CN114410841A - Primer and probe for detecting bovine epidemic heat and bovine epidemic heat virus and application thereof - Google Patents
Primer and probe for detecting bovine epidemic heat and bovine epidemic heat virus and application thereof Download PDFInfo
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- CN114410841A CN114410841A CN202210044975.9A CN202210044975A CN114410841A CN 114410841 A CN114410841 A CN 114410841A CN 202210044975 A CN202210044975 A CN 202210044975A CN 114410841 A CN114410841 A CN 114410841A
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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
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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
- C12Q1/68—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
Abstract
The invention discloses a primer and a probe for detecting bovine epidemic heat and bovine epidemic heat virus and application thereof, wherein the nucleotide sequence of the primer is shown as SEQ ID NO: 8 and SEQ ID NO: 9, the nucleotide sequence of the probe is shown as SEQ ID NO: 1 is shown. The invention utilizes ERA technology to combine the primer and the probe to establish a method and a kit for detecting the bovine epidemic fever virus. The amplification stage is matched with a fluorescence constant temperature amplification instrument, the target fragment can be effectively amplified under the constant temperature condition of 42 ℃ for 15 minutes, and the method has the advantages of short time, high sensitivity, strong specificity, good repeatability, low cost and the like, wherein the minimum detection limit is 1.0 multiplied by 102copies/mu L, can be used in short time in basic laboratory environmentThe clinical detection of BEFV with high precision has good application prospect.
Description
Technical Field
The invention relates to the technical field of virus detection, in particular to primers and probes for detecting bovine epidemic fever and bovine epidemic fever virus and application thereof.
Background
Bovine Ephemeral Fever (BEF) is a febrile acute infectious disease caused by Bovine Ephemeral Fever Virus (BEFV) virus. Characterized by high fever, dyspnea, lameness. The main infected animals are cows, cattle and buffalo, and sometimes deer. All cattle of different varieties, sexes and ages can be infected with the disease, and the cattle of 3-5 years old are most susceptible to the disease. The virus has only 1 serotype, is enveloped, is conical, is easy to be inactivated by alcohol and ether, and can be inactivated by high temperature or low pH value. But still has long-term infectivity at-70 ℃.
The main current prevention and treatment measures for bovine epidemic fever mainly include vaccination and inactivated vaccine for immune prevention and control, implementation of vector prevention and control, and enhancement of pasture environment and sanitation management. At present, the diagnosis method for BEFV mainly comprises the prior, domestic and mainly adopted trace serum neutralization test method, and because live virus is needed, the detection period is long, the diagnosis is slow, and the risk of virus pollution diffusion exists. The establishment of ELISA detection technology provides convenient conditions for screening bovine epidemic heat antibody, common PCR and fluorescent quantitative PCR in nucleic acid detection technology have the advantages of sensitivity, rapidness, specificity, high flux and the like, and the method is widely used for detecting various important epidemic pathogens, and LUX-TM fluorescent RT-PCR detection method has good detection accuracy and sensitivity, but expensive instruments are required for fluorescent quantitative detection, and the cost of detection reagents is high.
Therefore, the establishment of a rapid, efficient and low-cost molecular biological diagnosis method has important significance for the prevention and control of the bovine epidemic fever.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a primer and a probe for detecting bovine epidemic fever and bovine epidemic fever virus and application thereof.
The first purpose of the invention is to provide a primer for detecting bovine epizootic fever and/or bovine epizootic fever virus.
It is a second object of the present invention to provide probes for detecting bovine epizootic fever and/or bovine epizootic fever virus.
The third purpose of the invention is to provide the application of the primer and/or the probe in preparing a kit for detecting bovine epidemic heat and/or bovine epidemic fever virus.
The fourth purpose of the invention is to provide a kit for detecting bovine epizootic fever and/or bovine epizootic fever virus.
In order to achieve the purpose, the invention is realized by the following scheme:
a primer for detecting bovine ephemeral fever and/or bovine ephemeral fever virus, wherein the nucleotide sequence of the primer is shown as SEQ ID NO: 8 and SEQ ID NO: shown at 9.
An upstream primer: CAAGAGGGGGAATGGTTAAAAAGAATC (SEQ ID NO: 8);
a downstream primer: TGCCATTTAGGGTTACATTAAGCTTT (SEQ ID NO: 9).
A probe for detecting bovine ephemeral fever and/or bovine ephemeral fever virus, wherein the nucleotide sequence of the probe is as shown in SEQ ID NO: 1 is shown.
Probe BEFV-G-Probe: CCTGCTGGTGCTGTTTCTAATATAGCATGATTCCCGACATATTATGT (SEQ ID NO: 1).
Preferably, the fluorescent probe modified by the probe is as follows:
CCTGCTGGTGCTGTTTCTAATATAGCATGA//i6FAMdT//idSp//iBHQ1dT//CCCGACATATTATGT//iSpC3。
a primer probe composition for detecting bovine epidemic heat and/or bovine epidemic heat virus, which comprises the primer and/or the probe.
Preferably, said primer and said probe are comprised.
The primer and/or the probe are/is applied to preparation of a kit for detecting bovine epidemic heat and/or bovine epidemic fever virus.
Preferably, the kit is a nucleic acid amplification (ERA) kit.
A kit for detecting bovine ephemeral fever and/or bovine ephemeral fever virus contains the primer and/or the probe.
Preferably, the kit is a nucleic acid amplification (ERA) kit.
More preferably, an activator and/or a dissolving agent is contained.
Further preferably, the reaction temperature of the kit is 38-42 ℃.
Even more preferably, the reaction temperature of the kit is 42 ℃.
Further preferably, the reaction system of the kit: the nucleotide sequence is shown as SEQ ID NO: 1, 0.24-0.72 mu L of probe, and nucleotide sequence shown as SEQ ID NO: 8 and SEQ ID NO: 0.84-3.15 μ L of each primer shown in 9, 2 μ L of template, 1-3 μ L of activator, and 20 μ L, ddH of lytic agent2O make up to 50.0. mu.L.
Even more preferably, the nucleotide sequence is as set forth in seq id NO: 1, 0.48 mu L of probe and nucleotide sequence shown as SEQ ID NO: 8 and SEQ ID NO: 2.1. mu.L of each of the primers shown in 9, 2. mu.L of the template, 2. mu.L of the activator, and 20. mu. L, ddH of the lytic agent2O make up to 50.0. mu.L.
Most preferably, the kit for detecting the bovine epizootic fever and/or bovine epizootic fever virus comprises the following components:
the nucleotide is shown as SEQ ID NO: 8-9 of upstream and downstream primers and nucleotides shown in SEQ ID NO: 1, a probe, a lytic agent, and an activator.
Probe BEFV-G-Probe:
CCTGCTGGTGCTGTTTCTAATATAGCATGA//i6FAMdT//idSp//iBHQ1dT//CCCGACATATTATGT//iSpC3(SEQ ID NO：1)；
the upstream primer ERA-G-F4: CAAGAGGGGGAATGGTTAAAAAGAATC (SEQ ID NO: 8);
the downstream primer ERA-G-R4: TGCCATTTAGGGTTACATTAAGCTTT (SEQ ID NO: 9).
Second, use method
The reaction conditions are as follows:
the reaction temperature is 42 ℃;
reaction system: 2. mu.L of template, 20. mu.L of lytic reagent, 2. mu.L of activator (25mmol/mL), 2.1. mu.L of each of the solution (10. mu. mol/mL) of forward primer ERA-G-F4 and downstream primer ERA-G-R4, 0.48. mu.L of the solution (10. mu. mol/mL) of Probe BEFV-G-Probe, ddH2O was added to 50. mu.L.
Thirdly, judging the result
If the sample peak time is less than or equal to 15min or the Ct value is less than 35, the sample is judged to be positive, and the sample contains the bovine epidemic fever virus; if the sample peak time is more than or equal to 20min or the Ct value is more than or equal to 35, the sample is judged to be negative, and the sample does not contain the bovine epidemic fever virus.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes ERA technology to combine the primer and the probe to establish a method and a kit for detecting the bovine epidemic fever virus. The amplification stage is matched with a fluorescence constant temperature amplification instrument, the target fragment can be effectively amplified under the constant temperature condition of 42 ℃ for 15 minutes, and the method has the advantages of short time, high sensitivity, strong specificity, good repeatability, low cost and the like, wherein the minimum detection limit is 1.0 multiplied by 102The copes/mu L has no cross reaction with bovine sarcoidosis virus (LSDV), Bovine Viral Diarrhea Virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine mycoplasma, Sendai Virus (SV), Klebsiella pneumoniae and salmonella, can be applied to the clinical detection of BEFV with short time and high precision in the basic laboratory environment, and has good application prospect.
Drawings
FIG. 1 shows DNA template preparation.
FIG. 2 shows the selection of primers for the ERA reaction.
FIG. 3 shows the result of specific detection by the kit.
FIG. 4 shows the results of the sensitivity test of the kit.
FIG. 5 shows the result of the repetitive detection of the kit.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Primary reagents and instruments
The fluorescent nucleic acid amplification kit (ERA method) was purchased from Suzhou Xianda Gene technology, Inc. DL2000 Plus DNA Marker, AceQ Universal U + Probe Master Mix V2 was purchased from Nanjing Novowed Biotech, Inc. GenStar 2 × Taq PCR StarMix and Loading Dye were purchased from Guangzhou ConruningA biological company.
HS DNA Polymerase and pMD18-T Vector Cloning kit was purchased from TAKARA.
Viral strains and clinical samples
Bovine sarcoidosis virus (LSDV), Bovine Viral Diarrhea Virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), Mycoplasma bovis, Sendai Virus (SV), Klebsiella pneumoniae, Salmonella are all kept in the laboratory. 16 BEFV-positive nucleic acid samples were stored in this laboratory.
EXAMPLE 1 preparation of DNA Standard plasmid
First, experiment method
Using BEFV JM2020 virus cDNA as a template
And obtaining a G gene target fragment by using the HS DNA Polymerase kit through Polymerase chain reaction.
Reaction system: 5 XPrimeSTAR Buffer each 10. mu.L, dNTP Mix (10mM) each 1. mu.L, PrimeSTAR HS DNA Polymerase each 0.5. mu.L, upstream (G-F) primer (10. mu. mol/L) 2.5. mu.L, downstream (G-R) primer (10. mu. mol/L) 2.5. mu.L, template 30ng, ddH2O make up to 50. mu.L.
G-F：ATGTTCAGGGTCCTAATAAT，
G-R：TTAATGATCAAAGAATCTGT。
Reaction procedure: 3min at 98 ℃; 10s at 98 ℃, 15s at 55 ℃, 2min at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min.
The purified target fragment was ligated to pMD-18T vector and then sent to Biotechnology (Shanghai) GmbH for sequencing. After the sequencing is correct, the number of copies is converted according to the following formula:
second, experimental results
The specific product with the fragment of 1872bp is obtained by amplification. The amplified fragment was purified, cloned into a vector pMD18-T, tested by in vitro amplification to give the results (FIG. 1), and sequenced correctly, and designated pMD18T-BEFV-G as a DNA standard plasmid.
Example 2 primers for the ERA method
First, experiment method
1. Probe primer design
The BEFV whole genome sequence published on NCBI is collected, ERA primers and probes (Table 1) are designed, the specificity of the primers and probes is determined by BLAST comparison, and synthetic markers are sent to the company Biotechnology engineering (Shanghai) GmbH.
Table 1 ERA primer and probe sequences:
"i 6-FAMdT" is a 6-FAM fluorescently labeled dT nucleotide, "idSp" refers to a base deletion that incorporates a dSpace group, "iBHQ 1 dT" is a dT nucleotide labeled with a BHQ1 quencher group, "iSpC 3" refers to a connection blocking group C3 Space.
2. Amplification of ERA primers
(1) Using the constructed DNA standard plasmid pMD18T-BEFV-G constructed in example 1 as a template, each pair of ERA primers were amplified by the conventional polymerase chain reaction, respectively, and the amplification effects of the ERA primer pairs were compared.
Reaction system: 2 XTaq PCR Starmix 10. mu.L each, upstream and downstream primers (10. mu. mol/L) 0.4. mu.L each, template 30ng, ddH2O make up to 20. mu.L.
Reaction procedure: 94 ℃ for 2 min; 30s at 94 ℃, 30s at 59 ℃, 45s at 72 ℃ and 35 cycles; extension at 72 ℃ for 5 min.
(2) Using the constructed DNA standard plasmid pMD18T-BEFV-G constructed in example 1 as a template, each pair of ERA primers were subjected to a conventional ERA reaction, respectively, and the amplification effects of the ERA primer pairs were compared.
Reaction system: 20. mu.L of each dissolving agent (buffer), 2.1. mu.L of each upstream primer and downstream primer (10. mu. mol/L), 0.6. mu.L of probe, 30ng of template, 2. mu.L of each activator, ddH2O complement 50 μ L. Reaction procedure: FAM fluorescence signals were collected every 15s at 37 ℃ for 30 min. .
Second, experimental results
The results show (FIG. 2) that, by performing a general polymerase chain reaction amplification, each pair of primers was found to have good specificity; and performing conventional ERA reaction, performing fluorescence detection on each pair of ERA primers, and finding that the ERA-G-F4/R4(SEQ ID NO: 8-9) primer pair has high amplification fluorescence intensity and short peak starting time, is an optimal primer pair, and performing subsequent experiments.
EXAMPLE 3 reaction conditions of the ERA Process
First, experiment method
According to the ERA method reagent instructions, determination of temperature, probe concentration, primer concentration, activator concentration are the main factors affecting the reaction of the system. To determine the optimal level of the 4 factors in the ERA system, an orthogonal approach using L9(34) was used to design a protocol (Table 2) in which both primers and probe were 10. mu. mol/L.
TABLE 2L9(34) Experimental protocol
After performing the ERA reaction according to the protocol of Table 2 using the ERA-G-F4/R4(SEQ ID NO: 8-9) primer pair and probe (SEQ ID NO: 1) of example 2, each combination was repeated 4 times.
Second, experimental results
The results are shown in table 3, the amplification effects of the experimental results of different groups are obviously different, the analysis result obtains the optimal level of each factor, and the optimal reaction conditions are as follows:
the reaction temperature was 42 deg.C
Reaction system (50.0 μ L): 20. mu.L of lytic reagent, 2. mu.L of activator (25mmol/mL), 2.1. mu.L of upstream and downstream primer volumes (10. mu. mol/mL), 0.48. mu.L of probe solution (10. mu. mol/mL), ddH2O was added to 50. mu.L.
TABLE 3 range analysis table
Example 4 kit for detecting bovine epidemic fever by ERA method
A, make up
The nucleotide is shown as SEQ ID NO: 8-9 of upstream and downstream primers and nucleotides shown in SEQ ID NO: 8-9 of a probe, a dissolving agent and an activating agent.
Probe BEFV-G-Probe:
CCTGCTGGTGCTGTTTCTAATATAGCATGA//i6FAMdT//idSp//iBHQ1dT//CCCGACATATTATGT//iSpC3(SEQ ID NO：1)；
the upstream primer ERA-G-F4: CAAGAGGGGGAATGGTTAAAAAGAATC (SEQ ID NO: 8);
the downstream primer ERA-G-R4: TGCCATTTAGGGTTACATTAAGCTTT (SEQ ID NO: 9).
Second, use method
The reaction conditions are as follows:
the reaction temperature was 42 c,
reaction system (50.0 μ L): 2. mu.L of template DNA, 20. mu.L of lytic reagent, 2. mu.L of activator (25mmol/mL), 2.1. mu.L of upstream and downstream primer volumes (10. mu. mol/mL), 0.48. mu.L of probe solution (10. mu. mol/mL), ddH2O was added to 50. mu.L.
Thirdly, judging the result
If the sample peak time is less than or equal to 15min or the Ct value is less than 35, the sample is judged to be positive, and the sample contains the bovine epidemic fever virus; if the sample peak time is more than or equal to 20min or the Ct value is more than or equal to 35, the sample is judged to be negative, and the sample does not contain the bovine epidemic fever virus.
Example 5 specificity of kit for detecting bovine epidemic fever by ERA method
First, experiment method
Using the kit of example 4, the DNA or cDNA of LSDV, BVDV, SV, Mycoplasma bovis, Klebsiella pneumoniae was used as a control, and the presence or absence of an amplification curve was observed using distilled water as a blank control, to thereby determine the specificity of the method.
Second, experimental results
ERA method for DNA or cDNA of LSDV, BVDV, SV, mycoplasma bovis and Klebsiella pneumoniaeDetection of, ddH2O is a negative control, and the detection was performed using the kit of example 4. The results show (FIG. 3) that there was no specific amplification of DNA or cDNA from other 5 bovine diseases. It can be seen that the method can specifically amplify BEFV. The kit is proved to have good specificity to BEFV.
Example 5 sensitivity of kit for detecting bovine epidemic fever by ERA method
First, experiment method
pMD18T-BEFV-G DNA standard plasmid prepared in example 1 was diluted to 10-fold in 10-fold gradient6、105、104、103、102、101And 100copies/. mu.L. pMD18T-BEFV-G at each concentration gradient was used as a template, and distilled water was added as a blank. The kit of example 4 was used for detection to test its sensitivity.
Second, experimental results
At 1.0 × 107～1.0×100The plasmid pMD18T-BEFV-G at a copies/μ L concentration gradient was used as a template for detection using the kit of example 4. The results show that the time spent in the detection is within 7min, within 7.5min, within 8min, within 8.5min, within 9min, within 13min and within 15.5min, respectively, and is 1.0 × 101And 1.0X 100The concentration template was not amplified. The detection limits of the plasmid by the kit of example 4 were all 1.0X 102copies/. mu.L (FIG. 4).
Example 5 repeatability and stability of the kit for detecting bovine epizootic fever by ERA method
First, experiment method
pMD18T-BEFV-G DNA standard plasmid prepared in example 1 was diluted to 0 by 10-fold gradient5、104And 103copies/. mu.L. pMD18T-BEFV-G at each concentration gradient was used as a template, and distilled water was added as a blank. The assay was performed using the kit of example 4, with 5 replicates per concentration. And calculating the variation coefficient according to the appearance time of the fluorescence curve.
Second, experimental results
In order to evaluate the stability and the repeatability of the establishment method, 4 concentrations of plasmid templates are selected to perform 5 times of repeatability tests respectively, the variation coefficient results are all less than 10%, and the established kit for detecting the bovine epidemic fever by the ERA method has good repeatability (figure 5).
Example 6 detection of clinical samples of kit for detecting bovine epidemic fever by ERA method
First, experiment method
The results of 15 positive clinical DNA samples and 5 negative clinical DNA samples were used as templates for detection (ERA-BEFV) using the kit of example 4, while the real-time PCR method (real-time PCR-BEFV) detection was used as a control, and the results of both methods were analyzed by comparison.
The reaction system of real-time PCR is as follows:
480Probes Master (2 Xconc) 10. mu.L, 10. mu. mol/L of each of the forward primer and the reverse primer 1. mu.L, 0.5pL of the probe, ddH2O5.5. mu.L, template 2. mu.L, total volume 20. mu.L.
The reaction conditions of real-time PCR are as follows: 5min at 95 ℃; fluorescence was collected at 95 ℃ for 20s, 53 ℃ for 30s, 72 ℃ for 20s, 40 cycles, and 53 ℃.
Primers for real-time PCR were: an upstream primer: TCATTGATAAGAAGATGGC, the downstream primer is: TGGTTCCACAAAGATCATTC, the probe is: (FAM) AGCTTCCTCCTGCTGGTGC (BHQ 1).
Second, experimental results
The results show that the positive detection rate is 100%, and the detection rate of the real-time PCR method is 100%. The ERA method and real-time PCR method were consistent in detectable rate (Table 4).
Table 4:
it should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
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Claims (10)
1. A primer for detecting bovine ephemeral fever and/or bovine ephemeral fever virus, wherein the nucleotide sequence of the primer is as shown in SEQ ID NO: 8 and SEQ ID NO: shown at 9.
2. A probe for detecting bovine ephemeral fever and/or bovine ephemeral fever virus, wherein the nucleotide sequence of the probe is as set forth in SEQ ID NO: 1 is shown.
3. A primer probe composition for detecting bovine ephemeral fever and/or bovine ephemeral fever virus, comprising the primer of claim 1 and/or the probe of claim 2.
4. Use of the primer of claim 1 and/or the probe of claim 2 for the preparation of a kit for detecting bovine ephemeral fever and/or bovine ephemeral fever virus.
5. The kit of claim 4, wherein the kit is a nucleic acid amplification kit.
6. A kit for detecting bovine epizootic fever and/or bovine epizootic fever virus, comprising the primer of claim 1 and/or the probe of claim 2.
7. The kit of claim 6, wherein the kit is a nucleic acid amplification kit.
8. The kit of claim 7, further comprising an activator and/or a lytic agent.
9. The kit according to claim 7, wherein the reaction temperature of the kit is 38-42 ℃.
10. The kit according to claim 9, wherein the reaction temperature of the kit is 42 ℃.
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| CN117737069A (en) * | 2024-02-19 | 2024-03-22 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | BVDV specific crRNA based on CRISPR-Cas12a, and related kit and detection method thereof |
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