 42 DEG C.Add in 50 μ L phenol chloroforms solution (1:1), after mixing, 12000rpm centrifugations
1min removes supernatant, and 10 μ L is taken to carry out 1.5% agarose gel electrophoresis.
4. specificity and sensitivity analysis
It will take each 1mL of oropharyngeal swab specimen of the Influenza virus H1N1 positive and the H3N2 positives, after mixing, extraction virus
RNA takes 3 μ L RNA extracting solutions to be detected using multiple RT-RPA respectively.The results are shown in Figure 1, is expanded from H1N1 positive samples
The primer size gone out is respectively 279 and 216bp, consistent with expected results；Amplifying primer size from H3N2 positive samples is
As a result two bands of 394 and 279bp are consistent with expection；3 specific items are amplified from two kinds of mixing samples of H1N1 and H3N2
Band, respectively 216,279 and 394bp；For influenza B virus, Respiratory Syncytial Virus(RSV) A and B positive sample and feminine gender
Any band is not amplified in control.The result shows that designed primer has specificity, available for influenza A virus
The detection of Matrix, H1 and H3 gene and parting.
Gradient dilution synthesizing single-stranded RNA (Matrix, H1 and H3), dilution gradient is followed successively by 1 × 108、1×107、1×
106、 1×105、1×104、1×103、1×102Copies/ μ L, respectively take the 10 above-mentioned dilutions of μ L, and mixing takes 3 μ L as mould
Plate is detected using multiple RT-RPA amplification systems, after the completion of reaction, carries out 1.5% agarose gel electrophoresis analysis.Knot
Fruit is as shown in Fig. 2, the detection of multiple RT-RPA amplifications is limited to 104copies/reaction。
5. detection application
In December, 2015 is acquired to 19 throat swabs of Nanjing General Hospital, Nanjing Military Area Command, PLA Pediatric Clinic patient between in March, 2016
Sample extracts viral RNA using kit, RT-Nested PCR is respectively adopted and multiple RT-RPA methods are detected, as a result such as
Shown in Fig. 3, show that two methods testing result is consistent, can detect three Matrix genes and H1 gene masculine samples.
SEQUENCE LISTING
<110>Nanjing General Hospital, PLA Nanjing Region
<120>A kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings combine and its application
<130> 1
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213>Artificial sequence
<400> 1
ggctctcatg gaatggctaa agacaagac 29
<210> 2
<211> 31
<212> DNA
<213>Artificial sequence
<400> 2
ttgtatatga ggcccatgca actggcaagt g 31
<210> 3
<211> 31
<212> DNA
<213>Artificial sequence
<400> 3
tgttggttct attggaaaat gaaagaactt t 31
<210> 4
<211> 31
<212> DNA
<213>Artificial sequence
<400> 4
tgtttaattt tgcttcctct gagtattttg g 31
<210> 5
<211> 30
<212> DNA
<213>Artificial sequence
<400> 5
gaataagcat ctattggaca atagtaaaac 30
<210> 6
<211> 32
<212> DNA
<213>Artificial sequence
<400> 6
ccctcagaat tttgatgcct gaaaccgtac ca 32
<210> 7
<211> 279
<212> DNA
<213>Artificial sequence
<400> 7
ggctctcatg gaatggctaa agacaagacc aatcctgtca cctctgacta aggggatttt 60
ggggtttgtg ttcacgctca ccgtgcccag tgagcgagga ctgcagcgta gacgctttgt 120
ccaaaatgcc ctcaatggga atggggatcc aaataacatg gacagagcag ttaaactata 180
tagaaaactt aagagggaga taacattcca tggggccaaa gaaatagcac tcagttattc 240
tgctggtgca cttgccagtt gcatgggcct catatacaa 279
<210> 8
<211> 181
<212> DNA
<213>Artificial sequence
<400> 8
tgttggttct attggaaaat gaaagaactt tggactatca cgattcaaat gtgaagaact 60
tgtatgaaaa agtaagaaac cagttaaaaa acaatgccaa ggaaattgga aacggctgct 120
ttgaatttta ccacaaatgc gataacacgt gcatggaaag tgtcaaaaat gggacttatg 180
actacccaaa atactcagag gaagcaaaat taaaca 216
<210> 9
<211> 394
<212> DNA
<213>Artificial sequence
<400> 9
gaataagcat ctattggaca atagtaaaac cgggagacat acttttgatt aacagcacag 60
ggaatctaat tgctcctagg ggttacttca aaatacgaag tgggaaaagc tcaataatga 120
gatcagatgc acccattggc aaatgcaatt ctgaatgcat cactccaaat ggaagcattc 180
ccaatgacaa accattccaa aatgtaaaca ggatcacata cggggcctgt cccagatatg 240
ttaagcaaaa cactctgaaa ttggcaacag gaatgcgaaa tgtaccagag aaacaaacta 300
gaggcatatt tggcgcaata gcgggtttca tagaaaatgg ttgggaggga atggtggatg 360
gttggtacgg tttcaggcat caaaattctg aggg 394
<210> 10
<211> 46
<212> DNA
<213>Artificial sequence
<400> 10
gatcactaat acgactcact atagggcaga gacttgaaga tgtctt 46
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<400> 11
tgctgggagt cagcaatctg 20
<210> 12
<211> 46
<212> DNA
<213>Artificial sequence
<400> 12
gatcactaat acgactcact atagggggta gatggatggt acggtt 46
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<400> 13
cgacagttga atagatcgcc 20
<210> 14
<211> 46
<212> DNA
<213>Artificial sequence
<400> 14
gatcactaat acgactcact atagggttat gcctccctta ggtcac 46
<210> 15
<211> 21
<212> DNA
<213>Artificial sequence
<400> 15
ccctcagaat tttgatgcct g 21
<210> 16
<211> 427
<212> DNA
<213>Artificial sequence
<400> 16
gatcactaat acgactcact atagggcaga gacttgaaga tgtctttgct gggaaaaaca 60
cagatcttga ggctctcatg gaatggctaa agacaagacc aatcctgtca cctctgacta 120
aggggatttt ggggtttgtg ttcacgctca ccgtgcccag tgagcgagga ctgcagcgta 180
gacgctttgt ccaaaatgcc ctcaatggga atggggatcc aaataacatg gacagagcag 240
ttaaactata tagaaaactt aagagggaga taacattcca tggggccaaa gaaatagcac 300
tcagttattc tgctggtgca cttgccagtt gcatgggcct catatacaac aggatggggg 360
ctgtaaccac tgaagtggcc tttggcctgg tatgtgcaac atgtgaacag attgctgact 420
cccagca 427
<210> 17
<211> 556
<212> DNA
<213>Artificial sequence
<400> 17
gatcactaat acgactcact atagggggta gatggatggt acggttatca ccatcaaaat 60
gagcaggggt caggatatgc agccgacctg aagagcacac aaaatgccat tgacaagatt 120
actaacaaag taaattctgt tattgaaaag atgaatacac agttcacagc agtgggtaaa 180
gagttcaacc acctggaaaa aagaatagag aatttaaata aaaaagttga tgatggtttc 240
ctggacattt ggacttacaa tgccgaactg ttggttctat tggaaaatga aagaactttg 300
gactaccacg attcaaatgt gaagaacttg tatgaaaagg taagaaacca gttaaaaaac 360
aatgccaagg aaattggaaa cggctgcttt gaattttacc acaaatgcga taacacgtgc 420
atggaaagtg tcaaaaatgg gacttatgac tacccaaaat actcagagga agcaaaatta 480
aacagagaaa aaatagatgg ggtaaagctg gaatcaacaa ggatttacca gattttggcg 540
atctattcaa ctgtcg 556
<210> 18
<211> 420
<212> DNA
<213>Artificial sequence
<400> 18
gatcactaat acgactcact ataggggaat aagcatctat tggacaatag taaaaccggg 60
agacatactt ttgattaaca gcacagggaa tctaattgct cctaggggtt acttcaaaat 120
acgaagtggg aaaagctcaa taatgagatc agatgcaccc attggcaaat gcaattctga 180
atgcatcact ccaaatggaa gcattcccaa tgacaaacca ttccaaaatg taaacaggat 240
cacatacggg gcctgtccca gatatgttaa gcaaaacact ctgaaattgg caacaggaat 300
gcgaaatgta ccagagaaac aaactagagg catatttggc gcaatagcgg gtttcataga 360
aaatggttgg gagggaatgg tggatggttg gtacggtttc aggcatcaaa attctgaggg 420
Claims (8)
1. a kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings combine, which is characterized in that institute
It states multiple RT-RPA primers combination and includes the RT-RPA primers for detecting influenza A virus Matrix genes, forward primer
Sequence is as shown in SEQ ID NO.1, and reverse primer sequences are as shown in SEQ ID NO.2；For detecting influenza A virus H1 Asias
The RT-RPA primers of type HA genes, forward primer sequence is as shown in SEQ ID NO.3, reverse primer sequences such as SEQ ID
Shown in NO.4；For detecting the RT-RPA primers of influenza A virus H3 hypotype HA genes, forward primer sequence such as SEQ ID
Shown in NO.5, reverse primer sequences are as shown in SEQ ID NO.6.
2. a kind of multiple RT-RPA primer sets for being used for influenza A virus detection and H1 and H3 partings as described in claim 1
It closes, which is characterized in that each primer concentration of the multiple RT-RPA primers combination is 10 μM；Influenza A virus Matrix bases
Product Sequence expand through RT-RPA by, H1 hypotypes and H3 hypotype HA genes respectively such as SEQ ID NO.7, SEQ ID NO.8 and
Shown in SEQ ID NO.9.
3. contain a kind of multiple RT-RPA primers for being used for influenza A virus detection and H1 and H3 partings described in claim 1
The detection kit of combination, which is characterized in that the kit is detected for influenza A virus and H1 and H3 partings, kit
It is combined including multiple RT-RPA primers described in claim 1.
4. detection kit as claimed in claim 3, the kit also comprises the following components：Buffer solution, enzyme reaction solution, nothing
RNase water, reverse transcriptase, RNase inhibitor, magnesium acetate solution, negative control and positive control；The positive control is 1.0
×105The external synthesizing single-stranded RNA standard items of copies/ μ L；Negative control is normal person's throat swab of inactivation.
5. a kind of multiple RT-RPA primers for influenza A virus detection and H1 and H3 partings described in claim 1 combine
Application, which is characterized in that influenza A virus is detected using the multiple RT-RPA primers combination and sub- to H1 and H3
Type is identified.
A kind of 6. multiple RT-RPA methods for being used for influenza A virus detection and H1 and H3 partings, which is characterized in that the side
Method includes the following steps：
(1) sample to be tested total serum IgE is extracted；
(2) multiple RT-RPA primers combination described in claim 1 is designed and synthesized, to extract the total serum IgE of sample to be tested as mould
Plate carries out multiple RT-RPA amplifications；After RT-RPA amplifications, influenza A virus Matrix genes, H1 hypotypes and H3 hypotype HA bases
Because the Product Sequence through amplification is respectively as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9；
After (3) 42 DEG C of water-bath 4min, mixing, 42 DEG C of reaction 30min；It is detected again through agarose gel electrophoresis；According to electrophoresis result
Judged：If there is the band of 279bp sizes, contain influenza A virus in sample to be tested；If occur simultaneously 279bp and
Two bands of 216bp sizes then contain influenza A virus H1 hypotypes in sample to be tested；If occur 279bp simultaneously and 394bp is big
Two small bands then contain influenza A virus H3 hypotypes in sample to be tested；If occur 279bp, 216bp and 394bp tri- simultaneously
Band then contains influenza A virus H1 and H3 hypotype simultaneously in sample to be tested；If there is not the band of 279bp sizes, and
There is the band of 216bp or/and 394bp, then need sequence verification again.
7. a kind of multiple RT-RPA methods for being used for influenza A virus detection and H1 and H3 partings as claimed in claim 6,
It is characterized in that, the system of the RT-RPA amplifications is set as 50 μ L, system includes：29.5 μ L of buffer solution, 6.0 μ L of primer sets,
Without 8.0 μ L of RNase deionized waters, 0.5 μ L of reverse transcriptase, 0.5 μ L of RNase inhibitor, 3.0 μ L of RNA extracting solutions, added in after mixing
The magnesium acetate solution of 2.5 μ L.
8. a kind of multiple RT-RPA methods for being used for influenza A virus detection and H1 and H3 partings as claimed in claim 6,
It is characterized in that, in the step (2), marked with the RNA of influenza A virus Matrix genes, H1 hypotypes and H3 hypotype HA genes
Quasi- product are as positive control, and a concentration of the 1 × 10 of RNA standard items5copies/μL；Using normal person's throat swab of inactivation as the moon
Property control.
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| CN108841997A (en) * | 2018-06-29 | 2018-11-20 | 中山大学达安基因股份有限公司 | A kind of kit of Electrochemistry gene chip method detection common type influenza A virus |
| CN108977585A (en) * | 2018-09-10 | 2018-12-11 | 宁波怡和医药科技有限公司 | A kind of subtypes of influenza A virus genotyping detection method |
| CN109321677A (en) * | 2018-09-11 | 2019-02-12 | 迈克生物股份有限公司 | A kind of method of RPA combination immunofluorescence chromatography specific detection H1 swin flu hypotype |
| CN109797245A (en) * | 2019-02-21 | 2019-05-24 | 中国人民解放军东部战区总医院 | It is a kind of for detecting RPA primer, probe, kit and the application of influenza B virus |
| CN110468238A (en) * | 2019-09-11 | 2019-11-19 | 深圳市芯思微生物科技有限公司 | A kind of primed probe group, kit and the application of constant-temperature amplification detection A type and influenza B virus |
| CN111118219A (en) * | 2020-02-06 | 2020-05-08 | 广州普世利华科技有限公司 | RDA method and kit for rapidly detecting influenza A virus |
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| CN109797245A (en) * | 2019-02-21 | 2019-05-24 | 中国人民解放军东部战区总医院 | It is a kind of for detecting RPA primer, probe, kit and the application of influenza B virus |
| CN110468238A (en) * | 2019-09-11 | 2019-11-19 | 深圳市芯思微生物科技有限公司 | A kind of primed probe group, kit and the application of constant-temperature amplification detection A type and influenza B virus |
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| CN111118219A (en) * | 2020-02-06 | 2020-05-08 | 广州普世利华科技有限公司 | RDA method and kit for rapidly detecting influenza A virus |
| WO2021250617A1 (en) * | 2020-06-10 | 2021-12-16 | King Abdullah University Of Science And Technology | A rapid multiplex rpa based nanopore sequencing method for real-time detection and sequencing of multiple viral pathogens |
| CN112176106A (en) * | 2020-10-13 | 2021-01-05 | 华芯生物科技(武汉)有限公司 | Primer for detecting respiratory tract pathogenic microorganisms and application thereof |
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