CN116676422A - Primer probe combination for detecting bovine viral diarrhea virus, freeze-dried pellet and application thereof - Google Patents
Primer probe combination for detecting bovine viral diarrhea virus, freeze-dried pellet and application thereof Download PDFInfo
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Classifications
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- 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
- C12Q1/6851—Quantitative amplification
-
- 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
Abstract
The application discloses a primer probe combination for detecting bovine viral diarrhea virus, a freeze-dried pellet and application thereof. The primer probe combination comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.1, a reverse primer with a nucleotide sequence shown as SEQ ID NO.2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3. The freeze-dried pellet comprises the primer probe combination, a fluorescent quantitative PCR solution and a freeze-drying protection solution. The primer probe combination for detecting the bovine viral diarrhea virus is combined with the freeze-dried pellet technology, so that the primer probe combination for detecting the bovine viral diarrhea virus has high sensitivity and strong specificity, and the freeze-dried pellets can be stored at normal temperature, so that the transportation and storage cost is greatly reduced.
Description
Technical Field
The application belongs to the technical field of molecular biological detection, and particularly relates to a primer probe combination for detecting bovine viral diarrhea virus, a freeze-dried pellet and application thereof.
Background
Bovine viral diarrhea virus (Bovine Viral Diarrhea Virus, BVDV), bovine infectious rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (Bovine Respiratory Syncytial Virus, BRSV), mycoplasma bovis (mycorplasta bovis, M.b), niu Bushi bacillus (Bovine Brucella) are the major causative agents of Bovine disease, which seriously affect the performance of cattle after infection and cause death in cattle in severe cases. However, the clinical infection symptoms of BRSV, IBRV and BVDV are similar, and common mixed infection greatly improves the diagnosis and prevention difficulty of epidemic diseases.
In the existing clinical work of pastures, the problems of difficult diagnosis of a part of pathogens and the like widely exist, and detection methods such as colloidal gold test paper, ELISA and the like can provide rapid diagnosis and detect the pathogens from the protein level, but certain cross reaction and animal autoantibody interference exist, inaccurate diagnosis results are easy to appear, and vaccine immunity and virus infection are difficult to distinguish. Real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) can solve the above detection drawbacks. qPCR is a method for adding a fluorescent group into a PCR reaction system in DNA amplification reaction, monitoring the whole PCR process in real time by utilizing fluorescent signal accumulation, and finally quantitatively analyzing an unknown template through a standard curve. However, most of the conventional qPCR reagents are liquid reagents, which results in high costs for frozen transportation and storage in each detection unit or farm. Secondly, detection personnel need to be prepared at present when detecting, and a plurality of reagents need to be calculated and mixed, so that certain difficulty is provided for basic-layer cultivation personnel. In addition, repeated freeze thawing of the liquid reagent can affect the stability and accuracy of the assay.
The diagnostic reagent is made into the shape of freeze-dried beads by the freeze-drying technique, and is put into a designed reagent tray after freeze-drying, so that an operator can simply operate according to the requirement, and the rapid diagnosis and use of a user are facilitated. Compared with in-situ freeze-dried powder, the freeze-dried pellets are in a solid pellet state, so that whether detection reagents exist in the detection tube or not is facilitated to be observed, normal-temperature solid storage is achieved, cold chain transportation is not needed, the storage period of products is greatly prolonged, and the logistics cost is greatly reduced. When in use, the solid-state pellets have extremely strong resolubility, can be dissolved quickly when meeting sample liquid, and can be used as a normal PCR reagent, thus being the development trend of the current PCR reagent treatment. In addition, the freeze-dried pellets can realize industrialized low-cost mass production.
Disclosure of Invention
The application aims to: the first object of the application is to provide a primer probe combination for detecting bovine viral diarrhea virus; the second object of the application is to provide a freeze-dried pellet of the primer probe combination; a third object of the present application is to provide a method for preparing the above freeze-dried pellet; a fourth object of the present application is to provide the use of the primer probe combination and lyophilized pellet described above.
The technical scheme is as follows: the primer probe combination for detecting bovine viral diarrhea virus comprises a forward primer with a nucleotide sequence shown as SEQ ID NO.1, a reverse primer with a nucleotide sequence shown as SEQ ID NO.2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3.
The freeze-dried pellet for detecting bovine viral diarrhea virus comprises the primer probe combination, a fluorescent quantitative PCR solution and a freeze-drying protection solution.
Further, the volume ratio of the primer probe combination, the fluorescent quantitative PCR solution and the freeze-drying protection solution is 1:19:20-1:39:40.
Further, the addition amount of the forward primer and the reverse primer in the primer probe combination is 0.2-0.4 mu L respectively, and the addition amount of the probe is 0.1-0.2 mu L.
Further, the fluorescent quantitative PCR solution comprises Taq Pro U+ Multiple Probe qPCR Mix and RNase-free ddH 2 O。
Further, the freeze-drying protection solution comprises 0.01-0.1mol/L of phosphate buffer solution, 0.5-10g/L of bovine serum albumin, 0.1-1g/L of gelatin, 1-10g/L of polyethylene glycol 8000 and 0.5-5mL/L of Triton X.
Further, the diameter of the freeze-dried pellets is 3.6-3.8mm.
The preparation method of the freeze-dried pellet provided by the application comprises the following steps:
(1) Mixing phosphate buffer solution, bovine serum albumin, gelatin, polyethylene glycol 8000 and Triton X-100 to obtain freeze-drying protective solution;
(2) Taq Pro U+ Multiple Probe qPCR Mix and RNase-free ddH are taken 2 Mixing and preparing O to obtain a fluorescent quantitative PCR solution;
(3) And uniformly mixing the primer probe combination, the fluorescent quantitative PCR solution and the freeze-drying protection solution according to the volume ratio of 1:19:20-1:39:40, and freeze-drying to obtain the microsphere.
Further, the freeze-drying condition in the step (3) is that freeze-drying is firstly carried out for 1-2 hours at the temperature of-50 ℃, then vacuum drying is carried out for 2-3 hours, and then the temperature is gradually increased to 25 ℃, and the temperature is kept for 1-1.5 hours at the temperature of 10 ℃ per time.
The primer probe combination and the freeze-dried pellet are applied to preparation of a kit for detecting bovine viral diarrhea virus.
The beneficial effects are that: compared with the prior art, the application has the following remarkable advantages: the primer probe combination for detecting the bovine viral diarrhea virus is combined with the freeze-dried pellet technology, so that the primer probe combination for detecting the bovine viral diarrhea virus has high sensitivity and strong specificity, and the freeze-dried pellets can be stored at normal temperature, so that the transportation and storage cost is greatly reduced.
Drawings
FIG. 1 is an amplification curve of an actual sample detected using the freeze-dried pellet of the present application (wherein curves 1-2 are positive controls, curves 3-4 are actual samples to be detected, and curves 5-6 are negative controls);
FIG. 2 is an amplification curve of the lyophilized pellet of the present application with a liquid reagent for detecting bovine viral diarrhea virus (wherein curves 1-3 are the lyophilized pellets of the present application and 4-6 are the liquid reagents);
FIG. 3 shows amplification curves of the primer probe combinations and the control primer probe sets for detecting bovine viral diarrhea virus (wherein curves 1-3 are the primer probe combinations of the application, and 4-6 are the primer probe combinations of the control set);
FIG. 4 shows amplification curves of bovine viral diarrhea virus (bovine viral diarrhea virus) using the primer probe set and control probe set of the present application after diluting RNA of the sample to be tested 10-fold with the solution (wherein curves 1-2 are the 10-fold diluted samples detected by the primer probe set of the present study, and 3-4 are the 10-fold diluted samples detected by the control primer probe set).
Detailed Description
The technical scheme of the application is further described below with reference to the accompanying drawings.
Example 1 preparation of lyophilized pellets for detection of bovine viral diarrhea Virus
Reagent: phosphate buffer (cat# P5244) from Sigma-Aldrich 0.1mol, polyethylene glycol 8000 (cat# 25322-68-3) 1g and Triton X-100 (cat# 9036-19-5) 100mL; 25G of bovine serum albumin (cat# A80320) and 100G of gelatin (cat# G637) of Melin company; taq Pro U+ Multiple Probe qPCR Mix (product number: QN 23-01) 1mL and RNase-free ddH of Nanjinopran Biotech Co., ltd 2 O (cat# P07-01) 5mL. The primer probe combination of the application comprises SEQ ID NO. 1-6 which are synthesized by the division of biological engineering (Shanghai).
(1) Phosphate buffer solution (final concentration is 0.01 mol/L), bovine serum albumin (final concentration is 6 g/L), gelatin (0.5 g/L), polyethylene glycol 8000 (final concentration is 5 g/L) and Triton X-100 (final concentration is 2 mL/L) are mixed to prepare a freeze-drying protective solution, and the total system is 20 mu L;
(2) Taq Pro U+ Multiple Probe qPCR Mix 10. Mu.L and 9. Mu.L RNase Free ddH were taken 2 Mixing O to prepare a fluorescent quantitative PCR solution, wherein the total system is 19 mu L;
(3) The primer probe combination (comprising 0.4. Mu.L of forward primer with nucleotide sequence shown as SEQ ID NO.1 (5'-GGCCCACTGT ATTGCTACT-3'), 0.4. Mu.L of reverse primer with nucleotide sequence shown as SEQ ID NO.2 (5'-CTGGTCGTAA ACAGGTTCC-3') and 0.2. Mu.L of probe with nucleotide sequence shown as SEQ ID NO.3 (5'-CTGTACATGG CACATGGAGT TGA-3')), the fluorescent quantitative PCR solution and the freeze-drying protection solution were mixed uniformly. The test tube containing the mixed liquid is quickly immersed in liquid nitrogen, is taken out after standing for 10 seconds and is placed in a freeze dryer which is pre-cooled to 50 ℃, the vacuum freeze dryer is kept at 50 ℃ for 1.5 hours, then is vacuumized and dried for 3 hours, and then is gradually heated, and is kept at 10 ℃ for 1 hour every time until the temperature is raised to 25 ℃.
Example 2 actual sample detection
Taking 100 ng/. Mu.L of RNA solution of sample to be tested (stored in the center of nutrition and feed engineering of ruminant at Nanjing university of agriculture), diluting 100 ng/. Mu.L of positive control group (bovine viral diarrhea virus RNA) by 20 times to obtain 5 ng/. Mu.L of use solution, and setting up negative control group (RNase Free ddH) 2 O). 20. Mu.L of the use solution was mixed with the lyophilized pellet prepared in example 1, and PCR was performed after the lyophilized pellet was completely dissolved. Detection procedure: 50 ℃ for 10min;95℃for 3min,95℃for 10s,60℃for 30s,45 cycles. If the Ct value is less than or equal to 39, the sample to be tested is positive for bovine viral diarrhea virus; if the Ct value is more than 39 or the fluorescent amplification curve is not available, the sample to be tested is negative to bovine viral diarrhea virus.
As shown in FIG. 1, the number of cycles of detecting positive samples by the freeze-dried pellets of the primer probe set of the application is 17, the sample to be detected is 19, and the negative control group is more than 39, which indicates that the freeze-dried pellets of the application can rapidly detect RNA of bovine viral diarrhea virus.
Example 3 liquid detection reagent and lyophilized pellet reagent comparative experiments
The lyophilized pellet prepared in example 1 and the liquid detection reagent with the same components (the components are the same, the preparation steps are obtained by removing the lyophilization step in example 1) are used for detecting bovine viral diarrhea virus RNA. Detection procedure: 50 ℃ for 10min;95℃for 3min,95℃for 10s,60℃for 30s,45 cycles. The amplification results are shown in FIG. 2. The uniformity of the freeze-drying reagent amplification and the detection cycle number are superior to those of the liquid detection reagent, the cycle number of the freeze-drying pellet group is 17-21, and the cycle number of the liquid reagent group is 21-27. The lower the cycle number, the earlier the virus in the sample was detected, and the above results indicate that the virus detection ability of the primer probe set lyophilized pellet of the present application is better than that of the liquid reagent set.
Example 4 primer probe combinations comparative experiments
Bovine viral diarrhea virus RNA was detected using the lyophilized pellet prepared in example 1 and the control lyophilized pellet (the nucleotide sequence of the forward primer in the primer probe combination is SEQ ID NO.4 5'-TCAAGGAACC CCACCAGAT-3', the nucleotide sequence of the reverse primer is SEQ ID NO.5 5'-AGGCAAAGTC ATTTCTAGGT GCAA-3', and the nucleotide sequence of the probe is SEQ ID NO.6 5'-TGGCAAACTT ACCTATCGGT GACCCT-3'), respectively. Detection procedure: 50 ℃ for 10min;95℃for 3min,95℃for 10s,60℃for 30s,45 cycles. The amplification results are shown in FIG. 3. The cycle number of the freeze-dried pellet detection of the primer probe combination is 17, and the cycle number of the virus detected by the control primer probe group is 19, so that the result shows that the primer probe combination pellet of the application is superior to the freeze-dried pellet of the control primer probe group, and the result shows that the primer probe combination of the application can detect the virus RNA more quickly.
Example 5 comparative experiment after dilution of sample
The RNA use solution of the sample to be tested after 10-fold dilution was detected with the lyophilized pellet prepared in example 1 and the control lyophilized pellet (the nucleotide sequence of the forward primer in the primer probe combination is SEQ ID NO.4 5'-TCAAGGAACC CCACCAGAT-3', the nucleotide sequence of the reverse primer is SEQ ID NO.5 5'-AGGCAAAGTC ATTTCTAGGT GCAA-3', and the nucleotide sequence of the probe is SEQ ID NO.6 5'-TGGCAAACTT ACCTATCGGT GACCCT-3'), respectively, and the RNA amount was 10ng. Detection procedure: 50 ℃ for 10min;95℃for 3min,95℃for 10s,60℃for 30s,45 cycles. The amplification results are shown in FIG. 4. The cycle number of the freeze-dried pellet of the primer probe combination of the application is 17, and the cycle number of the virus detected by the control primer probe set is 31, and although the two groups can detect the virus, the cycle number required by the primer probe combination pellet of the application to detect the virus is lower than that of the control primer probe set, and in conclusion, the result shows that the primer probe combination pellet of the application is superior to the freeze-dried pellet of the control primer probe set. And the primer probe of the application can detect viruses with lower concentration.
Claims (10)
1. The primer probe combination for detecting the bovine viral diarrhea virus is characterized by comprising a forward primer with a nucleotide sequence shown as SEQ ID NO.1, a reverse primer with a nucleotide sequence shown as SEQ ID NO.2 and a probe with a nucleotide sequence shown as SEQ ID NO. 3.
2. A lyophilized pellet for detecting bovine viral diarrhea virus comprising the primer probe combination of claim 1, a fluorescent quantitative PCR solution, and a lyoprotection solution.
3. The lyophilized pellet of claim 2, wherein the primer probe combination, fluorescent quantitative PCR solution, and lyoprotection solution are in a volume ratio of 1:19:20-1:39:40.
4. The lyophilized pellet according to claim 2, wherein the amount of the forward primer and the reverse primer added in the primer probe combination is 0.2 to 0.4 μl each, and the amount of the probe added is 0.1 to 0.2 μl.
5. The lyophilized pellet of claim 2, wherein the fluorescent quantitative PCR solution comprises Taq Pro u+ Multiple Probe qPCR Mix and RNase-free ddH 2 O。
6. The lyophilized pellet of claim 2, wherein the lyoprotection solution comprises phosphate buffer 0.01-0.1mol/L, bovine serum albumin 0.5-10g/L, gelatin 0.1-1g/L, polyethylene glycol 8000 1-10g/L, triton X-100.5-5 mL/L.
7. The lyophilized pellet of claim 2, wherein the diameter of the lyophilized pellet is 3.6-3.8mm.
8. A method of preparing the lyophilized pellet of any one of claims 2-7, comprising the steps of:
(1) Mixing phosphate buffer solution, bovine serum albumin, gelatin, polyethylene glycol 8000 and Triton X-100 to obtain freeze-drying protective solution;
(2) Taq Pro U+ Multiple Probe qPCR Mix and RNase-free ddH are taken 2 O mixing configuration to obtain fluorescence quantificationA PCR solution;
(3) And uniformly mixing the primer probe combination, the fluorescent quantitative PCR solution and the freeze-drying protection solution according to the volume ratio of 1:19:20-1:39:40, and freeze-drying to obtain the microsphere.
9. The method according to claim 8, wherein the lyophilization conditions in step (3) are lyophilization at-50 ℃ for 1-2 hours, followed by vacuum drying for 2-3 hours, and further gradually heating to 25 ℃ and maintaining at 10 ℃ for 1-1.5 hours.
10. Use of the primer probe combination of claim 1 and the lyophilized pellet of claims 2-7 in the preparation of a kit for detecting bovine viral diarrhea virus.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117159480A (en) * | 2023-11-01 | 2023-12-05 | 江西赛基生物技术有限公司 | Recombinant human IFN-gamma protein freeze-dried pellet and preparation method and application thereof |
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| US20070259348A1 (en) * | 2005-05-03 | 2007-11-08 | Handylab, Inc. | Lyophilized pellets |
| CN114634996A (en) * | 2022-02-28 | 2022-06-17 | 广东海大畜牧兽医研究院有限公司 | Primer-probe combination for detecting bovine respiratory diseases, kit and application thereof |
| CN116042370A (en) * | 2022-12-16 | 2023-05-02 | 厦门宝太生物科技股份有限公司 | Novel coronavirus detection kit for home self-detection and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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