CN106868210A - Daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT PCR kits and detection method - Google Patents
Daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT PCR kits and detection method Download PDFInfo
- Publication number
- CN106868210A CN106868210A CN201710134679.7A CN201710134679A CN106868210A CN 106868210 A CN106868210 A CN 106868210A CN 201710134679 A CN201710134679 A CN 201710134679A CN 106868210 A CN106868210 A CN 106868210A
- Authority
- CN
- China
- Prior art keywords
- daffod
- virus
- concentration
- nysv
- nmv
- 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
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 a kind of daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT PCR kits and detection method, for quick detection daffod bar virus and narcissus mosaic virus simultaneously.The kit includes：NYSV forward primers, NYSV reverse primers, NYSV fluorescence probes, NMV forward primers, NMV reverse primers, NMV fluorescence probes, random primer, RT Buffer, RNase inhibiting factor, reverse transcriptase, dNTPs, TaqMan PCR Master Mix, positive control, negative control and RNase free ddH2O.The present invention is respectively for daffod bar virus, conserved sequence design specific primer and the fluorescence probe of narcissus mosaic virus gene, detect daffod bar virus and narcissus mosaic virus simultaneously using multiple fluorescence quantitative RT round pcrs, with sensitivity is high, the good remarkable advantage of high specificity, accuracy, the quick detection of daffod bar virus and narcissus mosaic virus in the Check and Examination of Port quarantine that is very suitable for passing in and out, agricultural production.
Description
Technical field
The present invention relates to a kind of daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT-PCR detection kit
And its detection method, belong to technical field of plant quarantine, it is adaptable to daffod bar in Check and Examination of Port of passing in and out quarantine, agricultural production
The quick detection of virus and narcissus mosaic virus.
Background technology
Narcissus is Narcissus（Narcissus）Herbaceos perennial, in China, cultivation history is long, is that China's tradition goes out
Mouth famous flower.Virosis is the important disease on narcissus, and virus infection narcissus can cause bulb to diminish, flower arrow reduces, fragrance becomes
The symptom such as light, bulb degeneration and plant dwarfing, causes the yield and quality of narcissus to decline.Daffod bar virus（NYSV）, narcissus
Mosaic virus（NMV）It is that two kinds of more serious viruses occur in narcissus production.NYSV belongs to marmor upsilon section
（Potyviridae）, Potyvirus（Potyvirus）Member, can be passed by aphid vector with non-persistent manner
Broadcast, it is also possible to propagated by juice.NYSV natural hosts are mainly the narcissus and jonquil of Amaryllidaceae, and frictional inoculation can infect
New Zealand spinach platymiscium.NYSV genomes are sense single stranded rna, and a length of 9650 nucleotides of complete sequence, virion is in bending,
Particle size about 755nm × 12nm.The NYSV symptoms such as floral leaf Huang bar, napiform root can be caused to diminish on infected narcissus plant,
Usually with narcissus mosaic virus Combined Infection.NYSV is almost distributed in the water all over the world such as Europe, North America, Australia and Asia
Celestial Main Cultivation area.NMV belongs to linear viral section（Flexiviridae）, potexvirus（Potexvirus）Member, it is main
Will be by juice contact transmission.The natural host that NMV has been reported is narcissus.NMV genomes are sense single stranded rna, and genome is complete
A length of 6955 nucleotides, virion is linear, particle size about 550nm × 13nm.After NMV infects narcissus, the initial stage draws
Play plant leaf or chlorisis spot occurs in bennet, the later stage causes floral leaf gradually obvious, and expands to larger patch, is caused when serious
The symptoms such as leaf curling, yellow and plant deformity.NMV is mainly distributed on the countries such as Holland, Britain and China.
It is control daffod bar virus（NYSV）And narcissus mosaic virus（NMV）Generation, propagation and the diffusion of virus, protection
Narcissus production safety, strengthens NYSV, NMV detection and is extremely important.At present, reported and apply more NYSV,
NMV detection methods are two methods of serology ELISA and conventional RT-PCR.Serology ELISA detection method needs to use costliness
Detection reagent, it is and high to the specificity of serum and purity requirement, while its sensitivity is relatively low with conventional RT-PCR compared with, examine
Survey result and false negative easily occur.Conventional RT-PCR method is simply possible to use in the single detection of NYSV or NMV on narcissus, it is impossible to for same
When detect two kinds of viruses of NYSV and NMV.Such as the Chinese invention patent of Patent No. ZL 201210195105.8《For detecting water
The kit and its detection method of celestial mosaic virus and daffod bar virus》, being capable of single detection NYSV or NMV, it is impossible to once
Experiment detects two kinds of viruses simultaneously.Compared with serology ELISA, conventional RT-PCR, detected using multiple fluorescence quantitative RT-PCR
NYSV, NMV, can not only realize that NYSV, NMV be single or detection of Combined Infection, and specific stronger, sensitivity is higher.
But up to the present, there is not yet dedicated for NYSV, NMV simultaneously detect multiple fluorescence quantitative RT-PCR detection method, more not
See special multiple fluorescence quantitative RT-PCR detection kit.
The content of the invention
Can be used in daffod bar virus it is an object of the invention to provide one kind, narcissus mosaic virus is single or compound invades
The multiple fluorescence quantitative RT-PCR detection kit and its detection method of detection are contaminated, defect present in prior art and not is overcome
Foot, can in pass in and out port and agricultural production daffod bar virus, narcissus mosaic virus carry out quickly, precise Identification, spy
It is not two kinds of Combined Infection identifications of virus, with high specificity, the remarkable advantage that sensitivity is high and accuracy is good.
Technical solution of the present invention is as follows：
（One）A kind of multiple fluorescence quantitative RT-PCR detection kit detected for daffod bar virus and narcissus mosaic virus,
The kit includes：
1）NYSV- forward primers：10 μm of ol/L, primer sequence is 5 '-TGGATGGAGAAGAACAAGTTGAATT-3 '；
2）NYSV- reverse primers：10 μm of ol/L, primer sequence is 5 '-GCCATTATCTGCCTGAGTGTAGGT-3 '；
3）NYSV- fluorescence probes：10 μm of ol/L, fluorescence probe sequence is 5 '-CY5-
CCACTTAAACCCATCATTGATCACGCCA-BHQ-3 ', the fluorescent reporter group of the end of fluorescence probe 5 ' mark is CY5,3 ' ends
The fluorescent quenching group of mark is BHQ；
4）NMV- forward primers：10 μm of ol/L, primer sequence is 5 '-CCATCAAACCCTGCGATCA-3 '；
5）NMV- reverse primers：10 μm of ol/L, primer sequence is 5 '-CAGACCACCTTGGCGAAGTAC-3 '；
6）NMV- fluorescence probes：10 μm of ol/L, fluorescence probe sequence is 5 '-FAM-TCACTCCGCGCCAGTTCTGCA-BHQ-
3 ', the fluorescent reporter group of the end of fluorescence probe 5 ' mark is FAM, and the fluorescent quenching group of 3 ' end marks is BHQ；
7）Random primer：100μmol/L；
8）RT Buffer：5×；
9）RNase inhibiting factor：40U/μL；
10）Reverse transcriptase：200U/μL；
11）dNTPs：10mmol/L；
12）TaqMan PCR Master Mix：2×；
13）Daffod bar virus, the positive control sample of narcissus mosaic virus；
14）Negative control sample without daffod bar virus, narcissus mosaic virus；
15）RNase-free ddH2O。
（Two）Using the detection of above-mentioned daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT-PCR kit
Method, comprises the following steps：
1）Reverse transcription reaction：It is 100 μm of μ L of the random primer of ol/L 1 that the μ L of testing sample total serum IgE 3, concentration are added in PCR pipe
With RNase-free ddH2O 7 μ L, 70 DEG C of water-baths 10min, rapid ice bath 5min, add following reagent：5× RT
The μ L of Buffer 5, concentration are the μ L of 10mmol/L dNTPs 2, concentration is the μ L of 200U/ μ L reverse transcriptase 1, concentration is 40U/ μ L RNA
Enzyme inhibition factor 1 μ L, 42 DEG C of water-bath 60min, are cooled to room temperature after 70 DEG C of water-bath 10min, synthesize cDNA；
2）Quantitative fluorescent PCR reacts：Take step 1）The μ L of cDNA 3 of synthesis, are 10 μm of ol/L NMV- positive by often pipe plus concentration
The μ L of primer 1, concentration are 10 μm of μ L of ol/L NMV- reverse primers 1, concentration is 10 μm of the μ L of ol/L NMV- fluorescence probes 0.75, concentration
For 10 μm of μ L of ol/L NYSV- forward primers 1, concentration for 10 μm of μ L of ol/L NYSV- reverse primers 1, concentration are 10 μm of ol/L
The μ L of NYSV- fluorescence probes 0.75,2 × TaqMan PCR Master Mix 12.5 μ L, ddH2The μ L of O 4, make the reaction cumulative volume be
25μL；Mixed reaction solution, 95 DEG C of 30s, then 95 DEG C of 15s, 60 DEG C of 1min, carry out 40 circulations altogether, and reaction terminates.
The present invention is according to daffod bar virus and the conserved sequence design specific primer and glimmering of narcissus mosaic virus gene
Light probe, establishes the multiple fluorescence quantitative RT-PCR method for daffod bar virus and narcissus mosaic virus quick detection.
For than prior art, daffod bar virus provided by the present invention and narcissus mosaic virus multiple fluorescence quantitative RT-PCR are examined
Test agent box and its detection method beneficial effect are：1）High specificity：Specific primer and fluorescence probe can during PCR
To realize the double control to target gene, daffod bar virus can not only be realized, narcissus mosaic virus is single or Combined Infection
Detection, additionally it is possible to daffod bar virus, narcissus mosaic virus and other non-specific virals are accurately distinguished；2）Sensitivity
It is high：Compared with serology ELISA, conventional RT-PCR detection method, fluorescence quantitative RT-RCR has sensitivity higher；3）Save
Sample, shortening detection cycle：It is daffod bar virus, water because multiple fluorescence quantitative RT-PCR can be detected on same sample
Celestial mosaic virus single virus infect, or two viruses infect simultaneously, therefore one-time detection can simultaneously detect two viruses,
Both saved detection sample, also shortened detection cycle, it is to avoid before two kinds of viruses of detection need to carry out common RT- twice
The shortcoming of PCR；4）It is easy to operate：The detection method that the present invention is provided is easy to operate, and traditional detection method efficiency can be overcome low
Deficiency.
Brief description of the drawings
Fig. 1 is the specific assay result of embodiment 3.Wherein 1：Daffod bar viral sample；2：Narcissus mosaic virus sample
Product；3：Narcissus degeneration viral sample；4：Narcissus cryptovirus sample；5：The common cryptovirus sample of narcissus；6：Negative control.
Fig. 2 is the sensitivity determination result of the NYSV of embodiment 4.Wherein 1：RNA 10-1Dilution；2：RNA 10-3Dilution；
3：RNA 10-5Dilution；4：RNA 10-7Dilution；5：RNA 10-9Dilution；6：Negative control.
Fig. 3 is the sensitivity determination result of the NMV of embodiment 4.Wherein 1：RNA 10-1Dilution；2：RNA 10-3Dilution；3：
RNA 10-5Dilution；4：RNA 10-7Dilution；5：RNA 10-9Dilution；6：Negative control.
Specific embodiment
Below by way of specific embodiment, the invention will be further described.
Embodiment 1：The configuration of daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT-PCR kit（10 times
Detection limit）
1）NYSV- forward primers：10 μm of ol/L, 1 pipe（20μL）；
2）NYSV- reverse primers：10 μm of ol/L, 1 pipe（20μL）；
3）NYSV- fluorescence probes：10 μm of ol/L, 1 pipe（20μL）；
4）NMV- forward primers：10 μm of ol/L, 1 pipe（20μL）；
5）NMV- reverse primers：10 μm of ol/L, 1 pipe（20μL）；
6）NMV- fluorescence probes：10 μm of ol/L, 1 pipe（20μL）；
7）Random primer：100 μm of ol/L, 1 pipe（20μL）；
8）RT Buffer：5 ×, 1 pipe（100μL）；
9）RNase inhibiting factor：40U/ μ L, 1 pipe（50μL）；
10）Reverse transcriptase：200U/ μ L, 1 pipe（20μL）；
11）dNTPs：10mmol/L, 1 pipe（20μL）；
12）TaqMan PCR Master Mix：2 ×, 1 pipe（150μL）；
13）Daffod bar virus, the positive control sample of narcissus mosaic virus, 1 pipe（50μL）；
14）Negative control sample without daffod bar virus, narcissus mosaic virus, 1 pipe（50μL）；
15）RNase-free ddH2O, 1 pipe（1mL）.
Embodiment 2：The detection method of daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT-PCR kit
The detection method of above-mentioned daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT-PCR kit, including it is following
Step：
1）Reverse transcription reaction：It is 100 μm of μ L of the random primer of ol/L 1 that the μ L of testing sample total serum IgE 3, concentration are added in PCR pipe
With RNase-free ddH2O 7 μ L, 70 DEG C of water-baths 10min, rapid ice bath 5min, add following reagent：5× RT
The μ L of Buffer 5, concentration are the μ L of 10mmol/L dNTPs 2, concentration is the μ L of 200U/ μ L reverse transcriptase 1, concentration is 40U/ μ L RNA
The μ L of enzyme inhibition factor 1.42 DEG C of water-bath 60min, are cooled to room temperature after 70 DEG C of water-bath 10min, synthesize cDNA；
2）Quantitative fluorescent PCR reacts：Take step 1）The μ L of cDNA 3 of synthesis, are 10 μm of ol/L NMV- positive by often pipe plus concentration
The μ L of primer 1, concentration are 10 μm of μ L of ol/L NMV- reverse primers 1, concentration is 10 μm of the μ L of ol/L NMV- fluorescence probes 0.75, concentration
For 10 μm of μ L of ol/L NYSV- forward primers 1, concentration for 10 μm of μ L of ol/L NYSV- reverse primers 1, concentration are 10 μm of ol/L
The μ L of NYSV- fluorescence probes 0.75,2 × TaqMan PCR Master Mix 12.5 μ L, ddH2The μ L of O 4, make the reaction cumulative volume be
25μL；Mixed reaction solution, 95 DEG C of 30s, then 95 DEG C of 15s, 60 DEG C of 1min, carry out 40 circulations altogether, and reaction terminates.
Embodiment 3：Daffod bar virus is special with narcissus mosaic virus multiple fluorescence quantitative RT-PCR detection kit
Property determine
1）RNA is extracted：Take sample 0.1g to be placed in mortar, add the grinding of 1mL PBST buffer solutions, 4 DEG C, 10000g is centrifuged 5min,
Take supernatant and supernatant is transferred quickly in the 1.5mL centrifuge tubes of sterilizing, add 1mL TrizoL reagents, acutely after vibration,
It is stored at room temperature 5min；4 DEG C, 12000g centrifugation 10min take supernatant；The μ L of chloroform 300 are added, 15s is acutely shaken, is stored at room temperature
5min, 4 DEG C, 12000g centrifugation 15min take upper strata aqueous phase；Isometric isopropanol is added, is stood at room temperature after reverse mixing
15min, 4 DEG C, 12000g centrifugation 10min abandon supernatant；Add 1mL 75% ethanol washing precipitation 2 times, 4 °C every time, 7500g from
Heart 3min, abandons supernatant；After RNA precipitate is dried, with 20 μ L RNase-free ddH2O dissolves；
2）Reverse transcription reaction：It is 100 μm of μ L of the random primer of ol/L 1 that the μ L of testing sample total serum IgE 3, concentration are added in PCR pipe
With RNase-free ddH2O 7 μ L, 70 DEG C of water-baths 10min, rapid ice bath 5min, add following reagent：5× RT
The μ L of Buffer 5, concentration are the μ L of 10mmol/L dNTPs 2, concentration is the μ L of 200U/ μ L reverse transcriptase 1, concentration is 40U/ μ L RNA
Enzyme inhibition factor 1 μ L, 42 DEG C of water-bath 60min, are cooled to room temperature after 70 DEG C of water-bath 10min, synthesize cDNA；
3）Quantitative fluorescent PCR reacts：Take step 1）The μ L of cDNA 3 of synthesis, are 10 μm of ol/L NMV- positive by often pipe plus concentration
The μ L of primer 1, concentration are 10 μm of μ L of ol/L NMV- reverse primers 1, concentration is 10 μm of the μ L of ol/L NMV- fluorescence probes 0.75, concentration
For 10 μm of μ L of ol/L NYSV- forward primers 1, concentration for 10 μm of μ L of ol/L NYSV- reverse primers 1, concentration are 10 μm of ol/L
The μ L of NYSV- fluorescence probes 0.75,2 × TaqMan PCR Master Mix 12.5 μ L, ddH2The μ L of O 4, make the reaction cumulative volume be
25μL；Mixed reaction solution, 95 DEG C of 30s, then 95 DEG C of 15s, 60 DEG C of 1min, carry out 40 circulations altogether, and reaction terminates.Root
According to amplification curve（Fig. 1）, only infect the typical amplification curve of sample generation of daffod bar virus, narcissus mosaic virus, other diseases
Malicious sample and the equal nothing of healthy sample, show that kit of the present invention has very strong specificity.
Embodiment 4：Daffod bar virus is sensitive with narcissus mosaic virus multiple fluorescence quantitative RT-PCR detection kit
Degree is determined
The RNA of daffod bar virus, narcissus mosaic virus sample is diluted to 10 respectively-1Again, 10-3Again, 10-5Again, 10-7Times,
10-9Again as template, detected by the method for embodiment 2, as a result seen Fig. 2 and Fig. 3.Knowable to Fig. 2 and Fig. 3, can detect
Dilution 10-9Daffod bar viral sample RNA and narcissus mosaic virus sample RNA again, shows that kit of the present invention has very high
Sensitivity.
Embodiment 5：Daffod bar virus, the detection of narcissus mosaic virus on domestic and international narcissus sample
Choose 30 parts of Chinese narcissus sample, 30 parts of the inward narcissus sample of intercept and capture（Holland, Britain and Japanese narcissus each 10
Part）, totally 60 parts of samples, are detected after extracting RNA by the method for embodiment 2, and experiment also uses single conventional RT-PCR simultaneously
Method is verified.Result can detect Simple infection daffod bar virus, water using the present invention from 60 parts of narcissus samples
Celestial mosaic virus has 23 parts, while infecting daffod bar virus, narcissus mosaic virus 2 kinds of viruses has 12 parts, above-mentioned detection
Result is consistent with conventional RT-PCR method validation result.
SEQUENCE LISTING
<110>Inspection ＆ Quarantine Technology Center of Fujian Entry-Exit Inspection ＆ Quar
<120>Daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT-PCR kit and detection method
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
tggatggaga agaacaagt tgaatt 25
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<400> 2
gccattatct gcctgagtgt aggt 24
<210> 3
<211> 28
<212> DNA
<213>Artificial sequence
<400> 3
ccacttaaac ccatcattga tcacgcca 28
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence
<400> 4
ccatcaaacc ctgcgatca 19
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<400> 5
cagaccacct tggcgaagta c 21
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
tcactccgcg ccagttctgc a 21
Claims (2)
1. a kind of daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT-PCR kit, it is characterised in that：It is described
Kit includes：
1）NYSV- forward primers：10 μm of ol/L, primer sequence is 5 '-TGGATGGAGAAGAACAAGTTGAATT-3 '；
2）NYSV- reverse primers：10 μm of ol/L, primer sequence is 5 '-GCCATTATCTGCCTGAGTGTAGGT-3 '；
3）NYSV- fluorescence probes：10 μm of ol/L, fluorescence probe sequence is 5 '-CY5-
CCACTTAAACCCATCATTGATCACGCCA-BHQ-3 ', the fluorescent reporter group of the end of fluorescence probe 5 ' mark is CY5,3 ' ends
The fluorescent quenching group of mark is BHQ；
4）NMV- forward primers：10 μm of ol/L, primer sequence is 5 '-CCATCAAACCCTGCGATCA-3 '；
5）NMV- reverse primers：10 μm of ol/L, primer sequence is 5 '-CAGACCACCTTGGCGAAGTAC-3 '；
6）NMV- fluorescence probes：10 μm of ol/L, fluorescence probe sequence is 5 '-FAM-TCACTCCGCGCCAGTTCTGCA-BHQ-
3 ', the fluorescent reporter group of the end of fluorescence probe 5 ' mark is FAM, and the fluorescent quenching group of 3 ' end marks is BHQ；
7）Random primer：100μmol/L；
8）RT Buffer：5×；
9）RNase inhibiting factor：40U/μL；
10）Reverse transcriptase：200U/μL；
11）dNTPs：10mmol/L；
12）TaqMan PCR Master Mix：2×；
13）Daffod bar virus, the positive control sample of narcissus mosaic virus；
14）Negative control sample without daffod bar virus, narcissus mosaic virus；
15）RNase-free ddH2O。
2. using the daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT-PCR kit described in claim 1
Detection method, it is characterised in that：It is comprised the following steps：
1）Reverse transcription reaction：Add testing sample in PCR pipe, the μ L of RNA 3, concentration be 100 μm of μ L of the random primer of ol/L 1 and
RNase-free ddH2O 7 μ L, 70 DEG C of water-baths 10min, rapid ice bath 5min, add following reagent：5× RT Buffer
5 μ L, concentration are the μ L of 10mmol/L dNTPs 2, concentration is the μ L of 200U/ μ L reverse transcriptase 1, concentration is that 40U/ μ L RNases suppress
The factor 1 μ L, 42 DEG C of water-bath 60min, are cooled to room temperature after 70 DEG C of water-bath 10min, synthesize cDNA；
2）Quantitative fluorescent PCR reacts：Take step 1）The μ L of cDNA 3 of synthesis, are 10 μm of ol/L NMV- positive by often pipe plus concentration
The μ L of primer 1, concentration are 10 μm of μ L of ol/L NMV- reverse primers 1, concentration is 10 μm of the μ L of ol/L NMV- fluorescence probes 0.75, concentration
For 10 μm of μ L of ol/L NYSV- forward primers 1, concentration for 10 μm of μ L of ol/L NYSV- reverse primers 1, concentration are 10 μm of ol/L
The μ L of NYSV- fluorescence probes 0.75,2 × TaqMan PCR Master Mix 12.5 μ L, ddH2The μ L of O 4, make the reaction cumulative volume be
25μL；Mixed reaction solution, 95 DEG C of 30s, then 95 DEG C of 15s, 60 DEG C of 1min, carry out 40 circulations altogether, and reaction terminates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710134679.7A CN106868210A (en) | 2017-03-08 | 2017-03-08 | Daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT PCR kits and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710134679.7A CN106868210A (en) | 2017-03-08 | 2017-03-08 | Daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT PCR kits and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106868210A true CN106868210A (en) | 2017-06-20 |
Family
ID=59171157
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710134679.7A Pending CN106868210A (en) | 2017-03-08 | 2017-03-08 | Daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT PCR kits and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106868210A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828918A (en) * | 2017-12-15 | 2018-03-23 | 福建出入境检验检疫局检验检疫技术中心 | Buddhist nun moistens dual the RT PCR detection kits and its detection method of cryptovirus and the common cryptovirus of narcissus |
| CN113215323A (en) * | 2021-06-07 | 2021-08-06 | 福建省农业科学院果树研究所 | Method for detecting BlShV by real-time fluorescent quantitative RT-PCR and kit used by method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690896A (en) * | 2012-05-16 | 2012-09-26 | 福建出入境检验检疫局检验检疫技术中心 | Kit for detecting narcissus mosaic virus and narcissus yellow stripe virus and detection method of kit |
-
2017
- 2017-03-08 CN CN201710134679.7A patent/CN106868210A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690896A (en) * | 2012-05-16 | 2012-09-26 | 福建出入境检验检疫局检验检疫技术中心 | Kit for detecting narcissus mosaic virus and narcissus yellow stripe virus and detection method of kit |
Non-Patent Citations (1)
| Title |
|---|
| J.JIN等: "Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828918A (en) * | 2017-12-15 | 2018-03-23 | 福建出入境检验检疫局检验检疫技术中心 | Buddhist nun moistens dual the RT PCR detection kits and its detection method of cryptovirus and the common cryptovirus of narcissus |
| CN113215323A (en) * | 2021-06-07 | 2021-08-06 | 福建省农业科学院果树研究所 | Method for detecting BlShV by real-time fluorescent quantitative RT-PCR and kit used by method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jiao et al. | Field detection of multiple RNA viruses/viroids in apple using a CRISPR/Cas12a‐based visual assay | |
| CN111004870B (en) | Novel coronavirus N gene nucleic acid detection kit | |
| Dai et al. | A multiplex reverse transcription PCR assay for simultaneous detection of five tobacco viruses in tobacco plants | |
| WO2018059195A1 (en) | Hrm detection primer, kit, and method for quickly identifying classical strain and variant strain of porcine epidemic diarrhea virus | |
| CN105506182B (en) | A kind of pig virus diarrhoea virus 5 weight RT-PCR detection kit | |
| Nie et al. | Visual detection of human enterovirus 71 subgenotype C4 and Coxsackievirus A16 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye | |
| Harper et al. | Detection of Tomato black ring virus by real-time one-step RT-PCR | |
| Zhang et al. | Oligonucleotide microarray with a minimal number of probes for the detection and identification of thirteen genera of plant viruses | |
| Kumar et al. | Establishment of a one-step reverse transcription recombinase polymerase amplification assay for the detection of potato virus S | |
| CN106967845A (en) | A kind of kit detected for pig Delta coronavirus and detection method | |
| CN111534637B (en) | Universal primer, probe and kit for enterovirus nucleic acid detection | |
| Abrahamian et al. | Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR | |
| CN110643745A (en) | Composition and kit for detecting feline calicivirus and feline parvovirus and application thereof | |
| CN104561382A (en) | Specific primers and probe for fluorescence RT-PCR detection for bluetongue virus-16 | |
| CN105441586A (en) | A-type H5N6 subtype avian influenza virus dual-channel real-time fluorescence PCR (polymerase chain reaction) detection kit and detection method | |
| CN106868210A (en) | Daffod bar virus and narcissus mosaic virus multiple fluorescence quantitative RT PCR kits and detection method | |
| Tiberini et al. | Development of a reverse transcription-loop-mediated isothermal amplification (LAMP) assay for the rapid detection of onion yellow dwarf virus | |
| CN102732642B (en) | Narcissus latent virus detection kit and detection method thereof | |
| CN104120192B (en) | Narcissus degenerates viral RT-Nested PCR detection kit and detection method thereof | |
| Wei et al. | Development of efficient, sensitive, and specific detection method for Encephalomyocarditis virus based on CRISPR/Cas13a | |
| CN109161611B (en) | Primer probe set and kit for jointly detecting banna virus, Canadenuovirus and Liaoning virus based on triple fluorescence PCR method | |
| CN102154519A (en) | General TaqMan real-time fluorescent quantitative polymerase chain reaction (PCR) detection kit for dengue virus | |
| CN112899397A (en) | Primer and probe for detecting dengue virus | |
| CN106939357A (en) | H4 hypotypes, H6 hypotypes and the triple RT PCR primer combinations of H9 hypotypes AIV, kit and its application | |
| CN102108421A (en) | Fluorescence quantitative polymerase chain reaction (PCR) kit for quickly detecting coxsackie virus type A16 |
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: 20170620 |
|
| RJ01 | Rejection of invention patent application after publication |