CN113981137B - Real-time fluorescence PCR primer, detection kit and application of African swine fever virus A137R gene - Google Patents
Real-time fluorescence PCR primer, detection kit and application of African swine fever virus A137R gene Download PDFInfo
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Abstract
The application discloses a fluorescent quantitative PCR kit for detecting African swine fever virus and application thereof. The fluorescent quantitative PCR kit contains primers shown in SEQ ID NO.1 and SEQ ID NO.2, a fluorescent quantitative PCR reaction reagent, a standard substance and a reference substance. The fluorescence quantitative PCR kit can detect African swine fever virus, has strong specificity, is only specifically combined with African swine fever virus, and has no cross reaction with other swine viruses; the sensitivity of the detection is high, and the standard substance is 1 multiplied by 10 1 ～1×10 8 The copy range has excellent linear relation, so that the African swine fever virus disease pig cotton swab and the virus carrying pig without clinical symptoms can be efficiently detected, and the detection efficiency is improved.
Description
Technical Field
The application belongs to the technical field of virus detection, and particularly relates to a fluorescence PCR primer, a detection kit and application of an African swine fever virus A137R gene.
Background
African Swine Fever (ASF) is an acute, virulent, highly contagious animal infectious disease caused by African Swine Fever Virus (ASFV), and the world animal health Organization (OIE) lists ASF as a legally reported animal epidemic, which is also listed as a major precaution animal epidemic in China. The disease has the advantages of short disease incidence process, wide infection range, high morbidity and mortality reaching 100 percent, great harm to pig industry, serious influence on the development of pig industry in China and international folk life, no effective method for controlling African swine fever at present, and the detection and elimination of infected animals are the only methods for controlling and eradicating ASF.
The A137R gene of ASFV can code p11.5 protein and is an important structural protein of ASFV. There are studies showing that p11.5 protein is expressed in high abundance in three susceptible cell lines that infect ASFV, suggesting that it has an important role in the viral replication cycle. Compared with the traditional quantitative technology (such as semi-quantitative PCR, competitive quantitative PCR and the like), the fluorescent quantitative PCR method has the advantages of good repeatability, special sensitivity, accurate quantification, simple and convenient operation, small pollution to samples, high automation degree and the like. The currently reported fluorescent quantification method is mainly designed for the African swine fever virus VP72, P54 and K205R genes, however, recent researches show that the conventional PCR sensitivity and specificity recommended by OIE are reduced, and presumably caused by nucleotide mismatch between the primer and the virus target genes.
Therefore, it is highly desirable to create a new ASFV fluorescent quantitative PCR detection reagent product and method that enables reliable detection of currently popular ASFV strains. A fluorescence detection reagent product for detecting African swine fever virus A137R gene and a using method thereof are not reported yet.
Disclosure of Invention
The application aims to: the application aims to solve the technical problem of providing a primer pair.
The application also solves the technical problem of providing a kit for detecting African swine fever virus.
The application finally solves the technical problem of a real-time fluorescence quantitative PCR detection method based on the coding gene A137R of ASFV P11.5 protein for non-diagnosis.
In order to achieve the above purpose, the application adopts the following technical scheme: in a first aspect of the application, a primer pair for ASFV fluorescent quantitative PCR detection is provided, the amplification target length is 116bp, and the nucleotide sequence of the primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2. The method comprises the following steps:
forward primer: p11.5-F:5'-CTTACCAAACTCGACCAGGAGG-3'; (SEQ ID NO. 1)
Reverse primer: p11.5-R:5'-GTGCATCGTTCCTCAGGGATT-3'; (SEQ ID NO. 2)
Further, an ASFV PCR amplification primer pair is provided, the primer pair is used for amplifying the full length of the coding gene A137R of ASFV P11.5 protein, the length of an amplification target product is 414bp, the sequence of the target product is shown as SEQ ID NO.9, and the nucleotide sequence of the primer pair is shown as SEQ ID NO.3-SEQ ID NO. 4. The method comprises the following steps:
forward primer: p11.5-F2:5'-ATGGAAGCAGTTCTTACCAAACTCG-3'; (SEQ ID NO. 3)
Reverse primer: p11.5-R2:5'-TTAGCCTTCTTTGATATTCATCTTGCC-3'; (SEQ ID NO. 4)
In a second aspect of the application, there is provided the use of the primer pair described above in the preparation of a kit for detecting African swine fever virus.
In a third aspect of the present application, there is provided a kit for fluorescent quantitative PCR detection comprising the primer pair described above.
Further, the fluorescent quantitative PCR detection kit further comprises: fluorescent quantitative PCR reaction reagent, standard substance and reference substance.
Wherein the fluorescent quantitative PCR reaction reagent comprises SYBR-Green fluorescent dye and RNase Free dH 2 O。
Wherein, the standard substance is prepared by the following method: PCR amplification is carried out by taking DNA of African swine fever virus as a template and using primers shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplification product, the length of the amplification product is 414bp, the target product sequence is shown in SEQ ID NO.9, gel electrophoresis is carried out on the amplification product, gel recovery and purification are carried out on the amplification product, the purified amplification product is connected with a pGEM-T carrier, the amplification product is converted into DH5 alpha competent cells, positive clones are screened, plasmid DNA is extracted, and the standard product is prepared.
Wherein the reference substances are divided into a positive reference substance and a negative reference substance, the positive reference substance is a DNA sample with ASFV DNA, and the negative reference substance is a DNA sample without ASFV DNA.
In a fourth aspect of the present application, there is provided the use of the above-described fluorescent quantitative PCR detection kit in any one of the following (1) to (3):
(1) Epidemiological investigation of African swine fever virus;
(2) Monitoring african swine fever virus contamination in blood or serum products;
(3) And (3) accurately detecting the copy number of the African swine fever virus and determining the infection process of the African swine fever virus.
In a fifth aspect, the present application provides a method for detecting african swine fever virus using the fluorescent quantitative PCR kit for non-diagnostic purposes, comprising the steps of:
(1) Carrying out gradient dilution on the standard substance, then carrying out fluorescence quantitative PCR reaction, and drawing a fluorescence quantitative standard curve according to the concentration and Ct value of the standard substance;
(2) Extracting genome DNA of a sample to be detected, performing fluorescent quantitative PCR reaction, collecting fluorescent signals of the sample to be detected, performing data processing on the fluorescent signals to obtain Ct values and amplification curves, and performing qualitative and quantitative detection on the sample to be detected, wherein the specific judging method comprises the following steps of: if the fluorescence signal of the sample to be detected exceeds a threshold value and Ct is less than or equal to 35.0, judging positive, otherwise judging negative.
Preferably, in step (1) and step (2), the procedure of the fluorescent quantitative PCR reaction is: pre-denaturation at 95℃for 30sec, 5sec at 95℃for 60℃for 30sec,40 cycles.
The beneficial effects are that: compared with the prior art, the application has the following advantages:
(1) Aiming at African swine fever virus, the application designs a fluorescent quantitative PCR kit capable of detecting the virus, which has strong specificity and is only specifically combined with African swine fever virus and does not have cross reaction with other common swine-origin viruses, such as Porcine Epidemic Diarrhea Virus (PEDV), porcine rotavirus (PoRV), transmissible gastroenteritis virus (TGEV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV 2), swine fever virus (CSFV) and the like.
(2) The sensitivity of the detection of the application is 1X 10 in the standard substance 1 ～1×10 8 Has excellent linear relation in the copying range and can be highThe method can effectively detect the African swine fever virus disease pig tissue and virus-carrying pigs without clinical symptoms, and improves the detection efficiency.
(3) The kit can monitor African swine fever virus infection so as to discover that an infected pig group adopts isolation or killing measures to reduce the loss caused by African swine fever virus infection; epidemiological investigation of African swine fever virus and monitoring of African swine fever virus contamination in blood or serum preparations can also be performed.
Drawings
FIG. 1 is an amplification curve of a real-time fluorescent quantitative PCR standard.
FIG. 2 is a real-time fluorescent quantitative PCR standard curve.
FIG. 3 is a specific assay of the real-time fluorescent quantitative PCR method.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
For fluorescent quantitative PCR detection, how to design the primer sequences is critical to ensure the effectiveness of the detection. Although there are many primer design software and primer design principles in the prior art, the primer with strong specificity and high sensitivity is designed and obtained simply not through the primer design software, which requires the skilled person to continuously select and optimize the target area by using the professional knowledge, then design the primer according to the optimized target area, and repeatedly screen, optimize and redesign the designed primer. The application optimizes the target area for primer design in the test process, and uses the sequence conservation area of the A137R gene of the African swine fever virus for primer design, thereby realizing the specificity detection of the virus. The African swine fever virus of the application is from animal biosafety third-level laboratory of vinca veterinary research institute of China academy of agricultural sciences.
In the design process, the reaction conditions of the common PCR and the fluorescent quantitative PCR are further fully considered, the mutual interference between the primers is avoided, and finally the primers are designed. Wherein, the fluorescent quantitative PCR detection primer:
forward primer: p11.5-F:5'-CTTACCAAACTCGACCAGGAGG-3'; (SEQ ID NO. 1)
Reverse primer: p11.5-R:5'-GTGCATCGTTCCTCAGGGATT-3'; (SEQ ID NO. 2)
In addition, common PCR amplification primers:
forward primer: p11.5-F2:5'-ATGGAAGCAGTTCTTACCAAACTCG-3'; (SEQ ID NO. 3)
Reverse primer: p11.5-R2:5'-TTAGCCTTCTTTGATATTCATCTTGCC-3'; (SEQ ID NO. 4)
In the test process, a plurality of groups of different primer pairs are designed according to other conserved region sequences of African swine fever virus A137R genes and other primer design principles, and the specificity and the amplification efficiency of the primer pairs are respectively detected after reaction so as to screen optimal primers for clinical detection. For example:
a first group:
forward primer: f:5'-TGAAATCCCTGAGGAACGATGCAC-3'; (SEQ ID NO. 5)
Reverse primer: r:5'-AACTTCATGCCAGGCGGTGTG-3'; (SEQ ID NO. 6)
Second group:
forward primer: f:5'-CATTTCGGGACATCGAGTGG-3'; (SEQ ID NO. 7)
Reverse primer: r:5'-ATCTTGCCGATGAGATTTCCCT-3'; (SEQ ID NO. 8)
As a result, it was found that the specificity and sensitivity of detection of the African swine fever virus using the primers of the present application (SEQ ID NO.1 and SEQ ID NO. 2) were optimal. The other primer pairs (SEQ ID NO.5, 6, 7 and 8) cannot effectively distinguish the African swine fever virus from other swine viruses, false positives or false negatives are easy to occur, such that amplification curves appear when the first group of primers (SEQ ID NO.5 and 6) detect the porcine rotavirus (PoRV), and the dissolution curves of the second group of primers (SEQ ID NO.7 and 8) have impurity peaks, and the products are nonspecific. SEQ ID NO.1 and SEQ ID NO.2 were therefore selected as detection primers for the present kit.
The application optimizes the fluorescent quantitative condition in the test process, reduces the operation to the maximum extent in the clinical application of the kit, ensures the sensitivity, reduces various pollution to the maximum extent and ensures the scientific and accurate result.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, the technical scheme of the present application will be described in detail with reference to specific embodiments.
The test materials used in the examples of the present application, which are not specifically described, are all conventional in the art and are commercially available. Specific experimental conditions and methods are not noted in the embodiment of the application, and the experimental guidelines (third edition) of molecular cloning are generally adopted according to conventional conditions, such as sambrook and other main editions, scientific press, 2002; D.L spelt et al, scientific press, 2001, guidance on cell experiments; or according to manufacturer recommended conditions.
Example 1: design of real-time fluorescent quantitative PCR primer
Specific primer sequences are designed aiming at the sequence conservation region of the coding gene A137R of the ASFV P11.5 protein, and the sequences are designed as follows:
forward primer: p11.5-F:5'-CTTACCAAACTCGACCAGGAGG-3'; (SEQ ID NO. 1)
Reverse primer: p11.5-R:5'-GTGCATCGTTCCTCAGGGATT-3'; (SEQ ID NO. 2)
Example 2: establishment of fluorescent quantitative PCR detection method
(1) Extraction of viral genomic DNA:
and extracting genome DNA of the sample to be detected by adopting a commercialized kit.
(2) And (3) establishing a fluorescent quantitative PCR reaction system:
the fluorescence quantification kit used was TB with the product number RR820A from Takara Corp Premix Ex Taq TM Reagent II, TB Green Premix Ex Taq II mu are added into 200 mu l fluorescent reaction tube in sequencel PCR Forward Primer (10. Mu.M) 0.8. Mu.l, PCR Reverse Primer (10. Mu.M) 0.8. Mu.l, 2. Mu.l of the genomic DNA of the sample to be tested extracted in step (1) were extracted using RNase Free dH 2 O was supplemented to 20. Mu.l system. Put in Light->96 (Roche Diagnostics) the reaction was carried out in a fluorescent quantitative PCR apparatus under conditions of 95℃for 30sec,95℃for 5sec,60℃for 30sec, and 40 cycles.
(3) Preparation of a standard curve:
in order to accurately quantify the copy number of the virus in the sample, a plasmid containing the amplified fragment of interest is prepared as a standard to draw a standard curve. Firstly, designing forward and reverse primers (shown as SEQ ID NO.3-SEQ ID NO. 4) of an A137R gene, amplifying an African swine fever virus genome to obtain an amplified product (shown as SEQ ID NO. 9) of 414bp, connecting the amplified product to a pGEM-T vector according to a classical molecular cloning operation method, naming the product as P11.5-T, screening positive clones, extracting plasmid DNA, and measuring the plasmid concentration by using a spectrophotometer to obtain a standard product. Performing 10-time gradient dilution on the standard substance, performing fluorescence quantitative PCR reaction, and drawing a fluorescence quantitative standard curve according to the concentration and Ct value of the standard substance; the method comprises the following steps: y= -3.276x+37.08, correlation coefficient R 2 =0.9987, exhibiting good linearity; where x represents the log of the copy number of the sample and Y represents the Ct value.
(4) And (3) judging a detection result:
and collecting fluorescent signals according to fluorescent quantitative PCR reaction, and processing data by using instrument software to obtain an amplification curve and Ct value. And taking a Ct value and an amplification curve corresponding to the lowest concentration of the standard substance as a judgment basis.
The result judging method comprises the following steps: if the fluorescence signal of the sample to be detected exceeds the threshold value and Ct is less than or equal to 35.0, and the amplification curve is smooth S-shaped, the sample to be detected can be judged to contain African swine fever virus.
According to the fluorescent quantitative standard curve, the African swine fever virus in the sample can be quantitatively measured.
Example 3: composition of the kit, optimization of experimental parameters, and investigation of specificity, sensitivity and repeatability
1. Composition of the kit:
the kit of the embodiment is a fluorescent quantitative kit and is used for detecting African swine fever virus. The kit comprises: forward primer (10. Mu.M) shown in SEQ ID No.1, reverse primer (10. Mu.M) shown in SEQ ID No.2, fluorescent quantitative PCR reagents, standards and controls were designed for example 1.
The standard substance is prepared by the following method:
amplifying genome of African swine fever virus by using primers shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplified product (SEQ ID NO. 9) of 414bp, connecting the amplified product to a pGEM-T vector, screening positive clones, and extracting plasmid DNA to obtain a standard product.
The fluorescent quantitative PCR reaction reagent comprises: SYBR-Green fluorescent dye and RNase Free dH 2 O。
The reference substances are positive reference substances and negative reference substances, wherein the positive reference substances are DNA samples with ASFV DNA, and the negative reference substances are DNA samples without ASFV DNA.
2. Optimization of experimental parameters
The fluorescent quantitative PCR reaction conditions of the primers (SEQ ID NO.1 and SEQ ID NO. 2) in the kit are optimized and explored, and the final concentration of the primers is respectively 0.2 mu M, 0.4 mu M, 0.6 mu M and 0.8 mu M, so that the result shows that the amplification efficiency is highest and is closest to 100% when the final concentration of the primers is 0.4 mu M, and therefore, 0.4 mu M is selected as the optimal final concentration of the primers. The annealing temperature was amplified at 58 ℃,60 ℃ and 62 ℃ respectively, and the results show that the annealing temperature was the best specific at 60 ℃, so 60 ℃ was selected as the best annealing temperature. The final reaction conditions are 95 ℃ pre-denaturation 30sec,95 ℃ 5sec,60 ℃ 30sec and 40 cycles, and the sensitivity and the detection effect of the kit are best.
3. Specificity, sensitivity and repeatability investigation of the kit
(1) Specificity test
The kit is used for carrying out a specificity test, and the kit is used for carrying out fluorescent quantification by taking DNA samples of Porcine Epidemic Diarrhea Virus (PEDV), porcine rotavirus (PoRV), porcine transmissible gastroenteritis virus (TGEV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV 2), swine fever virus (CSFV), african virus detected by the application and ASFV-free DNA as templates, so that the result shows that the DNA samples of Porcine Epidemic Diarrhea Virus (PEDV), porcine rotavirus (PoRV), porcine transmissible gastroenteritis virus (TGEV), porcine transmissible gastroenteritis virus (PRRSV), porcine circovirus type 2 (PCV 2), porcine Circovirus (CSFV) and ASFV-free DNA cannot be effectively amplified; only African swine fever virus was able to amplify efficiently.
The test results show that the specificity of the kit is 100%, the kit has stronger specificity, and the primers in the kit are specifically combined with African swine fever virus and have no cross reaction with other swine viruses.
(2) Repeatability test
The kit is applied to the repeatability test of the kit. Taking 1×10 1 ～1×10 8 The samples/μl of 8 dilutions of plasmid standard were subjected to intra-and inter-batch replicates, each with 3 replicates, and the Coefficient of Variation (CV) of both intra-and inter-batch replicates was less than 1%, indicating extremely high reproducibility.
(3) Sensitivity test
The standard substance is diluted into different concentrations, and the kit is adopted for detection, and meanwhile, common PCR detection is used as comparison. As a result, it was found that the kit of the present application was able to detect 10 copies of the standard, and was able to detect a copy number of 10 copies of the standard 1 ～10 8 There is an excellent linear relationship across the copy range. Whereas a minimum of 10 can be detected by ordinary PCR 4 Standard for copy number. The kit has the sensitivity of fluorescence quantitative PCR at least 1000 times of that of common PCR, and has higher sensitivity.
Example 4: clinical application experiment of the kit of the application
Viral genomic DNA (animal biosafety three-level laboratory of the national academy of agricultural sciences, vinca veterinarian research institute) was extracted from 60 cotton swab samples of pigs to be tested, and the results were detected and analyzed using the fluorescent quantitative PCR method established in example 2.
The result judging method comprises the following steps: if the fluorescence signal of the sample to be detected exceeds the threshold value and Ct is less than or equal to 35.0, and the amplification curve is smooth S-shaped, the sample to be detected can be judged to contain African swine fever virus.
And simultaneously compared with the fluorescent quantitative PCR method recommended by OIE aiming at the P72 gene.
The results show that 35 samples of 60 samples using the kit contain African swine fever virus, and the positive rate is 58%. And 33 samples in 60 samples are detected to contain African swine fever virus by using the fluorescence quantitative PCR method recommended by OIE for the P72 gene, the positive rate is 50%, wherein the Ct values of two negative samples are 33.21 and 30.70 respectively by using the PCR method, the result is positive, and the sample is positive by sequencing analysis. The accuracy of the detection result using the kit is 6% higher than that of the fluorescence quantitative PCR method recommended by OIE for the P72 gene, which shows that the PCR method is more reliable and is suitable for clinical use.
Sequence listing
<110> university of Yangzhou
<120> real-time fluorescence PCR primer and detection kit for African swine fever virus A137R gene and application
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Claims (7)
1. The application of the primer pair in preparing a kit for detecting African swine fever virus is characterized in that the nucleotide sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2.
2. The use according to claim 1, wherein the kit further comprises: fluorescent quantitative reaction reagent, standard substance and reference substance.
3. The use according to claim 2, wherein the standard is prepared by the following method: amplifying genome of African swine fever virus by using a primer pair shown in SEQ ID NO.3 and SEQ ID NO.4 to obtain an amplification product, connecting the amplification product to a vector, screening positive clones, and extracting plasmid DNA to obtain a standard product.
4. The use according to claim 3, wherein the nucleotide sequence of the amplification product is shown in SEQ ID No. 9.
5. The use according to claim 2, wherein the fluorescent quantitative reaction reagent comprises SYBR-Green fluorescent dye and RNase Free dH 2 O。
6. A method for detecting african swine fever virus for non-diagnostic purposes based on the kit of claims 1 to 5, comprising the steps of:
(1) Subjecting the standard substance of claim 3 to gradient dilution, then subjecting to fluorescence quantitative PCR reaction, and drawing a fluorescence quantitative standard curve according to the concentration and Ct value of the standard substance;
(2) Extracting total RNA of a sample to be detected, performing fluorescent quantitative PCR reaction, collecting fluorescent signals of the sample to be detected, performing data processing on the fluorescent signals to obtain Ct values and amplification curves, performing qualitative and quantitative detection on the sample to be detected, and judging positive if the fluorescent signals of the sample to be detected exceed a threshold value and Ct is less than or equal to 35.0, or judging negative.
7. The method of claim 6, wherein in step (1) and step (2), the procedure for the fluorescent PCR reaction is: pre-denaturation at 95℃for 30sec, 5sec at 95℃for 60℃for 30sec,40 cycles.
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