CN110846441B - Specific primer, probe and rapid detection kit for detecting macrobrachium flaviviridae-1 - Google Patents
Specific primer, probe and rapid detection kit for detecting macrobrachium flaviviridae-1 Download PDFInfo
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Abstract
The primer probe mixed liquid for detecting the shrimp flavivirus-1 consists of a primer group A and a Taqman fluorescent probe B, wherein a fluorescent reporter group is marked at the 5 'end of the Taqman fluorescent probe B, and a fluorescent quenching group is marked at the 3' end of the Taqman fluorescent probe B; the primer group A comprises a primer MrFV-q150F and a primer MrFV-q150R; the nucleotide sequence of the primer MrFV-q150F is shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the primer MrFV-q150R is shown in 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. A macrobrachium flavivirus-1 fluorescence quantitative detection kit comprises the mixed solution of the claim, and also comprises RT-qPCR reaction liquid, a positive quality control product and a negative quality control product, wherein the RT-qPCR reaction liquid is a probe method fluorescence quantitative one-step method RT-qPCR reaction liquid, the negative quality control product is sterilized normal saline, and the positive quality control product is a carrier containing a macrobrachium flavivirus-1 polymerase gene. The kit is used for detecting the shrimp flavivirus-1, and has high speed and strong specificity.
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 Macrobrachium nipponense flavivirus-1.
Background
Macrobrachium rosenbergii Flavivirus-1 (Macrobrachium rosenbergii Flavivivirus-1, mrFV-1) is a viral pathogen causing Macrobrachium rosenbergii in the breeding period, particularly Macrobrachium rosenbergii, often causes Macrobrachium rosenbergii 'viral iron shrimp character', causes premature gonads of the Macrobrachium rosenbergii in the breeding period, causes huge loss of the yield of the cultured Macrobrachium rosenbergii, and is also called as vacuole virus because the virus easily causes the cytoplasmosis of specific cell cytoplasm of the Macrobrachium rosenbergii. MrFV-1 is a single-stranded RNA virus that infects Macrobrachium rosenbergii larvae and adult shrimp. The 'viral iron shrimp symptom' of the macrobrachium rosenbergii occurs in a macrobrachium rosenbergii farm in Jiaxing Xiuzhou area in 2011 at the earliest time, and the macrobrachium rosenbergii has blue and long crayfish as disease symptoms, female crayfish hold eggs too early, and the macrobrachium rosenbergii grows and falls down slowly, so the macrobrachium rosenbergii is called as 'iron shrimp', 'cadre shrimp', 'long paw disease', and the like. According to epidemiological investigation, the virus is found to exist in slow-growing macrobrachium rosenbergii, and infection tests show that the macrobrachium rosenbergii flavivirus-1 can cause the slow growth of the macrobrachium rosenbergii. In addition, the virus can also infect the macrobrachium nipponensis and can also cause vacuolization lesion of specific cells of the macrobrachium nipponensis. The viral macrobrachium nipponensis is continuously generated in each macrobrachium rosenbergii breeding farm in China for years, and can be vertically spread through the virus-carrying macrobrachium nipponensis, so that the economic loss caused by the macrobrachium rosenbergii breeding and breeding industry in China cannot be estimated. Based on the discovered genomic sequence of the virus, the similarity to the reported flavivirus sequence is less than 40%, indicating a new virus of the Flaviviridae family. The virus is not reported at home and abroad, the genome sequence of the virus is also disclosed, and a detection kit and a detection method are not available so far, so that the development and development of the kit have innovation and uniqueness. The development and application of the virus detection kit can also play a role in early warning and prevention and control of macrobrachium rosenbergii diseases caused by the virus.
Disclosure of Invention
In view of the above, the invention firstly discloses a partial genome sequence of the shrimp flavivirus-1, and provides a specific primer, a probe and a rapid detection kit for detecting the shrimp flavivirus-1, the primer, the probe and the kit of the invention have strong specificity and high sensitivity, and can realize rapid, effective and accurate detection of the shrimp flavivirus-1; the kit is a one-step fluorescence quantitative detection kit, can judge the result under the closed condition, does not pollute the detection environment by the amplification product, can be used for monitoring and early-stage prevention of the flavivirus-1 disease of the macrobrachium nipponense, and can also be used for screening the non-flavivirus-1 macrobrachium rosenbergii breeding shrimps. The invention adopts a fluorescence quantitative technology to indirectly detect the carrying condition of the shrimp flavivirus-1 by detecting the membrane protein gene of the shrimp flavivirus-1, which is the kit for detecting the shrimp flavivirus-1 firstly developed at home and abroad at present; the development of the kit lays a foundation for monitoring and preventing the macrobrachium flaviviridae-1 disease.
In order to solve the technical problem, the invention discloses a primer probe mixed solution for detecting the shrimp flavivirus-1, which is characterized in that the mixed solution consists of a primer group A and a Taqman fluorescent probe B, the 5 'end of the probe B is marked with a fluorescent reporter group, the 3' end of the probe B is marked with a fluorescent quenching group, and the primer group A and the probe B are designed according to the sequence of the shrimp flavivirus-1 discovered by the inventor;
the primer group A comprises a primer MrFV-q150F and a primer MrFV-q150R;
the nucleotide sequence of the primer MrFV-q150F is as follows:
SEQ ID NO：1—5’-TGGCAAACTCTTAGGATTC-3’
the nucleotide sequence of the primer MrFV-q150R is as follows:
SEQ ID NO：2—5’-ACTGCACAATAGTCTTCG-3’
the nucleotide sequence of the Taqman fluorescent probe B is as follows:
SEQ ID NO：3—5’-CTACAACGCCAACCAGCAGAG-3’
the sequence of the macrobrachium flavivirus-1 referred to by the primer group and the probe design is as follows:
SEQ ID NO：4—
5’-TGGCAAACTCTTAGGATTCTGGAACCCCTTCTTGGCCCTCGTCATGGCGGCAGG AGTGGCTTACTTCTTCTTTGGAAGGAAAGTGGCCATTGTTGTCGCTCTGCTGGTTGGCGTTGTAGCGGTTTTCGGAGCCGAAGACTATTGTGCAGT-3’
further, the fluorescent reporter group is selected from one of 6-carboxyfluorescein (6-FAM), hexachloro-6-methylfluorescein (HEX), VIC fluorescent dye (VIC), tetrachloro-6-carboxyfluorescein (TET), carboxy-X-Rhodamine (ROX), 6-carboxytetramethylrhodamine (TAMRA), sulforhodamine (Texas Red), 6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein succinimidyl ester (JOE), cyanine 3 (Cy 3), cyanine 3.5 (Cy3.5), cyanine 5 (Cy 5), cyanine 5.5 (Cy5.5); the fluorescence quenching group is selected from one of 6-carboxytetramethylrhodamine (TAMRA), 4- (4-dimethylamino phenylazo) benzoic acid (DABCYL), black hole quencher 1 (BHQ 1), black hole quencher 2 (BHQ 2) and black hole quencher 3 (BHQ 3).
Further, the fluorescence reporter group is 6-carboxyfluorescein (6-FAM), and the fluorescence quencher group is a black hole quencher 1 (BHQ 1).
The invention also discloses a macrobrachium flavivirus-1 fluorescence quantitative detection kit which is characterized by comprising the primer group A and a Taqman fluorescent probe B,
the kit also comprises RT-qPCR reaction liquid, a positive quality control product and a negative quality control product which are independently packaged, wherein the RT-qPCR reaction liquid is RT-qPCR reaction liquid by a one-step probe method, the negative quality control product is sterilized physiological saline, and the positive quality control product is a carrier containing a macrobrachium flaviviridae-1 membrane protein gene.
The invention also discloses an application of the detection kit in detecting the macrobrachium flavivirus-1, which comprises the following steps:
adopting total RNA of a macrobrachium rosenbergii sample to be detected extracted by a commercial RNA extraction kit, adding 3uL of RNA into a PCR tube containing the primer probe mixed solution and the RT-q PCR reaction solution, uniformly mixing, and then carrying out one-step RT-qPCR, wherein the reaction conditions are as follows: at 42 deg.C for 20-30 min, at 95 deg.C for 5-8 min, at 95 deg.C for 10-15 sec, and at 60 deg.C for 30-40 sec for 38-42 cycles; the fluorescence signal collection is set as FAM, and the reaction is finished at 60 ℃;
and (5) judging a result: the detection channel does not have an S-shaped amplification curve and is judged to be negative to the Macrobrachium nipponensis flavivirus-1; the detection channel has an S-shaped amplification curve, the Ct value is less than or equal to 35, and the macrobrachium is judged to be positive by the flavivirus-1.
At present, no kit and technology for detecting the macrobrachium flavivirus-1 exist at home and abroad, so the detection technology of the kit has uniqueness and uniqueness, the kit can continuously carry out reverse transcription and qPCR in the same reaction tube, the operation is simple, the pollution can be effectively prevented, the amplification product can be monitored in real time in the reaction process, the detection sensitivity is greatly improved, and the electrophoresis step after PCR reaction is omitted. The technology of the kit is not the same as that of the prior art at home and abroad.
Of course, it is not necessary for any one product to achieve all of the above-described technical effects simultaneously in order to practice the present invention.
Drawings
FIG. 1 is a diagram showing the result of specific detection by the kit for quantitatively detecting Macrobrachium nipponensis flavivirus-1 by fluorescence; wherein, 1: macrobrachium flavivirus-1, 2: macrobrachium rosenbergii iridovirus, 3: macrobrachium rosenbergii bicistronic virus, 4: macrobrachium rosenbergii nodavirus, 5: macrobrachium rosenbergii newcastle virus, 6: prawn yellow head virus, 7: prawn white spot syndrome virus.
FIG. 2 is a diagram showing the sensitivity detection of the kit for the quantitative fluorescent detection of Macrobrachium nipponensis flavivirus-1; viral RNA dilutions were-1.10 -1 ；-2.10 -2 ；-3.10 -3 ；-4.10 -4 ；-5.10 -5 ；-6.10 -6 ；-7.10 -7 。
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 mixed liquid for detecting the shrimp flavivirus-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 MrFV-q150F and a primer MrFV-q150R; the nucleotide sequence of the primer MrFV-q150F is shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the primer MrFV-q150R is 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 fluorescent reporter group is selected from one of 6-carboxyfluorescein (6-FAM), hexachloro-6-methyl fluorescein (HEX), VIC fluorescent dye (VIC), tetrachloro-6-carboxyfluorescein (TET), carboxy-X-Rhodamine (ROX), 6-carboxytetramethylrhodamine (TAMRA), sulforhodamine (Texas Red), 6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxy fluorescein succinimidyl ester (JOE), cyanine 3 (Cy 3), cyanine 3.5 (Cy3.5), cyanine 5 (Cy 5), and cyanine 5.5 (Cy5.5); the fluorescence quenching group is selected from one of 6-carboxytetramethylrhodamine (TAMRA), 4- (4-dimethylamino phenylazo) benzoic acid (DABCYL), black hole quencher 1 (BHQ 1), black hole quencher 2 (BHQ 2) and black hole quencher 3 (BHQ 3).
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 | 6-FAM, TET, JOE, HEX, cy3 |
| TAMRA | 6-FAM, TET, JOE and HEX |
| BHQ1 | 6-FAM, TET, JOE, HEX, cy3 |
| BHQ2 | TAMRA, cy3, ROX, texas Red |
| BHQ3 | Cy5 or Cy5.5 |
More preferably, the fluorescent reporter group is 6-FAM; the fluorescence quenching group is BHQ1.
Example 2
The macrobrachium flaviviridae-1 fluorescence quantitative detection kit consists of RT-qPCR reaction liquid, a positive quality control product, a negative quality control product and primer probe mixed liquid which are packaged independently.
The primer probe mixed solution consists of a primer group A and a Taqman fluorescent probe B in the embodiment 1, wherein the final concentration of the upstream primer and the downstream primer of the primer group A is 0.2 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 6-FAM, and the 3' end of the nucleotide sequence of the Taqman fluorescent probe B is marked with BHQ1;
the negative quality control product is sterilized normal saline; the positive quality control product is a pseudovirus which is obtained by taking the purified macrobrachium flavivirus-1 genome as a template, carrying out PCR amplification by using a primer group A, connecting an amplification product with a vector, confirming the correctness through gene sequencing and then packaging;
the RT-qPCR reaction solution is a one-step probe method fluorescent quantitative RT-PCR reaction solution and contains AMV reverse transcriptase, hotstar Taq polymerase, antibody modified Anti Taq DNA polymerase, dNTPs and 20mmol/L Tris-HCl solution with the Mg2+ pH of 8.3.
Example 3
The macrobrachium flavivirus-1 fluorescence quantitative detection kit is applied, a sample to be detected is subjected to total RNA extraction by using a commercial RNA extraction kit, 3uL of RNA and 3uL of primer probe mixed solution of the embodiment 1 are added into a reaction tube, 19uL of RT-qPCR reaction solution is added, one-step fluorescence quantitative RT-qPCR is carried out after uniform mixing, and the reaction conditions are as follows: 40 cycles of 20 minutes at 42 ℃,5 minutes at 95 ℃, 10 seconds at 95 ℃ and 40 seconds at 60 ℃; the fluorescence signal collection is set as FAM, and the reaction is finished at 60 ℃;
and (5) judging a result:
if an S-shaped amplification curve appears in the detection channel and the Ct value is less than or equal to 35, judging that the macrobrachium flaviviridae-1 is positive; the detection channel has no S-shaped amplification curve, and the macrobrachium flavivirus-1 is judged to be negative; and when the detection channel has an S-shaped amplification curve and the Ct value is greater than 35, the detection needs to be carried out again, and if the Ct value is still greater than 35, the result is that the macrobrachium flaviviridae-1 is negative.
The primer group A and the probe B are designed aiming at the gene of the membrane protein of the flavivirus-1 of the macrobrachium, the result detection is carried out by adopting the fluorescent group capture of the specific probe, and the primer group A and the probe B have the characteristics of high sensitivity and convenient use and are also the universal standard of a kit for international detection.
Example 4 specificity of Macrobrachium nipponensis flavivirus-1 fluorescence quantitative detection kit
(1) RNA extraction:
adopting a viral RNA extraction kit (DP 315-R) of Tiangen to extract total RNA of collected 50mg of whole larvae of the flavivirus-1 positive macrobrachium rosenbergii, and taking the extracted RNA as a template to be detected.
(2) One-step fluorescent quantitative RT-qPCR:
19 mu L of RT-qPCR reaction liquid is respectively added into 9 reaction tubes, 3 mu L of primer probe mixed liquid is prepared into a reaction system, and then nucleic acids of the following pathogens are respectively added into the reaction tubes: macrobrachium rosenbergii flavivirus-1, macrobrachium rosenbergii iridovirus, macrobrachium rosenbergii dicistronic daughter virus, macrobrachium rosenbergii nodavirus, macrobrachium rosenbergii newcastle disease virus, prawn yellow head virus, and prawn white spot syndrome virus;
fluorescent quantitative RT-PCR reaction conditions: 20 minutes at 42 ℃,5 minutes at 95 ℃, 10 seconds at 95 ℃,40 seconds at 60 ℃ and 40 cycles.
An Mx3005P fluorescence quantitative PCR instrument is adopted, the fluorescence signal collection is set as FAM fluorescein, and the reaction is finished at 60 ℃.
Negative quality control substances (sterilized normal saline) and positive quality control substances (macrobrachium flaviviridae-1 membrane protein gene plasmids) are arranged in each batch of reaction.
(3) And (5) judging a result: if the detection channel does not have an S-shaped amplification curve, the detection channel is judged to be negative to the Macrobrachium nipponensis flavivirus-1; if an S-shaped amplification curve appears in the detection channel and the Ct value is less than or equal to 35, judging that the macrobrachium flaviviridae-1 is positive; and when the detection channel has an S-shaped amplification curve and the Ct value is greater than 35, the detection needs to be carried out again, and if the Ct value is still greater than 35, the result is that the macrobrachium flaviviridae-1 is negative.
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 shrimp flavivirus-1 membrane protein.
The result of the test for rapidly detecting the specificity of the macrobrachium flaviviruses-1 by adopting the kit and the method for quantitatively detecting the macrobrachium flaviviruses-1 by fluorescence is shown as an attached figure 1.
Example 5 sensitivity of Macrobrachium nipponensis flavivirus-1 fluorescent quantitative detection kit
(1) RNA extraction:
adopting a viral RNA extraction kit (DP 315-R) of Tiangen to extract total RNA of collected 50mg of macrobrachium rosenbergii flavivirus-1 positive macrobrachium rosenbergii larva whole shrimp samples, taking the extracted RNA as a template to be detected, and performing gradient dilution to 10 -7 。
(2) One-step fluorescent quantitative RT-qPCR:
respectively adding 19 mu L of RT-qPCR reaction liquid into 9 reaction tubes, preparing a reaction system by using 3 mu L of primer probe mixed liquid, and then respectively adding 3 mu L of viral nucleic acid RNA with each dilution into each reaction tube;
fluorescent quantitative RT-PCR reaction conditions: 20 minutes at 42 ℃,5 minutes at 95 ℃, 10 seconds at 95 ℃,40 seconds at 60 ℃ and 40 cycles.
An Mx3005P fluorescence quantitative PCR instrument is adopted, the fluorescence signal collection is set as FAM fluorescein, and the reaction is finished at 60 ℃.
(3) And (5) judging a result: the detection channel has no S-shaped amplification curve and is judged to be negative to the Macrobrachium nipponensis flavivirus-1; if the detection channel has an S-shaped amplification curve and the Ct value is less than or equal to 35, determining that the macrobrachium flaviviridae-1 is positive; and when the detection channel has an S-shaped amplification curve and the Ct value is greater than 35, the detection needs to be carried out again, and if the Ct value is still greater than 35, the result is that the macrobrachium flaviviridae-1 is negative.
FIG. 2 is a graph showing the detection sensitivity of Macrobrachium nipponensis flavivirus-1, which is an amplification graph of serial dilutions of Macrobrachium nipponensis flavivirus-1 RNA after one-step RT-qPCR, when the dilution is 10 added to the reaction system -1 -10 -6 All the viral RNAs of (1) were judged to be positive in amplification result.
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 macrobrachium flavivirus-1
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Claims (5)
1. The primer probe mixed liquid for detecting the shrimp flavivirus-1 is characterized by consisting of a primer group A and a Taqman fluorescent probe B, wherein the 5 'end of the Taqman fluorescent probe B is marked with a fluorescent reporter group, and the 3' end of the Taqman fluorescent probe B is marked with a fluorescent quenching group;
the primer group A comprises a primer MrFV-q150F and a primer MrFV-q150R;
the nucleotide sequence of the primer MrFV-q150F is shown in SEQ ID NO:1 is shown in the specification;
the nucleotide sequence of the primer MrFV-q150R is shown in 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 mixed solution according to claim 1, wherein the fluorescent reporter group is one selected from the group consisting of 6-carboxyfluorescein, hexachloro-6-methylfluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethylrhodamine, sulforhodamine, 6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5, and cyanine 5.5; the fluorescence quenching group is selected from one of 6-carboxytetramethylrhodamine, 4- (4-dimethylaminobenzazoxy) benzoic acid, a black hole quenching agent 1, a black hole quenching agent 2 and a black hole quenching agent 3.
3. The mixed solution of claim 1, wherein the fluorescence reporter group is 6-carboxyfluorescein and the fluorescence quencher group is a black hole quencher 1.
4. A macrobrachium flavivirus-1 fluorescence quantitative detection kit is characterized by comprising the mixed solution of claim 1, 2 or 3, RT-qPCR reaction solution, a positive quality control product and a negative quality control product, wherein the RT-qPCR reaction solution is the RT-qPCR reaction solution of a probe method fluorescence quantitative one-step method, the negative quality control product is sterilized normal saline, and the positive quality control product is a carrier containing a macrobrachium flavivirus-1 membrane protein gene.
5. Use of the kit according to claim 4 for the preparation of a product for detecting Macrobrachium nipponensis flavivirus-1, comprising the steps of:
adopting a commercial RNA extraction kit to extract total RNA of a macrobrachium rosenbergii sample to be detected, adding 3uL of RNA into a PCR tube containing the mixed solution and the RT-q PCR reaction solution, uniformly mixing, and then carrying out one-step RT-qPCR, wherein the reaction conditions are as follows: 38-42 cycles of 20-30 minutes at 42 deg.C, 5-8 minutes at 95 deg.C, 10-15 seconds at 95 deg.C, and 30-40 seconds at 60 deg.C; the fluorescence signal collection is set as 6-FAM, and the reaction is finished at 60 ℃;
and (5) judging a result: the detection channel does not have an S-shaped amplification curve and is judged to be negative to the macrobrachium flaviviridae-1; the detection channel has an S-shaped amplification curve, and the Ct value is less than or equal to 35, and the detection channel is judged to be the positive of the macrobrachium flavivirus-1; and when the detection channel has an S-shaped amplification curve and the Ct value is greater than 35, the detection needs to be carried out again, and if the Ct value is still greater than 35, the result is that the macrobrachium flaviviridae-1 is negative.
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| CN112094854B (en) * | 2020-10-29 | 2021-10-19 | 浙江省淡水水产研究所 | Specific primer, probe and kit for detecting pelodiscus sinensis flavivirus |
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