CN111500790A - Primer probe set for detecting African swine fever by fluorescent quantitative PCR and application thereof - Google Patents
Primer probe set for detecting African swine fever by fluorescent quantitative PCR and application thereof Download PDFInfo
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Abstract
The invention discloses a primer probe set for detecting African swine fever by fluorescence quantitative PCR (polymerase chain reaction) and application thereof, wherein the primers are MGF360-F and MGF360-R, the probe is MGF360-Taq Man, the nucleotide sequence of MGF360-F is shown as SEQ ID No.1, the nucleotide sequence of MGF360-R is shown as SEQ ID No.2, and the nucleotide sequence of MGF360-Taq Man is shown as SEQ ID No.3, and a method and a kit for detecting African swine fever by using the primer probe set are provided. The primer probe group has high sensitivity and specificity, makes up the defects of the prior art, and lays a solid technical reserve and theoretical foundation for the revival of the domestic pig industry.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer probe set for detecting African swine fever by fluorescence quantitative PCR and application thereof.
Background
African swine fever is an acute, virulent and highly contagious disease of domestic and wild pigs, and virulent strains can kill domestic pigs within about 5-14 days of infection, wherein the death rate is close to 100%, and no effective preventive vaccine or specific therapeutic drug is available. In 2018, 8 and 3, the Shenyang city Shenbei new district in Liaoning province of China reports the epidemic situation of the African swine fever, and the occurrence of the epidemic situation of the African swine fever brings great harm to the breeding industry of live pigs in China. In order to prevent the rapid spread of the African swine fever, early diagnosis and early treatment are the main methods for preventing and controlling the African swine fever at the present stage.
The PCR detection method is a detection method recommended by OIE authorities, has low requirement on the purity of pathogenic nucleic acid, is simple, convenient and quick to operate, has high sensitivity and specificity, and is particularly suitable for identifying pathogens which cannot be separated in vitro.
Real-time fluorescent quantitative PCR (qPCR) is a new quantitative test technology introduced by Applied Biosystems in 1996, and is characterized in that a PCR product is labeled and tracked through a fluorescent dye or a fluorescent labeled specific probe, the reaction process is monitored on line in real time, the product can be analyzed by combining with corresponding software, and the initial concentration of a sample template to be detected is calculated. In 2019, 1 month and 4 days, the first batch of ASFV on-site rapid detection reagents are published by the rural part of China agriculture, and more than 60 percent of the ASFV on-site rapid detection reagents adopt a real-time qPCR detection method. The method can detect the reaction process in real time, determine the absolute quantity of the template, has strong specificity and the like, so that the qPCR has higher utilization value in the actual ASFV investigation.
The TaqMan probe method is a quantitative qPCR technique with high specificity. The working principle of the kit is that a pair of PCR primers and a probe exist in a PCR reaction system, a 5 'end of the probe is marked with a reporter group, a 3' end of the probe is marked with a fluorescence quenching group, the probe is only specifically combined with a template, and the combining site of the probe is between the two primers. When the probe is complete, the fluorescence energy of the reporter group is absorbed by the quencher group, so the signal can not be collected by the instrument, as the reaction progresses, the Taq enzyme meets the probe, the probe is cut off by the activity of 3 '→ 5' exonuclease, so the fluorescence energy of the reporter group can not be absorbed by the quencher group, a fluorescence signal is generated, and the intensity of the signal represents the copy number of the template DNA.
The application adopts a TaqMan probe method for detection, a primer is designed according to a conservative gene fragment of ASFV, the ASFV can be specifically identified, compared with the fluorescence quantitative PCR recommended by OIE, the OIE is mainly designed based on an ASFV early diagnosis target B646L (p72) gene, and the application mainly aims at detecting the MFG360 gene of African swine fever virus.
Disclosure of Invention
The invention provides a primer, a probe and a method for detecting African swine fever by fluorescence quantitative PCR, which are used for detecting the African swine fever virus MGF360 gene by real-time fluorescence quantitative PCR. Makes up the defects of the prior art and lays a solid technical reserve and theoretical foundation for the reviving of the domestic pig industry.
The invention is realized by the following technical scheme;
a primer probe set for detecting African swine fever by fluorescent quantitative PCR is disclosed, wherein the primers are MGF360-F and MGF360-R, the probe is MGF360-Taq Man, the nucleotide sequence of MGF360-F is shown in SEQ ID NO.1, the nucleotide sequence of MGF360-R is shown in SEQ ID NO.2, and the nucleotide sequence of MGF360-Taq Man is shown in SEQ ID NO. 3.
The probe is characterized in that a reporter group is marked at the 5 'end, a fluorescence quenching group is marked at the 3' end, the reporter group is FAM, and the fluorescence quenching group is BHQ 1.
In another aspect of the invention, the application of the primer with the sequence shown in SEQ ID NO. 1-2 or the probe with the sequence shown in SEQ ID NO.3 in detecting African swine fever is provided.
In another aspect of the invention, a kit for detecting African swine fever by fluorescence quantitative PCR is provided, and the kit comprises a primer probe set with a nucleotide sequence shown as SEQ ID NO. 1-3.
The kit also comprises reagents of 2 × Pro TaqHS Probe Premix, MGF360-F, MGF360-R, MGF 360-Probe, DNase/RNase-Free deinized Water.
In another aspect of the present invention, there is provided a method for detecting african swine fever by fluorogenic quantitative PCR, comprising the steps of: viral genomic DNA was extracted using the kit, and amplification was performed according to the reaction procedure using the above primers and probes.
The reaction system is 25 mu L: 2 × Pro TaqHS Probe Premix 12.5 mu L, upstream and downstream primers (50 pmol/. mu. L) are 1 mu L respectively, a Probe (50 pmol/. mu. L) is 1 mu L, and a template DNA (2 mu L/RNase-Free deinized Water) is added to 25 mu L.
The reaction procedure is as follows: 2min at 50 ℃; pre-denaturation at 95 ℃ for 10 min; 95 ℃ for 15s, 55 ℃ for 1min, 40 cycles.
The invention has the beneficial effects that:
the real-time fluorescence PCR method designed for detecting the MGF360 gene of the African swine fever can carry out specificity detection on the MGF360 gene, can detect the reaction process in real time and determine the absolute amount of a template. The method has the advantages of high sensitivity, high specificity and high accuracy.
Drawings
FIG. 1 shows the result of PCR identification of the recombinant plasmid standard of the present invention, wherein M is D L2000 DNA Marker, 1 is MGF360 gene fragment, and 2 is negative control;
FIG. 2 is a MGF360 fluorescence quantitative PCR standard curve of the present invention;
FIG. 3 shows the results of the sensitivity test of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Main test materials:
a plasmid containing the MFG360 gene sequence (GenBank: MK333180.1, gene length 7559bp, nucleotide sequence position 27942-35500) was synthesized by the company.
Porcine reproductive and respiratory syndrome virus, circovirus, pseudorabies virus, parvovirus, Japanese encephalitis B, porcine epidemic diarrhea virus, classical swine fever virus vaccine and foot and mouth disease virus vaccine are stored in the laboratory.
DH5 α competent cells, a viral genome DNA extraction kit, a common agarose gel DNA recovery kit and a common plasmid mini-extraction kit are all purchased from Tiangen Biotechnology limited public, and pMD19-T vector, 2 × Taq PCR Master Mix, D L2000 DNA Marker and the like are purchased from Takara bioengineering (Dalian) Limited company.
Example 1 construction and characterization of recombinant plasmid standards
Based on the nucleic acid sequence of ASFV (MK333180.1), primers and fluorescent probes specific to MGF360 gene were designed (Table 1), and the primers and probes were synthesized by WTO Biotech GmbH and Shanghai Biotech, respectively.
TABLE 1 primer and TaqMan Probe sequence information
Based on the MFG360 gene sequence of ASFV in GenBank, the 143bp gene sequence of the objective fragment (the 27942-35500 nucleotide sequence of accession number MK333180.1) was artificially synthesized as the positive template for the subsequent experiments. And performing amplification culture on the template. The specific primer MGF360-F/MGF360-R is adopted to amplify the target gene, the target fragment is recovered after 1 percent agarose gel electrophoresis detection, the target fragment is cloned to a pMD19-T vector, and the positive clone product is sequenced and identified by Shanghai biological engineering technical service company Limited. Culturing the positive strain, and extracting the recombinant plasmid by using the plasmid extraction kit, wherein the recombinant plasmid is named as pMD19-ASFV-MGF 360.
Using a NanoDrop 2000 coreThe acid protein analyzer measures the concentration of the recombinant plasmid standard and calculates the copy number thereof according to the formula (plasmid concentration × 10): the copy number of the recombinant plasmid (copy/. mu. L)-9×6.02×1023) /(660 daltons/base ×), the copy number is calculated.
The results show that about 143bp target bands are respectively obtained by the strains through amplification (figure 1), PCR products are respectively cloned to pMD19-T vectors after being recovered and purified to construct recombinant plasmid standard products pMD19-ASFV-MGF360, through sequencing identification, compared with a reference sequence (accession number: MK333180.1), MGF360 gene mutation is not found, the results are in accordance with expectations, the plasmid standard products pMD19-ASFV-MGF360 are correctly constructed, and through calculation, the plasmid standard products are respectively 1.8 × 108Copy/. mu. L.
Example 2 fluorescent quantitative PCR Standard Curve
The constructed pMD19-T recombinant plasmid is used as a standard substance for pMD19-ASFV-MGF360 quantification, 10-fold gradient dilution is carried out on the standard substance according to the determined plasmid concentration, and 7 gradients (10) are carried out in total1～107) Diluting. And (3) performing fluorescent quantitative PCR by taking the recombinant plasmids with different concentrations as templates, recording the Ct value of each gradient standard substance, and establishing a quantitative standard curve of the corresponding relation between the copy number of the plasmid and the copy number of the plasmid.
A25 mu L reaction system is adopted, positive plasmids with the same concentration of 2 mu L are used as templates, primers and probes with different concentrations are selected, the optimal concentrations of the primers and the probes are optimized by a matrix method, the lowest Ct value and the highest fluorescence intensity increase value (delta Rn) of the reaction are obtained, the amplification efficiency and the sensitivity of the reaction are improved, the annealing temperature (55-62 ℃) is optimized, and meanwhile a blank control is set.
The optimized fluorescent quantitative PCR reaction system has a total volume of 25 mu L, which comprises 2 × Pro TaqHS Probe Premix 12.5 mu L, upstream and downstream primers (50 pmol/. mu. L) each 1 mu. L, a probe (50 pmol/. mu. L) 1 mu. L, template DNA 2 mu L/RNase-Free deinized Water added to 25 mu L, amplification is carried out by a Bio-Rad CFX96 fluorescent quantitative PCR instrument according to the following procedures of 50 ℃ for 2min, 95 ℃ for 10min, 95 ℃ for 15s, 55 ℃ for 1min, 40 cycles are carried out in the last two steps, fluorescent signal detection is carried out in the annealing stage, and a fluorescent channel is selected from FAM.
Optimized fluorescent quantitative PCR reaction system is utilized to carry out 7 dilutions (1.8 × 10)1～1.8×107) The quantitative standard substance is detected, and the standard curve is 1.8 × 101-1.8×107The copy number has a good linear relation, the correlation coefficient is 0.999, and a linear equation of the copy number and the Ct value of the standard product is obtained (figure 2).
Example 3 fluorescent quantitative PCR sensitivity detection
By ddH2O10-fold serial dilution of the standard to 7 gradients (1.8 × 10)1-1.8×107Copy/. mu. L) as a template, performing detection under optimal reaction conditions, and determining the minimum detection amount of the established fluorescent quantitative PCR detection method for evaluating the sensitivity of the method.
As shown in FIG. 3, the minimum detection amount of the established fluorescent quantitative PCR detection method was determined to be 1.8 × 100The method was found to have a high sensitivity by copying/. mu. L.
Example 4 specificity test
Extracting the genome DNA of the porcine reproductive and respiratory syndrome virus, the circovirus, the pseudorabies virus, the parvovirus, the Japanese encephalitis B, the porcine epidemic diarrhea virus, the classical swine fever virus vaccine and the foot-and-mouth disease virus vaccine stored in the laboratory, taking the extracted genome DNA as a template, taking a recombinant plasmid standard as a positive control, taking ddH as a positive control2O was used as a negative control and amplified using the fluorescent quantitative PCR method established in this study to assess the specificity of the method.
The extracted corresponding DNA samples of porcine reproductive and respiratory syndrome virus, circovirus, pseudorabies virus and the like and the recombinant plasmid sample are subjected to specificity test, negative control is set, amplification is carried out according to an optimized fluorescent quantitative PCR system and conditions, the result shows that only the recombinant plasmid sample is positive, no obvious amplification curve appears in other virus and negative control samples, and the detection results are negative, which shows that the method has good specificity.
Example 5 repeatability test
To evaluate the reproducibility of the detection method, 1.8 × 102～1.8×104Copies/. mu. L3 concentration of recombinant plasmid were tested, 3 replicates per concentration were performed, and the Ct values obtained were counted and the mean, standard deviation and coefficient of variation calculated.
TABLE 2 fluorescent quantitative PCR repeatability test results
As can be seen from the table 2, the fluorescence quantitative PCR detection method for the African swine fever MGF360 genotype established by the test has better repeatability, and can stably and reliably detect the African swine fever MGF360 genotype sample.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
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Claims (10)
1. A primer probe set for detecting African swine fever by fluorescent quantitative PCR is characterized in that a nucleotide sequence of a primer is shown as SEQ ID No. 1-2, and a nucleotide sequence of a probe is shown as SEQ ID No. 3.
2. The primer probe set for detecting African swine fever by fluorogenic quantitative PCR of claim 1, wherein the 5 'end of the probe is labeled with a reporter group, and the 3' end is labeled with a fluorescence quenching group.
3. The primer probe set for detecting African swine fever by fluorogenic quantitative PCR of claim 2, wherein the reporter group is FAM and the fluorescence quenching group is BHQ 1.
4. The primer probe set of claim 1, wherein the primer probe set is used for detecting African swine fever.
5. A kit for detecting African swine fever by fluorescent quantitative PCR is characterized by comprising a primer probe group with a nucleotide sequence shown as SEQ ID No. 1-3.
6. The kit of claim 6, wherein the kit comprises the reagents 2 × Pro Taq HSProbe Premix, MGF360-F, MGF360-R, MGF360-Taq Man, DNase/RNase-Free DeionizidWater.
7. A method for detecting African swine fever by fluorescent quantitative PCR, which is characterized in that viral genome DNA is extracted by using a kit, and PCR amplification is carried out by using the primer and the probe of claim 1 according to a reaction program.
8. The method of claim 7, wherein the reaction system is 25 μ L: 2 × ProTaqHS Probe Premix 12.5 μ L, upstream and downstream primers are 1 μ L, Probe 1 μ L, template DNA 2 μ L/RNase-Free deinized Water added to 25 μ L.
9. The method of claim 8, wherein the concentration of the primers and probes upstream and downstream of the PCR is 50pmol/μ L.
10. The method for detecting African swine fever according to claim 7, wherein the PCR reaction program is: 2min at 50 ℃; pre-denaturation at 95 ℃ for 10 min; 95 ℃ for 15s, 55 ℃ for 1min, 40 cycles.
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Application publication date: 20200807 |