CN106521032A - Nucleic acid amplification technology and application thereof in detection of mosquito-borne viruses - Google Patents
Nucleic acid amplification technology and application thereof in detection of mosquito-borne viruses Download PDFInfo
- Publication number
- CN106521032A CN106521032A CN201611089150.XA CN201611089150A CN106521032A CN 106521032 A CN106521032 A CN 106521032A CN 201611089150 A CN201611089150 A CN 201611089150A CN 106521032 A CN106521032 A CN 106521032A
- Authority
- CN
- China
- Prior art keywords
- sequence
- primer
- probe group
- primed probe
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a nucleic acid amplification technology and an application thereof in detection of mosquito-borne viruses. The invention provides a method for detecting whether n kinds of target RNA sequences are contained in a sample to be detected or not. The method sequentially comprises the following steps: 1, extracting the total RNA of the sample to be detected; 2, carrying out reverse transcription recombinase polymerase amplification; and 3, carrying out recombinase polymerase amplification, wherein n is a natural number. The invention also provides a primer probe combination. The combination comprises primers and probes represented by sequence 1 to sequence 18 in a sequence table. A micro-fluidic chip, recombinase polymerase amplification and nucleic acid sequence dependent amplification are combined to effectively solve the problem of detection limit reduction of the micro-fluidic chip. The invention further provides a kit used for detecting the mosquito-borne viruses. The kit can simultaneously complete detection of six varieties of the mosquito-borne viruses one time, and has the advantages of high sensitivity, good specificity, fastness, good convenience, and wide application values.
Description
Technical field
The present invention relates to a kind of nucleic acid amplification technologies and its application in carapuru virus detection.
Background technology
Polymerase chain reaction (Polymerase Chain Reaction, PCR) is the most frequently used one of current detection of nucleic acids
Detection technique is planted, its testing result is accurate, specificity is high, reproducible.Multiplex PCR is the one kind developed on the basis of PCR
The technology of multiple target genes is detected simultaneously can, at least two pairs or more primer in general multi-PRC reaction system,
Multiple nucleic acid fragments can be amplified simultaneously in primary first-order equation, be mainly used in detection or mirror while multiple pathogenic microorganisms simultaneously
It is fixed.But due to there is a plurality of PCR primer in multiplex PCR, easily produce between primer and interfere, tuple is more, the primer for including
More, the probability for producing interference from each other is bigger, and designing and developing more difficult this of process becomes the exploitation of multiplex PCR System Design
During subject matter.
In order to avoid carry out in same reaction that multi-primerses design causes it is huge design and develop difficulty, using micro-fluidic
Multiple reaction in same PCR pipe is reduced to the reaction of the substance in multiple miniature cavities by the physics point chamber technology of chip, and this is big
Simplify greatly the primer amplification system development process of multi objective.Microfluidic chip technology, is a kind of by physics point chamber, is realized high
The technology of flux detection, can complete the detection of multiple indexs in one-time detection simultaneously, solve multiple PCR primer well and set
A difficult problem in meter, micro-fluidic chip need not design multi-primerses, and which has bigger customization degree of freedom, flux enter as needed
Row is adjusted.But, although many flux system developments are enormously simplify by the way of physics point chamber, but while also generate one
More serious side effect.I.e. multi-cavity causes the shunting for adding sample form, causes its detection sensitivity to be deteriorated, for example, 24
Hole micro-fluidic chip, limited sample form have respectively enterd 24 reaction receptors, and its test limit have dropped 24 times, this to its
Application clinically is a test.
Carapuru virus is widely distributed in the whole world, is a kind of important infectious disease pathogens, general to be propagated by mosquito bite,
Spread speed is fast.In recent years, constantly there is related carapuru virus Epidemic outbreak of disease all over the world, mainly agree including zika virus, datum hole
Refined virus, dengue virus etc..These virus infection, can cause the similar symptoms such as heating, arthralgia, it is difficult to from clinical manifestation
It is upper to judge it is that virus infection.Therefore, the detection of nucleic acids of mosquito matchmaker correlated virus is most important.
The content of the invention
It is an object of the invention to provide a kind of nucleic acid amplification technologies and its application in carapuru virus detection.
Present invention firstly provides a kind of primer combination of probe, is following (a1) or (a2) or (a3):
(a1) by primed probe group I, primed probe group II, primed probe group III, primed probe group IV, primed probe group V
Constitute with primed probe group VI;
(a2) by the primed probe group I, the primed probe group II, the primed probe group III, the primed probe
Any two, any three, any four or any five in group IV, the primed probe group V and the primed probe group VI
Individual composition;
(a3) the primed probe group I, the primed probe group II, the primed probe group III, the primed probe group
IVth, the primed probe group V or the primed probe group VI;
The primed probe group I is made up of primer ZIKV-F, primer ZIKV-R and probe ZIKV-P;
The primer ZIKV-F is following (b1) or (b2);
(b1) single strand dna shown in the sequence 1 of sequence table;
(b2) sequence 1 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of identical function;
The primer ZIKV-R is following (b3) or (b4);
(b3) single strand dna shown in the sequence 2 of sequence table;
(b4) sequence 2 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of identical function;
The nucleotides sequence of the probe ZIKV-P is classified as (b5) as follows or (b6);
(b5) single strand dna as shown in the sequence 13 of sequence table;
(b6) sequence 13 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 13
There is the DNA molecular of identical function;
The primed probe group II is made up of primer CHIKV-F, primer CHIKV-R and probe CHIKV-P;
The primer CHIKV-F is following (c1) or (c2);
(c1) single strand dna shown in the sequence 3 of sequence table;
(c2) sequence 3 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 3
The DNA molecular of identical function;
The primer CHIKV-R is following (c3) or (c4);
(c3) single strand dna shown in the sequence 4 of sequence table;
(c4) sequence 4 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 4
The DNA molecular of identical function;
The nucleotides sequence of the probe CHIKV-P is classified as (c5) as follows or (c6);
(c5) single strand dna as shown in the sequence 14 of sequence table;
(c6) sequence 14 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 14
There is the DNA molecular of identical function;
The primed probe group III is made up of primer DENV1-F, primer DENV1-R and probe DENV1-P;
The primer DENV1-F is following (d1) or (d2);
(d1) single strand dna shown in the sequence 5 of sequence table;
(d2) sequence 5 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 5
The DNA molecular of identical function;
The primer DENV1-R is following (d3) or (d4);
(d3) single strand dna shown in the sequence 6 of sequence table;
(d4) sequence 6 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 6
The DNA molecular of identical function;
The nucleotides sequence of the probe DENV1-P is classified as (d5) as follows or (d6);
(d5) single strand dna as shown in the sequence 15 of sequence table;
(d6) sequence 15 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 15
There is the DNA molecular of identical function;
The primed probe group IV is made up of primer DENV2-F, primer DENV2-R and probe DENV2-P;
The primer DENV2-F is following (e1) or (e2);
(e1) single strand dna shown in the sequence 7 of sequence table;
(e2) sequence 7 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 7
The DNA molecular of identical function;
The primer DENV2-R is following (e3) or (e4);
(e3) single strand dna shown in the sequence 8 of sequence table;
(e4) sequence 8 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 8
The DNA molecular of identical function;
The nucleotides sequence of the probe DENV2-P is classified as (e5) as follows or (e6);
(e5) single strand dna as shown in the sequence 16 of sequence table;
(e6) sequence 16 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 16
There is the DNA molecular of identical function;
The primed probe group V is made up of primer DENV3-F, primer DENV3-R and probe DENV3-P;
The primer DENV3-F is following (f1) or (f2);
(f1) single strand dna shown in the sequence 9 of sequence table;
(f2) sequence 9 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 9
The DNA molecular of identical function;
The primer DENV3-R is following (f3) or (f4);
(f3) single strand dna shown in the sequence 10 of sequence table;
(f4) sequence 10 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 10
There is the DNA molecular of identical function;
The nucleotides sequence of the probe DENV3-P is classified as (f5) as follows or (f6);
(f5) single strand dna as shown in the sequence 17 of sequence table;
(f6) sequence 17 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 17
There is the DNA molecular of identical function;
The primed probe group VI is made up of primer DENV4-F, primer DENV4-R and probe DENV4-P;
The primer DENV4-F is following (g1) or (g2);
(g1) single strand dna shown in the sequence 11 of sequence table;
(g2) sequence 11 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 11
There is the DNA molecular of identical function;
The primer DENV4-R is following (g3) or (g4);
(g3) single strand dna shown in the sequence 12 of sequence table;
(g4) sequence 12 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 12
There is the DNA molecular of identical function;
The nucleotides sequence of the probe DENV4-P is classified as (g5) as follows or (g6);
(g5) single strand dna as shown in the sequence 18 of sequence table;
(g6) sequence 18 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 18
There is the DNA molecular of identical function.
In probe described in any of the above, 5 ' ends have fluorophor FAM, and 3 ' ends have quenching group BHQ1.
The present invention also protects a kind of special chip, is by described primed probe group I, primed probe group II, primed probe
Group III, primed probe group IV, primed probe group V and primed probe group VI are embedded in the differential responses chamber of micro-fluidic chip respectively
In obtain;
The function of the special chip is following (h1) or (h2):
(h1) it is used for identifying zika virus, Chikungunya virus, I type of dengue virus, II type of dengue virus, dengue fever
IV type of virus type iii or dengue virus;
(h2) whether zika virus and/or Chikungunya virus and/or Dengue calentura are contained in being used for detecting sample to be tested
IV type of malicious I type and/or II type of dengue virus and/or III type of dengue virus and/or dengue virus.
The primer and probe are specifically embedded in reaction tube by encapsulation reagent.The encapsulation reagent is by 3-10 mass parts fine jades
Lipolysaccharide, 1-5 mass parts saponin and 2-6 mass parts hydroxypropyl cellulose composition.The encapsulation reagent is specifically by 5 mass parts agar
Sugar, 3 mass parts saponin and 4 mass parts HPMC composition.
It is embedded with the preparation method of the micro-fluidic chip of primer and probe:1. will encapsulation reagent, primer, probe and sterilized water
Mixing, (in embedding liquid, the concentration for encapsulating reagent is 0.1g/100ml, and the concentration of primer is 0.4 μM, probe to obtain embedding liquid
Concentration is 0.2 μM);2. micro-fluidic chip is taken, 1 μ l embedding liquid in each reaction chamber, is added;3. take that 2. step obtain is micro-fluidic
Chip, is placed in clean super-clean bench and dries, then tabletting encapsulation punching press evacuation.
The present invention also protection includes the test kit first of the primer combination of probe.
The present invention also protection includes the test kit second of the micro-fluid chip.
The test kit second is also combined including primer pair;The primer pair is combined by primer pair I, primer pair II, primer pair
IIIth, primer pair IV, primer pair V and primer pair VI are constituted;The primer pair I is made up of primer ZIKV-F and primer ZIKV-R;Institute
State primer pair II to be made up of primer CHIKV-F and primer CHIKV-R;The primer pair III is by primer DENV1-F and primer
DENV1-R is constituted;The primer pair IV is made up of primer DENV2-F and primer DENV2-R;The primer pair V is by primer
DENV3-F and primer DENV3-R compositions;The primer pair VI is made up of primer DENV4-F and primer DENV4-R.
The primer pair combination is specifically in the form of primer mixed liquor.Primer mixture liquid by ZIKV-F, ZIKV-R,
CHIKV-F、CHIKV-R、DENV1-F、DENV1-R、DENV2-F、DENV2-R、DENV3-F、DENV3-R、DENV4-F、
DENV4-R and sterilized water composition;In primer mixture liquid, the concentration of ZIKV-F is 0.2 μM, the concentration of ZIKV-R is 0.2 μM,
The concentration of CHIKV-F is 0.15 μM, the concentration of CHIKV-R is 0.15 μM, the concentration of DENV1-F is 0.2 μM, DENV1-R it is dense
Spend the concentration for 0.2 μM, DENV2-F be 0.15 μM, the concentration of DENV2-R be 0.15 μM, the concentration of DENV3-F be 0.3 μM,
The concentration of DENV3-R is 0.3 μM, the concentration of DENV4-F is 0.25 μM, the concentration of DENV4-R is 0.25 μM.
The test kit second also includes also including reaction mixture BQ and enzyme mixation EQ.
Reaction mixture BQ:Tris-HCl buffer of the solvent for pH8.0,200mM;Solute and its concentration are as follows:DTT
50mM、MgCl280mM, KCl 450mM, DMSO 15% (volumn concentration), Sorbitol 1M, dNTP 2mM, rNTP
4mM。
Enzyme mixation EQ:Solvent is water;Solute and its concentration are as follows:AMV reverse transcriptase 1U/ μ l, t7 rna polymerase 5U/ μ
L, ribonuclease H 0.5U/ μ l, pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA 0.5 μ g/ μ l.
The test kit also includes RT-RPA reaction buffers, RT-RPA enzymes mixed powder and 280mM magnesium acetate aqueous solutions.
The test kit also includes RT-RPA reaction buffers, the reaction tube equipped with RT-RPA enzyme mixed powders and 280mM
Magnesium acetate aqueous solution.
The present invention also protects the application of the test kit first, is following (h1) or (h2):
(h1) it is used for identifying zika virus, Chikungunya virus, I type of dengue virus, II type of dengue virus, dengue fever
IV type of virus type iii or dengue virus;
(h2) whether zika virus and/or Chikungunya virus and/or Dengue calentura are contained in being used for detecting sample to be tested
IV type of malicious I type and/or II type of dengue virus and/or III type of dengue virus and/or dengue virus.
The present invention also protects the application of the test kit second, is following (h1) or (h2):
(h1) it is used for identifying zika virus, Chikungunya virus, I type of dengue virus, II type of dengue virus, dengue fever
IV type of virus type iii or dengue virus;
(h2) whether zika virus and/or Chikungunya virus and/or Dengue calentura are contained in being used for detecting sample to be tested
IV type of malicious I type and/or II type of dengue virus and/or III type of dengue virus and/or dengue virus.
Identify that whether virus to be measured be zika virus, Chikungunya virus, I type of dengue virus, step on using test kit second
Leather II type of fever virus, IV type of III type of dengue virus or dengue virus method it is as follows:
(1) reaction tube equipped with RT-RPA enzyme mixed powders is taken, 29.5 μ l RT-RPA reaction bufferings are sequentially added
Liquid, 5 μ l primer mixed liquors, 10 μ l solution to be measured and 3 μ l water mix homogeneously, are subsequently adding 2.5 μ l 280mM magnesium acetates water-soluble
Liquid is starting reaction;Reaction condition:37℃10min;
(2) reaction tube of step (1) is taken into, following enzyme-deactivating process is carried out:80℃2min;12 DEG C, 3min;
(3) product of 12 μ l reaction mixture BQ, 3 μ l enzyme mixations EQ and 10 μ l steps (2), mix homogeneously, injection are taken
Enter to be embedded with the micro-fluidic chip of primer and probe, seal Quick spin 30s after membrana oralis;
(4), after completing step (3), the micro-fluidic chip for being embedded with primer and probe is placed in into brilliant core RTisochip-
In A constant-temperature amplification micro-fluidic chip nucleic acids instruments, 41 DEG C of reaction 60min simultaneously complete real-time fluorescence scanning simultaneously.
Determine which kind of virus is virus to be measured be as follows according to the amplification curve of sample in each reaction chamber:If bag
The amplification curve for being embedded with sample in the reaction chamber of primed probe group I is S type positive amplification curves, and virus to be measured is zika virus;
If the amplification curve for being embedded with sample in the reaction chamber of primed probe group II is S type positive amplification curves, virus to be measured is base
Agree refined virus in hole;If the amplification curve for being embedded with sample in the reaction chamber of primed probe group III is S type positive amplification curves, treat
It is I type of dengue virus to survey virus;If the amplification curve for being embedded with sample in the reaction chamber of primed probe group IV is that S types are positive
Amplification curve, virus to be measured are II type of dengue virus;If being embedded with the amplification of sample in the reaction chamber of primed probe group V
Curve is S type positive amplification curves, and virus to be measured is III type of dengue virus;If being embedded with the reaction chamber of primed probe group VI
The amplification curve of middle sample is S type positive amplification curves, and virus to be measured is IV type of dengue virus.
Detect in sample to be tested whether contain zika virus and/or Chikungunya virus and/or Dengue using test kit second
The method of IV type of I type of fever virus and/or II type of dengue virus and/or III type of dengue virus and/or dengue virus is as follows:
(1) reaction tube equipped with RT-RPA enzyme mixed powders is taken, 29.5 μ l RT-RPA reaction bufferings are sequentially added
Liquid, 5 μ l primer mixed liquors, 10 μ l solution to be measured and 3 μ l water mix homogeneously, are subsequently adding 2.5 μ l 280mM magnesium acetates water-soluble
Liquid is starting reaction;Reaction condition:37℃10min;
(2) reaction tube of step (1) is taken into, following enzyme-deactivating process is carried out:80℃2min;12 DEG C, 3min;
(3) product of 12 μ l reaction mixture BQ, 3 μ l enzyme mixations EQ and 10 μ l steps (2), mix homogeneously, injection are taken
Enter to be embedded with the micro-fluidic chip of primer and probe, seal Quick spin 30s after membrana oralis;
(4), after completing step (3), the micro-fluidic chip for being embedded with primer and probe is placed in into brilliant core RTisochip-
In A constant-temperature amplification micro-fluidic chip nucleic acids instruments, 41 DEG C of reaction 60min simultaneously complete real-time fluorescence scanning simultaneously.
According to the amplification curve of sample in each reaction chamber determine as follows sample to be tested contain it is any or which
Plant virus:If the amplification curve for being embedded with sample in the reaction chamber of primed probe group I is S type positive amplification curves, test sample is treated
Originally contain zika virus;If the amplification curve for being embedded with sample in the reaction chamber of primed probe group II is that S types positive amplification is bent
Line, sample to be tested contain Chikungunya virus;If the amplification curve for being embedded with sample in the reaction chamber of primed probe group III is S
Type positive amplification curve, sample to be tested contain I type of dengue virus;If being embedded with sample in the reaction chamber of primed probe group IV
Amplification curve be S type positive amplification curves, sample to be tested contains II type of dengue virus;If being embedded with primed probe group V
Reaction chamber in the amplification curve of sample be S type positive amplification curves, sample to be tested contains III type of dengue virus;If embedding
The amplification curve for having sample in the reaction chamber of primed probe group VI is S type positive amplification curves, and sample to be tested contains Dengue calentura
Malicious IV type.
I type of dengue virus concretely DEN-1 Hawaii strain described in any of the above.Dengue virus described in any of the above
The concretely DEN-2NGC strains of II type.III type of dengue virus concretely DEN-3H87 strains described in any of the above.Any of the above
The dengue virus IV type concretely DEN-4H241 strains.
The present invention also protects in a kind of detection sample to be tested the whether method containing n kind target RNA sequences, include successively as
Lower step (reaction principle schematic diagram is shown in Fig. 1):
(1) extract the total serum IgE of sample to be tested;
(2) carry out reverse transcription recombinase polymeric enzymatic amplification;
(3) carry out recombinase polymeric enzymatic amplification;N is natural number.
N is more than 2 natural number.
The recombinase polymeric enzymatic amplification is carried out in micro-fluid chip, and each reaction chamber of micro-fluid chip is used for detecting
A kind of target RNA sequence.
The present invention also protects a kind of for detecting in sample to be tested the whether test kit containing n kind target RNA sequences, including
The reagent for carrying out reverse transcription recombinase polymeric enzymatic amplification and the reagent for carrying out recombinase polymeric enzymatic amplification.
The test kit also includes special chip;The special chip is respectively by for detecting the phase of every kind of target RNA
The primed probe group of DNA is answered to be embedded in what the differential responses chamber of micro-fluid chip obtained;The primed probe group is by pair of primers
With a probe composition.
The reaction condition of reverse transcription recombinase polymeric enzymatic amplification can be:37 DEG C -42 DEG C, 5-30min.
The reaction condition of reverse transcription recombinase polymeric enzymatic amplification is concretely:37 DEG C, 10min.
Reverse transcription recombinase polymeric enzymatic amplification can be the multiplex amplification using the multiple primer pairs for different target sequence,
Alternatively using the single amplification of a primer pair for a certain target sequence.The multiplex amplification is concretely adopted and is directed to
The sixfold amplification of 6 primer pairs of 6 kinds of target sequence.
After the completion of reverse transcription recombinase polymeric enzymatic amplification, before recombinase polymeric enzymatic amplification is carried out, also including following inactivation step
Suddenly:80 DEG C, 2min;12 DEG C, 3min.
The reaction condition of recombinase polymeric enzymatic amplification can be:41 DEG C, 30-60min.
The primer that the primer adopted in reverse transcription recombinase polymeric enzymatic amplification is adopted with recombinase polymeric enzymatic amplification, Ke Yiwei
Identical primer can also be different primer.
Should the target sequence containing recombinase polymeric enzymatic amplification in the target sequence of reverse transcription recombinase polymeric enzymatic amplification.
It is for the detection target sequence realized, of the invention by micro-fluidic chip, recombinase polymeric enzymatic amplification and nucleotide sequence
Dependent amplification combines, there is provided a kind of method of detection target nucleic acids, first passes through reverse transcription recombinase polymeric enzymatic amplification
(carrying out multiplex amplification in same system using the multi-primerses for multi-target fragment) realizes the richness nucleic acid-templated to RNA
Collection, then in micro-fluidic chip carries out Nucleic acid sequence amplification and (is respectively directed to each target fragments in each reaction chamber
Carry out single amplification), can effectively solve the problems, such as that micro-fluidic chip test limit is reduced, while have taking short, high flux
(detection is completed within 1 hour, such as within 30 minutes with the advantage of high detection limit;Can realize simultaneously to multiple target sequence
Detection, for example simultaneously realize the detection viral to 6 kinds;Test limit is copied up to unitss, is improve relative to prior art
100 times).
Recombinase polymeric enzymatic amplification is the thermostat extra income grown up on the basis of existing beyond body nucleic acid expands principle
Fast amplification of nucleic acid technology, recombinase polymeric enzymatic amplification only need to a pair simple primers and can be achieved with isothermal duplication, but recombinate
The amount of the product of enzymatic amplification is not mostly high, it is difficult to be detected.
Nucleic acid sequence amplification is by pair of primers mediation, in vitro specificity continuous homogeneous to single-stranded
RNA carries out the process of constant-temperature amplification, is to react under a stationary temperature, and response speed is exceedingly fast, generally the characteristics of maximum
An amplified reaction can be just completed in 20 minutes.The technology is usually used in the detection of nucleic acids of RNA viruses, and its detection specificity is good, clever
Sensitivity is high, uses it for micro-fluidic chip detection technique, is capable of achieving high flux, the quick detection of RNA viruses, but due to above-mentioned micro-
The restriction of fluidic chip so as to which the detection to trace dna is greatly limited.
Further, the invention provides a kind of test kit for detecting carapuru virus.The test kit that the present invention is provided, can
Disposably to complete the detection to six kinds of carapuru viruses simultaneously, have the advantages that sensitivity is high, specificity is good, rapid and convenient, tool
Have a wide range of applications.
Description of the drawings
Fig. 1 is reaction principle schematic diagram.
Fig. 2 is the result of zika virus standard substance in embodiment 3.
Fig. 3 is the result of Chikungunya virus standard substance in embodiment 3.
Fig. 4 is the result of I type standard substance of dengue virus in embodiment 3.
Fig. 5 is the result of II type standard substance of dengue virus in embodiment 3.
Fig. 6 is the result of III type standard substance of dengue virus in embodiment 3.
Fig. 7 is the result of IV type standard substance of dengue virus in embodiment 3.
Specific embodiment
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetitions and tests, as a result make even
Average.In embodiment, water used is the water without RNase.BSA is bovine serum albumin.
AMV reverse transcriptases:Promega companies, article No. M5108.T7 rna polymerase:Promega companies, article No. P4074.
Ribonuclease H:NEB companies, article No. M0297S.Pyrophosphatase:Sigma companies, article No. I5907.RNase inhibitor:
Promega companies, article No. N2611.
TABARTS01kit is commercial kit, sees network addresshttp://www.twistdx.co.uk/products/ general_rd_kits/twistamp_basic_rt/。
Agarose:Baygene companies, article No. 162090.
Ginsenoside:Sigma companies, article No.:65839.
The chemical structural formula of ginsenoside is shown in formula I.
Hydroxypropyl cellulose (average Mw~100000):Sigma companies, article No. 191884.
The chemical structural formula of hydroxypropyl cellulose is shown in formula II.
Embodiment 1, design and prepare each primer and probe
For detecting that the primer pair of zika virus (Zika Virus) constitutes (5 ' → 3 ') by following two primers:
ZIKV-F (sequence 1 of sequence table):TGCATWGGAGTCAGCAA;
ZIKV-R (sequence 2 of sequence table):AATTCTAATACGACTCACTATAGGGAGATAGGATCTTACCTCVGCCA.
For detecting that the primer pair of Chikungunya virus (Chikungunya virus) constitutes (5 ' by following two primers
→3’):
CHIKV-F (sequence 3 of sequence table):CCGACTCAACCATCCTGGAT;
CHIKV-R (sequence 4 of sequence table):AATTCTAATACGACTCACTATAGGGAGAGGCARACGCAGTGGTACTT
CCT。
For detecting that the primer pair of I type of dengue virus constitutes (5 ' → 3 ') by following two primers:
DENV1-F (sequence 5 of sequence table):CAAAAGGAAGTCGTGCAATA;
DENV1-R (sequence 6 of sequence table):AATTCTAATACGACTCACTATAGGGAGACTGAGTGAATTCTCTCTA
CTGAACC。
For detecting that the primer pair of II type of dengue virus constitutes (5 ' → 3 ') by following two primers:
DENV2-F (sequence 7 of sequence table):CAGGTTATGGCACTGTCACGAT;
DENV2-R (sequence 8 of sequence table):AATTCTAATACGACTCACTATAGGGAGACCATCTGCAGCAACACCA
TCTC。
For detecting that the primer pair of III type of dengue virus constitutes (5 ' → 3 ') by following two primers:
DENV3-F (sequence 9 of sequence table):GGACTGGACACACGCACCCA;
DENV3-R (sequence 10 of sequence table):
AATTCTAATACGACTCACTATAGGGAGACATGTCTCTACCTTCTCGACTTGTCT。
For detecting that the primer pair of IV type of dengue virus constitutes (5 ' → 3 ') by following two primers:
DENV4-F (sequence 11 of sequence table):TTGTCCTAATGATGCTGGTCG;
DENV4-R (sequence 12 of sequence table):AATTCTAATACGACTCACTATAGGGAGATCCACCTGAGACTCCTT
CCA。
For detecting the probe of zika virus for ZIKV-P, 13 institute of sequence of the nucleotide sequence such as sequence table of ZIKV-P
Show, 5 ' ends have fluorophor FAM, 3 ' ends have quenching group BHQ1.
ZIKV-P:5’-FAM-CGGACGTCAGGTGGGACYTGGGTTGATGTTGTCTTGGGCCTG-BHQ1-3'.
For detecting the probe of Chikungunya virus for CHIKV-P, the sequence of the nucleotide sequence such as sequence table of CHIKV-P
Shown in 14,5 ' ends have fluorophor FAM, and 3 ' ends have quenching group BHQ1.
CHIKV-P:5’-FAM-CGGACTCCGACATCATCCTCCTTGCTGGCGCCTG-BHQ1-3'.
For detecting the probe of I type of dengue virus for DENV1-P, the sequence of the nucleotide sequence such as sequence table of DENV1-P
Shown in row 15,5 ' ends have fluorophor FAM, and 3 ' ends have quenching group BHQ1.
DENV1-P:5’-FAM-CGGACTATGGTACATGTGGTTGGGAGCACGCGCCTG-BHQ1-3'.
For detecting the probe of II type of dengue virus for DENV2-P, the sequence of the nucleotide sequence such as sequence table of DENV2-P
Shown in row 16,5 ' ends have fluorophor FAM, and 3 ' ends have quenching group BHQ1.
DENV2-P:5’-FAM-CGGACCTCTCCGAGAACAGGCCTCGACTTCAAGCCTG-BHQ1-3'.
For detecting the probe of III type of dengue virus for DENV3-P, the sequence of the nucleotide sequence such as sequence table of DENV3-P
Shown in row 17,5 ' ends have fluorophor FAM, and 3 ' ends have quenching group BHQ1.
DENV3-P:5’-FAM-CGGACACCTGGATGTCGGCTGAAGGAGCTTGGCCTG-BHQ1-3'.
For detecting the probe of IV type of dengue virus for DENV4-P, the sequence of the nucleotide sequence such as sequence table of DENV4-P
Shown in row 18,5 ' ends have fluorophor FAM, and 3 ' ends have quenching group BHQ1.
DENV4-P:5’-FAM-CGGACTTCCTACTCCTACGCATCGCATTCCGGCCTG-BHQ1-3'.
For detecting that the primer pair of reference gene (RNaseP genes) constitutes (5 ' → 3 ') by following two primers:
RNaseP-F (sequence 19):AGATTTGGACCTGCGAGCG;
RNaseP-R (sequence 20):AATTCTAATACGACTCACTATAGGGAGAGAGCGGCTGTCTCCACAAGT.
For detecting the probe of reference gene (RNaseP genes):
RNaseP-P (sequence 21):5’-FAM-CGGACTTCTGACCTGAAGGCTCTGCGCGGCCTG-BHQ1-3'.
Above in the nucleotide sequence of each primer and probe, W represents A or T, V represent A or G or C, R represent G or A, Y generation
Table C or T.
The preparation of embodiment 2, carapuru virus detection kit
First, the composition of test kit
Test kit constitutes (each component independent packaging) by following component:
(1) primer mixture liquid is by ZIKV-F, ZIKV-R, CHIKV-F, CHIKV-R, DENV1-F, DENV1-R, DENV2-
F, DENV2-R, DENV3-F, DENV3-R, DENV4-F, DENV4-R and sterilized water composition;In primer mixture liquid, ZIKV-F's
Concentration is 0.2 μM, the concentration of ZIKV-R is 0.2 μM, the concentration of CHIKV-F is 0.15 μM, the concentration of CHIKV-R is 0.15 μM,
The concentration of DENV1-F is 0.2 μM, the concentration of DENV1-R is 0.2 μM, the concentration of DENV2-F is 0.15 μM, the concentration of DENV2-R
For 0.15 μM, DENV3-F concentration be 0.3 μM, the concentration of DENV3-R be 0.3 μM, the concentration of DENV4-F be 0.25 μM,
The concentration of DENV4-R is 0.25 μM.
(2) RT-RPA reaction buffers:TwistDx companies, the component of TABARTS01kit.
(3) reaction tube equipped with RT-RPA enzyme mixed powders (for 50 μ l systems):TwistDx companies,
The component of TABARTS01kit.
(4) 280mM magnesium acetates aqueous solution.
(5) reaction mixture BQ:Tris-HCl buffer of the solvent for pH8.0,200mM;Solute and its concentration are as follows:
DTT 50mM、MgCl280mM, KCl 450mM, DMSO 15% (volumn concentration), Sorbitol 1M, dNTP 2mM are (i.e.
The concentration of dATP, dTTP, dCTP and dGTP is 2mM), (i.e. the concentration of rATP, rTTP, rCTP and rGTP is rNTP 4mM
4mM)。
(6) enzyme mixation EQ:Solvent is water;Solute and its concentration are as follows:AMV reverse transcriptase 1U/ μ l, t7 rna polymerase
5U/ μ l, ribonuclease H 0.5U/ μ l, pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA 0.5 μ g/ μ l.
(7) it is embedded with the micro-fluidic chip of primer and probe.
Using the micro-fluidic chip (each hole is a reaction chamber, and the volume of each reaction chamber is 1.4 μ l) in 1 × 24 hole,
0.4 × 10 is embedded with 1# reaction chambers-6μmol ZIKV-F、0.4×10-6μm ol ZIKV-R and 0.2 × 10-6μmol ZIKV-
P, is embedded with 0.4 × 10 in 2# reaction chambers-6μmol CHIKV-F、0.4×10-6μm ol CHIKV-R and 0.2 × 10-6μmol
CHIKV-P, is embedded with 0.4 × 10 in 3# reaction chambers-6μmol DENV1-F、0.4×10-6μm ol DENV1-R and 0.2 × 10-6μ
Mol DENV1-P, are embedded with 0.4 × 10 in 4# reaction chambers-6μmol DENV2-F、0.4×10-6μm ol DENV2-R and 0.2 ×
10-6μm ol DENV2-P, are embedded with 0.4 × 10 in 5# reaction chambers-6μmol DENV3-F、0.4×10-6μm ol DENV3-R and
0.2×10-6μm ol DENV3-P, are embedded with 0.4 × 10 in 6# reaction chambers-6μmol DENV4-F、0.4×10-6μmol
DENV4-R and 0.2 × 10-6μm ol DENV4-P, are embedded with 0.4 × 10 in 7# reaction chambers-6μmol RNaseP-F、0.4×10-6
μm ol RNaseP-R and 0.2 × 10-6μm ol RNaseP-P, are embedded with negative controls (sterilized water) in 8# reaction chambers.
It is embedded with the preparation method of the micro-fluidic chip of primer and probe:1. will encapsulation reagent, primer, probe and sterilized water
Mixing, (in embedding liquid, the concentration for encapsulating reagent is 0.1g/100ml, and the concentration of primer is 0.4 μM, probe to obtain embedding liquid
Concentration is 0.2 μM);Encapsulation reagent is made up of 5 mass parts agaroses, 3 mass parts saponin and 4 mass parts HPMC;
2. micro-fluidic chip is taken, 1 μ l embedding liquid in each reaction chamber, is added;3. the micro-fluidic chip that 2. step obtains is taken, cleaning is placed in
Dry in super-clean bench, then tabletting encapsulation punching press evacuation.
Primer mixture liquid, RT-RPA reaction buffers, the reaction tube equipped with RT-RPA enzyme mixed powders and 280mM acetic acid
Magnesium aqueous solution is used for reverse transcription recombinase polymeric enzymatic amplification (RT-RPA).
Reaction mixture BQ, enzyme mixation EQ and be embedded with the micro-fluidic chip of primer and probe for nucleotide sequence rely on
Property amplification (NASBA).
2nd, the using method of test kit
Solution to be measured:Take biological specimen to be measured viral or to be measured (such as serum), extract nucleic acid, the nucleic acid solution for obtaining or
Its diluent is solution to be measured.When virus to be measured is DNA viruses, extracts nucleic acid and refer to extract genomic DNA.Virus to be measured
For RNA viruses when, extract nucleic acid refer to extract total serum IgE.Biological specimen to be measured, extracts nucleic acid and refers to extract total serum IgE.
1st, reverse transcription recombinase polymeric enzymatic amplification (pre- to expand)
A reaction tube equipped with RT-RPA enzyme mixed powders is taken, 29.5 μ l RT-RPA reaction buffers, 5 μ are sequentially added
L primer mixed liquors, 10 μ l solution to be measured and 3 μ l water mix homogeneously, are subsequently adding 2.5 μ l 280mM magnesium acetates aqueous solutions to open
Dynamic reaction.
Reaction condition:37℃10min;80 DEG C, 2min;12 DEG C, 3min.
80 DEG C, the purpose of 2min process is in order to inactivate the enzyme in reaction system, in order to avoid have an impact to subsequent step.
After completing reaction, whole system is named as pre- amplified production.
2nd, Nucleic acid sequence amplification
(1) 12 μ l reaction mixture BQ, 3 μ l enzyme mixations EQ and the pre- amplified productions of 10 μ l is taken, mix homogeneously is injected into bag
It is embedded with the micro-fluidic chip of primer and probe, seals Quick spin 30s after membrana oralis.
(2), after completing step (1), the micro-fluidic chip for being embedded with primer and probe is placed in into brilliant core RTisochip-
In A constant-temperature amplification micro-fluidic chip nucleic acids instruments, 41 DEG C of reaction 60min simultaneously complete real-time fluorescence scanning simultaneously.
3rd, result judges
After reaction terminates, determine which sample to be tested contains as follows according to the amplification curve of sample in each reaction chamber
A kind of or which kind virus:If the amplification curve of sample is S type positive amplification curves in 1# reaction chambers, sample to be tested contains stockaded village
Card virus;If the amplification curve of sample is S type positive amplification curves in 2# reaction chambers, sample to be tested contains chikungunya disease
Poison;If the amplification curve of sample is S type positive amplification curves in 3# reaction chambers, sample to be tested contains I type of dengue virus;Such as
In fruit 4# reaction chambers, the amplification curve of sample is S type positive amplification curves, and sample to be tested contains II type of dengue virus;If 5#
In reaction chamber, the amplification curve of sample is S type positive amplification curves, and sample to be tested contains III type of dengue virus;If 6# reacts
In chamber, the amplification curve of sample is S type positive amplification curves, and sample to be tested contains IV type of dengue virus.Sample to be tested is biology
The blood or tissue of sample, such as human or animal;RNaseP genes be house-keeping gene, almost human or animal institute in a organized way in
In all high level expressions, therefore 7# reaction chamber, the amplification curve of sample should be S type positive amplification curves.Sample in 8# reaction chambers
Amplification curve should be negative amplification curve.
After reaction terminates, determine which kind of virus to be measured is as follows according to the amplification curve of sample in each reaction chamber
Virus:If the amplification curve of sample is S type positive amplification curves in 1# reaction chambers, virus to be measured is zika virus;If 2#
In reaction chamber, the amplification curve of sample is S type positive amplification curves, and virus to be measured is Chikungunya virus;If in 3# reaction chambers
The amplification curve of sample is S type positive amplification curves, and virus to be measured is I type of dengue virus;If sample in 4# reaction chambers
Amplification curve is S type positive amplification curves, and virus to be measured is II type of dengue virus;If the amplification of sample is bent in 5# reaction chambers
Line is S type positive amplification curves, and virus to be measured is III type of dengue virus;If the amplification curve of sample is S in 6# reaction chambers
Type positive amplification curve, virus to be measured are IV type of dengue virus.In 8# reaction chambers, the amplification curve of sample should be negative amplification
Curve.
The contrast of embodiment 3, two kind of nucleic acid amplification method
First, standard substance are prepared
Prepare the RNA1 (single strand RNA molecule, zika virus standard substance) shown in the sequence 22 of sequence table.RNA1 is dissolved in into nothing
Bacterium water, then carries out gradient dilution with sterilized water, obtains concentration for 104copies/μl、103copies/μl、102copies/μ
The RNA1 diluents of l, 10copies/ μ l or 1copies/ μ l.
Prepare the RNA2 (single strand RNA molecule, Chikungunya virus standard substance) shown in the sequence 23 of sequence table.RNA2 is molten
In sterilized water, then gradient dilution is carried out with sterilized water, obtain concentration for 104copies/μl、103copies/μl、
102The RNA2 diluents of copies/ μ l, 10copies/ μ l or 1copies/ μ l.
Prepare the RNA3 (single strand RNA molecule, I type standard substance of dengue virus) shown in the sequence 24 of sequence table.By RNA3
Sterilized water is dissolved in, then gradient dilution is carried out with sterilized water, obtain concentration for 104copies/μl、103copies/μl、
102The RNA3 diluents of copies/ μ l, 10copies/ μ l or 1copies/ μ l.
Prepare the RNA4 (single strand RNA molecule, II type standard substance of dengue virus) shown in the sequence 25 of sequence table.By RNA4
Sterilized water is dissolved in, then gradient dilution is carried out with sterilized water, obtain concentration for 104copies/μl、103copies/μl、
102The RNA4 diluents of copies/ μ l, 10copies/ μ l or 1copies/ μ l.
Prepare the RNA5 (single strand RNA molecule, III type standard substance of dengue virus) shown in the sequence 26 of sequence table.By RNA5
Sterilized water is dissolved in, then gradient dilution is carried out with sterilized water, obtain concentration for 104copies/μl、103copies/μl、
102The RNA5 diluents of copies/ μ l, 10copies/ μ l or 1copies/ μ l.
Prepare the RNA6 (single strand RNA molecule, IV type standard substance of dengue virus) shown in the sequence 27 of sequence table.By RNA6
Sterilized water is dissolved in, then gradient dilution is carried out with sterilized water, obtain concentration for 104copies/μl、103copies/μl、
102The RNA6 diluents of copies/ μ l, 10copies/ μ l or 1copies/ μ l.
2nd, the contrast of two kinds of amplification methods
1st, the method for the present invention (RPA-NASBA methods)
Each diluent of step one preparation is taken as solution to be measured, using the step of embodiment 2 one test kit for preparing
Detected according to the step of embodiment 2 two method description.
2nd, existing method (NASBA methods)
Take each diluent of step one preparation, replace the pre- amplified productions of 10 μ l, according to the step of embodiment 2 two 2
Method is detected.
The result of zika virus standard substance is shown in Fig. 2.In Fig. 2, R represents RPA-NASBA methods, and N represents NASBA methods, 1 to 5 according to
The secondary diluent for representing nucleic acid concentration from high to low.
The result of Chikungunya virus standard substance is shown in Fig. 3.In Fig. 3, R represents RPA-NASBA methods, and N represents NASBA methods, 1 to
5 represent nucleic acid concentration diluent from high to low successively.
The result of I type standard substance of dengue virus is shown in Fig. 4.In Fig. 4, R represents RPA-NASBA methods, and N represents NASBA methods, and 1
Nucleic acid concentration diluent from high to low is represented successively to 5.
The result of II type standard substance of dengue virus is shown in Fig. 5.In Fig. 5, R represents RPA-NASBA methods, and N represents NASBA methods, and 1
Nucleic acid concentration diluent from high to low is represented successively to 5.
The result of III type standard substance of dengue virus is shown in Fig. 6.In Fig. 6, R represents RPA-NASBA methods, and N represents NASBA methods, and 1
Nucleic acid concentration diluent from high to low is represented successively to 5.
The result of IV type standard substance of dengue virus is shown in Fig. 7.In Fig. 7, R represents RPA-NASBA methods, and N represents NASBA methods, and 1
Nucleic acid concentration diluent from high to low is represented successively to 5.
As a result show, the test limit of micro-fluidic chip can greatly be improved using the nucleic acid amplification method of the present invention, for
This six kinds of carapuru virus detection limits at least improve two Concentraton gradient.Illustrating that the present invention is very big improves micro-fluidic chip
Minimum detectability, and shorten detection time.
Embodiment 4, accuracy and specificity
Virus difference to be measured is as follows:
Zika virus:Zhang Shuo, Li Dexin;Zika virus and zika virus disease;Viral journal, in January, 2016, volume 32
1 phase, 121-123.
Chikungunya virus:Wang Yujiao, Zhang Yunbo, section new Asia, Wang Yaling, Zhao Guiping;What Chikungunya virus infected examines
Disconnected Research progress;《Clinical Laboratory magazine》,2012(7):522-524.
I type of dengue virus (strain of DEN-1 Hawaii):Shu Liping, Zuo Li, Li Yongnian, Hao Mu, Chen Aying;Universal primer
Detection dengue virus NS1 genes and its enzyme action typing;《Guiyang Medical College journal》,2004,29(4):283-286.
II type of dengue virus (DEN-2NGC strains):Shu Liping, Zuo Li, Li Yongnian, Hao Mu, Chen Aying;Universal primer is examined
Survey dengue virus NS1 genes and its enzyme action typing;《Guiyang Medical College journal》,2004,29(4):283-286.
III type of dengue virus (DEN-3H87 strains):Shu Liping, Zuo Li, Li Yongnian, Hao Mu, Chen Aying;Universal primer is examined
Survey dengue virus NS1 genes and its enzyme action typing;《Guiyang Medical College journal》,2004,29(4):283-286.
IV type of dengue virus (DEN-4H241 strains):Shu Liping, Zuo Li, Li Yongnian, Hao Mu, Chen Aying;Universal primer is examined
Survey dengue virus NS1 genes and its enzyme action typing;《Guiyang Medical College journal》,2004,29(4):283-286.
Hepatitis B viruss:Zhang Juan, Chen Jianzong, Zhang Jinping, Jia Xin, Jiang Weifeng;The external resistance of hepatitis B disease of astragaloside
The effect of poison;The Fourth Military Medical University, 2008,28 (24):2291-2293.
Epstein-Barr virus (ebb virus):Wu Fan, Zhai Weiwei, Ge Liuying, Qi Yanwei, Gao Hui;245 human immunities
The detection case analysis of nerpes vinrus hominises' 1-4 types in defective viruss the infected's saliva;《West China Journal of Stomatology》,2012,
30(5):514-517。
Morbillivirus L 4 strain:Zhao great Peng, Yao Weimin, Wu Xiaojuan, Qi Fengchun, Wang Chunyi;Morbillivirus L 4 strain genome is complete
The measure of sequence;National biological product academic conference, 2007.
Bird flu H5 types virus is specially H5N1 subtype avian influenza virus A/duck/Guangdong/S1322/2010 (DK/
GD/S1322/2010):Lu Kunpeng, Cui Pengfei, Zhang Fang, Wang Jing, Xiao Hongbo;H5 subtype avian influenza virus HA protein monoclonals resist
The preparation and identification of body;《Chinese veterinary science》,2015(5):453-457.
Bird flu H7 types virus is specially H7N2 subtype avian influenza virus (Avian in fluenzavirus, AIV) A/
Chicken/Hebei/1/2002:Wang Chuanbin, Tian Kegong, Wang Hongwei, Sun Ming, meet the elegant tinkling of pieces of jade;China H_7N_2 subtype avian influenzas disease
Malicious CK/HB/1/02 strains isolation identification;《CHINA virus》,2005,20(6):632-636.
Human cytomegalic inclusion disease virus:Wang Min, hair Jianping;Technologies Development on Human Cytomegalovirus Detection;《Chinese biological engineering magazine》,
2007,27(2):103-107。
The nucleic acid for extracting each virus to be measured (when virus to be measured is DNA viruses, extracts genomic DNA;Virus to be measured is
During RNA viruses, total serum IgE is extracted), as solution to be measured, then using the step of embodiment 2 one test kit for preparing according to enforcement
The method description of the step of example 2 two is detected.
The total serum IgE of extraction zika virus, the total serum IgE of Chikungunya virus, the total serum IgE of I type of dengue virus, Dengue calentura
The total serum IgE of malicious II type, the total serum IgE of III type of dengue virus, the total serum IgE of IV type of dengue virus, then mix, obtain aggregate sample
This (concentration of wherein various viral nucleic acids is 100copies/ μ l), using mixing sample as solution to be measured, then using reality
Test kit prepared by the step of applying example 2 one is detected according to the step of embodiment 2 two method description.
Each duplicate detection to be measured 3 times, as a result unanimously.
Testing result is as shown in table 1.It can be seen from the results that the nucleic acid detection method of the present invention, its accuracy height, to non-
Without non-specific amplification, its accuracy can reach 100% to target gene.
1 accuracy of table and specific detection result
Sequence table
<120>A kind of nucleic acid amplification technologies and its application in carapuru virus detection
<130> GNCYX161842
<160> 27
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (6)
<223> w = a or t
<400> 1
tgcatwggag tcagcaa 17
<210> 2
<211> 47
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (43)
<223> v = a or g or c
<400> 2
aattctaata cgactcacta tagggagata ggatcttacc tcvgcca 47
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
ccgactcaac catcctggat 20
<210> 4
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (33)
<223> r = g or a
<400> 4
aattctaata cgactcacta tagggagagg caracgcagt ggtacttcct 50
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
caaaaggaag tcgtgcaata 20
<210> 6
<211> 53
<212> DNA
<213>Artificial sequence
<400> 6
aattctaata cgactcacta tagggagact gagtgaattc tctctactga acc 53
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<400> 7
caggttatgg cactgtcacg at 22
<210> 8
<211> 50
<212> DNA
<213>Artificial sequence
<400> 8
aattctaata cgactcacta tagggagacc atctgcagca acaccatctc 50
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
ggactggaca cacgcaccca 20
<210> 10
<211> 54
<212> DNA
<213>Artificial sequence
<400> 10
aattctaata cgactcacta tagggagaca tgtctctacc ttctcgactt gtct 54
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence
<400> 11
ttgtcctaat gatgctggtc g 21
<210> 12
<211> 48
<212> DNA
<213>Artificial sequence
<400> 12
aattctaata cgactcacta tagggagatc cacctgagac tccttcca 48
<210> 13
<211> 42
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (18)
<223> y = c or t
<400> 13
cggacgtcag gtgggacytg ggttgatgtt gtcttgggcc tg 42
<210> 14
<211> 34
<212> DNA
<213>Artificial sequence
<400> 14
cggactccga catcatcctc cttgctggcg cctg 34
<210> 15
<211> 36
<212> DNA
<213>Artificial sequence
<400> 15
cggactatgg tacatgtggt tgggagcacg cgcctg 36
<210> 16
<211> 37
<212> DNA
<213>Artificial sequence
<400> 16
cggacctctc cgagaacagg cctcgacttc aagcctg 37
<210> 17
<211> 36
<212> DNA
<213>Artificial sequence
<400> 17
cggacacctg gatgtcggct gaaggagctt ggcctg 36
<210> 18
<211> 36
<212> DNA
<213>Artificial sequence
<400> 18
cggacttcct actcctacgc atcgcattcc ggcctg 36
<210> 19
<211> 19
<212> DNA
<213>Artificial sequence
<400> 19
agatttggac ctgcgagcg 19
<210> 20
<211> 48
<212> DNA
<213>Artificial sequence
<400> 20
aattctaata cgactcacta tagggagaga gcggctgtct ccacaagt 48
<210> 21
<211> 33
<212> DNA
<213>Artificial sequence
<400> 21
cggacttctg acctgaaggc tctgcgcggc ctg 33
<210> 22
<211> 1506
<212> RNA
<213>Artificial sequence
<400> 22
aucaggugca uuggagucag caauagagac uucguggagg gcaugucagg ugggaccugg 60
guugauguug ucuuggaaca uggaggcugc guuaccguga uggcacagga caagccaaca 120
gucgacauag aguuggucac gacgacgguu aguaacaugg ccgagguaag auccuauugc 180
uacgaggcau cgauaucgga cauggcuucg gacagucguu gcccaacaca aggugaagcc 240
uaccuugaca agcaaucaga cacucaauau gucugcaaaa gaacauuagu ggacagaggu 300
uggggaaacg guuguggacu uuuuggcaaa gggagcuugg ugacaugugc caaguuuacg 360
uguucuaaga agaugaccgg gaagagcauu caaccggaaa aucuggagua ucggauaaug 420
cuaucagugc auggcuccca gcauagcggg augauuggau augaaacuga cgaagauaga 480
gcgaaagucg agguuacgcc uaauucacca agagcggaag caaccuuggg aggcuuugga 540
agcuuaggac uugacuguga accaaggaca ggccuugacu uuucagaucu guauuaccug 600
accaugaaca auaagcauug guuggugcac aaagaguggu uucaugacau cccauugccu 660
uggcaugcug gggcagacac cggaacucca cacuggaaca acaaagaggc auugguagaa 720
uucaaggaug cccacgccaa gaggcaaacc gucgucguuc uggggagcca ggaaggagcc 780
guucacacgg cucucgcugg agcucuagag gcugagaugg auggugcaaa gggaaggcug 840
uucucuggcc auuugaaaug ccgccuaaaa auggacaagc uuagauugaa gggcguguca 900
uauuccuugu gcacugcggc auucacauuc accaaggucc cagcugaaac acugcaugga 960
acagucacag uggaggugca guaugcaggg acagauggac ccugcaagau cccaguccag 1020
auggcggugg acaugcagac ccugacccca guuggaaggc ugauaaccgc caaccccgug 1080
auuacugaaa gcacugagaa cucaaagaug auguuggagc uugacccacc auuuggggau 1140
ucuuacauug ucauaggagu uggggacaag aaaaucaccc accacuggca uaggaguggu 1200
agcaccaucg gaaaggcauu ugaggccacu gugagaggcg ccaagagaau ggcaguccug 1260
ggggauacag ccugggacuu cggaucaguc gggggugugu ucaacucacu ggguaagggc 1320
auucaccaga uuuuuggagc agccuucaaa ucacuguuug gaggaauguc cugguucuca 1380
cagauccuca uaggcacgcu gcuagugugg uuagguuuga acacaaagaa uggaucuauc 1440
ucccucacau gcuuggcccu ggggggagug augaucuucc ucuccacggc uguuucugcu 1500
gacgug 1506
<210> 23
<211> 1681
<212> RNA
<213>Artificial sequence
<400> 23
auggcugcgu gagacacacg uagccuacca guuucuuacu gcucuacucu gcaaagcaag 60
agauuaagaa cccaucaugg auccugugua cguggacaua gacgcugaca gcgccuuuuu 120
gaaggcccug caacgugcgu accccauguu ugagguggaa ccuaggcagg ucacaccgaa 180
ugaccaugcu aaugcuagag cguucucgca ucuagcuaua aaacuaauag agcaggaaau 240
ugaucccgac ucaaccaucc uggauauugg uagugcgcca gcaaggagga ugaugucgga 300
caggaaguac cacugcguuu gcccgaugcg cagugcagaa gaucccgaga gacucgccaa 360
uuaugcgaga aagcuagcau cugccgcagg aaaaguccug gacagaaaca ucucuggaaa 420
gaucggggac uuacaagcag uaauggccgu gccagacacg gagacgccaa cauucugcuu 480
acacacagau guaucaugua gacagagagc agacgucgcg auauaccaag acgucuaugc 540
uguacacgca cccacgucgc uauaccacca ggcgauuaaa gggguccgau uggcguacug 600
gguaggguuu gacacaaccc cguucaugua caaugccaug gcgggugccu accccucaua 660
cucgacaaau ugggcagaug agcagguacu gaaggcuaag aacauaggau uauguucaac 720
agaccugacg gaagguagac gaggcaaauu gucuauuaug agaggaaaaa agcuagaacc 780
gugcgaccgu gugcuguucu caguaggguc aacgcucuac ccggaaagcc guaagcuacu 840
uaagagcugg caccuaccau cgguguucca uuuaaagggc aagcucagcu ucacaugccg 900
cugugauaca gugguuucgu gcgaaggcua cgucguuaag agaauaacga ugagcccagg 960
ccuuuacgga aaaaccacag gguaugcggu aacccaccac gcagacggau uccugaugug 1020
caagaccacc gacacgguug acggcgaaag agugucauuc ucggugugca cguacgugcc 1080
ggcgaccauu ugugaucaaa ugaccggcau ccuugcuaca gaagucacgc cggaggaugc 1140
acagaagcug uugguggggc ugaaccagag aauagugguu aacggcagaa cgcaacggaa 1200
uacgaacacc augaaaaacu auaugauucc cguggucgcc caagccuuca guaagugggc 1260
aaaggagugc cggaaagaca uggaagauga aaaacuccug ggggucagag aaagaacacu 1320
gaccugcugc ugucuauggg cauuuaagaa gcagaaaaca cacacggucu acaagaggcc 1380
ugauacccag ucaauucaga agguucaggc cgaguuugac agcuuugugg uaccgagccu 1440
guggucgucc ggguugucaa ucccguugag gacuagaauc aaaugguugu uaagcaaggu 1500
gccaaaaacc gaccugaccc cauacagcgg ggacgcccaa gaagcccggg acgcagaaaa 1560
agaagcagag gaagaacgag aagcagaacu gacucuugaa gcccuaccac cccuucaggc 1620
agcacaggaa gauguucagg ucgaaaucga cguggaacag cuugaggaca gagcgggugc 1680
a 1681
<210> 24
<211> 1101
<212> RNA
<213>Artificial sequence
<400> 24
auggagguga cagccaggug guuauggggu uuucucucua gaaacaaaaa acccagaauc 60
ugcacaagag aggaguucac aagaaaaguc aggucaaacg cagcuauugg agcaguguuc 120
guugaugaaa aucaauggaa cucagcaaaa gaggcagugg aagaugaacg guucugggac 180
cuugugcaca gagagaggga gcuucauaaa caaggaaaau gugccacgug ugucuacaac 240
augaugggaa agagagagaa aaaauuagga gaguucggaa aggcaaaagg aagucgcgca 300
auaugguaca ugugguuggg agcgcgcuuu uuagaguuug aagcccuugg uuucaugaau 360
gaagaucacu gguucagcag agagaauuca cucaguggag uggaaggaga aggacuccac 420
aaacuuggau acauacucag agacauauca aagauuccag ggggaaauau guaugcagau 480
gacacagccg gaugggacac aagaauaaca gaggaugauc uucagaauga ggccaaaauc 540
acugacauca uggaaccuga acaugcccua uuggccacgu caaucuuuaa gcuaaccuac 600
caaaacaagg uaguaagggu gcagagacca gcgaaaaaug gaaccgugau ggaugucaua 660
uccagacgug accagagagg aaguggacag guuggaaccu auggcuuaaa caccuucacc 720
aacauggagg cccaacuaau aagacaaaug gagucugagg gaaucuuuuc acccagcgaa 780
uuggaaaccc caaaucuagc cgaaagaguc cucgacuggu ugaaaaaaca uggcaccgag 840
aggcugaaaa gaauggcaau caguggagau gacugugugg ugaaaccaau cgaugacaga 900
uuugcaacag ccuuaacagc uuugaaugac augggaaagg uaagaaaaga cauaccgcaa 960
ugggaaccuu caaaaggaug gaaugauugg caacaagugc cuuucuguuc acaccauuuc 1020
caccagcuga uuaugaagga ugggagggag auaguggugc caugccgcaa ccaagaugaa 1080
cuuguaggua gggccagagu a 1101
<210> 25
<211> 1085
<212> RNA
<213>Artificial sequence
<400> 25
gucaucagaa ggggccugga aacaugucca gagaauugaa acuuggaucu ugagacaucc 60
aggcuucacc augauggcag caauccuggc auacaccaua ggaacgacac auuuccaaag 120
agcccugauu uucaucuuac ugacagcugu cacuccuuca augacaaugc guugcauagg 180
aaugucaaau agagacuuug uggaaggggu uucaggagga agcuggguug acauagucuu 240
agaacaugga agcuguguga cgacgauggc aaaaaacaaa ccaacauugg auuuugaacu 300
gauaaaaaca gaagccaaac agccugccac ccuaaggaag uacuguauag aggcaaagcu 360
aaccaacaca acaacagaau cucgcugccc aacacaaggg gaacccagcc uaaaugaaga 420
gcaggacaaa agguucgucu gcaaacacuc caugguagac agaggauggg gaaauggaug 480
uggacuauuu ggaaagggag gcauugugac cugugcuaug uucagaugca aaaagaacau 540
ggaaggaaaa guugugcaac cagaaaacuu ggaauacacc auugugauaa caccucacuc 600
aggggaagag caugcagucg gaaaugacac aggaaaacau ggcaaggaaa ucaaaauaac 660
accacagagu uccaucacag aagcagaauu gacagguuau ggcacuguca caauggagug 720
cucuccaaga acgggccucg acuucaauga gaugguguug cugcagaugg aaaauaaagc 780
uuggcuggug cacaggcaau gguuccuaga ccugccguua ccaugguugc ccggagcgga 840
cacacaaggg ucaaauugga uacagaaaga gacauugguc acuuucaaaa auccccaugc 900
gaagaaacag gauguuguug uuuuaggauc ccaagaaggg gccaugcaca cagcacuuac 960
aggggccaca gaaauccaaa ugucaucagg aaacuuacuc uucacaggac aucucaagug 1020
caggcugaga auggacaagc uacagcucaa aggaauguca uacucuaugu gcacaggaaa 1080
guuua 1085
<210> 26
<211> 1208
<212> RNA
<213>Artificial sequence
<400> 26
aguuguuagu cuacguggac cgacaagaac aguuucgacu cggaagcuug cuuaacguag 60
ugcugacagu uuuuuauuag agagcagauc ucugaugaac aaccaacgga agaagacggg 120
aaaaccgucu aucaauaugc ugaaacgcgu gagaaaccgu gugucaacug gaucacaguu 180
ggcgaagaga uucucaaaag gacugcugaa cggccaggga ccaaugaaau ugguuauggc 240
guucauagcu uuccucagau uucuagccau uccaccaaca gcaggagucu uggcuagaug 300
gggaaccuuc aagaagucgg gggccauuaa gguccugaaa ggcuucaaga aggagaucuc 360
aaacaugcug agcauaauca accaacggaa aaagacaucg cucugucuca ugaugauauu 420
gccagcagca cuugcuuucc acuugacuuc acgagaugga gagccgcgca ugauuguggg 480
gaagaaugaa agagguaaau cccuacuuuu uaagacagcc ucuggaauca acaugugcac 540
acucauagcc auggauuugg gagagaugug ugaugacacg gucacuuaca aaugccccca 600
cauuaccgaa guggaaccug aagacauuga cugcuggugc aaccuuacau caacaugggu 660
gacuuaugga acgugcaauc aagcuggaga gcauagacgc gacaagagau caguggcguu 720
agcuccccau gucggcaugg gacuggacac acgcacccaa accuggaugu cggcugaagg 780
agcuuggaga caagucgaga agguagagac augggcccuu aggcacccag gguucaccau 840
acuagcccua uuucucgccc auuacauagg cacuucccug acccagaagg ugguuauuuu 900
cauauuauua augcugguca ccccauccau gacaaugaga ugugugggag uaggaaacag 960
agauuuugug gaagggcuau caggagcuac guggguugac guggugcucg agcacggggg 1020
gugugugacu accauggcua agaacaagcc cacgcuggau auagagcuuc agaagaccga 1080
ggccacccaa cuggcgaccc uaaggaagcu augcauugag gggaaaauua ccaacauaac 1140
aacugacuca agauguccua cccaagggga agcgguuuug ccugaggagc aggaccagaa 1200
cuacgugu 1208
<210> 27
<211> 992
<212> RNA
<213>Artificial sequence
<400> 27
auggauguca ucggaagggg cuuggaagca ugcucagaga guagagagcu ggauacucag 60
aaacccagga uucgcgcucu uggcaggauu uauggcuuau augauugggc aaacaggaau 120
ccagcgaacu gucuucuuug uccuaaugau gcuggucgcc ccauccuacg gaaugcgaug 180
cguaggagua ggaaacagag acuuugugga aggagucuca gguggagcau gggucgaccu 240
ggugcuagaa cauggaggau gcgucacaac cauggcccag ggaaaaccaa ccuuggauuu 300
ugaacugacu aagacaacag ccaaggaagu ggcucuguua agaaccuauu gcauugaagc 360
cucaauauca aacauaacua cggcaacaag auguccaacg caaggagagc cuuaucugaa 420
agaggaacag gaccaacagu acauuugccg gagagaugug guagacagag gguggggcaa 480
uggcuguggc uuguuuggaa aaggaggagu ugugacaugu gcgaaguuuu cauguucggg 540
gaagauaaca ggcaauuugg uccaaauuga gaaccuugaa uacacagugg uuguaacagu 600
ccacaaugga gacacccaug caguaggaaa ugacacaucc aaucauggag uuacagccau 660
gauaacuccc aggucaccau cgguggaagu caaauugccg gacuauggag aacuaacacu 720
cgauugugaa cccaggucug gaauugacuu uaaugagaug auucugauga aaaugaaaaa 780
gaaaacaugg cucgugcaua agcaaugguu uuuggaucug ccucuuccau ggacagcagg 840
agcagacaca ucagagguuc acuggaauua caaagagaga auggugacau uuaagguucc 900
ucaugccaag agacaggaug ugacagugcu gggaucucag gaaggagcca ugcauucugc 960
ccucgcugga gccacagaag uggacuccgg ug 992
Claims (10)
1. primer combination of probe, is following (a1) or (a2) or (a3):
(a1) by primed probe group I, primed probe group II, primed probe group III, primed probe group IV, primed probe group V and draw
Thing probe groups VI are constituted;
(a2) by the primed probe group I, the primed probe group II, the primed probe group III, the primed probe group IV,
Any two in the primed probe group V and the primed probe group VI, any three, any four or any five groups
Into;
(a3) the primed probe group I, the primed probe group II, the primed probe group III, the primed probe group IV, institute
State primed probe group V or the primed probe group VI;
The primed probe group I is made up of primer ZIKV-F, primer ZIKV-R and probe ZIKV-P;
The primer ZIKV-F is following (b1) or (b2);
(b1) single strand dna shown in the sequence 1 of sequence table;
(b2) sequence 1 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 1 identical
The DNA molecular of function;
The primer ZIKV-R is following (b3) or (b4);
(b3) single strand dna shown in the sequence 2 of sequence table;
(b4) sequence 2 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 2 identical
The DNA molecular of function;
The nucleotides sequence of the probe ZIKV-P is classified as (b5) as follows or (b6);
(b5) single strand dna as shown in the sequence 13 of sequence table;
(b6) sequence 13 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 13
The DNA molecular of congenerous;
The primed probe group II is made up of primer CHIKV-F, primer CHIKV-R and probe CHIKV-P;
The primer CHIKV-F is following (c1) or (c2);
(c1) single strand dna shown in the sequence 3 of sequence table;
(c2) sequence 3 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 3 identical
The DNA molecular of function;
The primer CHIKV-R is following (c3) or (c4);
(c3) single strand dna shown in the sequence 4 of sequence table;
(c4) sequence 4 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 4 identical
The DNA molecular of function;
The nucleotides sequence of the probe CHIKV-P is classified as (c5) as follows or (c6);
(c5) single strand dna as shown in the sequence 14 of sequence table;
(c6) sequence 14 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 14
The DNA molecular of congenerous;
The primed probe group III is made up of primer DENV1-F, primer DENV1-R and probe DENV1-P;
The primer DENV1-F is following (d1) or (d2);
(d1) single strand dna shown in the sequence 5 of sequence table;
(d2) sequence 5 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 5 identical
The DNA molecular of function;
The primer DENV1-R is following (d3) or (d4);
(d3) single strand dna shown in the sequence 6 of sequence table;
(d4) sequence 6 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 6 identical
The DNA molecular of function;
The nucleotides sequence of the probe DENV1-P is classified as (d5) as follows or (d6);
(d5) single strand dna as shown in the sequence 15 of sequence table;
(d6) sequence 15 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 15
The DNA molecular of congenerous;
The primed probe group IV is made up of primer DENV2-F, primer DENV2-R and probe DENV2-P;
The primer DENV2-F is following (e1) or (e2);
(e1) single strand dna shown in the sequence 7 of sequence table;
(e2) sequence 7 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 7 identical
The DNA molecular of function;
The primer DENV2-R is following (e3) or (e4);
(e3) single strand dna shown in the sequence 8 of sequence table;
(e4) sequence 8 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 8 identical
The DNA molecular of function;
The nucleotides sequence of the probe DENV2-P is classified as (e5) as follows or (e6);
(e5) single strand dna as shown in the sequence 16 of sequence table;
(e6) sequence 16 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 16
The DNA molecular of congenerous;
The primed probe group V is made up of primer DENV3-F, primer DENV3-R and probe DENV3-P;
The primer DENV3-F is following (f1) or (f2);
(f1) single strand dna shown in the sequence 9 of sequence table;
(f2) sequence 9 is had through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 9 identical
The DNA molecular of function;
The primer DENV3-R is following (f3) or (f4);
(f3) single strand dna shown in the sequence 10 of sequence table;
(f4) sequence 10 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 10
The DNA molecular of congenerous;
The nucleotides sequence of the probe DENV3-P is classified as (f5) as follows or (f6);
(f5) single strand dna as shown in the sequence 17 of sequence table;
(f6) sequence 17 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 17
The DNA molecular of congenerous;
The primed probe group VI is made up of primer DENV4-F, primer DENV4-R and probe DENV4-P;
The primer DENV4-F is following (g1) or (g2);
(g1) single strand dna shown in the sequence 11 of sequence table;
(g2) sequence 11 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 11
The DNA molecular of congenerous;
The primer DENV4-R is following (g3) or (g4);
(g3) single strand dna shown in the sequence 12 of sequence table;
(g4) sequence 12 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 12
The DNA molecular of congenerous;
The nucleotides sequence of the probe DENV4-P is classified as (g5) as follows or (g6);
(g5) single strand dna as shown in the sequence 18 of sequence table;
(g6) sequence 18 had into phase through the replacement and/or disappearance and/or addition of one or several nucleotide and with sequence 18
The DNA molecular of congenerous.
2. a kind of special chip, is by the primed probe group I described in claim 1, primed probe group II, primed probe group
IIIth, primed probe group IV, primed probe group V and primed probe group VI are embedded in the differential responses chamber of micro-fluidic chip respectively
Obtain;
The function of the special chip is following (h1) or (h2):
(h1) it is used for identifying zika virus, Chikungunya virus, I type of dengue virus, II type of dengue virus, dengue virus
IV type of III type or dengue virus;
(h2) whether contain I type of zika virus and/or Chikungunya virus and/or dengue virus in being used for detecting sample to be tested
And/or IV type of II type of dengue virus and/or III type of dengue virus and/or dengue virus.
3. the test kit first of primer combination of probe described in claim 1 is included or including micro-fluid chip described in claim 2
Test kit second.
4. test kit second as claimed in claim 3, it is characterised in that:The test kit is also combined including primer pair;It is described to draw
Thing is made up of primer pair I, primer pair II, primer pair III, primer pair IV, primer pair V and primer pair VI to combination;
The primer pair I is made up of the primer ZIKV-F and primer ZIKV-R in the primed probe group I described in claim 1;
The primer pair II is made up of the primer CHIKV-F and primer CHIKV-R in the primed probe group II described in claim 1;
The primer pair III is made up of the primer DENV1-F and primer DENV1-R in the primed probe group III described in claim 1;
The primer pair IV is made up of the primer DENV2-F and primer DENV2-R in the primed probe group IV described in claim 1;
The primer pair V is made up of the primer DENV3-F and primer DENV3-R in the primed probe group V in claim 1;
The primer pair VI is made up of the primer DENV4-F and primer DENV4-R in the primed probe group VI in claim 1.
5. test kit second as claimed in claim 4, it is characterised in that:The test kit also includes that reaction mixture BQ and enzyme are mixed
Close liquid EQ;
Reaction mixture BQ:Tris-HCl buffer of the solvent for pH8.0,200mM;Solute and its concentration are as follows:DTT 50mM、
MgCl280mM, KCl 450mM, DMSO 15% (volumn concentration), Sorbitol 1M, dNTP 2mM, rNTP 4mM;
Enzyme mixation EQ:Solvent is water;Solute and its concentration are as follows:AMV reverse transcriptase 1U/ μ l, t7 rna polymerase 5U/ μ l, core
Ribonuclease T. H 0.5U/ μ l, pyrophosphatase 0.5U/ μ l, RNase inhibitor 5U/ μ l, BSA 0.5 μ g/ μ l.
6. in claim 3 to 5 arbitrary test kit application, be following (h1) or (h2):
(h1) it is used for identifying zika virus, Chikungunya virus, I type of dengue virus, II type of dengue virus, dengue virus
IV type of III type or dengue virus;
(h2) whether contain I type of zika virus and/or Chikungunya virus and/or dengue virus in being used for detecting sample to be tested
And/or IV type of II type of dengue virus and/or III type of dengue virus and/or dengue virus.
7. a kind of whether method containing n kind target RNA sequences in detection sample to be tested, in turn includes the following steps:
(1) extract the total serum IgE of sample to be tested;
(2) carry out reverse transcription recombinase polymeric enzymatic amplification;
(3) carry out recombinase polymeric enzymatic amplification;N is natural number.
8. method as claimed in claim 7, it is characterised in that:N is more than 2 natural number;The recombinase polymeric enzymatic amplification
Carry out in micro-fluid chip, each reaction chamber of micro-fluid chip is used for detecting a kind of target RNA sequence.
9. a kind of for detecting in sample to be tested the whether test kit containing n kind target RNA sequences, including carrying out reverse transcription restructuring
The reagent of enzymatic polymerization enzymatic amplification and the reagent for carrying out recombinase polymeric enzymatic amplification.
10. test kit as claimed in claim 9, it is characterised in that:The test kit also includes special chip;The special core
Piece is will to be embedded in the differential responses of micro-fluid chip for the primed probe group for detecting the corresponding DNA of every kind of target RNA respectively
What chamber obtained;Each primed probe group is made up of pair of primers and a probe.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201611089150.XA CN106521032B (en) | 2016-11-30 | 2016-11-30 | A kind of nucleic acid amplification technologies and its application in carapuru virus detection |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201611089150.XA CN106521032B (en) | 2016-11-30 | 2016-11-30 | A kind of nucleic acid amplification technologies and its application in carapuru virus detection |
Publications (2)
Publication Number | Publication Date |
---|---|
CN106521032A true CN106521032A (en) | 2017-03-22 |
CN106521032B CN106521032B (en) | 2019-09-06 |
Family
ID=58354654
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201611089150.XA Active CN106521032B (en) | 2016-11-30 | 2016-11-30 | A kind of nucleic acid amplification technologies and its application in carapuru virus detection |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106521032B (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106929610A (en) * | 2017-05-09 | 2017-07-07 | 广州和实生物技术有限公司 | A kind of instant Constant Temperature Detection kit of I IV type dengue fever virus |
CN108085418A (en) * | 2017-12-29 | 2018-05-29 | 博迪泰(厦门)生物科技有限公司 | A kind of primer sets, kit and detection method for quickly detection dengue virus |
CN109295260A (en) * | 2018-11-09 | 2019-02-01 | 辽宁佰昊生物科技有限公司 | For detecting and/or assisting detection to cause primer sets, reagent and the kit and detection method of hand-foot-and-mouth disease poison EV71 |
CN111560479A (en) * | 2020-05-29 | 2020-08-21 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Nucleic acid kit for dual detection of Zika virus and chikungunya virus by NASBA technology and detection method thereof |
CN111593143A (en) * | 2020-05-29 | 2020-08-28 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | NASBA technology based kit for detecting Zika virus nucleic acid based on NS3 gene and detection method thereof |
CN111621585A (en) * | 2020-04-16 | 2020-09-04 | 大连民族大学 | Rapid detection kit for simultaneously detecting multiple transgenic rape lines and application thereof |
CN112111599A (en) * | 2019-06-19 | 2020-12-22 | 台达电子国际(新加坡)私人有限公司 | Multiple detection kit and method for drogong virus and zika virus |
CN113025746A (en) * | 2019-12-09 | 2021-06-25 | 北京博奥晶典生物技术有限公司 | Method for detecting REV by NASBA and kit used by same |
CN114085891A (en) * | 2021-11-23 | 2022-02-25 | 广州达安基因股份有限公司 | Reverse transcription amplification system and method based on recombinase polymerase amplification technology |
WO2022244104A1 (en) * | 2021-05-18 | 2022-11-24 | BioSeeds株式会社 | Isothermal gene amplification method, gene detection method, virus detection method, and kit used therefor |
WO2024055627A1 (en) * | 2022-09-13 | 2024-03-21 | 上海宝藤生物医药科技股份有限公司 | Primer-molecular beacon combination for molecular typing of wild type and mutant type of monkey poxvirus and use thereof |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102965452A (en) * | 2012-09-25 | 2013-03-13 | 珠海国际旅行卫生保健中心 | Norovirus real-time isothermal amplification detection kit, its primers and probe |
CN103397105A (en) * | 2013-07-01 | 2013-11-20 | 上海海洋大学 | Kit for detecting GII type norovirus and applications thereof |
US20150203901A1 (en) * | 2012-09-14 | 2015-07-23 | Life Technologies Corporation | Compositions and methods for detection of salmonella species |
CN105112407A (en) * | 2015-08-03 | 2015-12-02 | 博奥生物集团有限公司 | Kit for detecting enterovirus and application of kit |
-
2016
- 2016-11-30 CN CN201611089150.XA patent/CN106521032B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150203901A1 (en) * | 2012-09-14 | 2015-07-23 | Life Technologies Corporation | Compositions and methods for detection of salmonella species |
CN102965452A (en) * | 2012-09-25 | 2013-03-13 | 珠海国际旅行卫生保健中心 | Norovirus real-time isothermal amplification detection kit, its primers and probe |
CN103397105A (en) * | 2013-07-01 | 2013-11-20 | 上海海洋大学 | Kit for detecting GII type norovirus and applications thereof |
CN105112407A (en) * | 2015-08-03 | 2015-12-02 | 博奥生物集团有限公司 | Kit for detecting enterovirus and application of kit |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106929610A (en) * | 2017-05-09 | 2017-07-07 | 广州和实生物技术有限公司 | A kind of instant Constant Temperature Detection kit of I IV type dengue fever virus |
CN108085418A (en) * | 2017-12-29 | 2018-05-29 | 博迪泰(厦门)生物科技有限公司 | A kind of primer sets, kit and detection method for quickly detection dengue virus |
CN109295260A (en) * | 2018-11-09 | 2019-02-01 | 辽宁佰昊生物科技有限公司 | For detecting and/or assisting detection to cause primer sets, reagent and the kit and detection method of hand-foot-and-mouth disease poison EV71 |
CN112111599A (en) * | 2019-06-19 | 2020-12-22 | 台达电子国际(新加坡)私人有限公司 | Multiple detection kit and method for drogong virus and zika virus |
CN113025746B (en) * | 2019-12-09 | 2022-06-07 | 北京博奥晶典生物技术有限公司 | Method for detecting REV by NASBA and kit used by same |
CN113025746A (en) * | 2019-12-09 | 2021-06-25 | 北京博奥晶典生物技术有限公司 | Method for detecting REV by NASBA and kit used by same |
CN111621585A (en) * | 2020-04-16 | 2020-09-04 | 大连民族大学 | Rapid detection kit for simultaneously detecting multiple transgenic rape lines and application thereof |
CN111593143A (en) * | 2020-05-29 | 2020-08-28 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | NASBA technology based kit for detecting Zika virus nucleic acid based on NS3 gene and detection method thereof |
CN111560479A (en) * | 2020-05-29 | 2020-08-21 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Nucleic acid kit for dual detection of Zika virus and chikungunya virus by NASBA technology and detection method thereof |
WO2022244104A1 (en) * | 2021-05-18 | 2022-11-24 | BioSeeds株式会社 | Isothermal gene amplification method, gene detection method, virus detection method, and kit used therefor |
CN114085891A (en) * | 2021-11-23 | 2022-02-25 | 广州达安基因股份有限公司 | Reverse transcription amplification system and method based on recombinase polymerase amplification technology |
WO2023093060A1 (en) * | 2021-11-23 | 2023-06-01 | 广州达安基因股份有限公司 | Reverse-transcription amplification system and method based on recombinase polymerase amplification technology |
WO2024055627A1 (en) * | 2022-09-13 | 2024-03-21 | 上海宝藤生物医药科技股份有限公司 | Primer-molecular beacon combination for molecular typing of wild type and mutant type of monkey poxvirus and use thereof |
Also Published As
Publication number | Publication date |
---|---|
CN106521032B (en) | 2019-09-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106521032B (en) | A kind of nucleic acid amplification technologies and its application in carapuru virus detection | |
CN105112559B (en) | A kind of kit and its application for being used to detect coronavirus | |
CN108456747A (en) | A kind of multiple PCR detection kit differentiating pig circular ring virus | |
CN108504775A (en) | A kind of detection A type, the kit of influenza B virus and its application method based on micro-fluidic chip | |
WO2016078553A1 (en) | Kit for detecting and typing dengue viruses by reverse transcription pcr and detection method thereof | |
CN106967845A (en) | A kind of kit detected for pig Delta coronavirus and detection method | |
CN114262759B (en) | PCR primer group and kit for combined detection of multiple respiratory viruses | |
CN115852052A (en) | Real-time fluorescent quantitative PCR primer probe combination for detecting CymRSV and method thereof | |
CN106222298B (en) | LAMP detection kit, detection method and application of RNA virus | |
CN114807444A (en) | Primer, kit and method for detecting African swine fever virus | |
CN107447043A (en) | The type PCR detection primers pair of pig circular ring virus 3 and kit | |
CN109161611B (en) | Primer probe set and kit for jointly detecting banna virus, Canadenuovirus and Liaoning virus based on triple fluorescence PCR method | |
CN105154584B (en) | A kind of the HRM non-marked detecting probe method and its primer and probe of quick differentiation PRRSV classical strains and variation strain | |
CN103146846A (en) | Single standard product-based four-color fluorogenic quantitative PCR (Polymerase Chain Reaction) method and kit | |
Xia et al. | Rapid detection of Banna virus by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) | |
CN104946796B (en) | The kit and detection method of tomato spotted wilf virus are detected for RT LAMP methods | |
CN107287352A (en) | The probe primer group and its method of duck enteritis virus and duck hepatitis virus quick detection | |
CN109609697A (en) | A kind of primer, probe and detection kit quickly detecting ERC group virus based on RAA fluorescence method | |
CN112941240B (en) | Primer pair, kit and method for detecting goose astrovirus and goose goblet virus | |
CN106521038B (en) | A kind of real-time fluorescence quantitative PCR detection methods of highly sensitive BHV 2 and kit | |
KR101236197B1 (en) | Differential detection of West nile virus and Japanese encephalitis virus | |
CN105039596B (en) | A kind of kit and its application for being used to detect parainfluenza virus | |
CN108130385A (en) | A kind of human cytomegalovirus kit for detecting nucleic acid | |
CN104894112B (en) | A kind of Japanese B encephalitis virus real-time fluorescence isothermal amplification detection kit and its primer and probe | |
Can-Can et al. | Multiplex Nested Solid Phase PCR-Array Chip for Simultaneous Detection of Highly Pathogenic Microorganisms |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |