CN108660253A - A kind of probe, primer, detection kit and detection method detecting worm borne virus based on suspension microballon array system - Google Patents
A kind of probe, primer, detection kit and detection method detecting worm borne virus based on suspension microballon array system Download PDFInfo
- Publication number
- CN108660253A CN108660253A CN201810346243.9A CN201810346243A CN108660253A CN 108660253 A CN108660253 A CN 108660253A CN 201810346243 A CN201810346243 A CN 201810346243A CN 108660253 A CN108660253 A CN 108660253A
- Authority
- CN
- China
- Prior art keywords
- suspension
- probe
- worm
- borne virus
- primer
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
-
- 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)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of probe, primer, detection kit and detection methods detecting worm borne virus based on suspension microballon array system.The present invention is by the way that the probe for detecting worm borne virus to be coupled from different encoding microbeads respectively, suspension micropearl array is obtained after mixing, then multiple RT PCR amplifications are carried out to sample to be tested with the specific primer of the present invention, PCR product is hybridized with suspension micropearl array, it is developed the color again with Streptavidin R phycoerythrin, instrument is read by suspension micropearl array and reads testing result, whether is judged in sample to be tested containing the worm borne virus detected.The probe and primer that worm borne virus is detected based on suspension microballon array system of the present invention, can detect 7 kinds of worm borne virus simultaneously, can greatly shorten detection time, improve detection efficiency and accuracy.
Description
Technical field：
The invention belongs to molecular biology fields, and in particular to one kind is situated between sick based on suspension microballon array system detection entomophila
Probe, primer, detection kit and the detection method of poison.
Background technology：
Worm borne virus refer to one kind using hematophagus as medium, a variety of serious diseases are propagated between vertebrate and people, animal
The virus of disease.In recent years in the dengue fever virus of world matto area and south China prevalence, zika virus infection, all
Belong to the virus that entomophila Jie propagates.The patient for having infected worm borne virus has points of resemblance in clinical symptoms, including fever, flesh
Meat arthralgia, fash, lymph enlargement, etc..Clinically mainly pass through enzyme-linked immunization or quantitative fluorescent PCR side at present
Method, single virus are diagnosed, therefore there is the risk failed to pinpoint a disease in diagnosis.
There are some reports with regard to being made some attempts on the high-throughput diagnostic of worm borne virus, these technologies are not wrapped mostly
The zika virus of newest generation is included, and is only faced with the cell strain of laboratory infection virus and a small number of in practical application
Bed sample is tested, and the availability of its technology of actual response is unable to.
Suspension bead array technologies are the one kind for belonging to biochip technology, it is can suspension flow in a liquid with one kind
Dynamic microballon fixes support probe for medium, and probe can be hybridized with the type of flow and sample of microballon；And it fixes
Probe on glass planar or nylon membrane plane is then not flowable, such suspension bead array technologies nucleic acid molecule
Hybridization kinetics will be better than the hybridization of solid state surface, and since microballon can be made by outer surface coated magnetic material
Obtain the automation mechanized operation easy to implement of suspension bead array technologies.
The technological core of suspension bead array technologies is the coding and decoding of microballon.Probe on surface is by having
The position relationship of the row and column of sequence realizes the addressing of probe；And in microballon suspension array technology, microballon is flowing, natural
It can not be just addressed by position, therefore can only be by being encoded to microballon itself.There are two types of microballons to compile in the world at present
Code technology is exactly two kinds of infra-red materials one is the fluorescence-encoded technology based on infra-red material of the Luminex companies in the U.S.
It is mixed according to different proportionings, is allowed to send out the fluorescence of different-waveband, achievees the purpose that encoding microbeads, referred to as fluorescence-encoded, example
After being mixed such as the B material of 99% A materials+1%, No. 1 microballon is encoded；After the B material mixing of 98% A materials+2%, coding 2
Number microballon, such as analogizes, can at least encode 100 kinds of microballons in this way.Another suspension bead technology is U.S. Applied
The two-dimensional encoded technology of BioCode companies and Taiwan PlexBio companies is by computer micro-processing technology, respectively rectangular
The trace that different directions are etched on the siliceous particles of body and cylinder generates horizontal and vertical two-dimensional encoded, codified
4000-16000 kind microballons.
Both suspension bead array technologies for clinical application, all may be used although respective coding techniques is different
With meet a testing inspection index it is more, be easy automation mechanized operation, the unit interval detection sample size more than the needs of.
Invention content：
The primary purpose of the present invention is that overcome the shortcomings of it is existing from nucleic acid level detect entomophila Jie's viral methods, provide
A kind of probe, primer, detection kit and detection method detecting worm borne virus based on suspension microballon array system.
The first purpose of the invention is to provide it is a kind of based on suspension microballon array system detect worm borne virus probe,
The probe sequence is as follows：
GAPDH-P：5 '-TGGGGAGTCCCTGCCACAC-3 ' (as shown in SEQ ID NO.1)；
DENV1-P：5 '-TCAGTRTGGAATAGGGTTT-3 ' (as shown in SEQ ID NO.2)；
DENV2-P：5 '-TCAACATAGAAGCAGAACC-3 ' (as shown in SEQ ID NO.3)；
DENV3-P：5 '-TATGGCTGAAACTCCGAG-3 ' (as shown in SEQ ID NO.4)；
DENV4-P：5 '-TCAATATGCTGAAACGC-3 ' (as shown in SEQ ID NO.5)；
WNV-P：5 '-AGCCAAGATCAGCATGCCAGC-3 ' (as shown in SEQ ID NO.6)；
JEV-P：5 '-TGACNATTCCTGCGGTTTTGGGG-3 ' (as shown in SEQ ID NO.7)；
TBE-P：5 '-CCCATYACYCCWgTgTCAC-3 ' (as shown in SEQ ID NO.8)；
YFV-P1：5 '-CTGGATGATCAAGGAAACAGCYTGCCTC-3 ' (as shown in SEQ ID NO.9)；
YFV-P2：5 '-CTGGATGATCAAAGAAACGGCYTGCCTC-3 ' (as shown in SEQ ID NO.10)；
Chikv-P：5 '-CTGCAACGTCACACAGATGAGGGA-3 ' (as shown in SEQ ID NO.11)；
Zika-P：5 '-TGCTGGTGTATGGGCACARCAC-3 ' (as shown in SEQ ID NO.12)；The probe
5 ' ends carry linking arm.Chemical condensation reaction can occur with-the COOH on microballon for the-NH2 on linking arm so that probe connects
Onto microballon.
The linking arm is preferably alkylamine linking arm.
The alkylamine linking arm is preferably C18-NH2 linking arms.
Second object of the present invention is to provide a kind of primer detecting worm borne virus based on suspension microballon array system,
The primer sequence is as follows：
GAPDH-F：5 '-CAAGCTCATTTCCTGGTATGACA-3 ' (as shown in SEQ ID NO.13), GAPDH-R：5’-
GGGAGATTCAGTGTGGTGGG-3 ' (as shown in SEQ ID NO.14)；
DENV1-F：5 '-CAATGGATGACAACAGAAGAYATG-3 ' (as shown in SEQ ID NO.15), DENV1-R：
5 '-TCCATCCATGGGTTTTCCTCTAT-3 ' (as shown in SEQ ID NO.16)；
DENV2-F：5 '-GCAGARACACAACATGGAACRATAGT-3 ' (as shown in SEQ ID NO.17), DENV2-R：
5 '-TGATGTARCTGTCYCCRAATGG-3 ' (as shown in SEQ ID NO.18)；
DENV3-F：5 '-ATGGAATGTGTGGGAGGTGG-3 ' (as shown in SEQ ID NO.19), DENV3-R：5’-
GGCTTTCTATCCARTAGCCCATG-3 ' (as shown in SEQ ID NO.20)；
DENV4-F：5 '-GCAGATCTCTGGAAAAATGAACCA-3 ' (as shown in SEQ ID NO.21), DENV4-R：
5 '-GAGAATCTCTTCACCAACCCYTG-3 ' (as shown in SEQ ID NO.22)；
NS2A-F：5 '-CCTTTTCAGYTGGGCCTTCTG-3 ' (as shown in SEQ ID NO.23), NS2A-R：5’-
CAGTGTAVGTVATRCCCCCAA-3 ' (as shown in SEQ ID NO.24)；
TBE-F：5 '-TggAYTTYAgACAggAANCRACACA-3 ' (as shown in SEQ ID NO.25), TBE-R：5’-
TCCAgAgRCTYTgRTCDgTgTggA-3 ' (as shown in SEQ ID NO.26)；
YFV-F：5 '-GAGGAAGGGTGTCTCCAGGAA-3 ' (as shown in SEQ ID NO.27), YFV-R：5’-
ACATGTTGGCATAGGCYTTGCT-3 ' (as shown in SEQ ID NO.28)；
Chikv-F：5 '-TGTACTGGCAGCAGCCACG-3 ' (as shown in SEQ ID NO.29), Chikv-R：5’-
ATAGGGCTGGCAGCAAATTC-3 ' (as shown in SEQ ID NO.30)；
Zika-F：5 '-ACGCTCAGAGTCCTYTCCATG-3 ' (as shown in SEQ ID NO.31), Zika-R：5’-
GTCGCTCCAKGGTYTCCATC-3 ' (as shown in SEQ ID NO.32)；
The primer GAPDH-R, DENV1-R, DENV2-R, DENV3-R, DENV4-R, NS2A-R, TBE-R, YFV-R,
5 ' the ends of Chikv-R and Zika-R are marked with biotin.
Third object of the present invention is to provide a kind of detections detecting worm borne virus based on suspension microballon array system
Kit, including the probe and primer that worm borne virus is detected based on suspension microballon array system.
The detection kit further includes 2 × OneStep RT-PCR Buffer, OneStep RT-PCR
EnzymeMix and microballon.
The microballon is preferably fluorescence-encoded micro-beads or two-dimensional encoded microballoon.The fluorescence-encoded beads are preferably
The microballon of the surface carboxyl groups modification of Luminex companies of the U.S..
Fourth object of the present invention is to provide a kind of detection detecting worm borne virus based on suspension microballon array system
Method includes the following steps：
1) preparation of suspension micropearl array：The probe is coupled from different coding microballs respectively, then will
It has been coupled the coding microball mixed in equal amounts of probe, has obtained suspension micropearl array；
2) sample PCR amplification：The total serum IgE for extracting sample to be tested is carried out using the total serum IgE of the primer pair sample to be tested
Multiplex RT-PCR amplification obtains PCR product；
3) PCR product is hybridized with suspension micropearl array, obtains hybrid product, then with Streptavidin R- algae red eggs
It develops the color in vain to hybrid product, instrument is read by suspension micropearl array and reads testing result, judges whether contain in sample to be tested
The worm borne virus detected.
The multiplex RT-PCR amplification, reaction system are preferably 25 μ L：Including 1 × OneStep RT-PCR
Buffer, the primer GAPDH-F, GAPDH-R, DENV1-F, DENV1-R, DENV2-F, DENV2-R, DENV3-F,
DENV3-R、DENV4-F、DENV4-R、NS2A-F、NS2A-R、TBE-F、TBE-R、YFV-F、YFV-R、Chikv-F、Chikv-
R, Zika-F and Zika-R is 0.4 μM each, 0.5 μ L of OneStep RT-PCR EnzymeMix, the 2 μ L of total serum IgE of sample to be tested,
Remaining is RNase-Free Water；Reaction condition is：45℃30min；95℃2min；94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s, 40
Secondary cycle；72℃5min.
The design of most critical of the present invention is：The present invention propagates the multiplexed PCR amplification of common virus by designing entomophila Jie
Primer and probe, can be compatible with two kinds of suspension microballon array systems to detect a variety of worm borne virus, in one experiment can be real
Now to 7 kinds of worm borne virus：Dengue fever virus (1-4 types), Japanese B encephalitis virus, russian spring-summer encephalitis virus, west nile virus,
The refined willing fever virus of flavivirus, datum hole, fork clip virus are detected.On the one hand the flow of detection worm borne virus is simplified,
On the other hand clinical testing cost can also be reduced.
Suspension microballon array system detection worm borne virus described in the invention is relatively equally detected in nucleic acid level at present
The advantages of detection methods such as the fluorescent quantitation of the virus such as dengue fever includes：1. 7 kinds of entomophilas can be completed in one experiment to be situated between
The detection of virus, can prevent missing inspection to the greatest extent, be conducive to clinic and targetedly treated；2. dividing single index
Detection on, the cost of reagent consumptive material is greatly lowered, therefore can reduce medical treatment cost；3. detection takes short, operation
Step is simple, about 8 hours of entire testing process；4. specificity is strong, the dengue fever virus of different serotypes can be especially detected.
Description of the drawings：
Fig. 1 is 2 type of dengue fever virus of the present invention detected based on suspension microballon array system in sample.
Fig. 2 is the zika virus of the present invention detected based on suspension microballon array system in sample.
Specific implementation mode：
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
The present invention propagates the multiplexed PCR amplification primer and probe of common virus by designing entomophila Jie, can be compatible with two kinds
Suspension microballon array system detects a variety of worm borne virus, can realize in one experiment to dengue fever virus (1-4 types), day
The refined willing fever virus of this japanese encephalitis virus, russian spring-summer encephalitis virus, west nile virus, flavivirus, datum hole, fork clip virus carry out
Detection.
Embodiment 1：
1, multiple PCR primer designs：
Above 7 kinds of virus is all RNA virus, therefore the multiple reverse transcription PCR of this 7 kinds of RNA virus designs in a pipe instead
It answers, this 7 kinds of viruses, and the corresponding PCR primer sequence of GAPDH genes of the people for internal reference is shown in Table 1.
All reverse primer (i.e. primer GAPDH-R, DENV1-R, DENV2-R, DENV3-R, DENV4-R, NS2A-R,
TBE-R, YFV-R, Chikv-R and Zika-R) 5 ' end be marked with biotin.Contain degeneracy base in the primer sequence of part,
Middle Y represents base C or T, and R represents base A or G, V represent bases G or A or C, and N represents base A or T or G or C, and D represents bases G
Or A or T, K represent bases G or T.
Table 1. detects the multiple PCR primer sequence of RNA virus
Note：F indicates forward primer；R indicates reverse primer
2, probe designs：
The present invention devises 12 probes, wherein yellow fever altogether for 7 kinds of worm borne virus and the reference gene of 1 people
2 probes of viral design.Probe sequence such as table 2.
2. probe sequence of table
| Probe title | Probe sequence (5 ' -3 ') | The pathogen detected |
| GAPDH-P | TGGGGAGTCCCTGCCACAC | The reference gene of people |
| DENV1-P | TCAGTRTGGAATAGGGTTT | 1 type of dengue fever virus |
| DENV2-P | TCAACATAGAAGCAGAACC | 2 type of dengue fever virus |
| DENV3-P | TATGGCTGAAACTCCGAG | 3 type of dengue fever virus |
| DENV4-P | TCAATATGCTGAAACGC | 4 type of dengue fever virus |
| WNV-P | AGCCAAGATCAGCATGCCAGC | West nile virus |
| JEV-P | TGACNATTCCTGCGGTTTTGGGG | Japanese B encephalitis virus |
| TBE-P | CCCATYACYCCWGTGTCAC | Russian spring-summer encephalitis virus |
| YFV-P1 | CTGGATGATCAAGGAAACAGCYTGCCTC | Flavivirus (probe 1) |
| YFV-P2 | CTGGATGATCAAAGAAACGGCYTGCCTC | Flavivirus (probe 2) |
| Chikv-P | CTGCAACGTCACACAGATGAGGGA | Chikungunya fever virus |
| Zika-P | TGCTGGTGTATGGGCACARCAC | Zika virus |
Note：5 ' end band C18-NH2 of all probes are modified
3, the preparation of suspension micropearl array：The probe of fluorescence-encoded micro-beads (or two-dimensional encoded microballoon) and table 2 is distinguished
Coupling, then (can will be coupled the coding microball of probe by the coding microball mixed in equal amounts for being coupled probe according to testing goal
Mixing), obtain suspension micropearl array.It is calculated according to 10,000 person-portion theoretical amounts, the magnetic bead of a kind of probe and a coding carries out occasionally
Connection, specific experiment step are：
A, dichloroethanes (EDC) powder that hermetically drying preserves is taken out from -20 DEG C, makes it restore to room temperature.
B, melt configured 100 μM good of probe solution, vortex 5s mixings and 3000rpm centrifugations 30s.
C, the magnetic bead (2-8 DEG C of storage) of soft vortex suspension ingredient inspection qualification, is ultrasonically treated 30-60s.
D, the 1.5mL low adsorption centrifuge tubes number of finishing (magnetic bead number+probe number) is taken to be fixed on magnetic frame, magnetic bead is soft
It reverse 5-10 time, avoids acutely shaking, pipettor absorption 12.5 × 10 after pressure-vaccum 3 times repeatedly6A (1mL) magnetic bead is in magnetic frame
Wall adds in corresponding centrifuge tube, adsorbs 1min.
E, carefully removing supernatant, (left hand pushes down pipe shaft toward magnetic frame direction, and the right hand slowly exhausts supernatant, and pipette tips should be use up always
Amount is by far from magnetic frame direction, if there is magnetic bead follow-up pipette tips, needing to blow and beat again uniformly, be again attempted to after it is adsorbed again)；
2- (N- morpholinoes) ethanesulfonic acid (MES) solution of 60 μ L 0.1M, pH=4.5 is added, is ultrasonically treated 30-60s, vortex 5s mixings.
F, 15 μ L, 100 μM of probes are added.
G, the EDC powder for weighing 10mg is vortexed as 1mL pure water, final concentration 10mg/mL in 1.5mL centrifuge tube pipes, is added
5s mixings take 10 μ L to be added in pipe coupling immediately, vortex 5s mixings.
H, pipe coupling room temperature, which is protected from light, is incubated 30min, during which every 10min vortex 5s mixings.
I, step G and H are repeated twice.
J, 0.02% Tween-20 of 1mL, vortex 5s mixings is added, 3000rpm centrifuges 1min, upper magnetic frame 1min.
K, it gently uncaps and carefully removes supernatant, 1mL 0.1%SDS are added, be vortexed 5 seconds and be resuspended, 3000rpm centrifugations
1min, upper magnetic frame 1min.
L, it gently uncaps and carefully removes supernatant, 500 1 × TE of μ L (pH8.0, including 10mM Tris-HCl and 1mM is added
EDTA it) is vortexed 5 seconds and is resuspended after supersound process 30-60s.
M, it takes 1 μ L to dilute 100 times and is counted (have automatic counter for counting also can) under the microscope with cell counting board, calculate
The rate of recovery calculates microballon sum.
N, the microballon of Luminex companies is mixed that (Applied BioCode are public according to every person-portion each microballon 2000
The microballon of department is mixed for 50 according to every person-portion each microballon), obtain suspension micropearl array；
O, it is kept in dark place for 2-8 DEG C, the term of validity 14 months.
4, Samples detection：
The sample that the present invention is detected is blood plasma (or serum, saliva etc.), and the commercialization of different company can be used
Kit-virus genom DNA/RNA extracts kits extract the total serum IgE of sample.The detection of RNA virus needs to do reverse transcription
PCR reacts, reverse transcription PCR reaction system such as table 3, reaction condition such as table 4.
The reaction system of 3. multiplex RT-PCR amplification RNA virus of table
4. multiple RT-PCR reaction condition of table
Take the prepared suspension micropearl array of step 3, in packing to 96 orifice plates, per 45 μ L of hole (including 12 kinds be coupled respectively
The encoding microbeads of 12 kinds of probes in table 2, each 2000 of each microballon), 5 μ L of PCR product are then added.In 95 DEG C of thermal denaturations
5min, 60 DEG C of hybridization 15min, obtains hybrid product, the 25 diluted Streptavidin R- of μ L 1 × TMAC hybridization solutions is added per hole
0.04 μ g of phycoerythrin (can Streptavidin R-PE be first configured to 50 with 1 × TMAC hybridization solutions × colour developing it is female
Liquid, 1 × TMAC hybridization solutions include 3M TMAC, the sarcosyl of mass volume ratio 0.1%, pH8.050mM Tris-
HCl and pH8.0 4mM EDTA), suction is beaten to mix well under 10-20,60 DEG C of colour developing 7min.
Instrument is read with the suspension micropearl array of Luminex companies to be decoded (if using Applied microballon
The microballon of BioCode companies then reads instrument with the suspension micropearl array of Applied BioCode companies and is decoded to microballon),
And on microballon hybridization signal reading.
By whether sick containing entomophila Jie detected in the Fluorescence Intensity Assays clinical sample of instrument software detection probe
Poison.The standard of interpretation is：The signal value ＞ 1000 of the reference gene GAPDH probes of people, illustrates Success in Experiment.Under the premise of herein,
When entomophila Jie Viral Probe ＜ 600, worm borne virus is feminine gender；When 600≤entomophila Jie's Viral Probe signal≤1000, weight is needed
Repetition measurement is tried or is verified with qPCR methods；When entomophila Jie Viral Probe ＞ 1000, worm borne virus is the positive.
5, sensitivity technique：
Take respectively clone there is the plasmid of the target fragment of above 11 kinds of virus subtypes to be after nucleic acid quantification
Row dilution, is then detected with the method for the present embodiment, it is found that the detection for 11 kinds of virus subtypes that the present invention is detected is sensitive
Degree is 2 × 103-1×104Copies/mL is suitable with the conventional reverse transcription PCR method sensitivity of single viral diagnosis.
6, specific detection
For 11 kinds of virus subtypes that the present invention is detected, can be collected into clinical samples uses clinical samples；It cannot collect
Clinical samples are arrived, by being incorporated into blood plasma after cell culture and virus, total serum IgE is extracted by the method for step 4 respectively and carries out
Hybridized with suspension micropearl array after multiplex RT-PCR amplification, it is found that the probe signals of only corresponding virus subtype are shown as sun
Property；Simultaneously also with the clinical sample for having infected other RNA virus clinically collected, including Respiratory Syncytial Virus(RSV), inclined tuberculosis
Poison, enterovirus, rotavirus, norovirus, rhinovirus, parainfluenza virus are detected, suspension array with the method for the present invention
On probe result be all negative, illustrate that the specificity that the present invention detects is high.
Embodiment 2：
Confirm according to the antigen method of method pair 10 and PCR method of embodiment 1 be the infection of 1 type of dengue fever virus the positive
Clinical sample is detected, and the results are shown in Table 5 for detection positive rate.
The positive rate of 1 type of primer pair dengue fever virus after the optimization of table 5.
As seen from Table 5 after designing the degeneracy base of primer, positive rate significantly improves, and can farthest examine
Go out positive sample.
Embodiment 3：
One have fever, DOMS symptom pregnant woman, with dengue antigens method have a blood test detection be dengue fever virus sun
Property, but do not know the dengue fever virus for that parting, it is determined as dengue fever 2 type virus (see Fig. 1) according to the method for embodiment 1,
As a result identical as antigen method and PCR method.
Embodiment 4：
One clinical symptoms infects for zika virus, and is the patient of zika virus infection by PCR method detection,
The equally detection of the method for the blood plasma present invention is zika virus infection (see Fig. 2).
Clinically suspect that the sample for having infected worm borne virus carries out using the method and kit pair 257 of the present invention
Detection, the rate of accuracy reached 100% compared with antigen method and PCR method.
As it can be seen that the present invention can detect 7 kinds of (11 hypotypes) worm borne virus, including dengue fever virus in one experiment
(1-4 types), Japanese B encephalitis virus, russian spring-summer encephalitis virus, west nile virus, flavivirus, the refined willing fever virus of datum hole, plug
Card virus is detected.Using method of the present invention and kit, can obtain a result within about 8 hours.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (9)
1. a kind of probe detecting worm borne virus based on suspension microballon array system, which is characterized in that the probe sequence
As follows：
GAPDH-P：5’-TGGGGAGTCCCTGCCACAC-3’；
DENV1-P：5’-TCAGTRTGGAATAGGGTTT-3’；
DENV2-P：5’-TCAACATAGAAGCAGAACC-3’；
DENV3-P：5’-TATGGCTGAAACTCCGAG-3’；
DENV4-P：5’-TCAATATGCTGAAACGC-3’；
WNV-P：5’-AGCCAAGATCAGCATGCCAGC-3’；
JEV-P：5’-TGACNATTCCTGCGGTTTTGGGG-3’；
TBE-P：5’-CCCATYACYCCWgTgTCAC-3’；
YFV-P1：5’-CTGGATGATCAAGGAAACAGCYTGCCTC-3’；
YFV-P2：5’-CTGGATGATCAAAGAAACGGCYTGCCTC-3’；
Chikv-P：5’-CTGCAACGTCACACAGATGAGGGA-3’；
Zika-P：5’-TGCTGGTGTATGGGCACARCAC-3’；
5 ' ends of the probe carry linking arm.
2. the probe according to claim 1 for detecting worm borne virus based on suspension microballon array system, which is characterized in that
The linking arm is alkylamine linking arm.
3. the probe according to claim 2 for detecting worm borne virus based on suspension microballon array system, which is characterized in that
The alkylamine linking arm is C18-NH2Linking arm.
4. a kind of primer detecting worm borne virus based on suspension microballon array system, which is characterized in that the primer sequence
As follows：
GAPDH-F：5 '-CAAGCTCATTTCCTGGTATGACA-3 ', GAPDH-R：5’-GGGAGATTCAGTGTGGTGGG-3’；
DENV1-F：5 '-CAATGGATGACAACAGAAGAYATG-3 ', DENV1-R：5’-
TCCATCCATGGGTTTTCCTCTAT-3’；
DENV2-F：5 '-GCAGARACACAACATGGAACRATAGT-3 ', DENV2-R：5’-
TGATGTARCTGTCYCCRAATGG-3’；
DENV3-F：5 '-ATGGAATGTGTGGGAGGTGG-3 ', DENV3-R：5’-GGCTTTCTATCCARTAGCCCATG-3’；
DENV4-F：5 '-GCAGATCTCTGGAAAAATGAACCA-3 ', DENV4-R：5’-
GAGAATCTCTTCACCAACCCYTG-3’；
NS2A-F：5 '-CCTTTTCAGYTGGGCCTTCTG-3 ', NS2A-R：5’-CAGTGTAVGTVATRCCCCCAA-3’；
TBE-F：5 '-TGGAYTTYAGACAGGAANCRACACA-3 ', TBE-R：5’-TCCAGAGRCTYTGRTCDGTGTGGA-
3’；
YFV-F：5 '-GAGGAAGGGTGTCTCCAGGAA-3 ', YFV-R：5’-ACATGTTGGCATAGGCYTTGCT-3’；
Chikv-F：5 '-TGTACTGGCAGCAGCCACG-3 ', Chikv-R：5’-ATAGGGCTGGCAGCAAATTC-3’；
Zika-F：5 '-ACGCTCAGAGTCCTYTCCATG-3 ', Zika-R：5’-GTCGCTCCAKGGTYTCCATC-3’；
The primer GAPDH-R, DENV1-R, DENV2-R, DENV3-R, DENV4-R, NS2A-R, TBE-R, YFV-R,
5 ' the ends of Chikv-R and Zika-R are marked with biotin.
5. a kind of detection kit detecting worm borne virus based on suspension microballon array system, which is characterized in that including right
It is required that described in 1 based on suspension microballon array system detect worm borne virus probe and claim 4 described in based on suspension
The primer of micropearl array system detectio worm borne virus.
6. detection kit according to claim 5, which is characterized in that further include 2 × OneStep RT-PCR
Buffer, OneStep RT-PCR EnzymeMix and microballon.
7. detection kit according to claim 6, which is characterized in that the microballon is fluorescence-encoded micro-beads or two
Tie up coding microball.
8. a kind of detection method detecting worm borne virus based on suspension microballon array system, which is characterized in that including following step
Suddenly：
1) preparation of suspension micropearl array：Probe described in claim 1 is coupled from different coding microballs respectively, so
It will be coupled the coding microball mixed in equal amounts of probe afterwards, obtained suspension micropearl array；
2) sample PCR amplification：The total serum IgE for extracting sample to be tested, utilizes the total of the primer pair sample to be tested described in claim 4
RNA carries out multiplex RT-PCR amplification, obtains PCR product；
3) PCR product is hybridized with suspension micropearl array, obtains hybrid product, then with Streptavidin R-PE pair
Whether hybrid product develops the color, and reads instrument by suspension micropearl array and reads testing result, judge in sample to be tested containing
The worm borne virus of detection.
9. detection method according to claim 8, which is characterized in that the multiplex RT-PCR amplification, reaction system
For 25 μ L：Including primer GAPDH-F, GAPDH-R, DENV1- described in 1 × OneStep RT-PCR Buffer, claim 4
F、DENV1-R、DENV2-F、DENV2-R、DENV3-F、DENV3-R、DENV4-F、DENV4-R、NS2A-F、NS2A-R、TBE-
F, TBE-R, YFV-F, YFV-R, Chikv-F, Chikv-R, Zika-F and Zika-R be 0.4 μM each, OneStep RT-PCR
The 2 μ L of total serum IgE of 0.5 μ L of EnzymeMix and sample to be tested, remaining is RNase-Free Water；Reaction condition is：45℃
30min；95℃ 2min；94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s, 40 cycles；72℃ 5min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810346243.9A CN108660253B (en) | 2018-04-17 | 2018-04-17 | Probe, primer, detection kit and detection method for detecting insect-mediated viruses based on suspended bead array system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810346243.9A CN108660253B (en) | 2018-04-17 | 2018-04-17 | Probe, primer, detection kit and detection method for detecting insect-mediated viruses based on suspended bead array system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108660253A true CN108660253A (en) | 2018-10-16 |
| CN108660253B CN108660253B (en) | 2022-05-31 |
Family
ID=63782299
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810346243.9A Active CN108660253B (en) | 2018-04-17 | 2018-04-17 | Probe, primer, detection kit and detection method for detecting insect-mediated viruses based on suspended bead array system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108660253B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2008302295B2 (en) * | 2007-09-18 | 2013-11-28 | Stanford University | Methods of treating a flaviviridae family viral infection and compositions for treating a flaviviridae family viral infection |
| CN103911463A (en) * | 2014-04-03 | 2014-07-09 | 河北国际旅行卫生保健中心 | Kit for detecting mosquito borne pathogens and detection method thereof |
| CN106755573A (en) * | 2016-12-07 | 2017-05-31 | 深圳澳东检验检测科技有限公司 | Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit |
| CN107254554A (en) * | 2017-07-05 | 2017-10-17 | 广州奥百阕谱生物科技有限公司 | Detect the suspension microballon array system of respiratory tract infection common causative |
| CN108265127A (en) * | 2018-04-01 | 2018-07-10 | 广州奥百阕谱生物科技有限公司 | Detect the suspension microballon array system of lead to diarrhea common causative |
-
2018
- 2018-04-17 CN CN201810346243.9A patent/CN108660253B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2008302295B2 (en) * | 2007-09-18 | 2013-11-28 | Stanford University | Methods of treating a flaviviridae family viral infection and compositions for treating a flaviviridae family viral infection |
| CN103911463A (en) * | 2014-04-03 | 2014-07-09 | 河北国际旅行卫生保健中心 | Kit for detecting mosquito borne pathogens and detection method thereof |
| CN106755573A (en) * | 2016-12-07 | 2017-05-31 | 深圳澳东检验检测科技有限公司 | Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit |
| CN107254554A (en) * | 2017-07-05 | 2017-10-17 | 广州奥百阕谱生物科技有限公司 | Detect the suspension microballon array system of respiratory tract infection common causative |
| CN108265127A (en) * | 2018-04-01 | 2018-07-10 | 广州奥百阕谱生物科技有限公司 | Detect the suspension microballon array system of lead to diarrhea common causative |
Non-Patent Citations (4)
| Title |
|---|
| SI-QING LIU等: "Development and evaluation of one-step multiplex real-time RT-PCR assay for simultaneous detection of Zika virus and Chikungunya virus", 《J MED VIROL.》 * |
| 石莹等: "5种重要虫媒病毒液相蛋白芯片多重检测方法的建立及应用", 《中国卫生检验杂志》 * |
| 罗渊: "五种虫媒病毒悬浮芯片检测方法的建立", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
| 骆明勇等: "条形码磁珠液相芯片在地中海贫血基因检测中的应用", 《热带医学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108660253B (en) | 2022-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Reslova et al. | xMAP technology: applications in detection of pathogens | |
| Fooks et al. | Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century | |
| CN104812915A (en) | Assay for the parallel detection of biological material based on PCR | |
| Fernandes et al. | Recent advances in point of care testing for COVID-19 detection | |
| Malik et al. | Advances in diagnostic approaches for viral etiologies of diarrhea: from the lab to the field | |
| Alhamid et al. | SARS-CoV-2 detection methods: A comprehensive review | |
| CN112941078B (en) | Aptamer for detecting novel coronavirus SARS-CoV-2S1 protein, screening method and use thereof | |
| CN105779649A (en) | Immune PCR reagent kit for detecting avian leukemia virus | |
| TW201300421A (en) | Reagents and methods for PRRSV detection | |
| CN107254554A (en) | Detect the suspension microballon array system of respiratory tract infection common causative | |
| CN111575406A (en) | Novel coronavirus rapid screening method and kit based on nucleic acid isothermal amplification | |
| Ma et al. | Rapid detection of avian influenza A virus (H7N9) by lateral flow dipstick recombinase polymerase amplification | |
| Yan et al. | Virome sequencing of the human intestinal mucosal–luminal interface | |
| CN105886663A (en) | Locked nucleic acid sensitivity-enhanced fluorescent quantitative PCR (polymerase chain reaction) detection reagent kit for wild strains of porcine pseudorabies viruses | |
| CN103210093B (en) | Method for detecting digestive tract pathogens | |
| CN106191311A (en) | A kind of quick detection Cavia porcellus LCMV, SV, PVM, Reo 3 virus multiple liquid phase method for gene chip and reagent | |
| CN106636454A (en) | Real-time fluorescence multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for simultaneously detecting human coronaviruses 229E, OC43, NL63 and HKU1 | |
| Asghar et al. | Advancements in testing strategies for COVID-19 | |
| Barker et al. | Advances in molecular diagnostics and treatment of feline infectious peritonitis | |
| CN108265127A (en) | Detect the suspension microballon array system of lead to diarrhea common causative | |
| Wang et al. | Advances and future perspective on detection technology of human norovirus | |
| CN108660253A (en) | A kind of probe, primer, detection kit and detection method detecting worm borne virus based on suspension microballon array system | |
| Mercader et al. | Development and use of an endpoint titration assay to characterize mumps IgG avidity following measles, mumps, and rubella vaccination and wild-type mumps infection | |
| Shuryaeva et al. | Development and application of LAMP assays for the detection of enteric adenoviruses in feces | |
| CN101429545A (en) | Method for detecting Shigella by using suspension chip technology |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |