CN108977578A - Detect the kit and its method of H7N9 avian influenza virus - Google Patents
Detect the kit and its method of H7N9 avian influenza virus Download PDFInfo
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Abstract
The invention belongs to technical field of biological, and in particular to a kind of kit and its method for detecting H7N9 avian influenza virus.The kit includes the first forward primer, the first reverse primer and the first probe for expanding H7 gene, and the second forward primer, the second reverse primer and the second probe for expanding N9 gene；Wherein, the sequence of first forward primer is as shown in SEQ ID NO.1, the sequence of first reverse primer is as shown in SEQ ID NO.2, the sequence of first probe is as shown in SEQ ID NO.3, the sequence of second forward primer is as shown in SEQ ID NO.4, the sequence of second reverse primer is as shown in SEQ ID NO.5, and the sequence of second probe is as shown in SEQ ID NO.6.The kit can be applied to clinical immediately quick detection H7N9 avian influenza virus.
Description
Technical field
The invention belongs to technical field of biological, and in particular to it is a kind of detect H7N9 avian influenza virus kit and its
Method.
Background technique
Bird flu is a kind of Arbo infectious disease for seriously endangering humans and animals health, by avian influenza virus (Avian
Influenza virus, AIV) cause.Cause severe pneumonia, conjunctivitis, Respiratory Distress Syndrome(RDS) (ARDS), purulence after people's infection
Toxication etc., after birds infection body temperature rise rapidly, poor appetite, diarrhea, incoordination, death etc., be to cause animal husbandry great
One of the main reason for loss.The disease is widely current in countries in the world, and Asia, Africa, Australia, Europe and South and North America are all
It has been reported that.Bird flu not only causes huge economic loss, but also seriously threatens human health, while causing to ruin to tourist industry
Going out property strike, also result in ball ecological environment and further deteriorate, higher case fatality rate, it is difficult to predict localized epidemics and
The new cases constantly occurred bring huge difficult problem to current infectious disease prevention and control, by World Organization for Animal Health (OIE)
It is classified as A class infectious disease, our country is listed as a kind of infectious disease, the World Health Organization (WHO) thinks that the disease may be to the mankind
One of maximum disease of potential threat, by global highest attention.
Due to avian influenza virus antigen variation etc., the mankind is caused to recognize insufficient and lag to novel reassortant virus, it is past
Toward can not quickly detect pathogen in its outbreak of epidemic early stage, cause Delay in Diagnosis that the infected's aggravation is caused even to jeopardize life
Life, meanwhile, to big multimutation and new avian influenza virus without natural immunity, the above reason causes viral rapid sufferer
It spreads and spreads epidemic situation sharply, therefore, the early stage fast accurate detection of avian flu substance is particularly important.Currently, fowl
The detection method of influenza virus is more, mainly there is etiological diagnosis, serodiagnosis, molecular biological testing etc., these
Method has played important function during avian flu virus detection, but there is also some shortcomings and disadvantage, such as instrument and equipment
It is huge, testing cost is high, time-consuming, pre-treatment is cumbersome, needs the laboratory P Ш, to the professional more demanding etc. of operator, difficult
To realize that the fast accurate of sample detects demand, it is even more impossible to quickly detect pathogen in bird flu outbreak of epidemic early stage.
Recombinase polymeric enzymatic amplification (recombinase polymerase amplification, RPA) technology be by
Piepenburg et al. proposed that reaction relies primarily on recombinase, single-stranded DNA binding protein (SSB) and chain in 2006 first
Archaeal dna polymerase is replaced to realize, firstly, recombinase in conjunction with upper reverse primer, finds homologous double-stranded DNA, once positioning, occurs
Chain exchange, single-stranded binding albumen (SSB) and parent's chain are bound, and the template strand of itself and disengaging is prevented to interact, then,
Archaeal dna polymerase forms two double-stranded DNAs, such circulating repetition and then realization from 3 ' end starting templated synthesis of upper reverse primer
Amplification.Entire reaction system is by bacteriophage recombinase Uvs X and its confactor Uvs Y, oligonucleotide primer, archaeal dna polymerase, list
The composition such as chain DNA binding protein (gp32), buffer.RPA is different from PCR, is not required to complicated reaction process, can be 37 DEG C~39
Under DEG C steady temperature, amplification is can be completed in reaction 20min or so.RPA is quickly detected in combination with lateral flow immunochromatography technique
Pathogenic microorganism, it is main using the primer with biotin labeling and probe with Fluoresceincarboxylic acid (FAM) label and target nucleic acid into
Row amplified reaction makes final amplified production while carrying FAM and biotin mark.Sidestream chromatography test strips front end is coated with band
The nano-scale gold particle of FAM antibody is coated with biotin antibody in detection line, when reaction solution enters test strips, has FAM and biology
The amplified production of element will form biotin antibody-nucleic acid-nano-scale gold particle by antigen-antibody combination in detection line
Ternary complex simultaneously develops the color, and non-hybridized FAM label probe forms the two-spot compound for being free of biotin, is incorporated in containing anti-
It does not develop the color on the nature controlling line of FAM.When designing RPA primed probe, need to hold progress biotin labeling, probe in reverse primer 5 '
5 ' end fluorophor labels, have an abasic site (THF), THF, that is, tetrahydrofuran can at 30 away from fluorophor bases
By the identification of nfo ribozyme, cutting, extend under archaeal dna polymerase effect, eventually forms simultaneous with fluorophor and the double marks of biotin
The RPA amplified production of note can be caught by amplified production by chromatography membrane diffusion by the antibody that biotin ligand and fluorophor mark
It obtains, is formed and have coloured detection line.The detection method time-consuming is short, 10min or so energy visual observation, naked eyes interpretation.
Different from PCR reaction, RPA technology still belongs to starting conceptual phase, and the design of primer, probe is without special software, entirely
It carries out groping to screen according to RPA technical principle.
Summary of the invention
It is an object of the invention to overcome the above-mentioned deficiency of the prior art, a kind of examination for detecting H7N9 avian influenza virus is provided
Agent box and its method, it is intended to solve that existing avian flu virus detection is at high cost, time-consuming, it is difficult to realize the fast accurate inspection of sample
The technical issues of survey.
For achieving the above object, The technical solution adopted by the invention is as follows:
One aspect of the present invention provides a kind of kit for detecting H7N9 avian influenza virus, and the kit includes for expanding
The first forward primer, the first reverse primer and the first probe of H7 gene, and the second forward primer for expanding N9 gene,
Second reverse primer and the second probe；First forward primer, the first reverse primer, the first probe, the second forward primer,
Two reverse primers and the second probe are based on RPA Technology design；
Wherein, the sequence of first forward primer is as shown in SEQ ID NO.1, and the sequence of first reverse primer is such as
Shown in SEQ ID NO.2, the sequence of first probe is as shown in SEQ ID NO.3, and the sequence of second forward primer is such as
Shown in SEQ ID NO.4, the sequence of second reverse primer is as shown in SEQ ID NO.5, and the sequence of second probe is such as
Shown in SEQ ID NO.6.
Another aspect of the present invention provides a kind of method for detecting H7N9 avian influenza virus, includes the following steps:
Extract sample RNA；
It is cDNA by the sample RNA reverse transcription；
The cDNA is mixed with the reagent in the kit of the above embodiment of the present invention, RPA reaction is carried out, is reacted
Liquid；
The reaction solution is added to flow measurement chromatograph test strip to detect.
Provided by the present invention for detecting the kit of H7N9 avian influenza virus, contain the primer based on RPA Technology design
And probe combinations, these primer and probes are obtained by multiple groups optimization, screening, with high sensitivity, high specificity, repeatability
Good feature, with other subtype avian influenza virus no cross reactions such as H1N1, H3N2, H5N6 and H9N2, therefore, which can
H7N9 avian influenza virus is quickly detected immediately applied to clinical.
Method provided by the present invention for detecting H7N9 avian influenza virus is based on RPA technology and Sidestream chromatography technology
The method for the quick detection H7N9 avian influenza virus established, this method had not only had the high sensitivity of molecular Biological Detection, but also tool
Have the advantages that the specificity of immunology detection is good, easy to operate, does not need PCR instrument, fluorescence quantitative PCR instrument, electrophoresis apparatus, electrophoresis tank
Etc. complex and expensives instrument and equipment, can expand under constant temperature conditions, test strips Visual retrieval, really realize portable scene
Rapid nucleic acid detection is particularly suitable for laboratories and clinical H7N9 avian influenza virus rapid screening detection.
Detailed description of the invention
Fig. 1 is the selection result figure of the RPA primer of H7 gene in the embodiment of the present invention 2, probe groups, in which: 1 is primer
Nfo-H7-F, nfo-H7-R and probe nfo-H7-P group, 2 be primer nfo-H7-F, nfo-H7-R1 and probe nfo-H7-P group, 3
For nfo-H7-F, nfo-H7-R2 and probe nfo-H7-P group；A is that H7N9 avian influenza virus cDNA, b are negative control；
Fig. 2 is the selection result figure of the RPA primer of N9 gene in the embodiment of the present invention 2, probe groups, wherein 1 is primer
Nfo-N9-F, nfo-N9-R and probe nfo-N9-P group, 2 be primer nfo-N9-F, nfo-N9-R1 and probe nfo-N9-P group, 3
For nfo-N9-F, nfo-N9-R2 and probe nfo-N9-P group；A is that H7N9 avian influenza virus cDNA, b are negative control；
Fig. 3 is the efficiency assay result figure that RPA- Sidestream chromatography detects H7N9 avian influenza virus in the embodiment of the present invention 3,
Wherein a be H7 RPA reaction solution in Sidestream chromatography test strips as a result, b is N9 RPA reaction solution in Sidestream chromatography test strips
As a result, c be H7 RPA reaction solution in Sidestream chromatography test strips in 1~10min different time points as a result, d is N9 RPA
The result of reaction solution different time points in 1~10min in Sidestream chromatography test strips；
Fig. 4 is the sensitivity test result figure that RPA- Sidestream chromatography detects H7N9 avian influenza virus in the embodiment of the present invention 3,
Wherein a be H7 difference dilution RPA reaction solution in Sidestream chromatography test strips as a result, b is N9 difference dilution RPA reaction solution
It is in Sidestream chromatography test strips as a result, c be H7 difference dilution RPA reaction solution on 3% Ago-Gel electrophoresis as a result, d
For N9 difference dilution RPA reaction solution electrophoresis result on 3% Ago-Gel；
Fig. 5 is the specific test result figure that RPA- Sidestream chromatography detects H7N9 avian influenza virus in the embodiment of the present invention 3,
Wherein a be H1, H3, H5, H7 and H9 RPA reaction solution in Sidestream chromatography test strips as a result, b is N1, N2, N6 and N9 RPA
Reaction solution in Sidestream chromatography test strips as a result, c be H1, H3, H5, H7 and H9 RPA reaction solution on 3% Ago-Gel
Electrophoresis result, d N1, N2, N6 and N9 RPA reaction solution electrophoresis result on 3% Ago-Gel.
Specific embodiment
In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain
The present invention is not intended to limit the present invention.
It is to be appreciated that term " first ", " second " are used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Explicitly or implicitly include one or more of the features.
On the one hand, the embodiment of the invention provides a kind of kit for detecting H7N9 avian influenza virus, the kit packets
Include the first forward primer, the first reverse primer and the first probe for expanding H7 gene, and for expanding N9 gene
Two forward primers, the second reverse primer and the second probe；First forward primer, the first reverse primer, the first probe, second
Forward primer, the second reverse primer and the second probe are based on RPA Technology design；
Wherein, the sequence of first forward primer is as shown in SEQ ID NO.1, and the sequence of first reverse primer is such as
Shown in SEQ ID NO.2, the sequence of first probe is as shown in SEQ ID NO.3, and the sequence of second forward primer is such as
Shown in SEQ ID NO.4, the sequence of second reverse primer is as shown in SEQ ID NO.5, and the sequence of second probe is such as
Shown in SEQ ID NO.6.
The kit provided in an embodiment of the present invention for being used to detect H7N9 avian influenza virus, containing based on RPA Technology design
Primer and probe combination, these primer and probes by multiple groups optimization, screening obtain, with high sensitivity, high specificity,
Reproducible feature, with other subtype avian influenza virus no cross reactions such as H1N1, H3N2, H5N6 and H9N2, therefore, the examination
Agent box can be applied to clinical immediately quick detection H7N9 avian influenza virus.
Further, 5 ' ends of first reverse primer and 5 ' ends of second reverse primer are respectively connected with biology
Element, 5 ' ends of first probe are connected with the first fluorophor, and 5 ' ends of second probe are connected with the second fluorophor.
First fluorophor and second fluorophor are FAM.
Further, these primer and probes in the kit may be dissolved in primed probe liquid, primed probe liquid
In: forward primer and reverse primer final concentration are 0.4 μm of ol/L, final concentration of 0.12 μm of ol/L of probe.Further, institute
Stating kit further includes flow measurement chromatograph test strip.The kit further includes reaction buffer, product dilution, magnesium acetate buffering
Liquid and water (such as aseptic double-distilled water).It further comprise eight union of enzymatic amplification.
Further, the kit includes positive reference substance and/or negative controls.The positive reference substance includes:
The weight that sequence shown in sequence shown in SEQ ID NO.11 and SEQ ID NO.12 is connect with pET6xHN-N carrier respectively
Group plasmid.Wherein, it is pET6xHN-N-H7 for H7 gene, is the nucleotide expanded by SEQ ID No.7 and SEQ ID No.8
The recombinant plasmid that sequence fragment (SEQ ID No.11) obtains after being connected with pET6xHN-N carrier；It is for N9 gene
PET6xHN-N-N9 is the nucleotide sequence fragment (SEQ ID No.12) expanded by SEQ ID No.9 and SEQ ID No.10
The recombinant plasmid obtained after being connected with pET6xHN-N carrier, connection method are fields routine techniques.Above-mentioned positive control
Can product for examining corresponding reaction system and reaction condition normal reaction.And the negative controls are that water is (such as sterile double
Steam water).
Particular sequence in kit of the embodiment of the present invention is as follows:
First forward primer nfo-H7-F (SEQ ID NO.1):
5'-TTCTATGCAGAAATGAAATGGCTCCTGTCAA-3'；
First reverse primer nfo-H7-R (SEQ ID NO.2):
5'-[Biotin]AGCTGGGCTTTTTCTTGTATTTTTATATGACTTAG-3'；
First probe nfo-H7-P (SEQ ID NO.3):
5’-[FAM]AAATGAAATGGCTCCTGTCAAACACAGATA[THF]TGCTGCATTCCCGCA[C3-
spacer]-3'；
Second forward primer nfo-N9-F (SEQ ID NO.4):
5'-ATAGACCCAGTAGCAATGACACACACTAGTCA-3'；
Second reverse primer nfo-N9-R (SEQ ID NO.5):
5'-[Biotin]TTATTATTACCTGGATAAGGGTCATTACACT-3'；
Second probe sequence nfo-N9-P (SEQ ID NO.6):
5’-[FAM]CACTAGTCAATATATATGCAGTCCTGTTCTA[THF]AGACAGTCCCCGACCGA[C3-
spacer]-3’。
H7-F (SEQ ID NO.7): 5 '-AGAAATGAAATGGCTCCTGTCAA-3 '；
H7-R (SEQ ID NO.8): 5 '-GGTTTTTTCTTGTATTTTTATATGACTTAG-3 '；
N9-F (SEQ ID NO.9): 5 '-TGGCAATGACACACACTAGTCAGT-3 '；
N9-R (SEQ ID NO.10): 5 '-ATTACCTGGATAAGGGTCGTTACACT-3 '；
SEQ ID NO.11:
AGAAATGAAATGGCTCCTGTCAAACACAGATAATGCTGCATTCCCGCAGATGACTAAGTCATATAAAAATACAAGAA
AAAACC；
SEQ ID NO.12:
TGGCAATGACACACACTAGTCAGTATATATATGCAGTCCTGTTCTTACAGACAATCCCCGACCGAATGACCCAAATA
TAGGTAAGTGTAACGACCCTTATCCAGGTAAT。
On the other hand, the embodiment of the invention also provides a kind of methods for detecting H7N9 avian influenza virus, including walk as follows
It is rapid:
S01: sample RNA is extracted；
S02: being cDNA by the sample RNA reverse transcription；
S03: the cDNA is mixed with the reagent in the kit of the above embodiment of the present invention, is carried out RPA reaction, is obtained
Reaction solution；
S04: the reaction solution is added to flow measurement chromatograph test strip and is detected.
Method provided in an embodiment of the present invention for detecting H7N9 avian influenza virus is based on RPA technology and effluent layer
The method for the quick detection H7N9 avian influenza virus that analysis technology is established, this method had both had the highly sensitive of molecular Biological Detection
Degree, and have the advantages that the specificity of immunology detection is good, easy to operate, do not need PCR instrument, fluorescence quantitative PCR instrument, electrophoresis
The instrument and equipment of the complex and expensives such as instrument, electrophoresis tank can expand, test strips Visual retrieval under constant temperature conditions, really realize just
The live Rapid nucleic acid detection for taking formula is particularly suitable for laboratories and clinical H7N9 avian influenza virus rapid screening inspection
It surveys.
Specifically, H7N9 avian flu is extracted according to the operating procedure of viral nucleic acid DNA/RNA extracts kit specification
Poison RNA, according to reverse transcription reagent box specification operating procedure by RNA reverse transcription be cDNA；Using cDNA as template, add reaction slow
Fliud flushing, aseptic double-distilled water and magnesium acetate buffer carry out RPA amplification, preferred RPA amplification condition into eight unions of amplification are as follows:
37-42 DEG C of temperature, time 18-20min (in a specific embodiment, can be taken out and be mixed, then 39 DEG C after first 39 DEG C of incubations 4min
16min is incubated, amplified reaction is terminated).After reaction, 2 μ L are extracted reaction solution, are diluted with 30 μ L product dilutions, after mixing, from
Sidestream chromatography test strips are added in adding mouth, and on the horizontal plane that room temperature is put it clean, result is read in 10min or so observation；Observation determines
As a result, result is the positive when the quality control region of Sidestream chromatography test strips and detection zone all bright band occur, show to contain in sample
H7N9 avian influenza virus nucleic acid；When Sidestream chromatography test strips only have quality control region bright band occur, and detection zone does not have bright band, result is
It is negative.
Further, quality >=32fg of the cDNA；Method i.e. provided in an embodiment of the present invention can detecte at least
The H7N9 avian influenza virus of 32fg.Preferably, the system of RPA reaction contains 29.5 μ L of reaction buffer, H7 or N9 in terms of 50 μ L
Corresponding 4.8 μ L of primed probe liquid, 10 μ L of aseptic double-distilled water, the cDNA of sample to be tested or using positive criteria plasmid as positive control
It again or is respectively 3.2 μ L, 2.5 μ L of magnesium acetate buffer by blank control of aseptic double-distilled water.
The present invention successively carried out test of many times, and it is further detailed as reference pair invention progress now to lift A partial experiment result
Thin description, is described in detail combined with specific embodiments below.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.Nfo Kits is purchased from TwistDX company, and PCRD Nucleic Acid is purchased from Abingdon Health, instead
Transcript reagent box (Code No.6210A) and viral DNA/RNA extracts kit (Code No.9766) are purchased from precious biological work
Journey (Dalian) Co., Ltd, RPA primer and probe are synthesized by Sangon Biotech (Shanghai) Co., Ltd..In embodiment not
The experimental method of detailed noted provisos, usually according to conventional condition (as the condition in " molecular cloning: laboratory manual " walks
Suddenly it is operated) or according to actual conditions proposed by reagent or instrument manufacturing manufacturer.
Embodiment 1
The preparation of positive criteria plasmid
1. the design and synthesis of primer
By the Genebank database of NCBI and in conjunction with documents and materials, to H7N9 avian influenza virus H7 and N9 gene order
It compares and analyzes, separately designs 1 pair of specific universal primer, be named as SEQ ID No.7 and SEQ ID No.8, SEQ ID
No.9 and SEQ ID No.10, amplifies target gene fragment respectively, and for the building of positive criteria plasmid, all primers are by giving birth to
The synthesis of work bioengineering (Shanghai) limited liability company.
2.H7N9 the preparation of avian influenza virus cDNA
The RNA of H7N9 avian influenza virus is extracted according to the operating procedure of viral nucleic acid DNA/RNA extracts kit specification,
According to reverse transcription reagent box specification operating procedure by RNA reverse transcription be cDNA.
3. the preparation of positive plasmid
Using the cDNA of above-mentioned acquisition as template, PCR amplification is carried out, reaction system is 25 μ L:RNase Free dH2O14.5
μ L, dNTP Mixture2 μ L, 5 × Prime STAR Buffer5 μ L, Prime STAR HS0.5 μ L, 1 μ L of cDNA template, 10 μ
1 μ L of 1 μ L of mol/L forward primer and reverse primer.Response procedures are 98 DEG C of initial denaturation 3min, then 98 DEG C of 10s, 55 DEG C of 15s, 72
DEG C 15s, 35 circulations；72 DEG C of extension 4min, 4 DEG C of preservations.
The pcr amplification product of H7 gene is 83bp, and the pcr amplification product of N9 is 109bp.Through 1% agarose gel electrophoresis,
It is separately recovered, connect, converts with pET6xHN-N carrier, the single colonie that picking grows fine expands in 5mLLB fluid nutrient medium
Plasmid is extracted in a small amount after big culture.PCR verifying is carried out with corresponding primer respectively, identifies that correct plasmid send precious bioengineering
The sequencing identification of (Dalian) Co., Ltd, identifies that correct plasmid is respectively designated as pET6xHN-N-H7, pET6xHN-N-N9.
Embodiment 2
Detect the kit of H7N9 avian influenza virus and its optimization and foundation of method
The design of 1.RPA primer and probe
The key of RPA amplification success or failure is that the design of primer and probe, RPA technology still belong to starting conceptual phase, primer, spy
Principle of the design of needle without special software or maturation.PCR primer is generally less than 30bp, is not suitable for RPA amplification, RPA primer one
As be 30-35bp, primer is too short to reduce recombination fraction, influence amplification rate and detection sensitivity, and primer length is too long is easy
Form secondary structure.5 ' 3~5 nucleotide in end avoid the occurrence of multiple G, and 3 ' ends should have GC, avoid multiple phase same cores continuously occur
Thuja acid, G/C content account for 30~70%, and primer is avoided to form dimer and secondary structure, therefore, the primer of RPA amplification need by
A large amount of experiment is screened and is optimized.
RPA probe length will avoid the occurrence of as far as possible dimer and secondary structure in 46~52bp.The end of probe 5 ' FAM base
Group's label, it is intermediate to replace G or C base with THF, and G or C to 5 ' holds at least 30 bases, to 3 ' end at least 15 bases, 3 ' ends
It is modified with C3-spacer.
The present embodiment has separately designed a series of RPA primer and probe for H7 and N9 gene, is specifically shown in Table 1.
Table 1
Remarks: ● indicate Biotin modification；■ indicates FAM label；Base expression with mark of emphasis is replaced by THF；◆ table
Show that C3-spacer is modified；
2. screening primer and probe using RPA amplified reaction
The RNA of H7N9 avian influenza virus is extracted according to the operating procedure of viral nucleic acid DNA/RNA extracts kit specification,
According to reverse transcription reagent box specification operating procedure by RNA reverse transcription be cDNA, using cDNA be template with PRA method expand H7 with
N9 gene order, the specific steps are as follows:
(1) each 2.1 μ L of forward and reverse primer (10 μm of ol/L), 0.6 μ L (10 μ of probe of each detection gene are added in EP pipe
Mol/L), 29.5 μ L, cDNA3.2 μ L, ddH of reaction buffer2O10 μ L, of short duration vortex mix, and each pair of primed probe group is all provided with
Vertical negative control.
(2) it will be transferred to from after in 47.5 μ L mixed reaction solution winks in eight union of Twist Ampnfo containing freeze-drying enzyme powder,
Piping and druming to freeze-dried powder is completely dissolved.
(3) 2.5 μ L magnesium acetate (280mmol/L) buffers are added into each reaction tube, are put into 39 DEG C after mixing at once
In thermostat water bath, 4min is incubated.
(4) after 4min, reaction tube is taken out, continues to incubate 16min after mixing rapidly.
(5) after reaction, 2 μ L are extracted reaction solution, are diluted with 30 μ L product dilutions, after mixing, from adding mouth joining side
Chromatograph test strip is flowed, on the horizontal plane that room temperature is put it clean, result is read in 10min or so observation.
3. the selection result of primer and probe
The primed probe group of H7 and N9 gene shown in table 1 is detected, such as Fig. 1 and Fig. 2 after RAP is expanded by test strips
It is shown, nfo-H7-F, nfo-H7-R and nfo-H7-P primed probe group of H7 gene, nfo-N9-F, nfo-N9-R of N9 gene
RPA product with nfo-N9-P primed probe group, this 2 groups of primed probe groups is the positive, and test strips go out in quality control region and detection zone
Existing band, and negative control RPA product does not occur band in detection zone, only there are band, while other primed probes in quality control region
The detection zone of group does not occur band, only band occurs in quality control region, shows that these primed probe groups may not apply to the inspection of RPA
It surveys.
Embodiment 3
Detect validity, sensitivity, repeatability, the specific detection of H7N9 avian influenza virus kit and its method
1. detecting the efficiency assay of H7N9 avian influenza virus kit and its method, sensitivity analysis and repeatability detection
The efficiency assay of H7N9 avian influenza virus kit and its method is detected, the RPA reaction solution of H7 is in Sidestream chromatography
Result in test strips is shown in Fig. 3 a, and result of the RPA reaction solution of N9 in Sidestream chromatography test strips is shown in Fig. 3 b, the RPA reaction of H7
The result of different time points is shown in Fig. 3 c to liquid in 1~10min in Sidestream chromatography test strips, and the RPA reaction solution of N9 is in Sidestream chromatography
The result of different time points is shown in Fig. 3 d in 1~10min in test strips, the results showed that, the present invention detects the examination of H7N9 avian influenza virus
Agent box and its method are effective；H7N9 fowl is extracted according to the operating procedure of viral nucleic acid DNA/RNA extracts kit specification
The RNA of influenza virus, according to reverse transcription reagent box specification operating procedure by RNA reverse transcription be cDNA, by cDNA template
(1000ng/ μ L H7N9cDNA) from 1 to 10-10(3200ng to 0.32fg) serial dilution.It is carried out respectively with the RPA condition of optimization
RPA amplification, use nfo-H7-F, nfo-H7-R and nfo-H7-P primed probe group of H7 gene, the use nfo-N9-F of N9 gene,
Nfo-N9-R and nfo-N9-P primed probe group, reaction system are as follows: forward and reverse each 2.1 μ L of primer (10 μm of ol/L), 0.6 μ L of probe
(10 μm of ol/L), reaction buffer 29.5 μ L, cDNA3.2 μ L, ddH2O10 μ L is shifted after mixing the of short duration vortex of reaction solution
Into eight union of TwistAmpnfo containing freeze-drying enzyme powder, piping and druming is completely dissolved to freeze-dried powder, is added into each reaction tube
2.5 μ L magnesium acetate (280mmol/L) buffers, are put at once in 39 DEG C of thermostat water baths after mixing, take out instead after incubating 4min
Ying Guan is mixed continue to incubate 16min rapidly.
The template concentrations of each gradient of above-mentioned 2 groups of primed probe groups do three repetitions, pass through RPA Sidestream chromatography test strips
Detection, batch interior repeatability of verifying RPA Sidestream chromatography test strips method；In addition, being repeated once at interval of 2d, 3 weights are carried out altogether
It is multiple, verifying RPA Sidestream chromatography test strips method batch between repeatability.RPA product obtained carries out Sidestream chromatography test paper respectively
Item detection, is as a result shown in Fig. 4；Fig. 4 shows that 2 groups of primed probe groups can detect 10-8Dilution (32fg, 4 × 103Copy)
cDNA。
2. detecting the specificity analysis of H7N9 avian influenza virus kit and its method
According to viral nucleic acid DNA/RNA extracts kit specification operating procedure extract avian influenza virus H1N1, H3N2,
The RNA of H5N6, H7N9 and H9N2, according to reverse transcription reagent box specification operating procedure by RNA reverse transcription be cDNA.
With nfo-H7-F, nfo-H7-R, nfo-H7-P and nfo-N9-F of above-mentioned steps screening, nfo-N9-R, nfo-N9-
Two groups of primed probe groups of P are primer and probe, respectively using the cDNA of H1N1, H3N2, H5N6, H7N9 and H9N2 as template, with excellent
The RPA condition of change carries out RPA amplification respectively, test is repeated 3 times, and carries out detection H7N9 avian influenza virus kit and its method
Specificity analysis.
All RPA product carries out the detection of Sidestream chromatography test strips, as shown in figure 5, wherein a be with H1N1, H3N2,
The cDNA of H5N6, H7N9 and H9N2 are template, using nfo-H7-F, nfo-H7-R and nfo-H7-P primed probe group as primer and spy
Needle, RPA reaction solution in Sidestream chromatography test strips as a result, b be using the cDNA of H1N1, H3N2, H5N6, H7N9 and H9N2 as mould
Plate, using nfo-N9-F, nfo-N9-R and nfo-N9-P primed probe group as primer and probe, RPA reaction solution is tried in Sidestream chromatography
It is on paper slip as a result, c be using the cDNA of H1N1, H3N2, H5N6, H7N9 and H9N2 as template, with nfo-H7-F, nfo-H7-R and
Nfo-H7-P primed probe group be primer and probe, RPA reaction solution on 3% Ago-Gel electrophoresis as a result, d be with H1N1,
The cDNA of H3N2, H5N6, H7N9 and H9N2 are template, are to draw with nfo-N9-F, nfo-N9-R and nfo-N9-P primed probe group
Object and probe, RPA reaction solution electrophoresis result on 3% Ago-Gel.From the results, it was seen that nfo-H7-F, nfo-H7-R,
Nfo-H7-P and nfo-N9-F, nfo-N9-R, nfo-N9-P primed probe group amplification H7N9 avian influenza virus specific are good, with it
It detects viral no cross reaction.Therefore, the H7N9 avian influenza virus primed probe group-specific through preferably going out is strong, can be used for examining
H7N9 avian influenza virus kit and its method are surveyed, suitable for detecting the H7N9 avian influenza virus unknown sample.
Embodiment 4
Detect the assembling and sensitivity, repetitive test of H7N9 avian influenza virus kit
By each 420 μ L of primer nfo-H7-F and nfo-H7-R (10 μm of ol/L), 120 μ L of probe nfo-H7-P (10 μm of ol/
L it) mixes, packing 100 μ L/ pipe, the primed probe liquid as detection H7 gene；By each 420 μ of primer nfo-N9-F and nfo-N9-R
L (10 μm of ol/L), 120 μ L of probe nfo-N9-P (10 μm of ol/L) mixing, packing 100 μ L/ pipe, as drawing for detection N9 gene
Object probe liquid；Standard positive plasmid pET6xHN-N-H7 and pET6xHN-N-N9 dispense 50 μ L/ pipe respectively, as positive control；
Aseptic double-distilled water dispenses 100 μ L/ pipe, uses as reaction system or negative control is supplied；Then with eight union of enzymatic amplification, react slow
Fliud flushing, magnesium acetate buffer, product dilution and Sidestream chromatography test strips are assembled into detection H7N9 avian influenza virus kit.
Assembled detection H7N9 avian influenza virus kit is utilized, sensitivity technique is carried out to H7N9 avian influenza virus,
Reaction system are as follows: 4.8 μ L of primed probe liquid, reaction buffer 29.5 μ L, template cDNA3.2 μ L, ddH2O10 μ L is anti-by mixing
Liquid is answered to be added in eight union of enzymatic amplification, piping and druming is completely dissolved to freeze-dried powder, and 2.5 μ L magnesium acetates are added into each reaction tube
(280mmol/L) buffer sets 39 DEG C of 4min immediately after mixing, take out and continue 39 DEG C of 16min after mixing rapidly.Do three weights
It is multiple, take 2 μ LRPA amplified productions to be diluted with 30 μ L product dilutions, sidestream immune chromatograph test strip is detected, the results showed that,
H7N9 avian influenza virus cDNA can be detected in the detection H7N9 avian influenza virus kit assembled, and kit of the invention is available
In the detection of H7N9 avian influenza virus.
Embodiment 5
Detect the clinical sample detection of H7N9 avian influenza virus kit
Sample to be tested is to be flowed through this laboratory using the H7N9 fowl that Real time reverse transcription polymerase chain reaction method detected
21 parts of Influenza Virus positive sample, 29 parts of negative sample.According to the operating procedure of viral nucleic acid DNA/RNA extracts kit specification
Extract H7N9 avian influenza virus RNA, according to reverse transcription reagent box specification operating procedure by RNA reverse transcription be cDNA.Respectively
Using the cDNA of 50 parts of samples as template, using positive plasmid as positive control, ddH2O is negative control, is assembled using the present invention
Detect the detection that H7N9 avian influenza virus kit carries out H7N9 avian influenza virus.Reaction system are as follows: 4.8 μ L of primed probe liquid,
Reaction buffer 29.5 μ L, template cDNA3.2 μ L, ddH2Mixed reaction solution is added in eight union of enzymatic amplification, blows by O10 μ L
It beats and is completely dissolved to freeze-dried powder, 2.5 μ L magnesium acetate (280mmol/L) buffers are added into each reaction tube, after mixing immediately
39 DEG C of 4min are set, takes out and continues 39 DEG C of 16min after mixing rapidly.2 μ LRPA amplified productions are taken to be diluted with 30 μ L product dilutions,
Sidestream immune chromatograph test strip is detected.If bright band all occur in the quality control region and detection zone of Sidestream chromatography test strips, show
Contain H7N9 avian influenza virus in sample to be tested；If Sidestream chromatography test strips only have quality control region bright band occur, and detection zone do not have it is bright
Band then shows in sample without H7N9 avian influenza virus.
It is detected through detection H7N9 avian influenza virus kit of the invention, in the sample 21 of the H7N9 avian influenza virus positive
Part, 29 parts of the sample being negative, this result and reverse transcriptase polymerase chain reaction testing result are completely the same.Show of the invention
Primer, probe, kit and the detection method for detecting H7N9 avian influenza virus are effective, can be used for the clinic of H7N9 avian influenza virus
Fast accurate detection.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
SEQUENCE LISTING
<110>Zhongshan University
<120>kit and its method of H7N9 avian influenza virus are detected
<130> 2018
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 31
<212> DNA
<213>artificial synthesized
<400> 1
ttctatgcag aaatgaaatg gctcctgtca a 31
<210> 2
<211> 35
<212> DNA
<213>artificial synthesized
<400> 2
agctgggctt tttcttgtat ttttatatga cttag 35
<210> 3
<211> 45
<212> DNA
<213>artificial synthesized
<400> 3
aaatgaaatg gctcctgtca aacacagata tgctgcattc ccgca 45
<210> 4
<211> 32
<212> DNA
<213>artificial synthesized
<400> 4
atagacccag tagcaatgac acacactagt ca 32
<210> 5
<211> 31
<212> DNA
<213>artificial synthesized
<400> 5
ttattattac ctggataagg gtcattacac t 31
<210> 6
<211> 48
<212> DNA
<213>artificial synthesized
<400> 6
cactagtcaa tatatatgca gtcctgttct aagacagtcc ccgaccga 48
<210> 7
<211> 23
<212> DNA
<213>artificial synthesized
<400> 7
agaaatgaaa tggctcctgt caa 23
<210> 8
<211> 30
<212> DNA
<213>artificial synthesized
<400> 8
ggttttttct tgtattttta tatgacttag 30
<210> 9
<211> 24
<212> DNA
<213>artificial synthesized
<400> 9
tggcaatgac acacactagt cagt 24
<210> 10
<211> 26
<212> DNA
<213>artificial synthesized
<400> 10
attacctgga taagggtcgt tacact 26
<210> 11
<211> 83
<212> DNA
<213>artificial synthesized
<400> 11
agaaatgaaa tggctcctgt caaacacaga taatgctgca ttcccgcaga tgactaagtc 60
atataaaaat acaagaaaaa acc 83
<210> 12
<211> 109
<212> DNA
<213>artificial synthesized
<400> 12
tggcaatgac acacactagt cagtatatat atgcagtcct gttcttacag acaatccccg 60
accgaatgac ccaaatatag gtaagtgtaa cgacccttat ccaggtaat 109
Claims (10)
1. a kind of kit for detecting H7N9 avian influenza virus, which is characterized in that the kit includes for expanding H7 gene
The first forward primer, the first reverse primer and the first probe, and it is the second forward primer for expanding N9 gene, second anti-
To primer and the second probe；First forward primer, the first reverse primer, the first probe, the second forward primer, second are reversely
Primer and the second probe are based on RPA Technology design；
Wherein, the sequence of first forward primer is as shown in SEQ ID NO.1, the sequence such as SEQ of first reverse primer
Shown in ID NO.2, the sequence of first probe is as shown in SEQ ID NO.3, the sequence such as SEQ of second forward primer
Shown in ID NO.4, the sequence of second reverse primer is as shown in SEQ ID NO.5, the sequence such as SEQ of second probe
Shown in ID NO.6.
2. kit as described in claim 1, which is characterized in that 5 ' ends of first reverse primer and described second are reversely
5 ' ends of primer are respectively connected with biotin, and 5 ' ends of first probe are connected with the first fluorophor, second probe
5 ' ends are connected with the second fluorophor.
3. kit as claimed in claim 2, which is characterized in that first fluorophor and second fluorophor are equal
For FAM.
4. kit as described in claim 1, which is characterized in that the kit further includes flow measurement chromatograph test strip.
5. kit as described in claim 1, which is characterized in that the kit further includes reaction buffer, product dilution
Liquid, magnesium acetate buffer and water.
6. kit as described in claim 1, which is characterized in that the kit further includes positive reference substance and/or feminine gender
Reference substance.
7. kit as claimed in claim 6, which is characterized in that the positive reference substance includes: shown in SEQ ID NO.11
Sequence and SEQ ID NO.12 shown in the recombinant plasmid that is connect respectively with pET6xHN-N carrier of sequence；The feminine gender
Reference substance is water.
8. a kind of method for detecting H7N9 avian influenza virus, which comprises the steps of:
Extract sample RNA；
It is cDNA by the sample RNA reverse transcription；
The cDNA is mixed with the reagent in the described in any item kits of claim 1-7, carries out RPA reaction, is obtained anti-
Answer liquid；
The reaction solution is added to flow measurement chromatograph test strip to detect.
9. method according to claim 8, which is characterized in that the condition of the RPA reaction includes: 37-42 DEG C of temperature, the time
18-20min。
10. method according to claim 8, which is characterized in that quality >=32fg of the cDNA.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819738A (en) * | 2019-11-20 | 2020-02-21 | 拱北海关技术中心 | Detection method of highly pathogenic H7N9 avian influenza virus, and primer and probe thereof |
| CN110938708A (en) * | 2019-10-15 | 2020-03-31 | 湖北省疾病预防控制中心 | Kit for detecting H7N9 avian influenza virus based on isothermal amplification technology and application thereof |
| CN111304364A (en) * | 2020-01-19 | 2020-06-19 | 河北农业大学 | Primer and probe combination for detecting avian influenza virus, kit and detection method |
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| CN110938708B (en) * | 2019-10-15 | 2023-03-24 | 湖北省疾病预防控制中心 | Kit for detecting H7N9 avian influenza virus based on isothermal amplification technology and application thereof |
| CN110819738A (en) * | 2019-11-20 | 2020-02-21 | 拱北海关技术中心 | Detection method of highly pathogenic H7N9 avian influenza virus, and primer and probe thereof |
| CN111304364A (en) * | 2020-01-19 | 2020-06-19 | 河北农业大学 | Primer and probe combination for detecting avian influenza virus, kit and detection method |
| CN111304364B (en) * | 2020-01-19 | 2023-11-14 | 河北农业大学 | Primer and probe combination for detecting avian influenza virus, kit and detection method |
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Application publication date: 20181211 |