CN112760416A - African swine fever virus fluorescent PCR detection reagent, kit and detection method - Google Patents
African swine fever virus fluorescent PCR detection reagent, kit and detection method Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and discloses a fluorescence PCR detection reagent, a kit and a detection method for African swine fever virus. The detection reagent comprises specific primers A-F and A-R and probes A-P; the 5 'end of the probe is marked with a fluorescence reporter group HEX, and the 3' end of the probe is marked with a fluorescence quenching group BHQ 1. The detection kit provided by the invention has the advantages that the specific primers and probe sequences adopted by the detection kit are short, the amplification efficiency is high, the mismatching probability is low, and the detection efficiency, accuracy and stability can be obviously enhanced; the established fluorescence PCR detection method has high sensitivity and strong specificity.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a fluorescence PCR detection reagent, a kit and a detection method for African swine fever virus.
Background
African Swine Fever (ASF) is a highly contagious disease of pigs, the fatality rate can reach 100%, and the African Swine Fever Virus (ASFV) causes and spreads. The disease progresses very rapidly, beginning in africa and rolling to almost the entire world in less than 1 century. ASF is an animal epidemic that seriously harms the "world economy" and is also a group A animal epidemic prescribed by the world animal health Organization (OIE).
The African swine fever virus is the only member of the ASF-like virus family, the diameter of the virus particle is 175-215 nanometers, the virus particle is 20-face-symmetric and has an envelope, the genome is twin-stranded linear DNA, and the size is 170-190 kb. The virus mainly takes macrophage as an infection target cell, and takes the virus protein with a plurality of specific genes and a complex mechanism to participate in functions of ASFV adsorption, invasion of host cells, virus replication and assembly, host immune defense system avoidance and the like, so that the virus can more effectively infect the host cells, and acute, febrile and hemorrhagic clinical symptoms and pathological damage of pigs are caused. In addition, the African swine fever has a complex infection circulation mode, recessively infected pigs, live pig products polluted by swine fever viruses and products thereof are important transmission media for the African swine fever introduced into a healthy area, and the viruses can also infect wild pigs and soft ticks and can be circularly transmitted among domestic pigs, wild pigs, soft ticks and domestic pigs.
At present, an effective vaccine prevention and treatment method for ASF does not exist, prevention and control are carried out by means of early diagnosis and regional prevention and control management in a short period, a strict monitoring plan is adopted, and once the situation is found, a large-range killing mode is adopted to cut off the infection source. The serological detection methods such as HAD, ELISA, IFA and the like recommended by OIE are long in time consumption, complex in operation and not suitable for rapid detection of African swine fever viruses. The Real-time Quantitative PCR (Real-time Quantitative PCR qPCR) has the advantages of simple operation, rapidness, convenience, high sensitivity, good repeatability and low pollution rate, and is widely applied to the field of gene detection.
Patent CN 108300808A discloses a fluorescent PCR detection kit for African swine fever virus, a preparation method and a use method thereof. The kit comprises ASFV-reaction liquid, DNase mixed liquid, ASFV-internal standard, ASFV-positive control, ASFV-negative control, nucleic acid releasing agent and ASFV-quantitative reference substance, wherein the ASFV-reaction liquid comprises: primer: ASF03F03 and ASF03R 03; an internal standard primer: IPC05F01 and IPC05R 01; probe ASF03P and internal standard probe IPC 05P; the primers and probes correspond to a highly conserved fragment of the p72 gene of African swine fever virus. The primers ASF03F03 and ASF03R03 and the probe ASF03P adopted in the patent have excellent sensitivity and specificity, and the possible missing detection caused by primer probe mismatching is greatly reduced. By matching with internal standard primers IPC05F01 and IPC05R01 and an internal standard probe IPC05P, whether the whole PCR reaction system is normal can be monitored, so that the wrong judgment result caused by improper use is avoided.
Patent CN 109593893A discloses a fluorescence PCR rapid detection kit for African swine fever virus, which comprises specific primers ASFV-F and ASFV-R, and a TaqMan probe ASFV-P, wherein the 5 'end of the probe is marked with a report fluorescent dye HEX, and the 3' end is marked with a fluorescence quenching group BHQ-1. The invention selects a p72 gene (coding B646L structural protein) with highly conserved African swine fever virus as a target gene, designs specific primers and probes and establishes an ASFV specific fluorescence PCR detection method.
The primers and the probes adopted by the detection kit have good specificity although the p72 gene highly conserved in the African swine fever virus is used as a target gene. However, there is still room for further improvement in the amplification efficiency, detection accuracy and stability.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention mainly aims to provide a fluorescent PCR detection reagent for African swine fever virus.
The invention also aims to provide a fluorescence PCR detection kit for the African swine fever virus.
The invention also aims to provide a preparation method of the African swine fever virus fluorescent PCR detection kit.
The invention further aims to provide a fluorescence PCR detection method of African swine fever virus.
The purpose of the invention is realized by the following technical scheme:
a fluorescence PCR detection reagent for African swine fever virus comprises specific primers A-F and A-R, probes A-P and Taq enzyme; the 5 'end of the probe is marked with a fluorescence reporter group HEX, and the 3' end of the probe is marked with a fluorescence quenching group BHQ 1;
wherein the specific primers and probe sequences are as follows:
A-F：5′-CCCAGGRGATAAAATGACTG-3′；
A-R：5′-CACTRGTTCCCTCCACCGATA-3′；
A-P：5′-HEX-TCCTGGCCRACCAAGTGCTT-BHQ1-3′。
a fluorescence PCR detection kit for African swine fever virus comprises a PCR reaction solution consisting of the detection reagent, and a negative control and a positive control.
Further, the African swine fever virus fluorescent PCR detection kit also comprises a nucleic acid releasing agent.
Further, the content of each reagent in the African swine fever virus fluorescent PCR detection kit is as follows:
nucleic acid amplification reagents:
1000. mu.L of PCR reaction solution;
comparison products:
The preparation method of the African swine fever virus fluorescent PCR detection kit comprises the following steps:
(1) designing specific primers A-F and A-R and a probe A-P by taking VP72 gene of African swine fever virus as a target gene;
(2) synthesizing VP72 gene of African swine fever virus, cloning to pUC57 vector to obtain pUC57-VP72 recombinant plasmid; then transforming to competent cell culture, extracting plasmid as positive control;
(3) the pUC57 plasmid was transformed into competent cells for culture, and the plasmid was extracted as a negative control.
Further, the competent cell is DH5 α competent cell.
A fluorescence PCR detection method of African swine fever virus comprises the following steps:
(1) adding the PCR reaction solution into different PCR reaction tubes;
(2) respectively adding a sample to be detected, a negative control and a positive control into different PCR reaction tubes in the step (1), tightly covering a tube cover, moving to a fluorescence PCR instrument for PCR amplification, and collecting a fluorescence signal to set the fluorescence signal as HEX fluorescein;
(3) quality control standard: negative control should have no reported Ct value, and positive control should have a Ct value of 32.0 or less; otherwise, the experiment is regarded as invalid;
(4) and (5) judging a result: samples with no reported Ct value were negative; the sample with the Ct value less than or equal to 38 is positive; the Ct value is more than 38 and less than 42, the sample with the typical amplification curve needs repeated detection, the Ct value is negative without reporting in the repeated detection result, otherwise, the Ct value is positive.
Further, the amount of the PCR reaction solution added to the PCR reaction tube in the step (1) is 20. mu.L, and the amount of the sample to be tested, the negative control and the positive control added in the step (2) are 5. mu.L, respectively.
Further, when the sample to be detected in the step (2) is blood or serum, diluting the sample with normal saline or purified water, centrifuging the diluted sample, taking supernatant as a template, and adding the template into a PCR reaction tube for detection; when the sample to be detected is saliva or a nasal swab, extracting a sample DNA template by adopting a column extraction method or a magnetic bead method, and adding the sample DNA template into a PCR reaction tube for detection; and when the sample to be detected is a tissue, extracting a sample DNA template by adopting a nucleic acid releasing agent, and adding the sample DNA template into a PCR reaction tube for detection.
Further, the PCR amplification conditions in step (2) are as follows:
primary activation at 95 ℃ for 30 sec; denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 30sec, fluorescence signal acquisition, and 45 cycles.
Compared with the prior art, the invention has the beneficial effects that:
the detection kit provided by the invention has the advantages of short sequence of the adopted specific primers and probes, high amplification efficiency, low mismatching probability and capability of obviously enhancing the detection efficiency, accuracy and stability.
Drawings
FIG. 1 is a diagram showing the results of PCR identification of the transformation product of the recombinant plasmid pUC57-VP72 in example 1;
FIG. 2 is a graph showing the result of the fluorescent PCR assay of the negative control in example 1;
FIG. 3 is a graph showing the results of the sensitivity test in the real-time fluorescent PCR detection method for African swine fever virus in example 1.
FIG. 4 is a graph showing the results of the real-time fluorescence PCR assay for African swine fever virus in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
The preparation method of the African swine fever virus fluorescent PCR detection kit provided by the embodiment comprises the following specific steps:
(1) preparing primers and probes:
a pair of specific primers A-F and A-R and a fluorescent probe A-P designed based on the African swine fever virus VP72 gene (GenBank: KJ195685.1) were synthesized by TaKaRa company. The 5 'end of the probe is marked with a fluorescence reporter group HEX, and the 3' end of the probe is marked with a fluorescence quenching group BHQ 1;
wherein the sequences of the synthesized specific primers and probes are as follows:
A-F：5′-CCCAGGRGATAAAATGACTG-3′(SEQ ID NO：1)；
A-R：5′-CACTRGTTCCCTCCACCGATA-3′(SEQ ID NO：2)；
A-P：5′-HEX-TCCTGGCCRACCAAGTGCTT-BHQ1-3′(SEQ ID NO：3)。
(2) preparation of positive control:
1. synthesis of African swine fever virus VP72 gene (GenBank: KJ195685.1)
According to the published gene sequence of VP72 gene (GenBank: KJ195685.1), Beijing Rui Boxing Ke Biotech Co., Ltd is requested to synthesize the gene, and the synthesized gene is cloned into pUC57 vector to obtain pUC57-VP72 recombinant plasmid, which provides a sequencing report.
2. Transformation of recombinant plasmid pUC57-VP72 carrying VP72 gene of African swine fever virus
mu.L of DH 5. alpha. competent cells were thawed on ice bath, 1. mu.L of pUC57-VP72 was added, gently blown and mixed, and ice-cooled for 30 min. The bacterial solution was placed in a 42 ℃ water bath for 90 seconds with heat shock and immediately placed in an ice bath for 2 minutes. Then, 0.9mL of LB medium was added and incubated at 37 ℃ for 1 hour on a constant temperature shaker at 200 rpm. Centrifuging the bacterial solution at 4,000rpm/min for 3 minutes, leaving 200 mul of supernatant to disperse the thalli, uniformly coating the thalli on the surface of an agar plate containing ampicillin resistance, and inversely culturing the plate at 37 ℃ for 12-16 hours.
3. Results of PCR identification
The bacterial liquid was taken for PCR identification, and as a result, a band appeared at about 1941bp, as shown in FIG. 1, and the size coincided with the size of the target gene fragment.
4. Sequencing identification results
After the plasmid is extracted from the bacterial liquid which is positive in PCR detection by using a plasmid extraction kit, the bacterial liquid is sent to a sequencing company for sequencing, and the sequencing result is 100% consistent with the template sequence, which shows that the plasmid can be used as a recombinant plasmid for preparing positive control.
(3) Preparation of negative control:
plasmid pUC57 was transformed into DH 5. alpha. competent cells, which were cultured in LB solid medium (ampicillin resistant) at 37 ℃ for 12 to 16 hours, and many white colonies were observed. Randomly picking single white colony to respectively inoculate LB liquid culture medium (ampicillin resistance), shaking and culturing for 12-16 hours at 37 ℃ by a shaking table. As shown in FIG. 2, the fluorescent PCR-identified bacterial suspension showed no 1.9kb band, which was negative for VP72 gene.
(4) The kit comprises the following components: the prepared primers and probes and a commercially available Taq enzyme form a PCR reaction solution, and a 1000 mu LPCR reaction solution, 1000 mu L negative control and 20 mu L positive control form a detection kit.
(5) The PCR amplification conditions were optimized experimentally as follows: primary activation at 95 ℃ for 30 sec; denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 30sec, fluorescence signal acquisition, and 45 cycles.
(6) Sensitivity test (detection limit):
using the established PCR detection conditions to carry out the detection on 10-10 pairs5The fluorescence PCR detection is carried out on the African swine fever virus plasmid DNA template with copy number, the result shows that the detection limit reaches 10 copies, and the result is shown in figure 3.
(7) And (3) specific test:
the established real-time fluorescence PCR detection method for the African swine fever virus is used for respectively detecting swine fever live vaccine nucleic acid, highly pathogenic porcine reproductive and respiratory syndrome virus vaccine nucleic acid, pseudorabies live vaccine nucleic acid, porcine epidemic diarrhea virus culture solution nucleic acid, porcine circovirus type 2 culture solution nucleic acid and normal porcine lymph node 10% suspension nucleic acid, and the specificity of the method is determined. The results are all negative, which indicates that the detection method has high specificity, and the African swine fever virus nucleic acid is detected while other virus nucleic acids which are easy to cross-infect are not detected.
(8) The results of the sensitivity comparison with the conventional PCR method:
the general fluorescence PCR detection method for African swine fever viruses established by the invention and the general PCR method are used for 10-10 pairs6Copy number of plasmid DNA was detected and the sensitivity of the 2 methods was compared. The result shows that the sensitivity of the common PCR method can reach 103Copying, the sensitivity of the real-time fluorescence PCR detection method for the African swine fever virus can reach 10 copies, and the result is shown in figure 4.
Example 2
The fluorescence PCR detection method of the African swine fever virus comprises the following specific steps:
firstly, sample collection, storage and transportation
1. Collecting:
1.1 live pig samples 5ml of anticoagulated blood or serum was collected aseptically.
1.2 collecting the spleen, lung, kidney, tonsil, lymph node, muscle and other tissue samples of dead pigs aseptically from the necropsy samples of pigs dead of illness or slaughterhouse.
1.3 collecting excrement, feed and sewage samples of places relevant to sick pigs in the peripheral environment polluted by the sick pigs.
2. And (3) storage: the sample to be tested is preserved for no more than 24 hours at the temperature of 2-8 ℃; the storage at-20 ℃ should not exceed three months; long-term storage at-70 deg.C or below; the sample to be tested should be protected from repeated freezing and thawing.
3. And (3) transportation: and (5) carrying out transportation by adding ice in an ice kettle or adding ice in a foam box and sealing.
Second, sample treatment (sample treatment zone)
If the sample is liquid (blood or serum), 200 microliters (area between the serum and the precipitate) is directly diluted by 5 times with normal saline or purified water, uniformly mixed by shaking, centrifuged for 2 minutes at 3000-. If the liquid sample is saliva, the nasal swab suggests to use a column extraction method or a magnetic bead method to extract the sample DNA, so as to improve the detection accuracy. If the sample is tissue, 5-10mg of tissue (about half of mung bean) is extracted by a nucleic acid releasing agent, centrifugation is carried out for 2 minutes at 3000-5000 revolutions, and the supernatant is taken as a template for direct detection. The negative and positive controls were loaded directly without treatment.
Third, preparation of amplification reagents (preparation zone before PCR)
And (3) taking the ASFV PCR reaction solution out of the kit, melting at room temperature, shaking and uniformly mixing, adding 20 mu L of the ASFV PCR reaction solution into the set n PCR reaction tubes, and transferring the ASFV PCR reaction solution to a sample processing area.
Sample adding (sample processing zone)
And (3) respectively adding the DNA template extracted in the step two, the negative control and the positive control into the set n PCR reaction tubes by 5 mu L, tightly covering tube covers, transferring the PCR reaction tubes to a detection area, placing the PCR reaction tubes on a PCR instrument, and recording the arrangement sequence of the samples.
Fifth, PCR amplification (detection zone)
Setting circulation conditions:
1. 95℃:30sec；
2.95 ℃ 5sec, 60 ℃ 30sec (fluorescence collected): and 45, circulating. The reaction system was 25 ul.
Selecting instrument detection channels: the fluorescence signal was collected and set to HEX (VIC) fluorescein.
Quality control standard
1. The Ct value of the negative control should be equal to none.
2. The Ct value of the positive control should be 32.0 or less. Otherwise, this experiment is considered invalid. (Ct value should be based on the number of cycles at the inflection point of the sigmoid curve).
Seventh, result judgment
Samples with a Ct value of none were negative.
The sample with Ct value less than or equal to 38.0 is positive.
Samples with Ct values greater than 38.0 and less than 42 and typical amplification trends are recommended for rework. And if the Ct value of the redo result is none, the redo result is negative, otherwise, the redo result is positive.
The detection method of the embodiment is adopted to detect and verify clinical negative samples and clinical simulation positive samples
(1) Clinical sample testing
Nucleic acid DNA is extracted from 40 clinical samples, and the extracted nucleic acid is subjected to African swine fever virus nucleic acid detection, wherein the CT value of the detection result is none, and the detection result is negative to the African swine fever virus nucleic acid.
(2) Clinical simulation positive sample test
The African swine fever virus vp72 gene in the prepared clinical simulation positive sample is detected, 31 clinical simulation positive samples prepared according to the result are subjected to fluorescent quantitative PCR detection, and the detection result is shown in table 1, which indicates that the clinical simulation positive samples are qualified in inspection.
TABLE 1 test results for clinical simulation positive samples
Note: "+" indicates that the test result was positive.
(3) Detection and verification of clinical negative sample and clinical simulation positive sample
The detection method provided by the invention is used for detecting 40 clinical negative samples stored in a refrigerator at the temperature of-80 ℃ and 15 extracted clinical simulation positive samples, wherein 55 samples with known negative and positive are counted. The results in Table 2 show that the established method can detect all African swine fever virus positive samples, and false positive results do not occur, which indicates that the established method is reliable and practical.
TABLE 2 test results of clinical samples
Note: "+" indicates a positive test result, and "-" indicates a negative test result.
The results show that the kit can detect the amount of DNA by aiming at the African swine fever virus real-time fluorescence PCR detection method, and the limit reaches 10 copies; the detection results of highly pathogenic porcine reproductive and respiratory syndrome virus vaccine nucleic acid, pseudorabies live vaccine nucleic acid, porcine epidemic diarrhea virus culture solution nucleic acid, porcine circovirus type 2 culture solution nucleic acid and normal porcine lymph node 10% suspension nucleic acid are negative, and no cross reaction is found, which indicates that the method has high sensitivity and strong specificity. Compared with the conventional PCR method recommended by OIE, the fluorescent PCR method established by the invention has the advantages that the sensitivity of the fluorescent PCR can be improved by 100 times compared with the conventional PCR method. False positive and false negative results are not found in the detection of 55 clinical negative samples and clinical simulation positive samples, and the practicability of the method is further verified.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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<120> African swine fever virus fluorescence PCR detection reagent, kit and detection method
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Claims (9)
1. A fluorescent PCR detection reagent for African swine fever virus is characterized in that: comprises specific primers A-F and A-R, probes A-P and Taq enzyme; the 5 'end of the probe is marked with a fluorescence reporter group HEX, and the 3' end of the probe is marked with a fluorescence quenching group BHQ 1;
wherein the specific primers and probe sequences are as follows:
A-F：5′-CCCAGGRGATAAAATGACTG-3′；
A-R：5′-CACTRGTTCCCTCCACCGATA-3′；
A-P：5′-HEX-TCCTGGCCRACCAAGTGCTT-BHQ1-3′。
2. a fluorescence PCR detection kit for African swine fever virus is characterized in that: comprising a PCR reaction solution comprising the detection reagent according to claim 1, and a negative control and a positive control.
3. The African swine fever virus fluorescent PCR detection kit according to claim 2, wherein: the African swine fever virus fluorescent PCR detection kit comprises the following reagents in parts by weight:
nucleic acid amplification reagents:
1000. mu.L of PCR reaction solution;
comparison products:
negative control 1000. mu.L;
positive control 20. mu.L.
4. The preparation method of the African swine fever virus fluorescent PCR detection kit according to any one of claims 2-3, characterized by comprising the following steps:
(1) designing specific primers A-F and A-R and a probe A-P by taking VP72 gene of African swine fever virus as a target gene;
(2) synthesizing VP72 gene of African swine fever virus, cloning to pUC57 vector to obtain pUC57-VP72 recombinant plasmid; then transforming to competent cell culture, extracting plasmid as positive control;
(3) the pUC57 plasmid was transformed into competent cells for culture, and the plasmid was extracted as a negative control.
5. The preparation method of the African swine fever virus fluorescent PCR detection kit according to claim 4, which is characterized in that: the competent cell is DH5 alpha competent cell.
6. A fluorescence PCR detection method of African swine fever virus is characterized by comprising the following steps:
(1) adding the PCR reaction solution into different PCR reaction tubes;
(2) respectively adding a sample to be detected, a negative control and a positive control into different PCR reaction tubes in the step (1), tightly covering a tube cover, moving to a fluorescence PCR instrument for PCR amplification, and collecting a fluorescence signal to set the fluorescence signal as HEX fluorescein;
(3) quality control standard: negative control should have no reported Ct value, and positive control should have a Ct value of 32.0 or less; otherwise, the experiment is regarded as invalid;
(4) and (5) judging a result: samples with no reported Ct value were negative; the sample with the Ct value less than or equal to 38 is positive; the Ct value is more than 38 and less than 42, the sample with the typical amplification curve needs repeated detection, the Ct value is negative without reporting in the repeated detection result, otherwise, the Ct value is positive.
7. The fluorescent PCR detection method for African swine fever virus according to claim 6, wherein the fluorescent PCR detection method comprises the following steps: the amount of the PCR reaction solution added into the PCR reaction tube in the step (1) is 20 muL, and the amount of the sample to be detected, the negative control and the positive control added in the step (2) is 5 muL.
8. The fluorescent PCR detection method for African swine fever virus according to claim 6, wherein the fluorescent PCR detection method comprises the following steps: when the sample to be detected in the step (2) is blood or serum, diluting the sample with normal saline or purified water, centrifuging the diluted sample, taking supernatant as a template, and adding the template into a PCR reaction tube for detection; when the sample to be detected is saliva or a nasal swab, extracting a sample DNA template by adopting a column extraction method or a magnetic bead method, and adding the sample DNA template into a PCR reaction tube for detection; and when the sample to be detected is a tissue, extracting a sample DNA template by adopting a nucleic acid releasing agent, and adding the sample DNA template into a PCR reaction tube for detection.
9. The fluorescent PCR detection method for African swine fever virus according to claim 6, wherein the fluorescent PCR detection method comprises the following steps: the PCR amplification conditions in the step (2) are as follows:
primary activation at 95 ℃ for 30 sec; denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 30sec, fluorescence signal acquisition, and 45 cycles.
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