CN106755573A - Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit - Google Patents
Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit Download PDFInfo
- Publication number
- CN106755573A CN106755573A CN201611116443.2A CN201611116443A CN106755573A CN 106755573 A CN106755573 A CN 106755573A CN 201611116443 A CN201611116443 A CN 201611116443A CN 106755573 A CN106755573 A CN 106755573A
- Authority
- CN
- China
- Prior art keywords
- seq
- detection
- fever virus
- 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.)
- Pending
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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- 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)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the technical field of molecular biological detection of insect-borne infectious disease, zika virus, dengue fever virus, triple real-time fluorescence RT PCR detection methods of chikungunya fever virus, primer and probe and kit are disclosed.Primer includes SEQ ID No.1, SEQ ID No.2, SEQ ID No. 4, SEQ ID No.5, SEQ ID No.7, the sequence of SEQ ID No.8, and probe includes SEQ ID No.3, SEQ ID No.6, the sequence of SEQ ID No.9.Kit includes：Primed probe mixed liquor, RT PCR reaction solutions, enzyme mixation, positive criteria product.The present invention is by optimizing reaction formula of liquid and primer probe sequence, using triple real-time fluorescence RT PCR amplification techniques can in same tube reaction liquid quick detection zika virus, dengue fever virus and chikungunya fever virus, without separately individually detection, compared with normal PCR detection method, operating procedure is reduced, can quickly, accurately detect whether, containing these three viruses, to shorten detection time, detection efficiency is improved, it is cost-effective.
Description
Technical field
The present invention relates to the technical field of molecular biological detection of insect-borne infectious disease, and in particular to zika virus, dengue fever
Virus, triple real-time fluorescent RT-PCR method for detecting of chikungunya fever virus, primer and probe and kit.
Background technology
Zika virus disease is also referred to as stockaded village's card heat（Zika fever）, it is by zika virus（Zika Virus, ZIKV）Cause simultaneously
A kind of viral disease propagated by mosquito matchmaker.Zika virus disease disease symptom：Slight heating, fash, conjunctivitis, muscle and pass
Section pain, malaise and headache etc., these symptoms are typically lasted for 2 ~ 7 days.There is research display zika virus disease to be likely to result in
The complication of nerve and self immune system, such as actue infectious polyradiculoneuritis（Guillian-Barr syndrome）, pregnant woman's sense
Dye zika virus may then cause baby's microcephalus.Since in May, 2015, after Brazil finds that the first makes a definite diagnosis stockaded village's card case,
The whole world more than 1,500,000 people of existing more than 40 countries infection zika virus simultaneously triggers other multiple country's introduced cases, China north
There is the report that zika virus infects Jing Shi, Guangdong Province and Taiwan.
Dengue fever（Dengue fever, DF）It is by dengue fever virus（Dengue virus, DENV）The class urgency for causing
Sexually transmitted disease, the disease is main to be propagated by Aedes aegypti and aedes albopictus, and typical disease symptom is to have a fever, have a headache and arthralgia etc.,
Bleeding and shock even lethality are presented under critical conditions.Dengue fever is a kind of being widely current in the torrid zone or subtropical zone
Disease, dengue fever virus is small-sized flavivirus, and I, I, II, III, IV type are divided into according to E protein antigenicity difference in serology.
It is reported that dengue fever occurs repeatedly breaking out phenomenon in recent years, relevant statistics show：Malaysian cases of dengue fever in 2014
Add up to surpass 60,000；Nearly case 30,000 is made a definite diagnosis altogether by Sri Lanka in 2015, and the common many cases of confirmed cases 70,000 of Brazil, China is wide
East saves many cases of confirmed cases 30,000.
Chikungunya fever（Chikungunya fever）It is by CHIK（Chikungunya virus,
CHIKV）It is causing, through her mosquito-borne acute infectious disease, current Major Epidemic is in Southeast Asia and African Territories.The sick incubation period
Generally 2 ~ 4 days, also it is 7 ~ 12 days.Principal pathogenetic symptom has：Unexpected heating, shiver with cold, trunk fash, severe joint are bitterly
With headache etc., can be with nausea, vomiting, photophobia, conjunctival congestion, stomachache or bleeding etc..The current Major Epidemic of the disease and the Indian Ocean
Area, China also has the sick epidemic situation to report.
At present, because fluorescent RT-PCR technology has, Idiotype is strong, sensitivity is high, reproducible, detection time is short and inspection
The advantages of survey expense is relatively low, is widely used when clinical great amount of samples is detected.And stockaded village's card disease has been developed on the market
The related substance kit for detecting nucleic acid product of poison, dengue fever virus, chikungunya fever virus, but to zika virus, Dengue
Fever virus, the product of the triple detection of nucleic acids of chikungunya fever virus yet there are no report.
The content of the invention
It is an object of the invention to provide specific good, sensitivity it is high, can simultaneously detect zika virus, dengue fever virus, base
Agree the primer and probe of refined fever virus in hole.
Zika virus, dengue fever virus, Chikungunya fever can be simultaneously detected it is a further object of the present invention to provide one kind
The detection method of virus.
Zika virus, dengue fever virus, Chikungunya fever can be simultaneously detected it is a further object of the present invention to provide one kind
The kit of virus.
To reach one of above-mentioned purpose, the present invention uses following technical scheme：
A kind of primer of the triple real-time fluorescence RT-PCRs detection for zika virus, dengue fever virus, chikungunya fever virus
And probe, including：
Two primers of detection zika virus, its base sequence is respectively SEQ ID No.1 and SEQ ID No.2；
The universal probe of one detection zika virus, its base sequence is SEQ ID No.3；
The universal primer of two detection dengue fever virus, its base sequence is respectively SEQ ID No. 4 and SEQ ID No.5；
The universal probe of one detection dengue fever virus, its base sequence is SEQ ID No.6；
Two primers of detection chikungunya fever virus, its base sequence is respectively SEQ ID No.7 and SEQ ID No.8；
One probe of detection chikungunya fever virus, its base sequence is SEQ ID No.9.
Further, the 5 ' ends of the SEQ ID No.3 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching base
Group；5 ' the ends of the SEQ ID No.6 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group；The SEQ ID
5 ' the ends of No.9 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
Further, the primer and probe also include：
Target primer in two detections, its base sequence is respectively SEQ ID No.10 and SEQ ID No.11；
Target probe in one detection, its base sequence is SEQ ID No.12；
5 ' the ends of the SEQ ID No.12 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
A kind of triple real-time fluorescence RT-PCRs detection for zika virus, dengue fever virus, chikungunya fever virus
Kit, including：
Two primers of detection zika virus, its base sequence is respectively SEQ ID No.1 and SEQ ID No.2；
The universal probe of one detection zika virus, its base sequence is SEQ ID No.3；
The universal primer of two detection dengue fever virus, its base sequence is respectively SEQ ID No. 4 and SEQ ID No.5；
The universal probe of one detection dengue fever virus, its base sequence is SEQ ID No.6；
Two primers of detection chikungunya fever virus, its base sequence is respectively SEQ ID No.7 and SEQ ID No.8；
One probe of detection chikungunya fever virus, its base sequence is SEQ ID No.9.
Further, the 5 ' ends of the SEQ ID No.3 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching base
Group；5 ' the ends of the SEQ ID No.6 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group；The SEQ ID
5 ' the ends of No.9 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
Further, the kit also includes：
Target primer in two detections, its base sequence is respectively SEQ ID No.10 and SEQ ID No.11；
Target probe in one detection, its base sequence is SEQ ID No.12；
5 ' the ends of the SEQ ID No.12 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
Above primer and probe composition primed probe mixed liquor.
Further, the kit also includes：RT-PCR reaction solutions, enzyme mixation, positive criteria product.
Further, the RT-PCR reaction solutions include：RT-PCR buffer solutions, magnesium chloride, triphosphoric acid dezyribonucleoside
Acid blend and PCR reinforcing agents.
Preferably, the PCR buffer solutions include Tris-HCl（pH8.0）, ammonium sulfate, potassium chloride and Triton X-100.
Preferably, the PCR reinforcing agents are selected from tetramethyl ammonium chloride（Tetramethylammonium chloride,
TMAC）, carnitine（L-carnitine）, trehalose（D-(+)-trehalose）With the one kind in nonionic detergent NP-40 or
It is various.
Further, the enzyme mixation includes Taq archaeal dna polymerases, M-MLV reverse transcriptases and RNase inhibitor；Institute
It is the DNA molecular fragment synthesized according to viral gene conserved sequence to state positive criteria product.
Preferably, the saltant type Taq archaeal dna polymerases are resistant to up to 5 ~ 10% whole blood, 0.2 ~ 0.4 μ g/mL corruption
Grow acid, or 2.6 ~ 5.2 mM lactoferrins.
Further, the kit also includes internal standard, is inside designated as a kind of MS2 bacteriophage coat protein bags of prokaryotic expression
The RNA particles of dress, RNA molecule contain amplification of internal standard needed for sequence, the base composition of sequence is SEQ No. 13.
A kind of detection zika virus, dengue fever virus, the method for chikungunya fever virus, comprise the following steps：
S1, sample process：The serum sample that will be gathered, extracts the geneome RNA of sample, standby；
S2, amplifing reagent prepare：13 μ L RT-PCR reaction solutions, 3 μ L enzyme mixations, 4 μ L primed probes mixed liquors, 5 μ L samples are molten
Liquid, reaction system cumulative volume is 25 μ L；
S3, RT-PCR amplification program：55 DEG C, 30min；95 DEG C, 3min；（95 DEG C, 10s；55 DEG C, 25s）× 45 circulations；
S4, result judgement.
The result judgement is specifically：FAM, ROX, CY5 passage without amplification curve, HEX channel C t value≤35.0, as a result
It is judged to feminine gender；ROX channel C t value≤35.0, and there is obvious Exponential growth stage, represent that sample zika virus is the positive；
FAM channel C t value≤35.0, and there is obvious Exponential growth stage, represent that sample dengue fever virus is positive；CY5 channel C t values≤
35.0, and there is obvious Exponential growth stage, represent sample Chikungunya fever virus-positive；Four passages without amplification curve,
Illustrate that amplification is invalid.Reform detection of the Ct values of Viral diagnosis result between 35.0-40.0, if Ct values still less than
40, and curve has obvious Exponential growth stage, can report that sample is positive, otherwise report that sample is negative or detected sample nucleic acid
Levels are less than the detection method test limit.
The invention has the advantages that：
The present invention uses triple real-time fluorescence RT-PCR amplification techniques can be with by optimizing reaction formula of liquid and primer probe sequence
Quick detection zika virus, dengue fever virus and chikungunya fever virus in same tube reaction liquid, without separately independent
Detection, reduces operating procedure, can quickly, accurately detect whether to contain zika virus, dengue fever virus, Chikungunya fever
Virus, greatly shortens detection time, improves detection efficiency, and cost-effective, does not exist serological cross reaction in course of reaction.
Compared with normal PCR detection method, detection method reduces operating procedure, shortens detection time, drop
Low testing cost, and sensitivity and specificity are suitable with conventional method, and with the analysis spirit consistent with normal PCR detection
Sensitivity.
The entomophila caused by zika virus, dengue fever virus, chikungunya fever virus is passed using kit of the invention
The preventing and treating of epidemic situation of catching an illness, early screening, diagnosis provide accurate, easy, effective monitoring means, while also to insect-borne infectious disease
The epidemiological study of poison is significant.
It is of the invention crucial including two parts：One is creatively to search out suitable primer and probe, can overcome and draw
Thing and probe by multiplicity sampling complicated factor influenceed and primer and probe form labyrinth and cause to interfere with each other etc. because
The influence of element, it is possible to achieve zika virus, dengue fever virus, the triple samples of chikungunya fever virus are carried out in same reaction tube
RT-PCR augmentation detections, using the inventive method to the worm that is caused by zika virus, dengue fever virus, chikungunya fever virus
The preventing and treating of matchmaker's epidemic, early screening, diagnosis provide accurate, easy, effective monitoring means；Two is that reaction is used
Reagent optimize, especially RT-PCR reaction solutions are optimized, and making one kind can efficiently carry out zika virus, step on
Leather fever virus, the agent prescription of the triple real-time fluorescence RT-PCR augmentation detections of chikungunya fever virus.
Brief description of the drawings
Fig. 1 is the zika virus sensitive amplification testing result of embodiment 3；
Fig. 2 is embodiment 4 to zika virus sample specific detection result；
Fig. 3 is embodiment 4 to dengue fever virus sample specific detection result；
Fig. 4 is embodiment 4 to Chikungunya fever Virus Sample specific detection result.
Specific embodiment
With reference to specific embodiment, the present invention is described further.
Embodiment 1
The design of primer and probe
Zika virus whole in Primary Reference GenBank databases, dengue fever virus and chikungunya when primed probe is designed
The gene order of fever virus, using the softwares such as the BLAST instruments on NCBI websites, the PrimerExpress of DNAStar, ABI with
And the conserved sequence of analysis software zika virus, dengue fever virus and chikungunya fever virus that designed, designed is write, and
Specific primer and probe are designed according to the conserved sequence for obtaining, the interaction between analysis primer, optimizes and screening is drawn
Thing probe combinations, the cross reaction reduced to greatest extent between Flavivirus is produced, to ensure high specific, the high sensitivity of reagent
With high gene type coverage rate.Resulting primer and probe is as shown in table 1：
TableZika virus, dengue fever virus and chikungunya fever virus and internal standard detection primer probe sequence
5 ' the ends of SEQ ID No.3 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group；SEQ ID No.6's
5 ' ends are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group；5 ' the ends of SEQ ID No.9 are marked with fluorescence report
Group is accused, 3 ' ends are marked with fluorescent quenching group；5 ' the ends of SEQ ID No.12 are marked with fluorescent reporter group, and 3 ' ends are marked with
Fluorescent quenching group.
Embodiment 2
Zika virus, dengue fever virus, the clinical sample detection of the triple real-time fluorescence RT-PCRs of chikungunya fever virus
1. sample treatment：Take a certain amount of serum sample and add 3 times of methyl alcohol of volume, stand five minutes after mixing, 3000r/min
Centrifugation 15min, choosing supernatant carries out nucleic acid extraction viral RNA.Viral RNA extracts the Reansy using QIAGEN companies of Germany
Mini Kit, are extracted by kit specification, obtain viral RNA.10 times of gradient dilutions are being carried out with LB buffer solutions respectively and is being marked
Note.
2. reagent prepares：
RT-PCR reaction solutions are constituted：RT-PCR buffer solutions, magnesium chloride, triphosphate deoxyribose nucleotide mixture and PCR
Reinforcing agent；PCR buffer solutions include Tris-HCl（pH8.0）, ammonium sulfate, potassium chloride and Triton X-100；PCR reinforcing agents are selected from
One or more in tetramethyl ammonium chloride, carnitine, trehalose and nonionic detergent NP-40.
Primed probe mixed liquor includes the primer and probe of embodiment 1.
Enzyme mixation is constituted：The M-MLV of saltant type Taq archaeal dna polymerases, high thermal stability for multiplicity sampling is inverse
Transcriptase and RNase inhibitor（RNasin）；Saltant type Taq archaeal dna polymerases be resistant to up to 5 ~ 10% whole blood, 0.2 ~
0.4 μ g/mL humic acid, or 2.6 ~ 5.2 mM lactoferrins.
Positive criteria product is the DNA molecular fragment synthesized according to viral gene conserved sequence, card disease in stockaded village's in positive criteria product
Malicious concentration is 2.85 × 1014The universal concentration of copies/mL, dengue fever virus is 4.77 × 1014Copies/mL, chikungunya
Fever virus concentration is 4.61 × 1014copies/mL；10 times of gradient dilution positive criteria products are standby.
Above RT-PCR reaction solutions, primed probe mixed liquor, enzyme mixation, positive criteria product composition kit, kit
Internal standard can also be included.
A kind of RNA particles of the MS2 bacteriophage coat proteins packaging of prokaryotic expression are inside designated as, RNA molecule contains internal standard expansion
Sequence needed for increasing, the base composition of sequence is SEQ No. 13：5’-ATATGTCCTAGAAGAGATGAGTTGCAT
ACCAGGAGGAAGGATGTATGCAGATGACACTGCTGGCTGGGACACCCGCATCAGCAGGTTTGATCTGGAGAAT
GAAGCTCTAATCACCAACCAAATGGAG-3’。
3. RT-PCR amplification programs：
Quantitative real time PCR Instrument（ABI7500）, refrigerated centrifuge（5804R, Eppendorf）.
55 DEG C, 30min；
95 DEG C, 3min；
95 DEG C, 5s；
55 DEG C, 40s；
4. interpretation of result：Without amplification curve, HEX channel C t value≤35.0, result judgement is feminine gender to FAM, ROX, CY5 passage；
ROX channel C t value≤35.0, and there is obvious Exponential growth stage, represent that sample zika virus is the positive；FAM channel C t values≤
35.0, and there is obvious Exponential growth stage, represent that sample dengue fever virus is positive；CY5 channel C t value≤35.0, and occur bright
Aobvious Exponential growth stage, represents sample Chikungunya fever virus-positive；Four passages without amplification curve, illustrate amplification without
Effect.Reform detection of the Ct values of Viral diagnosis result between 35.0-40.0, if Ct values are still less than 40, and curve has substantially
Exponential growth stage, can report that sample is positive, otherwise report that sample is negative or detected sample nucleic acid levels be less than should
Detection method test limit.
15 parts of clinical serum pattern detection result such as table 2 below.
The clinical serum pattern detection result of table 2
Note："+" represents positive, and "-" represents negative.
Result shows：Zika virus, dengue fever virus, Chikungunya fever virus-virus shown in embodiment 1 is triple in real time
The detection kit of fluorescence RT-PCR is tried with commercially available zika virus, dengue fever virus, chikungunya fever virus substance RT-PCR
The testing result contrast that agent box is obtained（It is temporarily triple in real time without zika virus, dengue fever virus, Chikungunya fever virus-virus at present
The detection kit of fluorescence RT-PCR）：The inventive method detects 14 parts of the positive, and sample is without crossover phenomenon；Commercially available substance detection
Kit detects 14 parts of the positive, but sample cross phenomenon occurs in sample number 5,13.Comparing result shows：The present invention is triple
Real-time fluorescence PCR detection method has with consistent testing result with substance PCR detection method, but from sample cross result,
The inventive method is better than substance detection kit.
Embodiment 3
The analysis of zika virus, dengue fever virus, the triple real-time fluorescence RT-PCRs of chikungunya fever virus and substance conventional reagent
Remolding sensitivity compared with
Standard items strain sample of nucleic acid to zika virus is diluted to the nucleic acid concentration of 0.001 LD50；According to side in embodiment 2
Method and step carry out zika virus, dengue fever virus, the triple real-time fluorescence RT-PCRs of chikungunya fever virus and substance and routinely try
Agent augmentation detection compares, each concentration duplicate detection 50 times.Result is as shown in table 3 and Fig. 1.
The sensitivity for analysis result of table 3
| Detection method | Total sample number（Part） | Positive sample（Part） | Recall rate（%） |
| Triple real-time fluorescence RT-PCR kits | 50 | 48 | 96 |
| Conventional substance RT-PCR kit | 50 | 44 | 88 |
Knowable to table 3 and result shown in Fig. 1, the analysis spirit of triple real-time fluorescence RT-PCR method detection zika viruses of the invention
Sensitivity is 96%, the sensitivity for analysis position 88% of conventional reagent substance RT-PCR detection kit.Result explanation is triple glimmering in real time
The sensitivity for analysis of light RT-PCR method detection zika virus compares with the sensitivity for analysis of substance conventional RT-PCR, San Chongshi
When fluorescence RT-PCR sensitivity for analysis it is higher.
Embodiment 4
The triple real-time fluorescent RT-PCR detection reagent box specificity of zika virus, dengue fever virus, chikungunya fever virus
To being separately added into stockaded village in zika virus, dengue fever virus, the triple real-time fluorescence RT-PCR kits of chikungunya fever virus
Card virus, dengue fever virus, Chikungunya fever virus-positive standard items diluted sample are expanded, according to different fluorescence labelings
Amplification curve detects the specificity of this kit.Result can be seen that zika virus, Dengue as shown in Fig. 2 ~ 4 from Fig. 2,3,4
Fever virus and the triple real-time fluorescence PT-PCR specific detection no cross reactions of chikungunya fever virus.The above results show this examination
Agent box to zika virus, dengue fever virus, the triple real-time fluorescent RT-PCR detection reagent boxes of chikungunya fever virus specificity
Well.
The above, specific embodiment only of the invention, but protection scope of the present invention is not limited thereto, and it is any
Belong to those skilled in the art the invention discloses technical scope in, the change or replacement that can be readily occurred in, all should
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be defined by scope of the claims.
SEQUENCE LISTING
<110>Shenzhen Aodong Inspection ＆ Testing Technology Co., Ltd.
<120>Zika virus, dengue fever virus, the RT-PCR detection method of chikungunya fever virus, primer and probe and reagent
Box
<130>
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
ttgcatacca ggaggaagg 19
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
agcttcattc tccagatcaa ac 22
<210> 3
<211> 28
<212> DNA
<213>Artificial sequence
<400> 3
gatgacactg ctggctggga cacccgca 28
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
tggaaggact agaggttaga g 21
<210> 5
<211> 15
<212> DNA
<213>Artificial sequence
<400> 5
gcgttctgtg cctgg 15
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
cgctgggaga gaccagagat c 21
<210> 7
<211> 19
<212> DNA
<213>Artificial sequence
<400> 7
gcaggaaatt gaccccgac 19
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence
<400> 8
tatccgacat catcctcc 18
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence
<400> 9
cctggatatc ggcagtgcgc c 21
<210> 10
<211> 21
<212> DNA
<213>Artificial sequence
<400> 10
aatcctgtca cctctgacta a 21
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence
<400> 11
gcattttgga caaagcgtct a 21
<210> 12
<211> 21
<212> DNA
<213>Artificial sequence
<400> 12
cctgccgact gccgtgagcc a 21
<210> 13
<211> 127
<212> DNA
<213>Artificial sequence
<400> 13
atatgtccta gaagagatga gttgcatacc aggaggaagg atgtatgcag atgacactgc 60
tggctgggac acccgcatca gcaggtttga tctggagaat gaagctctaa tcaccaacca 120
aatggag 127
Claims (10)
1. what a kind of triple real-time fluorescence RT-PCRs for zika virus, dengue fever virus, chikungunya fever virus were detected draws
Thing and probe, it is characterised in that including：
Two primers of detection zika virus, its base sequence is respectively SEQ ID No.1 and SEQ ID No.2；
The universal probe of one detection zika virus, its base sequence is SEQ ID No.3；
The universal primer of two detection dengue fever virus, its base sequence is respectively SEQ ID No. 4 and SEQ ID No.5；
The universal probe of one detection dengue fever virus, its base sequence is SEQ ID No.6；
Two primers of detection chikungunya fever virus, its base sequence is respectively SEQ ID No.7 and SEQ ID No.8；
One probe of detection chikungunya fever virus, its base sequence is SEQ ID No.9.
2. primer according to claim 1 and probe, it is characterised in that the 5 ' ends of the SEQ ID No.3 are marked with glimmering
Light reporter group, 3 ' ends are marked with fluorescent quenching group；5 ' the ends of the SEQ ID No.6 are marked with fluorescent reporter group, 3 '
End is marked with fluorescent quenching group；5 ' the ends of the SEQ ID No.9 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescence and quench
Go out group.
3. primer according to claim 1 and probe, it is characterised in that also include：
Target primer in two detections, its base sequence is respectively SEQ ID No.10 and SEQ ID No.11；
Target probe in one detection, its base sequence is SEQ ID No.12；
5 ' the ends of the SEQ ID No.12 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
4. the examination of a kind of triple real-time fluorescence RT-PCRs detection for zika virus, dengue fever virus, chikungunya fever virus
Agent box, it is characterised in that the kit includes：
Two primers of detection zika virus, its base sequence is respectively SEQ ID No.1 and SEQ ID No.2；
The universal probe of one detection zika virus, its base sequence is SEQ ID No.3；
The universal primer of two detection dengue fever virus, its base sequence is respectively SEQ ID No. 4 and SEQ ID No.5；
The universal probe of one detection dengue fever virus, its base sequence is SEQ ID No.6；
Two primers of detection chikungunya fever virus, its base sequence is respectively SEQ ID No.7 and SEQ ID No.8；
One probe of detection chikungunya fever virus, its base sequence is SEQ ID No.9.
5. kit according to claim 4, it is characterised in that the 5 ' ends of the SEQ ID No.3 are marked with fluorescence report
Group is accused, 3 ' ends are marked with fluorescent quenching group；5 ' the ends of the SEQ ID No.6 are marked with fluorescent reporter group, 3 ' end marks
Note has fluorescent quenching group；5 ' the ends of the SEQ ID No.9 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching base
Group.
6. kit according to claim 4, it is characterised in that also include：
Target primer in two detections, its base sequence is respectively SEQ ID No.10 and SEQ ID No.11；
Target probe in one detection, its base sequence is SEQ ID No.12；
5 ' the ends of the SEQ ID No.12 are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
7. kit according to claim 4, it is characterised in that also include：RT-PCR reaction solutions, enzyme mixation, the positive
Standard items.
8. kit according to claim 7, it is characterised in that the RT-PCR reaction solutions include：RT-PCR buffer solutions,
Magnesium chloride, triphosphate deoxyribose nucleotide mixture and PCR reinforcing agents.
9. kit according to claim 7, it is characterised in that the enzyme mixation includes Taq archaeal dna polymerases, M-
MLV reverse transcriptases and RNase inhibitor；The positive criteria product is the DNA molecular piece synthesized according to viral gene conserved sequence
Section.
10. it is a kind of to detect zika virus, dengue fever virus, the method for chikungunya fever virus, it is characterised in that including following step
Suddenly：
S1, sample process：The serum sample that will be gathered, extracts the geneome RNA of sample, standby；
S2, amplifing reagent prepare：13 μ L RT-PCR reaction solutions, 3 μ L enzyme mixations, 4 μ L primed probes mixed liquors, 5 μ L samples are molten
Liquid, reaction system cumulative volume is 25 μ L；
S3, carry out RT-PCR amplifications with primer described in claim 1 and probe：55 DEG C, 30min；95 DEG C, 3min；（95 DEG C,
10s；55 DEG C, 25s）× 45 circulations；
S4, result judgement.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611116443.2A CN106755573A (en) | 2016-12-07 | 2016-12-07 | Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611116443.2A CN106755573A (en) | 2016-12-07 | 2016-12-07 | Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106755573A true CN106755573A (en) | 2017-05-31 |
Family
ID=58882217
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611116443.2A Pending CN106755573A (en) | 2016-12-07 | 2016-12-07 | Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755573A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660253A (en) * | 2018-04-17 | 2018-10-16 | 广东省妇幼保健院（广东省妇产医院、广东省儿童医院） | A kind of probe, primer, detection kit and detection method detecting worm borne virus based on suspension microballon array system |
| CN108707693A (en) * | 2018-02-13 | 2018-10-26 | 连云港出入境检验检疫局检验检疫综合技术中心（江苏国际旅行卫生保健中心连云港分中心、连云港出入境检验检疫局口岸门诊部） | A kind of yellow fever virus, chikungunya virus rapid fluorescence PCR detection kit |
| CN110066889A (en) * | 2019-05-21 | 2019-07-30 | 四川国际旅行卫生保健中心 | Quick detection primer group, kit and its application of the GeXP of four kinds of Flavivirus virus are detected simultaneously |
| CN110305985A (en) * | 2019-06-21 | 2019-10-08 | 中国疾病预防控制中心病毒病预防控制所 | A method of utilizing triple real-time fluorescence quantitative RT-PCRs detection zika virus, chikungunya virus and Ma Yaluo virus |
| CN110724767A (en) * | 2019-11-13 | 2020-01-24 | 中国疾病预防控制中心病毒病预防控制所 | Triple real-time fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for detecting dengue fever, chikungunya and Zika viruses |
| CN111763772A (en) * | 2020-08-13 | 2020-10-13 | 中国医学科学院输血研究所 | Premixed fluorescent PCR (polymerase chain reaction) detection reagent and kit for mosquito-borne infectious disease pathogens |
| 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 |
| CN112111598A (en) * | 2019-06-19 | 2020-12-22 | 台达电子国际（新加坡）私人有限公司 | Primer pair, kit and method for detecting zika virus |
| CN112760419A (en) * | 2021-02-04 | 2021-05-07 | 杭州遂曾生物技术有限公司 | Triple detection card box for dengue, Zika and chikungunya viruses |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140529A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | New dengue virus fluorescence quantitative polymerase chain reaction detection method and dengue virus polymerase chain reaction detection system |
| CN102191338A (en) * | 2010-12-24 | 2011-09-21 | 中国检验检疫科学研究院 | Fluorescence quantitative polymerase chain reaction (PCR) new method for detecting multiple viruses of yellow fever, dengue fever and epidemicencephalitis B and multiple virus detection PCR system |
| CN103025893A (en) * | 2010-07-29 | 2013-04-03 | 彼格泰格私人有限公司 | Probes and primers for detection of dengue |
| CN103305633A (en) * | 2013-06-14 | 2013-09-18 | 浙江国际旅行卫生保健中心 | Multiplex-fluorescence PCR (Polymerase Chain Reaction) detection kit and application thereof |
| CN105483289A (en) * | 2015-12-29 | 2016-04-13 | 深圳澳东检验检测科技有限公司 | Enterovirus triplex direct fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit and reaction system |
-
2016
- 2016-12-07 CN CN201611116443.2A patent/CN106755573A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025893A (en) * | 2010-07-29 | 2013-04-03 | 彼格泰格私人有限公司 | Probes and primers for detection of dengue |
| CN102140529A (en) * | 2010-12-24 | 2011-08-03 | 中国检验检疫科学研究院 | New dengue virus fluorescence quantitative polymerase chain reaction detection method and dengue virus polymerase chain reaction detection system |
| CN102191338A (en) * | 2010-12-24 | 2011-09-21 | 中国检验检疫科学研究院 | Fluorescence quantitative polymerase chain reaction (PCR) new method for detecting multiple viruses of yellow fever, dengue fever and epidemicencephalitis B and multiple virus detection PCR system |
| CN103305633A (en) * | 2013-06-14 | 2013-09-18 | 浙江国际旅行卫生保健中心 | Multiplex-fluorescence PCR (Polymerase Chain Reaction) detection kit and application thereof |
| CN105483289A (en) * | 2015-12-29 | 2016-04-13 | 深圳澳东检验检测科技有限公司 | Enterovirus triplex direct fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit and reaction system |
Non-Patent Citations (2)
| Title |
|---|
| JESSE J.WAGGONER ET AL: "Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika,Chikungunya, and Dengue Viruses", 《EMERGING INFECTIOUS DISEASES》 * |
| KANTI PABBARAJU ET AL: "Simultaneous detection of Zika, Chikungunya and Dengue viruses by amultiplex real-time RT-PCR assay", 《JOURNAL OF CLINICAL VIROLOGY》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108707693A (en) * | 2018-02-13 | 2018-10-26 | 连云港出入境检验检疫局检验检疫综合技术中心（江苏国际旅行卫生保健中心连云港分中心、连云港出入境检验检疫局口岸门诊部） | A kind of yellow fever virus, chikungunya virus rapid fluorescence PCR detection kit |
| CN108660253A (en) * | 2018-04-17 | 2018-10-16 | 广东省妇幼保健院（广东省妇产医院、广东省儿童医院） | A kind of probe, primer, detection kit and detection method detecting worm borne virus based on suspension microballon array system |
| CN108660253B (en) * | 2018-04-17 | 2022-05-31 | 广东省妇幼保健院（广东省妇产医院、广东省儿童医院） | Probe, primer, detection kit and detection method for detecting insect-mediated viruses based on suspended bead array system |
| CN110066889A (en) * | 2019-05-21 | 2019-07-30 | 四川国际旅行卫生保健中心 | Quick detection primer group, kit and its application of the GeXP of four kinds of Flavivirus virus are detected simultaneously |
| CN110066889B (en) * | 2019-05-21 | 2023-11-24 | 四川国际旅行卫生保健中心 | GeXP rapid detection primer group for simultaneously detecting four yellow fever viruses, kit 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 |
| CN112111598A (en) * | 2019-06-19 | 2020-12-22 | 台达电子国际（新加坡）私人有限公司 | Primer pair, kit and method for detecting zika virus |
| CN110305985A (en) * | 2019-06-21 | 2019-10-08 | 中国疾病预防控制中心病毒病预防控制所 | A method of utilizing triple real-time fluorescence quantitative RT-PCRs detection zika virus, chikungunya virus and Ma Yaluo virus |
| CN110305985B (en) * | 2019-06-21 | 2022-05-17 | 中国疾病预防控制中心病毒病预防控制所 | Method for detecting Zika virus, chikungunya virus and Mayaro virus by utilizing triple real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) |
| CN110724767A (en) * | 2019-11-13 | 2020-01-24 | 中国疾病预防控制中心病毒病预防控制所 | Triple real-time fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for detecting dengue fever, chikungunya and Zika viruses |
| CN111763772A (en) * | 2020-08-13 | 2020-10-13 | 中国医学科学院输血研究所 | Premixed fluorescent PCR (polymerase chain reaction) detection reagent and kit for mosquito-borne infectious disease pathogens |
| CN112760419A (en) * | 2021-02-04 | 2021-05-07 | 杭州遂曾生物技术有限公司 | Triple detection card box for dengue, Zika and chikungunya viruses |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106755573A (en) | Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit | |
| CN111004870B (en) | Novel coronavirus N gene nucleic acid detection kit | |
| WO2016082691A1 (en) | Kit for rt-pcr detection of chikungunya and test method thereof | |
| CN111334608B (en) | Dual-fluorescence freeze-drying microchip, kit and method for detecting novel coronavirus 2019-nCoV | |
| CN106086242A (en) | A kind of test kit detected for Flavivirus fast typing and virus load | |
| CN109777893A (en) | The invisible hepatitis B detection kit of droplet type digital pcr | |
| CN106755571A (en) | It is a kind of for detecting zika virus, dengue fever virus and the kit of CHIK | |
| CN105483283B (en) | Hepatitis C Virus HCV real-time fluorescence nucleic acid isothermal amplification detection kit | |
| CN107058632A (en) | A kind of multiplex PCR detects three kinds of diarrhea virus kits and its detection method | |
| CN107488749A (en) | A kind of LAMP primer and detection method for detecting the type of pig circular ring virus 3 | |
| Wang et al. | Development of a reverse transcription loop-mediated isothermal amplification assay for rapid and visual detection of Sugarcane streak mosaic virus in sugarcane | |
| CN107142334A (en) | The viruliferous RT LAMP detection methods of mulberry arteries and veins and its primer sets, kit and application | |
| CN108796127A (en) | A kind of kit and its detection method of specific detection lily mottle virus | |
| CN111321247B (en) | Freeze-drying microporous plate, kit and method for identifying African swine fever virus, swine fever wild strain and swine fever lapinized attenuated vaccine strain | |
| CN110317904A (en) | A kind of LAMP method and its primer combination of specific detection African swine fever virus | |
| 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 | |
| CN108330211A (en) | A group rotavirus/astrovirus/intestinal adenovirus nucleic acid multiple fluorescence PCR detection reagent box and application thereof | |
| Jiang et al. | Sap-direct RT-PCR for the rapid detection of coleus blumei viroids of the genus Coleviroid from natural host plants | |
| CN113005230A (en) | Asia Zika virus RT-RIA/CRISPR-Cas12a detection kit and detection method thereof | |
| CN109593886A (en) | A kind of hbv nucleic acid immue quantitative detection reagent box | |
| CN110295255B (en) | RT-LAMP-LFD-based rapid detection method for detecting kiwi chlorotic and ringspot related viruses | |
| CN108531647A (en) | A kind of zika virus one-step method fluorescence rt-PCR detection methods and kit | |
| CN105525036B (en) | A kind of hepatitis b virus hbv real-time fluorescence nucleic acid isothermal amplification detection kit | |
| WO2018169550A1 (en) | Real-time rt-pcr assay for detection of dengue, chikungunya, and zika viruses | |
| CN107400735A (en) | Detect the real-time fluorescent quantitative RT-PCR method of zika virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170531 |
|
| RJ01 | Rejection of invention patent application after publication |