CN108315478A - The probe and kit of RAA Fluorometric assay hydrophobins - Google Patents
The probe and kit of RAA Fluorometric assay hydrophobins Download PDFInfo
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- CN108315478A CN108315478A CN201810149632.2A CN201810149632A CN108315478A CN 108315478 A CN108315478 A CN 108315478A CN 201810149632 A CN201810149632 A CN 201810149632A CN 108315478 A CN108315478 A CN 108315478A
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Abstract
The present invention discloses a kind of detection hydrophobin RAA Fluorometric assay kits, and detection kit includes following components：The general reaction reagent of RAA basic fluorescences, reaction buffer, probe and primer mixture, negative quality-control product, positive quality control product and critical positive quality control product.The probe is 5 ' terminal sequences (fluorescent reporter group) (THF) (quenching group), 3 ' terminal sequence；This kit can directly detect hydrophobin DNA, be used especially for the detection lower sample of hydrophobin DNA content；This kit carries out isothermal duplication detection at 30~42 DEG C, and 5~20min completes detection.Kit is easy to operate, and cooperation miniature instrument, easy to carry has the advantages that quick, sensitive, accurate, reproducible；It is suitble to base and Site Detection, the good application prospect of tool.
Description
Technical field
The invention belongs to molecular Biological Detection fields, and in particular to a kind of detection hydrophobin RAA Fluorometric assays
Probe, primer and kit.
Background technology
Rabies are that most holy terror beast suffers from one of infectious disease altogether in the world.Its cause of disease rabies viruses (Rabies
Virus), belong to rhabdovirus (Rhabdovidae), Lyssavirus (Lyssavirus).Rabies viruses mainly passes through breakage
Skin or mucous membrane enter human body, through being advanced into central nervous system on nerve endings.Clinical manifestation is predominantly acute, carries out
Property, almost irreversible encephalomyelitis.For people once falling ill, 100% is dead.Rabies have become the highest infectious disease disease in China
Kind.Wild animal is rabic major storage host, and dog cat, domestic animal are the main infection sources of human rabies.It is complete according to statistics
The annual rabies death toll of ball is more than 60,000, wherein most case appears in Asia and Africa, and the annual rabies in Asia
Case accounts for about the 56% of global total cases.Recently as the increase of the pets such as cat, dog, which rises year by year
[13].The national reporting of infections of legal communicable diseases of ministry of Health of China publication claims, China's rabies number journey ascendant trend over the years,
It is to be only second to India, is to be endangered more serious one of country by rabies.Therefore, rabic prevention and control have become urgent appoint
Business.
Detection to hydrophobin, current method have：Fluorescent antibody test (FAT), quick rabies enzyme are immune to examine
Disconnected method (ELISA) mouse intracranial inoculation separation rabies viruses method (MIT), cell culture isolation technics (CIT), RT-PCR etc..Its
Middle FAT is detection hydrophobin " goldstandard ", but needs the fluorescent labeled antibody of expensive fluorescence microscope and high quality,
And fluorescent positive and feminine gender are not easy to recognize, it is necessary to experienced operating personnel.MIT needs 1 week or more time that could complete
Experiment, and need to just can determine that result with FAT.CIT needs to carry out in the laboratory for having cell culture condition, it is also desirable to coordinate FAT
It just can determine that result.ELISA method is simple, quick, but poor repeatability, and sensitivity is inadequate, and the viruses such as animal throat swab are contained
Less sample is measured, recall rate is low.
RT-PCR is now widely used for the detection of hydrophobin nucleic acid.With traditional virus purification or immunology detection
Method is compared, and RT-PCR has the characteristics that high specific, high sensitivity, therefore is more broadly used for rabic conventional detection and is divided
Sub- epidemiological study.RT-PCR applies also for the sample of the non-detectable corrupt samples and liquid of FAT, such as saliva and
Cerebrospinal fluid.In inspection to suspicious living animal sample, the especially inspection of samples such as zoogenetic infection early stage saliva hair, RT-
PCR has unrivaled advantage compared with conventional method.However, the method not only needs relevant expensive instrument and equipment and needs
There is professional technician's operation, therefore is very limited in the detection application of base and scene；The integral experiment time is in 2-4
Hour, and PCR is cumbersome, is easy to happen cross contamination.
Invention content
In order to solve in prior art detection method time-consuming and laborious, long time period and efficiency is low, need professional and
The problems such as expensive instrument, false positive rate is high, the invention discloses a kind of RT-RAA (Reverse Transcription-
Recombinase Aided Amplification) method detection hydrophobin primed probe and kit.With specificity
By force, high sensitivity, quick and convenient feature have larger application value.An embodiment of the present invention provides one kind for detecting
Primer, fluorescence probe and the kit of hydrophobin nucleic acid.The kit and detection method of the present invention uses RAA technologies, gram
Above disadvantage has been taken, has had the characteristics that short detection time, high sensitivity, high specificity, easy to operate.It only need to be anti-at 39 DEG C
Answering 5~20 minutes can analysis result.Have the characteristics that quick, sensitive, easy to operate.
Specifically, technical solutions according to the invention are as follows：
First, the present invention discloses a kind of probe of RAA Fluorometric assays hydrophobin, and the probe is：
5 ' terminal sequences (fluorescent reporter group) (THF) (quenching group), 3 ' terminal sequence；Wherein：
As shown in SEQ ID NO.3, sequence information is the nucleotide sequence of the 5 ' terminal sequence
CTTTGAAGCCTGAGATTATCGTGGATCAA；
As shown in SEQ ID NO.4, sequence information is the nucleotide sequence of the 3 ' terminal sequence
GAGTACAAGTACCCTG。
Therefore, entire probe sequence can also be expressed as：5 '-SEQ ID NO.3 (fluorescent reporter group) (THF) (are quenched
Group) SEQ ID NO.4-3 '.
Wherein, the THF is tetrahydrofuran residue；The fluorescent reporter group can select：FAM、HEX、TET、JOE、
Any one of VIC, ROX, Cy3 or Cy5, the quenching group can select：TAMRA、Eclipse、BHQ1、BHQ2、BHQ3
Or any one of DABCYL.
5’-CTTTGAAGCCTGAGATTATCGTGGATCAATATGAGTACAAGTACCCTG-3’
5’-CTTTGAAGCCTGAGATTATCGTGGATCAA(FAM-dT)(THF)(BHQ-dT)
GAGTACAAGTACCCTG-3’
It is specifically used in an embodiment of the present invention probe, design is characterized in that：The probe is using modification
Group is modified；The modification group includes fluorescent reporter group and fluorescent quenching group；Fluorescent reporter group modification is being visited
On position of the needle sequence from the ends 5' base number 30bp；Fluorescent quenching group is modified in position of the probe sequence from the ends 3' base number 17bp
It sets, 1 base positions is spaced between fluorescent reporter group and quenching group, tetrahydrofuran residue replaces the 31st bit base A.This
The primer that invention provides is suitable for RAA Fluorometric assays, and can accurately detect hydrophobin, and specificity reaches
100%.
Another aspect of the present invention is to disclose a kind of hydrophobin RAA Fluorometric assay kits comprising above
The probe and following primer：
Sense primer：5’-CAATGGATGCCGACAAGATTGTRTTCAAAGT-3’(SEQ ID No.1)；
Downstream primer：5’-CTGCYAARTAGGAACATACRTCATCAGGATCT-3’(SEQ ID No.2).
For a kind of hydrophobin RAA Fluorometric assay kits described in technical solution above, the present invention is to probe
It is not limited with storage concentration of the primer in kit, for the accuracy of experiment, under normal circumstances, the probe uses
When final concentration of 0.02-0.05mM in the reaction system, when the sense primer and downstream primer use in the reaction system
Final concentration of 0.05-0.1mM.
For a kind of hydrophobin RAA Fluorometric assay kits described in technical solution above, the kit is also
Including the general reaction reagent of reagent RAA basic fluorescences and reaction buffer.Wherein：The general reaction examination of the RAA basic fluorescences
Agent is the freeze-dried powder by frozen drying, is provided by Jiangsu Qi Tian genes bio tech ltd；It is that commodity article No. is
The basic reaction unit of F00001.Those skilled in the art can also select it according to the principle of RAA Fluorometric assay methods
He can be as the product of the general reaction reagent of RAA basic fluorescences as replacement.
For a kind of hydrophobin RAA Fluorometric assay kits described in technical solution above, the reaction is slow
Fliud flushing is made of following reagent：Storage concentration at this to it in kit does not limit, for the accuracy of experiment, generally
In the case of, the Tris-HCl buffer solutions, a concentration of using a concentration of 500mmol/L of each component in the reaction buffer
The PEG20000 that the MgAc and mass fraction of 250mmol/L is 10%.Preferably, the reaction buffer PH7.6.
For a kind of hydrophobin RAA Fluorometric assay kits described in technical solution above, the kit is also
Including following reagent：Negative quality-control product, positive quality control product and/or critical positive quality control product；
The positive quality control product contains the genomic DNA fragment for hydrophobin, the storage at this to it in kit
Concentration does not limit, for the accuracy of experiment, under normal circumstances, the use a concentration of 1.0 × 10 of positive quality control product5IU/mL；
Any one concentration of this concentration can also be selected above as positive quality control product.
The critical positive quality control product is the genomic DNA fragment containing hydrophobin, at this to it in kit
Storage concentration does not limit, for the accuracy of experiment, under normal circumstances, the use a concentration of 1.0 of critical positive quality control product ×
102IU/mL；
The feminine gender quality-control product is the reagent of the genomic fragment without containing hydrophobin, is typically chosen ddH2O or pure
Change water.
The kit easily and accurately identifies the presence of hydrophobin, and easy to operate, detection time is short, it is only necessary to 5~
20min can be completed, and so that DNA is untwisted without passing through high temperature, and only isothermal duplication need to be carried out at 30~42 DEG C can be completed detection.
The detection method is quick, high-throughput, reduces and differentiates time and testing cost, research shows that provided by the invention be based on RAA
The kit of Fluorometric assay hydrophobin has the characteristics that high specificity, specific up to 100%.Sensitivity is 102IU/
mL。
Meanwhile the kit provided by the invention based on RAA Fluorometric assay hydrophobins, it does not need large-scale instrument and sets
It is standby, it is suitable for Site Detection and suitable for large-scale screening.
The method for detecting hydrophobin using mentioned reagent box, includes the following steps：
(1) DNA of sample to be tested is extracted；DNA is extracted by normal method, may be used has the certified products of sale to extract on the market
Kit extracts the DNA of sample to be tested, such as Xi'an it is grand, QIAGEN kits, Tiangeng kit, and automatic nucleic acid can also be used
Extraction apparatus carries out DNA extractions；
(2) reaction Buffer is prepared：47 μ L reaction buffers are taken, the mixture of 2 μ L probes and primer is added, it is fully mixed
；Prepared 49 μ L reagents are added in the general reaction reagent freeze-dried powder of RAA basic fluorescences, it is made fully to dissolve and mix
；It is template to add extracted 1 μ L samples DNA, and total volume is 50 μ L；
(3) it is put into detection FAM fluorescence detection equipments and carries out real-time RAA fluorescence methods reaction；Real-time RAA fluorescence methods react item
Part is：39 DEG C of reaction 20min；
(4) interpretation of result：FAM fluorescence detection equipments detect that signal obviously increases within 20min, show amplification and sentence
It is set to the positive；FAM fluorescent instruments signal is determined as feminine gender without increase within 20min.
Description of the drawings
Fig. 1 is the genomic DNA plasmids detection result of various concentration hydrophobin；
Fig. 2 is the testing result of hydrophobin sample rna；
Fig. 3 is the specific detection result of hydrophobin.
Specific implementation mode
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to embodiment party of the present invention
Formula is described in further detail.But present disclosure is not limited thereto.
Primed probe and DNA plasmid are closed by Sangon Biotech (Shanghai) Co., Ltd. in following embodiment
At；
The present invention provides a kind of primer based on RAA Fluorometric assay hydrophobins, the primer includes that upstream is drawn
Object and downstream primer, the sense primer have the nucleotide sequence as shown in SEQ ID No.1 in sequence table；Draw in the downstream
Object has the nucleotide sequence as shown in SEQ ID No.2 in sequence table.
In the present invention, SEQ ID NO. in the plasmid dna sequence such as sequence table contained by the positive quality control product5It is shown：
CAATGGATGCCGACAAGATTGTATTCAAAGTCAATAATCAGGTGGTCTCTTTGAAGCCTGAGATTATCG
TGGATCAATATGAGTACAAGTACCCTGCTATCAAAGATTTGAAAAAGCCCTGTATAACCCTAGGGAAAGCCCCCGAC
TTAAACAAAGCATACAAGTCAGTTTTATCAGGCATGAATGCTGCCAAACTAGATCCTGATGATGTATGTTCCTACTT
GGCAGCAGCAATGCAGTTCTT
Sangon Biotech (Shanghai) Co., Ltd. is entrusted according to the above sequence) limited liability company carries out synthetic DNA plasmid, and plasmid is big
Small 244bp；
The design carried out using RAA technologies primer and probe design principle is determined by screening and evaluating
Sense primer：5’-CAATGGATGCCGACAAGATTGTRTTCAAAGT-3’(SEQ ID No.1)；
Downstream primer：5’-CTGCYAARTAGGAACATACRTCATCAGGATCT-3’(SEQ ID No.2).
The present invention also provides a kind of kits based on RAA Fluorometric assay hydrophobins, including the bases RT-RAA
The general reaction reagent of fluorescence, reaction buffer, negative quality-control product, positive quality control product, critical positive quality control product, probe and above-mentioned side
Primer described in case；
The probe has nucleotide sequence as follows：
5’-CTTTGAAGCCTGAGATTATCGTGGATCAATATGAGTACAAGTACCCTG-3’。
Kit provided by the invention includes primer.The primer includes sense primer and downstream primer.Sense primer and
The concentration of downstream primer is preferably independently 0.05~0.1mmol/L, more preferably 0.08mmol/L.The primer is by giving birth to work biology
Engineering (Shanghai) limited liability company is synthesized.
Kit provided by the invention includes probe.The concentration of the probe is preferably 0.02~0.05mmol/L, more excellent
It is selected as 0.04mmol/L.The probe has the nucleotide sequence as shown in SEQ ID No.3 in sequence table.In the present invention, institute
Probe is stated preferably to be modified using modification group.The modification group preferably includes fluorescent reporter group and fluorescent quenching base
Group.The method of modifying of the probe preferably includes：Between fluorescent reporter group modification within the probe；Fluorescent reporter group modification is being visited
On position of the needle sequence from the ends 5' base number 30bp；Fluorescent quenching group is modified in position of the probe sequence from the ends 3' base number 17bp
It sets, 1 base positions is spaced between fluorescent reporter group and quenching group, tetrahydrofuran residue replaces the 31st bit base A.
In the present invention, the fluorescent reporter group preferably includes FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5.
The fluorescent quenching group preferably includes TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.In the present invention in embodiment
Use FAM (6-Carboxyfluorescein) for fluorescent reporter group, BHQ (black hole quencher；
phosphate:3 ' phosphate to block elongation) it is fluorescent quenching group.Probe sequence after modification is：
5’-CTTTGAAGCCTGAGATTATCGTGGATCAA(FAM-dT)(THF)(BHQ-dT)
GAGTACAAGTACCCTG-3 ', wherein FAM is fluorescent reporter group, and BHQ is fluorescent quenching group, and THF is that tetrahydrofuran is residual
Base.The probe is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Kit provided by the invention includes the general reaction reagent of RT-RAA basic fluorescences.The RT-RAA basic fluorescences are logical
It is preferably the freeze-dried powder by frozen drying with reaction reagent.The general reaction of RT-RAA basic fluorescences in the embodiment of the present invention
Reagent is purchased from Jiangsu Qi Tian genes bio tech ltd, article No. F00001.
Kit provided by the invention includes reaction buffer.The reaction buffer preferably includes the group of following content
Point：The Tris-HCl buffer solutions of a concentration of 500mmol/L, the MgAc of a concentration of 250mmol/L and mass fraction are 10%
PEG20000.In the present invention, the concentration of the Tris-HCl buffer solutions, MgAc and PEG20000 is final concentration.The present invention couple
The source of the Tris-HCl buffer solutions, MgAc and PEG20000 is not particularly limited, using conventional commercial product.This hair
Tris-HCl's and PEG20000 described in bright embodiment is purchased from Sigma-Aldrich, and MgAc is tried purchased from traditional Chinese medicines Shanghai.
Kit provided by the invention includes positive quality control product.Hydrophobin is preferably comprised in the positive quality control product
Genomic DNA.The concentration of the genomic DNA of the hydrophobin is preferably 1 × 105IU/mL.Described in the embodiment of the present invention
Positive quality control product is cultivated and is extracted by recombinant plasmid switching Escherichia coli, and the sequence of plasmid is shown in SEQ ID in sequence table
NO.45。
Kit provided by the invention includes critical positive quality control product.Mad dog is preferably comprised in the critical positive quality control product
The genomic DNA of virus.The concentration of the genomic DNA of the hydrophobin is preferably 1 × 103IU/mL.The present invention is implemented
Critical positive quality control product described in example is cultivated and is extracted by recombinant plasmid switching Escherichia coli.
Kit provided by the invention includes negative quality-control product.The feminine gender quality-control product is preferably ddH2O or purified water.
In the present invention, the application method of the kit of the RAA Fluorometric assay hydrophobins described in said program, it is excellent
Choosing includes the following steps：
(1) DNA for extracting sample to be tested, obtains DNA extracting solutions；
(2) it is general anti-to be added to RT-RAA basic fluorescences after mixing 47 μ L reaction buffers with 1 μ L probes and 1 μ L primers
It answers in reagent and mixes；Obtain reaction mixture；
(3) reaction mixture obtained in the DNA extracting solutions obtained in step (1) described in 1 μ L and the step (2) is mixed
It closes, obtained reaction system carries out constant-temperature amplification, detects fluorescence signal；
The condition of the constant-temperature amplification is：37~42 DEG C of 5~20min of reaction；
(4) fluorescence detection equipment detects that signal obviously increases, generally increased fluorescent value be background values 30% with
Upper is positive.
In the present invention, the RNA of sample to be tested is extracted, obtains RNA extracting solutions.Do not have to the method for extracting RNA in the present invention
Particular determination is extracted using conventional method, can also be used it is commercially available extraction RNA kits extraction sample to be tested RNA or
RNA extractions are carried out using automatic instrument for extracting nucleic acid.In the present invention, the sample to be tested is the RNA of hydrophobin sample extraction.
In the present invention, it is anti-that the fluoroscopic examination preferably carries out real-time RAA fluorescence methods using detection FAM fluorescence detection equipments
It answers, FAM fluorescence detection equipments detect that signal obviously increases within 20min, and increased fluorescent value reaches the 30% of background values
It is the positive above；FAM fluorescent instruments signal is determined as feminine gender without increase within 20min.The present invention is to FAM fluorescence detectors
Source be not particularly limited.The RAA- provided using Jiangsu Qi Tian genes bio tech ltd in the embodiment of the present invention
F1620 fluorescence detectors.
Below in conjunction with the embodiment in the present invention, the technical solution in the present invention is clearly and completely described.It is aobvious
So, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
Embodiment 1
Probe is modified using fluorescent reporter group (FAM) and fluorescent quenching group (BHQ)；
Probe after modification is：5 '-CTTTGAAGCCTGAGATTATCGTGGATCAA (FAM-dT) (THF) (BHQ-dT)
GAGTACAAGTACCCTG-3’
Kit forms are as shown in table 1：
1 Kit components table of table
Recombinant plasmid switching Escherichia coli cultivate and extract 10105 μ L of hydrophobin recombinant dna plasmid are diluted to
The working standard of different gradients, respectively：
Working standard 1 contains 1.0 × 107IU/mL hydrophobin recombinant dna plasmids.
Working standard 2 contains 1.0 × 106IU/mL hydrophobin recombinant dna plasmids
Working standard 3 is positive quality control product.
Working standard 4 contains 1.0 × 104IU/mL hydrophobin recombinant dna plasmids.
Working standard 5 contains 1.0 × 103IU/mL hydrophobin recombinant dna plasmids.
Working standard 6 contains 1.0 × 102IU/mL hydrophobin recombinant dna plasmids.
The preparation of reaction buffer：376 μ L reaction buffers are drawn, the mixture (probe of 16 μ L probes and primer is added
A concentration of 0.05mmol/, a concentration of 1mmol/L of primer), mix well；The buffer solution after mixing is obtained, draws mixing every time
49 μ L of buffer solution afterwards are added separately in the general reaction reagent pipe of 7 RT-RAA basic fluorescences, and freeze-dried powder is made fully to dissolve simultaneously
It is mixed；
1 μ L feminine genders quality-control product, mark are separately added into 7 prepared general reaction reagent test tubes of RT-RAA basic fluorescences
Quasi- work product 6, standard work product 5, standard work product 4, standard work product 3, standard work product 2, standard work product 1 are template,
Each reaction tube is fully mixed, and each reaction tube total volume is 50 μ L.Reaction tube is put into fluorescence detector, setting is anti-
It is 39 DEG C to answer temperature, is reacted 20 minutes.Testing result is as shown in Fig. 1.As a result show obviously there is within most fast 2 minutes amplification, 10 points
All standard work product have amplification, sensitivity minimization that can reach 1.0 × 10 in clock2IU/mL, and facing final kit
Boundary's positive quality control product concentration is determined as 1.0 × 102IU/mL。
Embodiment 2
Primed probe and positive quality control product sequence are same as Example 1.
Kit forms are as shown in table 2：
2 Kit components table of table
Samples sources and RNA extractions
Hydrophobin sample cDNA is provided by Liaoning international travel health care center.Rabies vacciness is by the Zhejiang world
Clean travelling health center provides.
From rabies vacciness extract RNA use Tiangeng RNA extracts kits, operation by extraction specification in method into
Row, the RNA of extraction are preserved in -80 DEG C of refrigerators.
Reaction buffer is prepared：
Three reaction tubes are taken, following operation is pressed respectively, draws 47 μ L reaction buffers, the mixed of 2 μ L probes and primer is added
Object is closed, is fully mixed；49 μ L reagents of buffer solution after being mixed are added in the general reaction reagent pipe of RT-RAA basic fluorescences, are made
Freeze-dried powder is fully dissolved and is mixed；
1 μ L feminine genders quality-control product, 1 μ L hydrophobin samples are separately added into 3 prepared fluorescence reaction Reagent Tubes
The sample rna that cDNA, 1 μ L have been extracted is template, and each reaction tube is fully mixed, and each reaction tube total volume is 50 μ L；
Reaction tube is put into fluorescence detector, reaction temperature is set as 39 DEG C, reacts 20 minutes.Testing result such as attached drawing
Shown in 2, as a result show obviously there is amplification 6 minutes.
Embodiment 3
Primed probe and positive quality control product sequence are same as Example 1.
Kit forms are as shown in table 3：
3 Kit components table of table
Samples sources and RNA extractions
Rabies vacciness is provided by Zhejiang International Travel Healthcare Center.
From rabies vacciness extract RNA use Tiangeng RNA extracts kits, operation by extraction specification in method into
Row, the RNA of extraction are preserved in -80 DEG C of refrigerators.
Blood fluke sample is japonice ovum, and Echinococcus hydatid cyst sample is the tissue samples for infecting Echinococcus hydatid cyst, and liver fluke sample is infection
The fish sample of liver fluke, DNA extractions extract Tissue kit and special DNA extracts kit respectively using QIAGEN kits,
Extraction operation step is carried out by kit specification, and the DNA extracted is saved backup at -80 DEG C, and sample above is saved blood by Jiangsu
Fluke disease study on prevention is provided.
Reaction buffer is prepared：
5 reaction tubes are taken, following operation is pressed respectively, draws 47 μ L reaction buffers, the mixing of 2 μ L probes and primer is added
Object is fully mixed；49 μ L reagents of buffer solution after being mixed are added in the general reaction reagent pipe of RT-RAA basic fluorescences, make jelly
Dry powder is fully dissolved and is mixed；
1 μ L feminine genders quality-control product is separately added into 5 prepared fluorescence reaction Reagent Tubes, the rabies that 1 μ L have been extracted
It is template that RNA, 1 μ L japonice ovums DNA, 1 μ L Echinococcus hydatid cysts sample DNA, 1 μ L liver fluke sample DNAs are extracted in vaccine, each to react
Pipe is fully mixed, and each reaction tube total volume is 50 μ L.
Reaction tube is put into fluorescence detector, reaction temperature is set as 39 DEG C, reacts 20 minutes.Testing result such as attached drawing
Shown in 3.As a result display only has rabies vacciness sample rna obviously to have amplification, other samples and negative quality-control product do not expand,
It is feminine gender.
The foregoing is merely the preferable implementation examples of the present invention, are not intended to limit the invention, for the art
For those of ordinary skill, various improvements and modifications may be made without departing from the principle of the present invention, all in this hair
Within bright spirit and principle, any modification, equivalent replacement, improvement and so on should be included in protection scope of the present invention
Within.
Sequence table
<110>Wu Bin
<120>The probe and kit of RAA Fluorometric assay hydrophobins
<130> 2015
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213>Artificial sequence
<400> 1
ctttgaagcc tgagattatc gtggatcaa 29
<210> 2
<211> 16
<212> DNA
<213>Artificial sequence
<400> 2
gagtacaagt accctg 16
<210> 3
<211> 31
<212> DNA
<213>Artificial sequence
<400> 3
caatggatgc cgacaagatt gtrttcaaag t 31
<210> 4
<211> 32
<212> DNA
<213>Artificial sequence
<400> 4
ctgcyaarta ggaacatacr tcatcaggat ct 32
<210> 5
<211> 244
<212> DNA
<213>Artificial sequence
<400> 5
caatggatgc cgacaagatt gtattcaaag tcaataatca ggtggtctct ttgaagcctg 60
agattatcgt ggatcaatat gagtacaagt accctgctat caaagatttg aaaaagccct 120
gtataaccct agggaaagcc cccgacttaa acaaagcata caagtcagtt ttatcaggca 180
tgaatgctgc caaactagat cctgatgatg tatgttccta cttggcagca gcaatgcagt 240
tctt 244
Claims (6)
- The probe of 1.RAA Fluorometric assay hydrophobins, which is characterized in thatThe probe is 5 ' terminal sequences (fluorescent reporter group) (THF) (quenching group), 3 ' terminal sequence；Wherein：The nucleotide sequence of the 5 ' terminal sequence is as shown in SEQ ID NO.3；The nucleotide sequence of the 3 ' terminal sequence is as shown in SEQ ID NO.4；The THF is tetrahydrofuran residue；The fluorescent reporter group is selected from any one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5；The quenching group is selected from any one of TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
- 2. a kind of hydrophobin RAA Fluorometric assay kits, which is characterized in that the kit includes claim 1 institute The probe and following primer stated：The nucleotide sequence of sense primer is as shown in SEQ ID NO.1；The nucleotides sequence of downstream primer Row are as shown in SEQ ID NO.2.
- 3. hydrophobin RAA Fluorometric assay kits according to claim 2, which is characterized in that the probe Final concentration of 0.02-0.05mM, the sense primer and downstream primer final concentration are respectively 0.05-0.1mM.
- 4. hydrophobin RAA Fluorometric assay kits according to claim 2, which is characterized in that the kit Further include the general reaction reagent of reagent RAA basic fluorescences and reaction buffer.
- 5. hydrophobin RAA Fluorometric assay kits according to claim 4, which is characterized in that the reaction is slow Fliud flushing includes following content component：The Tris-HCl buffer solutions of a concentration of 500mmol/L, the MgAc of a concentration of 250mmol/L and The PEG20000 that mass fraction is 10%, buffer solution ph 7.6.
- 6. hydrophobin RAA Fluorometric assay kits according to claim 2, which is characterized in that the kit It further include following reagent：Negative quality-control product, positive quality control product and/or critical positive quality control product；The positive quality control product contains the genomic DNA fragment for hydrophobin, uses a concentration of 1.0 × 105IU/mL；The critical positive quality control product is the genomic DNA fragment containing hydrophobin, uses a concentration of 1.0 × 102IU/mL；The feminine gender quality-control product is the reagent of the genomic DNA fragment without containing hydrophobin.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609697A (en) * | 2019-01-04 | 2019-04-12 | 浙江国际旅行卫生保健中心（浙江出入境检验检疫局口岸门诊部） | A kind of primer, probe and detection kit quickly detecting ERC group virus based on RAA fluorescence method |
| CN110157823A (en) * | 2019-05-24 | 2019-08-23 | 中国人民解放军疾病预防控制中心 | Primed probe group, kit and its application of RAA Fluorometric assay Yersinia pestis |
| CN110592285A (en) * | 2019-10-21 | 2019-12-20 | 中国动物卫生与流行病学中心 | RAA primer probe for detecting Nipah virus and detection method |
| CN112795704A (en) * | 2021-03-09 | 2021-05-14 | 中国农业大学 | RAA primer pair, probe and kit for detecting porcine pseudorabies virus and application of RAA primer pair, probe and kit |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565758A (en) * | 2009-06-02 | 2009-10-28 | 武汉三利生物技术有限公司 | Rabies virus detecting fluorescence quantitative PCR kit and application thereof |
| CN102816756A (en) * | 2012-09-07 | 2012-12-12 | 江苏奇天基因生物科技有限公司 | Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method |
| CN105087825A (en) * | 2015-07-01 | 2015-11-25 | 浙江泰晶生物科技有限公司 | Method, reagent, primer and probe for quickly detecting Ebola viruses under constant-temperature and isothermal conditions |
| CN107201412A (en) * | 2017-07-25 | 2017-09-26 | 广州和实生物技术有限公司 | A kind of hydrophobin parting Constant Temperature Detection kit |
| CN107557496A (en) * | 2017-10-13 | 2018-01-09 | 宁波国际旅行卫生保健中心 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect MERS CoV viruses |
| CN107574262A (en) * | 2017-10-25 | 2018-01-12 | 宁波国际旅行卫生保健中心 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect A (H 1 N 1) virus |
| CN107619885A (en) * | 2017-10-25 | 2018-01-23 | 宁波国际旅行卫生保健中心 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect dengue virus |
-
2018
- 2018-02-13 CN CN201810149632.2A patent/CN108315478A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565758A (en) * | 2009-06-02 | 2009-10-28 | 武汉三利生物技术有限公司 | Rabies virus detecting fluorescence quantitative PCR kit and application thereof |
| CN102816756A (en) * | 2012-09-07 | 2012-12-12 | 江苏奇天基因生物科技有限公司 | Isothermal nucleic acid amplification reaction reagent and isothermal nucleic acid amplification method |
| CN105087825A (en) * | 2015-07-01 | 2015-11-25 | 浙江泰晶生物科技有限公司 | Method, reagent, primer and probe for quickly detecting Ebola viruses under constant-temperature and isothermal conditions |
| CN107201412A (en) * | 2017-07-25 | 2017-09-26 | 广州和实生物技术有限公司 | A kind of hydrophobin parting Constant Temperature Detection kit |
| CN107557496A (en) * | 2017-10-13 | 2018-01-09 | 宁波国际旅行卫生保健中心 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect MERS CoV viruses |
| CN107574262A (en) * | 2017-10-25 | 2018-01-12 | 宁波国际旅行卫生保健中心 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect A (H 1 N 1) virus |
| CN107619885A (en) * | 2017-10-25 | 2018-01-23 | 宁波国际旅行卫生保健中心 | A kind of fluorescence RT RAA primers, probe and detection method for being used to detect dengue virus |
Non-Patent Citations (3)
| Title |
|---|
| 曹墨菊: "《植物生物技术概论》", 30 October 2014 * |
| 许运斌等: "一步法Taqman荧光定量RT-PCR方法检测狂犬病病毒", 《中国畜牧兽医学会兽医公共卫生学分会第二次学术研讨会论文集》 * |
| 高志强等: "狂犬病病毒快速荧光RT-PCR 检测方法研究与应用", 《中国兽医杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609697A (en) * | 2019-01-04 | 2019-04-12 | 浙江国际旅行卫生保健中心（浙江出入境检验检疫局口岸门诊部） | A kind of primer, probe and detection kit quickly detecting ERC group virus based on RAA fluorescence method |
| CN110157823A (en) * | 2019-05-24 | 2019-08-23 | 中国人民解放军疾病预防控制中心 | Primed probe group, kit and its application of RAA Fluorometric assay Yersinia pestis |
| CN110592285A (en) * | 2019-10-21 | 2019-12-20 | 中国动物卫生与流行病学中心 | RAA primer probe for detecting Nipah virus and detection method |
| CN112795704A (en) * | 2021-03-09 | 2021-05-14 | 中国农业大学 | RAA primer pair, probe and kit for detecting porcine pseudorabies virus and application of RAA primer pair, probe and kit |
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