CN113186356B - Specific primer, probe and rapid detection kit for detecting pelteobagrus fulvidraco calico virus-1 - Google Patents
Specific primer, probe and rapid detection kit for detecting pelteobagrus fulvidraco calico virus-1 Download PDFInfo
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Abstract
The invention discloses a specific primer, a probe and a rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1, wherein the fluorescent quantitative detection kit for the pelteobagrus fulvidraco calicivirus-1 comprises a specific primer group A and a Taqman fluorescent probe B; the invention adopts a fluorescence quantification technology to detect the pelteobagrus fulvidraco calicivirus-1 by detecting the capsid protein gene of the pelteobagrus fulvidraco calicivirus-1, which is the primer group and the kit for detecting the pelteobagrus fulvidraco calicivirus-1 which are firstly developed at home and abroad at present. The development of the kit lays a foundation for monitoring and preventing the pelteobagrus fulvidraco calicivirus-1 disease.
Description
Technical Field
The invention belongs to the field of rapid detection of target RNA fragments, and particularly relates to a specific primer, a probe and a rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1.
Background
The Yellow catfish Calicivirus-1 (YCCV-1) is a potential pathogenic pathogen of Yellow catfish. The compound has certain pathogenicity on the pelteobagrus fulvidraco, often causes body surface bleeding and enteritis of the pelteobagrus fulvidraco, and has bleeding spots in the livers of a small number of diseased fishes. The disease caused by the compound has great harm to the yellow catfish breeding industry. YCCV-1 is a single-stranded RNA virus, belongs to the Caliciviridae family, has an undetermined classification status, and is found through genome comparison that the virus with the highest homology with the virus genome is Japanese sea eel Calicivirus (Conger japonica Calicivirus), and the homology is 38 percent, which indicates that the pelteobagrus fulvidraco Calicivirus-1 is a brand new virus and is different from any other virus in the known Caliciviridae family. The virus is separated from the yellow catfish in 2018 month 6 by the inventor, the genome of the virus is firstly obtained by the inventor, the genome comprises two coding frames, and the total length is 7149 bp. The disease incidence of the yellow catfish affects the economic income of the majority of fish people, and in recent years, the disease of the yellow catfish causes huge economic loss to the majority of farmers. The pseudobagrus fulvidraco calicivirus-1 is not reported at home and abroad, and the pseudobagrus fulvidraco does not have a detection kit and a detection method report so far, which seriously hinders the early warning and prevention work of diseases caused by the pseudobagrus fulvidraco, so that the development of the virus detection kit and the research of a detection technology are urgent.
Disclosure of Invention
In view of the above, the invention provides a specific primer, a probe and a rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1, aiming at the problems, the primer, the probe and the kit have strong specificity and high sensitivity, and can realize rapid, effective and accurate detection of the pelteobagrus fulvidraco calicivirus-1; the kit is a fluorescence quantitative detection kit, the result is judged under the closed condition, the amplification product does not pollute the detection environment, and the kit can be used for monitoring and early warning and preventing the pelteobagrus fulvidraco goblet virus-1 disease. The invention adopts a fluorescence quantitative technology to detect the pelteobagrus fulvidraco calicivirus-1 by detecting the capsid protein gene of the pelteobagrus fulvidraco calicivirus-1, which is a kit for detecting the pelteobagrus fulvidraco calicivirus-1, which is firstly developed at home and abroad at present; the development of the kit lays a foundation for monitoring and preventing the pelteobagrus fulvidraco calicivirus-1 disease.
In order to solve the technical problems, the invention also discloses a primer probe mixture for detecting the pelteobagrus fulvidraco calicivirus-1, which consists of a primer group A and a Taqman fluorescent probe B, wherein the 5 'end of the probe B is marked with a fluorescent reporter group, and the 3' end of the probe B is marked with a fluorescent quenching group.
Further, the primer set A comprises a primer YCCV-1-q183F and a primer YCCV-1-q 183R;
the nucleotide sequence of the primer YCCV-1-q183F is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the primer YCCV-1-q183R is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the probe B is shown as SEQ ID NO: 3, respectively.
Further, the fluorescence reporter group is selected from one or more of 6-carboxyfluorescein, hexachloro-6-methyl fluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethylrhodamine, sulforhodamine, 6-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxy fluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5 and cyanine 5.5; the fluorescence quenching group is selected from one or more of 6-carboxytetramethyl rhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, a black hole quenching agent 1, a black hole quenching agent 2 or a black hole quenching agent 3.
The invention also discloses a fluorescence quantitative detection kit for the pelteobagrus fulvidraco calico virus-1, which comprises a specific primer group A and a Taqman fluorescent probe B.
Furthermore, HEX is marked at the 5 'end of the nucleotide sequence of the Taqman fluorescent probe B, and BHQ1 is marked at the 3' end.
Further, the kit also comprises a virus nucleic acid extracting solution, a one-step RT-qPCR reaction solution, a positive quality control product and a negative quality control product which are independently packaged, wherein the virus nucleic acid extracting solution is a buffer solution containing 4mol/L of guanidinium isothiocyanate, 0.5% of sodium dodecyl sarcosinate, 0.1mmol/L of beta mercaptoethanol, 25mmol/L of sodium citrate, 20 mu g of glycogen and pH8.0, the one-step RT-qPCR reaction solution is a probe method fluorescent quantitative one-step RT-qPCR reaction solution, the negative quality control product is sterilized physiological saline, and the positive quality control product is a carrier containing the capsulavirus-1 capsid protein gene of the pelteobagrus fulvidraco.
The invention also discloses an application of the fluorescence quantitative detection kit for detecting the pelteobagrus fulvidraco calicivirus-1 in detecting the pelteobagrus fulvidraco calicivirus-1, which comprises the following steps:
extracting virus RNA from a sample to be detected by using a virus nucleic acid extracting solution, respectively adding the virus RNA into a primer probe mixed solution, then adding a one-step method RT-qPCR reaction solution, uniformly mixing, and then carrying out fluorescent quantitative PCR under the conditions of 42 ℃ for 20 minutes and 95 ℃ for 5-10 minutes; then reacting at 95 ℃ for 5-15 seconds and at 60 ℃ for 20-45 seconds for 38-42 cycles; setting HEX during the collection of the fluorescence signal, and setting the collection of the fluorescence signal at 60 ℃;
and (5) judging a result: if the S-type amplification curve does not appear in the detection channel, the pelteobagrus fulvidraco goblet virus is judged to be negative-1; if the detection channel has an S-type amplification curve and the Ct value is less than or equal to 35, the pelteobagrus fulvidraco calicivirus-1 is judged to be positive.
Compared with the prior art, the invention can obtain the following technical effects:
1) the primer group provided by the invention is used for detecting the pelteobagrus fulvidraco calico virus-1, and the existence of the target gene can be judged according to whether amplification is carried out or not because of high specificity;
2) the rapid diagnosis kit of the invention utilizes the fluorescence quantitative technology to rapidly detect the pelteobagrus fulvidraco calicivirus-1, the detection sensitivity is high, and the amplification template only needs 25.8 virus copies;
3) the rapid diagnostic kit disclosed by the invention is rapid and efficient in amplification, can complete amplification in less than 1 hour, and is high in efficiency;
4) the rapid diagnosis kit is simple to operate, the virus nucleic acid extracting solution is adopted to extract the virus nucleic acid, the requirement on the technical quality of detection personnel is low, a rapid screening system with low cost can be established, and the on-site high-flux rapid detection is realized;
5) the rapid diagnosis kit not only enables the qualitative detection of the pelteobagrus fulvidraco calicivirus-1 to be simpler, more convenient and faster, has high specificity and high sensitivity, but also is the first kit for detecting the pelteobagrus fulvidraco calicivirus-1, fills the gap that the pelteobagrus fulvidraco calicivirus-1 has no detection method, and has very high scientific research and economic values.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a specific detection diagram of the fluorescence quantitative detection kit for Pelteobagrus fulvidraco calicivirus-1 of the invention; wherein, 1: aeromonas hydrophila; 2: crucian herpes virus; 3: carp edema virus; 4: yellow catfish arterivirus 5: grass carp hemorrhagic disease virus; 6: koi herpesvirus; 7: pelteobagrus fulvidraco calicivirus-1; NTC: normal pelteobagrus fulvidraco RNA negative control;
FIG. 2 is a sensitivity detection diagram of the fluorescence quantitative detection kit for Pelteobagrus fulvidraco calicivirus-1 of the invention; wherein 1 to 8 each represent 2.58X 107copies、2.58×106copies、2.58×105copies、2.58×104copies、2.58×103copies、2.58×102copies、2.58×101copies、2.58×100copies。
Detailed Description
The following detailed description of the embodiments of the present invention will be provided with reference to the accompanying drawings and examples, so that how to implement the embodiments of the present invention by using technical means to solve the technical problems and achieve the technical effects can be fully understood and implemented. The reagents and materials used in the following are well known to those skilled in the art unless otherwise specified.
Example 1
The primer probe mixture for detecting the pelteobagrus fulvidraco calico virus-1 consists of a primer group A and a Taqman fluorescent probe B, wherein the 5 'end of the probe B is marked with a fluorescent reporter group, and the 3' end of the probe B is marked with a fluorescent quenching group.
The primer group A comprises a primer YCCV-1-q183F and a primer YCCV-1-q 183R;
the primer YCCV-1-q183F has a sequence shown in SEQ ID NO: 1 is shown in the specification;
the primer YCCV-1-q183R has a sequence shown in SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the probe B is shown as SEQ ID NO: 3, respectively.
Wherein, the fluorescence reporter group is selected from one or more of 6-carboxyl fluorescein, hexachloro-6-methyl fluorescein, VIC fluorescent dye, tetrachloro-6-carboxyl fluorescein, carboxyl-X-rhodamine, 6-carboxyl tetramethyl rhodamine, sulforhodamine, 6-carboxyl-4 ', 5' -dichloro-2 ', 7' -dimethoxy fluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5 and cyanine 5.5; the fluorescence quenching group is selected from one or more of 6-carboxytetramethyl rhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, a black hole quenching agent 1, a black hole quenching agent 2 or a black hole quenching agent 3.
More preferably, the fluorescent reporter and the fluorescent quencher are selected as shown in Table 1 below.
TABLE 1
| Fluorescence quenching group | Fluorescent reporter groups |
| DABCYL | At least one of 6-FAM, TET, JOE, HEX and Cy3 |
| TAMRA | At least one of 6-FAM, TET, JOE and HEX |
| BHQ1 | At least one of 6-FAM, TET, JOE, HEX and Cy3 |
| BHQ2 | At least one of TAMRA, Cy3, ROX and Texas Red |
| BHQ3 | Cy5 or Cy5.5 |
Most preferably, the fluorescent reporter is HEX; the fluorescence quenching group is BHQ 1.
Example 2
The fluorescence quantitative detection kit for the pelteobagrus fulvidraco calicivirus-1 comprises a virus nucleic acid extracting solution, a one-step RT-qPCR reaction solution, a positive quality control product, a negative quality control product and a primer probe mixed solution which are independently packaged.
The primer probe mixed solution consists of a primer group A and a Taqman fluorescent probe B, wherein the final concentrations of the upstream primer and the downstream primer of the primer group A are both 0.15 mu M, and the final concentration of the probe is 0.1 mu M.
The 5 'end of the nucleotide sequence of the Taqman fluorescent probe B is marked with HEX, and the 3' end of the nucleotide sequence of the Taqman fluorescent probe B is marked with BHQ 1;
the negative quality control product is sterilized normal saline without RNase; the positive quality control product is pseudovirus which is obtained by taking a purified pelteobagrus fulvidraco calicivirus-1 genome as a template, performing RT-PCR amplification by using a primer group A, connecting an amplification product with a vector, and packaging after the correctness is confirmed by gene sequencing;
the virus nucleic acid extracting solution is a buffer solution containing 4mol/L guanidine isothiocyanate, 0.5 percent sodium dodecyl sarcosine, 0.1mmol/L beta mercaptoethanol, 25mmol/L sodium citrate, 20 mu g glycogen and pH8.0;
the one-step RT-qPCR reaction solution is a probe method fluorescent quantitative RT-PCR reaction solution and contains AMV reverse transcriptase, PCR enzyme, dNTP and Mg2+。
Example 3
The application of the fluorescence quantitative detection kit for the pelteobagrus fulvidraco calico-1 virus is that 0.05g of a sample to be detected is added with 1mL of virus nucleic acid extract for full grinding, the sample is centrifuged at 12000r/min for 10min, the supernatant is taken for 800uL, 400uL of ethanol is added, after the room temperature is 5min, the sample is centrifuged at 12000r/min for 5min, after the precipitate is washed once by 75% of ethanol, after the precipitate is dried at the room temperature for 5min, 50uL of DEPC (diethylpyrocarbonate) is added for dissolving RNA, 5uL is taken and added into a reaction tube, then 2uL of primer probe mixed solution is added, 10uL of one-step RT-qPCR reaction solution is added, 3uL of DEPC water is added, and after uniform mixing, the fluorescence quantitative PCR is carried out, wherein the reaction conditions are that the temperature is 42 ℃ for 20 min and the reaction is carried out at the temperature of 95 ℃ for 5-10 min; then reacting for 5-15 seconds at 95 ℃ and 20-45 seconds at 60 ℃ for 45 cycles; setting HEX during the collection of the fluorescence signal, and setting the collection of the fluorescence signal at 60 ℃;
and (5) judging a result:
if the S-type amplification curve does not appear in the detection channel, the pelteobagrus fulvidraco goblet virus is judged to be negative-1; if the detection channel has an S-type amplification curve and the Ct value is less than or equal to 35, the pelteobagrus fulvidraco calicivirus-1 is judged to be positive.
The primer group A and the probe B are designed aiming at the capsid protein gene of the pelteobagrus fulvidraco calicivirus-1, the result detection is carried out by adopting the fluorescent group capture of the specific probe, and the kit has the characteristics of high sensitivity and convenient use and is also the universal standard of the international detection kit.
Example 4 application example of fluorescence quantitative detection kit for Pelteobagrus fulvidraco Californis virus-1
(1) RNA extraction:
taking 10-20mg of pelteobagrus fulvidraco liver or spleen tissue into a 1.5mL centrifuge tube, adding 1mL of virus nucleic acid extracting solution for full grinding, centrifuging at 12000r/min for 10min, taking 800uL of supernatant into a new 1.5mL centrifuge tube, adding 400uL of ethanol, centrifuging at 12000r/min for 5min after room temperature is 5min, washing precipitates with 75% ethanol once, drying at room temperature for 5min, adding 50uL of DEPC water to dissolve RNA for later use.
(2)RT-qPCR：
10 mu L of RT-PCR reaction liquid, 2 mu L of primer-probe mixed liquid and 3uL of DEPC water are respectively added into 8 reaction tubes to prepare a reaction system, and then 5.0 mu L of template to be detected is respectively added.
Fluorescent quantitative PCR reaction conditions: 20 minutes at 42 ℃; 10 minutes at 95 ℃; 95 ℃, 15 seconds, 60 ℃, 45 seconds, 40 cycles.
A LightCycler 96System fluorescent quantitative PCR instrument is adopted, HEX fluorescein is set during fluorescent signal collection, and the fluorescent signal collection is set at 60 ℃.
Negative quality control products (sterilized normal saline) and positive quality control products (pseudovirus of pelteobagrus fulvidraco calicivirus-1) are set in each batch of reaction.
(3) And (5) judging a result: the detection channel has no S-type amplification curve and is judged to be negative to the pelteobagrus fulvidraco calicivirus-1; if the S-type amplification curve appears in the detection channel and the Ct value is less than or equal to 35, the pelteobagrus fulvidraco calicivirus-1 is judged to be positive.
And (5) result verification: and (3) carrying out gene sequencing on the amplification product with positive detection, wherein the sequencing result is consistent with the gene sequence of the capsid protein of the pelteobagrus fulvidraco calicivirus-1.
The specificity and sensitivity test results of the rapid detection of the pelteobagrus fulvidraco calicivirus-1 by adopting the fluorescence quantitative detection kit and the method for the pelteobagrus fulvidraco calicivirus-1 are respectively shown in the attached drawings 1 and 2.
FIG. 1 is a specific detection diagram of Pelteobagrus fulvidraco calicivirus-1, as shown in the figure, after Real-time RT-PCR quantitative detection, only Pelteobagrus fulvidraco calicivirus-1 has a specific amplification curve, and other viruses and bacteria do not have amplification curves, which indicates that the method has good specificity.
FIG. 2 is a diagram of the sensitivity detection of Pelteobagrus fulvidraco calico-1, as shown in the figure, the amplification diagram of serial dilution of Pelteobagrus fulvidraco calico-1 RNA after Real-time RT-PCR quantitative detection, when 25.8 copies of virus RNA are added into the reaction system, the amplification and color development result is positive.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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<120> specific primer, probe and rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1
<130> 2021
<141> 2021-06-02
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gtattcagtt tggtgatggt atcatcaatc tgtttacttt cgatgggctt ctaacagcag 2700
cagtagtcca acaagaagat gaggcccctg tggacataat aattcgtccc aggagacaga 2760
taccaaattc aatctggcaa cttgttatca atgtgacagc atcaacaatt ggtgtcgtct 2820
cggcgtgtat ggcgagcttt cgccgttttg acgagtctcc taccgatgag gaggaggagt 2880
tcgctgagcg gcggaggccg gtcgcacgca ttagacagag tgacatgact ctgaagaatg 2940
cccgcaatcc aaatttgtgg aatgcaacaa gttggggtga tgaaatggag gaaagtgacc 3000
caatcatgct ccacactgtg gagtccttgc ggaccagtat tttgccggta acggcggaaa 3060
atggggtccg caagggctgg gcgttctctc atgagtcggg gatagttgtc aatagtcatg 3120
tcatctcatc tggctgccgg gttggccctc gaacagttgg gccctgtaaa acaacagttg 3180
tgaaaaactc tgatttttgt tttctccatc cccaggccga tccaggatac aagaaatgta 3240
aggcaccttt caccattccc tatgttggtg aaaagatcac tcaagttggt ttcgatggta 3300
gtctcaaggt tgcccgagtc gtgaaaaaac agagcctagt catcagtggg acaccgcggg 3360
tggtttggct agcagacatc attgagaaac cacagggtag tgagaaatct atcccgggtg 3420
attgtggcct tccttgggtc agacaaattg gcaccaatta tgaacttgtt gggcttcaca 3480
ctggcattta tgggacaatg atcatggtga cccctgttcc attgatgcca tcagagcatg 3540
gtgcaacttc taagaataag acctacctct atcgcactca gtattttggg atagaagaaa 3600
aagttattga catgaaacca gccccaaaat gcggggtttt caatggagtg gaagaggatt 3660
gtgagatgct gatacgtcgg tgtctacaac ccttttatga aactccgaac cccgcttttg 3720
catctcaaga agccttccaa gcgacgtgtg attatatcgc tatgaaagtc aataaaccca 3780
aacggtggtc gatcatcgcc gccgtaaaat ctctagacca aacaacatca gctggcccag 3840
cgtatggtgt gacaaaagac aaagtcttca acccggatgg gtctgtcaac ccacgttatg 3900
caggaatttg gcgaactggg ttgaggggtc caccagatgc cgggtgtaaa gttcatatca 3960
aagatgagat gagaccacgt cggaagtatg agggtggtaa ctcccgaccc atcttttgtt 4020
tcaacgtcca cgcggttgcg cgtgtgaaag cccttatggg tgatttgttg atgcagttga 4080
tggagacggt gggtgaccac ccttatgcgg tgggtgttgc tcctggaact gggacgtggc 4140
acaacattgc caaggagctt gaaaaatggc caattttggt tgatgcagat tttcaacgtt 4200
gggattcatg catcggccca aatttgatga aacaagccta tatggccctg acgcaccctc 4260
ttgaacctga ggatcgcgac agggcacgta ttgactatgc aacaattgtc cacccaatca 4320
ctcaatttgg cccaacaaga tgtggtctcc ctagtggcat ttgtggaaca tcccacttga 4380
actgtacagt ccacttgcta ctggtgaatg acaccttgat tcgtaacaac aagccttgcc 4440
ttggggaacc aggctgcccc atagcattct tttgctatgg agatgacttc gtggctgggg 4500
tcaagaacag tgaaaccctt cagatgctca ttgaagggtg gaaacgttta gggttccatg 4560
ccactaacgc aatgaagact ggacctccag aagcaacacg tctggaagat atccgtttct 4620
tgaaacgggc attcaaaaaa gacgagagtg ggttctaccg agcccctctt gaagagtcta 4680
gcctttggcg cgccctcagt ttctcccgtc aacacgtctc atacgattac acgggggaaa 4740
tggaggttgc gccattgtcg ggggttcgtc acacttcaat tttgcaagca gtactcagtg 4800
aagcttggca acatggtgaa acagggtaca agaagtgtgc tgatcgacta attaaatggt 4860
ccaaaaaggt ccatgaacgt ttgccgatcg cgattcctcc ttattcaagg ttccaaccaa 4920
gtgaaattct ctgcggatct gatcaattat ctttttctga caattctcat ccaagctttt 4980
tctgtgttcc acattcaaac gaaatggacg ctccaatggt tgttgatgtt cccgccactg 5040
gcgaaaactc agggttggtt gttgggcccg tgggggcggt agtccctgtg gctgcaccaa 5100
cagttggcgc tgagacaatg gctggtatga ctggtggaac tggcggttgt attgacccag 5160
ccatacgtgg caaatttgta gtcgtcccag gcggaattgt ctcggttagc accgaaactg 5220
ttccaggaac gaagatcttc caaattaaag ttgagccagg cataaacctc tatacacgtt 5280
atttgaaggc aatgtacaat gcttgggctg gcggtttcaa gattatgacc gtaattggtg 5340
caaacaactt cattggcggg aaattcctca tcgttttcac gccacctcaa atcaaccctg 5400
aaaattattc acttgaagct cttaccggtt tcccttctgt catcttggat attcgtgaaa 5460
tggacaatgt catattggat tgcaccgaca taaagcgtgt aatgtggcat cccactaacg 5520
acaattcgac tgatggcttt gctgggtggt tttctgtttt caccttagtt aacctcaaca 5580
ccgctggaac tggaccaatc actttggatc tacgtttctt ctcaacacca agcgacaact 5640
ttaattttga aatgttgatc ccccctgtag aagttggggc aggagcgcaa ttggaggtct 5700
gggcagcgct tgagggaaac aatctttcac tccctgggac ccaagcgagg gatggctccc 5760
ccactgaaac ccttatcgca ctttcaaaga acgcgagtgt tgttacgggg aatatgtggg 5820
ctggggtcaa ccctttggtt ggtacaccag ttggtgccat tccgtccgga ggtgtttcgg 5880
gtggctaccc agttaagctc ttggccacag gtgtggcaaa tgagtactgg gtggcatgtc 5940
tcgacgctga tggtaacatc tgggaggatg acacacaggg ttctgccaat ctgcctgagt 6000
tttggccctc atcagccccg ttaattggag ctggtgtggc agtgaaacgg tggtcagcaa 6060
cgggagacgc cgcagtgact ttaggcacct ggaagcccca aacatctgtg gggataatca 6120
aaccaagagt ctcaatgacc agtggcgtgt caagttcaat cacttacatg ctatactgga 6180
ttccagacac aattatgaac aatcaatcag gaactttcaa gccagtgtat acccccccca 6240
atggggagag cctcgtggtg tatggtggtc cgggaggact taaccttaca tcattgagtt 6300
tgtcaacaat ggaatcaatg tactaccgca agaataagcc cccggttgga ccgggagaag 6360
ccctcctctt tgcagtcaaa gacgccacaa ctttaactgt gatgcaggtg aaacttcact 6420
ccaatggcgt tctaaccact ggtggtgttt caacggcagt tcaatggatg gctccaatag 6480
tcttcaccta tgttggtgtc gtctctgaga actatctgtt gaacccacca gctggcggga 6540
cgtcatcaag tcttcttgac ctaacagaac gcatcacaga atggcagggg cagctatcgg 6600
agcacaattg ggatcctcaa tcatcggagg cgctttccaa cttggctctg ctgctctcgg 6660
aggcaagatt gcagccgaga acaacctcca gctccagtca cgtgaccagt cgtatggctt 6720
gcggctactc tccgaccaca aatcggagct tagccaggca ggcttgccga cgtacttggc 6780
atactctgga ggaggaggag gaggaggatt agctagccaa aaatacatga tcaaatcagg 6840
ctatggcatg agtgtctcaa cacacgcaaa tgctccaaat tcattcactt cattaacaaa 6900
tttctcaggc acaacattaa agaaatcaaa tgttaagaaa acaactgggc aactaaaccc 6960
atccactaca aattttgaaa acccaaatta tggtatccaa atgagcaatg ttaattattc 7020
aagacgcgac gagtttccga aagtttactt tcacgggatc gccgaatcaa ctgtttaaat 7080
ttttacttgt gttattatct tttaaatttt gtaatcatta aatgcatgct taatagtaaa 7140
ttaatccgt 7149
Claims (5)
1. The primer probe mixture for detecting the pelteobagrus fulvidraco calicivirus-1 is characterized by consisting of a primer group A and a Taqman fluorescent probe B, wherein a fluorescence reporter group is marked at the 5 'end of the Taqman fluorescent probe B, and a fluorescence quenching group is marked at the 3' end of the Taqman fluorescent probe B;
the primer group A comprises a primer YCCV-1-q183F and a primer YCCV-1-q 183R;
the nucleotide sequence of the primer YCCV-1-q183F is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the primer YCCV-1-q183R is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the Taqman fluorescent probe B is shown as SEQ ID NO: 3, respectively.
2. The fluorescence quantitative detection probe B of Pelteobagrus fulvidraco calico virus-1 according to claim 1, characterized in that the fluorescence reporter group is selected from one or more of 6-carboxyfluorescein, hexachloro-6-methyl fluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethyl rhodamine, sulforhodamine, 6-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxy fluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5 and cyanine 5.5; the fluorescence quenching group is selected from one or more of 6-carboxytetramethyl rhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, a black hole quenching agent 1, a black hole quenching agent 2 or a black hole quenching agent 3.
3. A fluorescence quantitative detection kit for Pelteobagrus fulvidraco calico virus-1 is characterized by comprising a specific primer group A and a Taqman fluorescent probe B; the primer group A comprises a primer YCCV-1-q183F and a primer YCCV-1-q 183R;
the nucleotide sequence of the primer YCCV-1-q183F is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the primer YCCV-1-q183R is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the Taqman fluorescent probe B is shown as SEQ ID NO: 3, respectively.
4. The fluorescence quantitative detection kit for detecting pelteobagrus fulvidraco calico virus-1 according to claim 3, characterized in that HEX is marked at the 5 'end of the nucleotide sequence of the Taqman fluorescent probe B, and BHQ1 is marked at the 3' end.
5. The fluorescence quantitative detection kit for detecting the pelteobagrus fulvidraco calicivirus-1 according to claim 3, characterized in that the kit further comprises a virus nucleic acid extracting solution, a one-step RT-qPCR reaction solution, a positive quality control product and a negative quality control product which are independently packaged, wherein the virus nucleic acid extracting solution is a buffer solution containing 4mol/L guanidinium isothiocyanate, 0.5% sodium dodecyl sarcosinate, 0.1mmol/L beta mercaptoethanol, 25mmol/L sodium citrate, 20 μ g glycogen and pH8.0, the PCR reaction solution is a probe method fluorescence quantitative PCR reaction solution, the negative quality control product is sterilized physiological saline, and the positive quality control product is a carrier containing the pelteobagrus fulvidraco calicivirus-1 capsid protein gene.
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Application publication date: 20210730 Assignee: WUHAN HUAYANG ANIMAL PHARMACEUTICAL Co.,Ltd. Assignor: ZHEJIANG INSTITUTE OF FRESH WATER FISHERIES Contract record no.: X2022330000666 Denomination of invention: Specific Primers, Probes and Rapid Detection Kit for Detection of Pelteobagrus fulvidraco Calicivirus-1 Granted publication date: 20220520 License type: Common License Record date: 20221121 |
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