CN112899398A - Fluorescent PCR (polymerase chain reaction) detection kit for African swine fever virus and use method thereof - Google Patents
Fluorescent PCR (polymerase chain reaction) detection kit for African swine fever virus and use method thereof Download PDFInfo
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Abstract
The invention provides a fluorescence PCR detection kit for African swine fever virus, which comprises a PCR reaction solution A, PCR, a reaction solution B, a negative control, a weak positive control and a strong positive control; the PCR reaction solution A comprises two groups of primer probes and a PCR buffer solution; the primer probe comprises an African swine fever primer probe and an internal standard primer probe; the African swine fever primer probe is designed by taking the gene vp72 of the African swine fever virus genome as a target gene; designing an internal standard primer probe by taking a telomere gene sequence of the pig as a target region; the PCR reaction solution B comprises DNA polymerase and UNG enzyme; the negative control is a recombinant plasmid containing a target gene fragment of a porcine telomere gene; the weak positive control is ASFV plasmid solution with the concentration of 10 copies/mu L; the strong positive control is 10 concentration3copies/. mu.LASFV plasmid solution. The fluorescence of the African swine fever virus of the inventionThe PCR detection kit has the lowest detection limit of 0.5 copy/. mu.L.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a fluorescent PCR detection kit for African swine fever virus and a using method thereof.
Background
African Swine Fever (ASF) is an acute, hemorrhagic and virulent viral infectious disease caused by African Swine Fever Virus (ASFV), which is a type of animal epidemic disease that must be declared by the world animal health Organization (OIE) regulations and causes huge economic loss to countries in the epidemic area.
ASFV is the only arbovirus DNA virus belonging to the genus African swine fever virus (Asfarviridae) of the family African swine fever virus (Asfivirus), and it has some characteristics similar to those of iridoviridae and poxviridae. ASF is clinically characterized by high fever, extensive bleeding of internal organs, splenomegaly and black color, and is one of the most serious viral diseases affecting the pig industry. Currently, there is no effective vaccine and specific therapeutic drug for preventing and treating ASF for african swine fever, and therefore, it poses a significant threat to the swine industry and global food safety.
The diagnosis technology of African swine fever virus is mainly divided into virus antigen detection and specific antibody detection. The incubation period of ASFV contact infection is 4-19d, the first 2d infected animals with clinical symptoms begin to spread the virus, and the 7-9d serum after infection turns positive. It is demonstrated that early diagnosis of ASF is more important for monitoring antigens than for detecting antibodies. In order to rapidly detect the antigen, molecular diagnostic techniques have become the main diagnostic tool.
However, since the wild strain of African swine fever virus can only proliferate in primary pig cells and the isolation and culture time is long, according to the management requirement of Ministry of agriculture on the experimental activities of pathogenic microorganisms of highly pathogenic animals, the wild strain of African swine fever virus needs to be carried out in a P3-grade laboratory under the condition of biological safety protection, and many local farms do not have such detection experimental conditions. Therefore, the detection of African swine fever virus is mainly based on nucleic acid PCR detection technology at present.
PCR detection techniques are divided into fluorescence quantitative PCR and common PCR. Compared with the common PCR, the fluorescence quantitative PCR has the advantages of no need of an electrophoresis process, stronger specificity, effective solution of the PCR pollution problem, avoidance of harm brought to experimenters by organic solvents, more suitability for epidemic disease monitoring and mainstream direction of epidemic disease detection.
A great number of researchers have studied the fluorescent quantitative PCR detection method of African swine fever, and constructed the detection method of various virus genes. In 2003, King and the like use a TaqMan probe to construct a real-time PCR detection method for detecting African swine fever for the first time, and introduce an OIE manual in 2012, wherein the method takes VP72 as a target point, and the detection limit is 10-100 copies/mu L. In 2020, Rongcaixia and the like design TaqMan fluorescent PCR primers and probes according to an ASFV CD2v gene sequence, the detection limit is 10 copies/mu L, and the TaqMan fluorescent PCR primers and probes do not have cross reaction with 5 swine disease viruses such as pseudorabies virus, porcine parvovirus, porcine circovirus type 2, classical swine fever virus, porcine reproductive and respiratory syndrome virus and the like. In 2020, primers and probes are designed and synthesized by the machilus pauhoi and the like according to a conserved sequence of an encoding gene D117L of an ASFV SY18 strain p17 protein, a TaqMan fluorescence quantitative PCR detection method is established, the detection limit is 10 copies/mu L, and the TaqMan fluorescence quantitative PCR detection method has no cross reaction with 4 swine fever viruses, porcine reproductive and respiratory syndrome viruses, porcine epidemic diarrhea viruses, pseudorabies viruses and the like. In 2020, 1 pair of specific primers and 1 specific probe are designed and synthesized by Wu ya Mac et al according to the related gene sequence of ASFV P27, a TaqMan real-time fluorescence quantitative PCR method is established, the detection limit is 10 copies/mu L, and the TaqMan real-time fluorescence quantitative PCR method does not generate cross reaction with 4 porcine disease pathogens such as porcine epidemic diarrhea virus, porcine delta coronavirus, highly pathogenic porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus and the like. The fluorescence PCR detection technology can detect ASFV, but the minimum detection limit is 10 copies/mu L, the sensitivity is not enough, the pathogen participating in the specificity research is too few, and the specificity is not strong. Therefore, it is urgently needed to develop a fluorescent quantitative PCR detection method capable of detecting and accurately extracting teeth in advance.
Disclosure of Invention
The invention aims to solve the technical problem that the sensitivity of a kit for detecting African swine fever viruses in the prior art is low, and provides a fluorescent PCR detection kit for African swine fever viruses and a use method thereof, wherein the minimum detection limit can reach 0.5 copy/mu L.
The invention provides a fluorescence PCR detection kit for African swine fever virus, which comprises a PCR reaction solution A, PCR, a reaction solution B, a negative control, a weak positive control and a strong positive control;
the PCR reaction solution A comprises two groups of primer probes and a PCR buffer solution; the primer probe comprises an African swine fever primer and an internal standard primer; the African swine fever primer probe is designed by taking the gene vp72 of the African swine fever virus genome as a target gene; designing an internal standard primer probe by taking a telomere gene sequence of the pig as a target region;
the PCR reaction solution B comprises DNA polymerase and UNG enzyme;
negative control is recombinant plasmid solution containing internal standard, namely target gene fragment of the porcine telomere gene;
the weak positive control is ASFV plasmid solution with the concentration of 10 copies/mu L;
the strong positive control is 10 concentration3copies/. mu.L ASFV plasmid solution.
The fluorescence PCR detection kit for African swine fever virus, as a preferred mode,
the African swine fever primer comprises:
an upstream primer: 5'-AAAAGCGCAACTTAATCCA-3', respectively;
a downstream primer: 5'-ATTACAGCTGTAATGGACCTCA-3', respectively;
and (3) probe: 5 '-FAM-AGCGCAAGAGGGGGCT-BHQ 1-3';
the internal standard primer comprises:
an upstream primer 5'-CCTACTGGCCGTAGCATTCC-3';
a downstream primer 5'-GAGGATGTGGCTGGTCGTAG-3';
probe 5 '-ROX-GCGGCTACGGGATTTTGATAAGT-BHQ 2-3'.
The use method of the fluorescence PCR detection kit for African swine fever virus, which is disclosed by the invention, as a preferred mode, comprises the following steps:
s1, DNA sample collection: extracting a DNA sample from an experimental sample;
s2, preparation of PCR reagents: weighing PCR reaction liquid A and PCR reaction liquid B in a kit according to a volume ratio of 14:1, uniformly mixing, and subpackaging into PCR reaction tubes to obtain a PCR reagent;
s3, sample adding: respectively adding a DNA sample with the same volume as the PCR reagent, a negative control and a positive control into a PCR reaction tube, and carrying out vortex centrifugation for 2-10 s;
s4, PCR amplification detection: carrying out amplification detection on the PCR reaction tube on a fluorescent PCR instrument;
s5, judging quality: whether the experimental result is valid is judged according to the following conditions,
judging a first condition: negative control FAM channel has no signal, and the Ct value of ROX channel is less than 35;
and judging a second condition: the Ct value of the weak positive control FAM channel is less than or equal to 36, and the ROX channel has no signal;
judging a third condition: the Ct value of the strong positive control FAM channel is less than or equal to 26, and the ROX channel has no signal;
if the above three determination conditions are satisfied simultaneously, the experimental result is yes, and the process proceeds to step S6; when any one of the two judgment conditions is not met, judging the experimental result to be no, and returning to the step S3;
s6, judging the result:
positive judgment conditions: the Ct value of the FAM channel is less than or equal to 38 and an amplification curve is formed;
negative judgment conditions: the FAM channel has no signal, and the Ct value of the ROX channel is less than 35, so that an amplification curve is obtained.
As a preferred mode, in the step S1, the experimental samples comprise serum, plasma, anticoagulation blood, lymph nodes, spleen, tonsil, muscle, lung, liver, oral fluid and environmental samples.
In the method for using the fluorescence PCR detection kit for African swine fever virus, as a preferred mode, the step S6 further comprises the following judgment processes:
and (3) judging conditions by combining samples: the FAM channel and the ROX channel both have no signal, and the process proceeds to step S7;
rechecking judgment conditions: FAM channel 38 < Ct value < 40, and proceeds to step S8.
The use method of the fluorescence PCR detection kit for African swine fever virus, which is disclosed by the invention, as a preferred mode, further comprises the following steps:
s7, if the experimental sample is serum, plasma, anticoagulation blood, lymph node, spleen, tonsil, muscle, lung and liver, the judgment result is invalid, and the step S1 is returned; if the experimental sample is oral liquid, prompting that the quality of the collected sample has a problem or the nucleic acid is possibly degraded, and returning to the step S1; if the experimental sample is the environmental sample, judging that the result is invalid, and returning to the step S1;
s8, rechecking the sample, and if the Ct value of the FAM channel is less than 40, judging the sample to be positive; if the FAM channel is rechecked and has no Ct value, the FAM channel is judged to be negative.
The application method of the fluorescence PCR detection kit for African swine fever virus, which is disclosed by the invention, is a preferable mode, and the minimum detection limit of the fluorescence PCR detection kit is 0.5 copy/mu L.
The invention has the beneficial effects that:
(1) the sensitivity is higher, and the lowest detection limit can reach 0.5 copy/mu L;
(2) and the internal standard containing the pig genome is added, the ASFV target gene and the internal standard pig gene are amplified simultaneously, and the quality of the whole detection process can be monitored.
Drawings
FIG. 1 is a flow chart of a method for using a fluorescent PCR detection kit for African swine fever virus.
FIG. 2 shows the results of the sensitivity test in example 3;
FIG. 3 shows the results of the sensitivity test of 30.5 copy/. mu.L positive sample in example;
FIG. 4 shows the results of the sensitivity test of 30.1 copy/. mu.L positive sample in example.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
A fluorescence PCR detection kit for African swine fever virus comprises a PCR reaction solution A, PCR, a negative control, a weak positive control and a strong positive control;
the PCR reaction solution A comprises two groups of primer probes and a PCR buffer solution; the primer probe comprises an African swine fever primer probe and an internal standard primer probe; the African swine fever primer probe is designed by taking the gene vp72 of the African swine fever virus genome as a target gene; designing an internal standard primer probe by taking a telomere gene sequence of the pig as a target region; wherein
The African swine fever primer comprises:
an upstream primer: 5'-AAAAGCGCAACTTAATCCA-3', respectively;
a downstream primer: 5'-ATTACAGCTGTAATGGACCTCA-3', respectively;
and (3) probe: 5 '-FAM-AGCGCAAGAGGGGGCT-BHQ 1-3';
the internal standard primer comprises:
an upstream primer 5'-CCTACTGGCCGTAGCATTCC-3';
a downstream primer 5'-GAGGATGTGGCTGGTCGTAG-3';
probe 5 '-ROX-GCGGCTACGGGATTTTGATAAGT-BHQ 2-3'
The PCR reaction solution B comprises DNA polymerase and UNG enzyme;
negative control is a recombinant plasmid containing an internal standard, namely a pig telomere gene target gene fragment;
the weak positive control is ASFV plasmid solution with the concentration of 10 copies/mu L;
the strong positive control is 10 concentration3copies/. mu.L ASFV plasmid solution.
As shown in fig. 1, the present embodiment includes the following steps:
s1, DNA sample collection: extracting a DNA sample from an experimental sample, wherein the experimental sample is one of serum, plasma, anticoagulation blood, lymph node, spleen, tonsil, muscle, lung, liver, oral fluid and environmental sample;
s2, preparation of PCR reagents: weighing 14 microliter of PCR reaction solution A and 1 microliter of PCR reaction solution B in the kit, uniformly mixing, and subpackaging into PCR reaction tubes to obtain a PCR reagent;
s3, sample adding: adding 15 mu L of DNA sample, negative control and positive control into a PCR reaction tube respectively, and carrying out vortex centrifugation for 2-10 s;
s4, PCR amplification detection: carrying out amplification detection on the PCR reaction tube on a fluorescent PCR instrument; the concentration of an upstream primer and a downstream primer of the African swine fever primer is 400nmol/L, and the concentration of a probe is 200 nmol/L; the concentrations of an upstream primer and a downstream primer of the internal standard primer are 200nmol/L, and the concentration of a probe is 100 nmol/L;
the cycle parameters for PCR amplification detection were set as follows:
s5, judging quality: whether the experimental result is valid is judged according to the following conditions,
judging a first condition: negative control FAM channel has no signal, and the Ct value of ROX channel is less than 35;
and judging a second condition: the Ct value of the weak positive control FAM channel is less than or equal to 36, and the ROX channel has no signal;
judging a third condition: ct value of the strong contrast FAM channel is less than or equal to 26, and the ROX channel has no signal;
if the above three determination conditions are satisfied simultaneously, the experimental result is yes, and the process proceeds to step S6; when any one of the two judgment conditions is not met, judging the experimental result to be no, and returning to the step S3;
s6, judging the result:
positive judgment conditions: the Ct value of the FAM channel is less than or equal to 38 and an amplification curve is formed;
negative judgment conditions: the FAM channel has no signal, and the Ct value of the ROX channel is less than 35, so that an amplification curve is obtained.
Example 2
The embodiment comprises the following steps:
s1, DNA sample collection: extracting a DNA sample from an experimental sample, wherein the experimental sample is one of serum, plasma, anticoagulation blood, lymph node, spleen, tonsil, muscle, lung, liver, oral fluid and environmental sample;
s2, preparation of PCR reagents: weighing 14 microliter of PCR reaction solution A and 1 microliter of PCR reaction solution B in the kit, uniformly mixing, and subpackaging into PCR reaction tubes to obtain a PCR reagent;
s3, sample adding: adding 15 mu L of DNA sample, negative control and positive control into a PCR reaction tube respectively, and carrying out vortex centrifugation for 2-10 s;
s4, PCR amplification detection: carrying out amplification detection on the PCR reaction tube on a fluorescent PCR instrument; the concentration of an upstream primer and a downstream primer of the African swine fever primer is 400nmol/L, and the concentration of a probe is 200 nmol/L; the concentrations of an upstream primer and a downstream primer of the internal standard primer are 200nmol/L, and the concentration of a probe is 100 nmol/L;
the cycle parameters for PCR amplification detection were set as follows:
s5, judging quality: whether the experimental result is valid is judged according to the following conditions,
judging a first condition: negative control FAM channel has no signal, and the Ct value of ROX channel is less than 35;
and judging a second condition: the Ct value of the weak positive control FAM channel is less than or equal to 36, and the ROX channel has no signal;
judging a third condition: the Ct value of the strong positive control FAM channel is less than or equal to 26, and the ROX channel has no signal;
if the above three determination conditions are satisfied simultaneously, the experimental result is yes, and the process proceeds to step S6; when any one of the two judgment conditions is not met, judging the experimental result to be no, and returning to the step S3;
s6, judging the result:
positive judgment conditions: the Ct value of the FAM channel is less than or equal to 38 and an amplification curve is formed;
negative judgment conditions: the FAM channel has no signal, and the Ct value of the ROX channel is less than 35, and an amplification curve is formed;
and (3) judging conditions by combining samples: the FAM channel and the ROX channel both have no signal, and the process proceeds to step S7;
rechecking judgment conditions: the FAM channel 38 < Ct value < 40, and the step S8 is entered;
s7, if the experimental sample is serum, plasma, anticoagulation blood, lymph node, spleen, tonsil, muscle, lung and liver, the judgment result is invalid, and the step S1 is returned; if the experimental sample is oral liquid, prompting that the quality of the collected sample has a problem or the nucleic acid is possibly degraded, and returning to the step S1; if the experimental sample is the environmental sample, judging that the result is invalid, and returning to the step S1;
s8, rechecking the sample, and if the Ct value of the FAM channel is less than 40, judging the sample to be positive; if the FAM channel is rechecked and has no Ct value, the FAM channel is judged to be negative.
Example 3
And (3) sensitivity detection: samples were diluted to 4 gradients for experimental detection. And (4) repeatedly detecting for 20 times by using two gradients near the detection limit respectively to determine the sensitivity of the kit. The results of the experiments are shown in the following table,
| concentration of | The result of the detection |
| 100copies/μL | Positive for |
| 10copies/μL | Positive for |
| 1copy/μL | Positive for |
| 0.5copy/μL | Positive for |
| 0.1copy/μL | Positive/negative |
As shown in FIG. 2, the detection results of the positive samples in the gradient dilution indicate that all the positive samples with the concentration of 1-100 copies/. mu.L can be detected to be positive; at a concentration of 102All positive samples of copies/mL could not be detected as positive.
As shown in FIG. 3, the result was positive in the case of 0.5 copy/. mu.L positive sample by repeating the test 10 times.
As shown in FIG. 4, the results of repeated tests of 0.1 copy/. mu.L positive samples were not all positive for 10 times.
The positive sample is positive after repeated detection for 10 times of 0.5 copy/. mu.L, so the detection limit of the kit is set.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A fluorescence PCR detection kit for African swine fever virus is characterized in that: comprises a PCR reaction solution A, PCR reaction solution B, a negative control, a weak positive control and a strong positive control;
the PCR reaction solution A comprises two groups of primer probes and a PCR buffer solution; the primer probe comprises an African swine fever primer probe and an internal standard primer probe; the African swine fever primer probe is designed by taking the gene vp72 of the African swine fever virus genome as a target gene; the internal standard primer probe is designed by taking a telomere gene sequence of the pig as a target region;
the PCR reaction solution B comprises DNA polymerase and UNG enzyme;
the negative control is a recombinant plasmid solution containing an internal standard, and the internal standard is a target gene fragment of a porcine telomere gene;
the weak positive control is ASFV plasmid solution with the concentration of 10 copies/mu L;
the strong positive control is at a concentration of 103copies/. mu.L ASFV plasmid solution.
2. The fluorescence PCR detection kit for African swine fever virus according to claim 1, wherein the fluorescence PCR detection kit comprises:
the African swine fever primer comprises:
an upstream primer: 5'-AAAAGCGCAACTTAATCCA-3', respectively;
a downstream primer: 5'-ATTACAGCTGTAATGGACCTCA-3', respectively;
and (3) probe: 5 '-FAM-AGCGCAAGAGGGGGCT-BHQ 1-3';
the internal standard primer comprises:
an upstream primer 5'-CCTACTGGCCGTAGCATTCC-3';
a downstream primer 5'-GAGGATGTGGCTGGTCGTAG-3';
probe 5 '-ROX-GCGGCTACGGGATTTTGATAAGT-BHQ 2-3'.
3. The use method of the fluorescence PCR detection kit for African swine fever virus according to any one of claims 1-2, comprising the following steps:
s1, DNA sample collection: extracting a DNA sample from an experimental sample;
s2, preparation of PCR reagents: weighing the PCR reaction solution A and the PCR reaction solution B according to a volume ratio of 14:1 in the kit, uniformly mixing, and subpackaging into PCR reaction tubes to obtain the PCR reagent;
s3, sample adding: respectively adding the DNA sample, the negative control and the positive control with the same volume as the PCR reagent into the PCR reaction tube, and carrying out vortex centrifugation for 2-10 s;
s4, PCR amplification detection: carrying out amplification detection on the PCR reaction tube on a fluorescent PCR instrument;
s5, judging quality: whether the experimental result is valid is judged according to the following conditions,
judging a first condition: the negative control FAM channel has no signal, and the Ct value of the ROX channel is less than 35;
and judging a second condition: the Ct value of the weak positive control FAM channel is less than or equal to 36, and the ROX channel has no signal;
judging a third condition: the Ct value of the strong positive control FAM channel is less than or equal to 26, and the ROX channel has no signal;
if the above three determination conditions are satisfied simultaneously, the experimental result is yes, and the process proceeds to step S6; when any one of the two judgment conditions is not met, judging the experimental result to be no, and returning to the step S3;
s6, judging the result:
positive judgment conditions: the Ct value of the FAM channel is less than or equal to 38 and an amplification curve is formed;
negative judgment conditions: the FAM channel has no signal, and the Ct value of the ROX channel is less than 35, so that an amplification curve is obtained.
4. The use method of the African swine fever virus fluorescent PCR detection kit according to claim 3, characterized in that: the experimental sample in the step S1 includes serum, plasma, anticoagulation blood, lymph node, spleen, tonsil, muscle, lung, liver, oral fluid, and environmental sample.
5. The use method of the African swine fever virus fluorescent PCR detection kit according to claim 4, characterized in that: step S6 further includes the following judgment process:
and (3) judging conditions by combining samples: the FAM channel and the ROX channel both have no signal, and the process proceeds to step S7;
rechecking judgment conditions: FAM channel 38 < Ct value < 40, and proceeds to step S8.
6. The use method of the African swine fever virus fluorescent PCR detection kit according to claim 5, characterized in that: further comprising the steps of:
s7, if the experimental sample is serum, plasma, anticoagulation blood, lymph node, spleen, tonsil, muscle, lung and liver, the judgment result is invalid, and the step S1 is returned; if the experimental sample is oral liquid, prompting that the quality of the collected sample has a problem or the nucleic acid is possibly degraded, and returning to the step S1; if the experimental sample is the environmental sample, judging that the result is invalid, and returning to the step S1;
s8, rechecking the sample, and if the Ct value of the FAM channel is less than 40, judging the sample to be positive; if the FAM channel is rechecked and has no Ct value, the FAM channel is judged to be negative.
7. The use method of the African swine fever virus fluorescent PCR detection kit according to claim 3, characterized in that: the minimum detection limit of the fluorescent PCR detection kit is 0.5 copy/. mu.L.
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