CN111593137A - Fluorescent quantitative PCR detection reagent and kit for African swine fever virus - Google Patents
Fluorescent quantitative PCR detection reagent and kit for African swine fever virus Download PDFInfo
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Abstract
The invention discloses a fluorescence quantitative PCR detection reagent and a kit for African swine fever virus, wherein a pair of sequences are respectively shown as SEQ ID NO: 2. SEQ ID NO: 3, and the nucleotide sequence shown in SEQ ID No.4 as a probe, wherein the 5 'end of the probe is provided with a FAM fluorescent reporter group marked by a fluorescent dye, and the 3' end of the probe is provided with a BQ1 fluorescent quenching group marked by a quenching fluorescent dye. The sequence constructed by the invention is SEQ ID NO: 1 as the positive quality control of real-time fluorescent quantitative PCR reaction; positive plasmid dilution 106The primer is used as a positive control, the sample lysis solution is 1 × TE (pH 8.0), the annealing temperature is 60 ℃, the primer concentration and the probe concentration are respectively 0.8 mu mol/L and 0.3 mu mol/L, and the invention has the advantages of high sensitivity, strong specificity, rapid diagnosis, high flux,Simple operation, good repeatability, high automation degree, easy standardized operation, high biological safety of the test and the like.
Description
Technical Field
The invention belongs to the technical field of biological detection, relates to a PCR detection reagent and a kit, and particularly relates to a fluorescence quantitative PCR detection reagent and a kit for African swine fever virus.
Background
African Swine Fever (ASF) is an acute, hemorrhagic, virulent infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs and various wild pigs (African wild pigs, European wild pigs, etc.). The world animal health Organization (OIE) classifies the animal infectious disease as a type A animal infectious disease, and the disease is also a type of animal epidemic disease which is mainly prevented in China. The swine fever virus is characterized by short morbidity process, the mortality rate of the most acute and acute infections is up to 100%, the clinical manifestations are fever (up to 40-42 ℃), the heartbeat is accelerated, the breathing is difficult, partial cough, serous or mucoid purulent secretion exists in eyes and nose, the skin is cyanotic, lymph nodes, kidneys and gastrointestinal mucosa are obviously bleeding, the clinical symptoms of African swine fever are similar to those of swine fever, and the African swine fever virus can be confirmed from blood, tissue fluid, viscera and other excreta of infected pigs by means of laboratory monitoring.
The OIE recommends that the method for detecting the African swine fever is Polymerase Chain Reaction (PCR), and the national standard method for detecting the African swine fever mainly comprises PCR and enzyme-linked immunosorbent assay (ELISA). The existing detection methods have the defects of long time, low sensitivity and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a detection reagent and a kit with high sensitivity, strong specificity, rapid diagnosis, high flux, simple operation, good repeatability, high automation degree, easy standardized operation and high biological safety of tests aiming at the defects of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a fluorescent quantitative PCR detection reagent of African swine fever virus, the detection reagent is to take partial target fragment (170 bp) of the VP72 gene of the African swine fever virus as a target, and comprises:
the upstream primer is SEQ ID NO: 2;
the downstream primer is SEQ ID NO: 3;
the probe is a nucleotide sequence shown as SEQ ID No. 4.
Wherein the 5 'end of the probe is provided with a FAM fluorescent reporter group labeled by a fluorescent dye, and the 3' end of the probe is provided with a BQ1 fluorescent quenching group labeled by a quenching fluorescent dye.
Intercepting 170bp of a VP72 gene of an African swine fever E75 strain (Genebank: FN 557520.1) as a target fragment, and amplifying a target nucleotide sequence of SEQ ID NO: 4, respectively.
A fluorescence quantitative PCR detection kit for African swine fever virus comprises fluorescence PCR reaction liquid, sample lysate, positive control and negative control.
A fluorescence quantitative PCR detection kit for African swine fever virus can also comprise sterile normal saline.
Wherein the fluorescent PCR reaction solution comprises:
the upstream primer is SEQ ID NO: 2;
the downstream primer is SEQ ID NO: 3;
the probe is a nucleotide sequence shown as SEQ ID No. 4;
the positive control is a nucleic acid comprising SEQ ID NO: 1, or a plasmid having the sequence shown in 1.
Wherein the 5 'end of the probe is provided with a FAM fluorescent reporter group labeled by a fluorescent dye, and the 3' end of the probe is provided with a BQ1 fluorescent quenching group labeled by a quenching fluorescent dye.
Wherein, the positive control is that the positive plasmid is diluted by 106And (4) doubling.
Wherein the negative control is SPF pig serum.
Wherein the sample lysate is 1 × TE (pH 8.0).
Wherein the 1 XTE (pH 8.0) preparation comprises the following steps:
(1) preparation of 1M Tris-HCl (pH 8.0): weighing 6.06g of Tris alkali, adding 40 mL of ultrapure water for dissolution, dropwise adding concentrated HCl of about 2.1 mL for adjusting the pH value to 8.0, and metering to 50 mL.
(2) Formulation of 0.5M EDTA (pH 8.0): weighing EDTA-Na2·2H2O9.306 g, 35 mL of ultrapure water, vigorously stirred, and then diluted with about 1g of strong sodium oxide particles, adjusting the pH to 8.0, and diluting to 50 mL (EDTA-Na)2·2H2O needs to be dissolved by adding strong sodium oxide to adjust pH to 8.0).
(3) Preparation of 1 XTE (pH 8.0): 1 mL of 1M Tris-HCl (pH 8.0), 2mL of 0.5M EDTA (pH 8.0), and 100 mL of ultrapure water to constant volume.
The real-time fluorescence PCR detection kit for African swine fever obtained by the invention is a method for monitoring the whole PCR process in real time by adding a fluorescent group in a PCR reaction system and utilizing fluorescent signal accumulation, and finally performing calculation analysis on an unknown template through software, and has the outstanding advantages of high sensitivity, strong specificity, rapid diagnosis, high flux, simple operation, good repeatability, high degree of automation, easy standardized operation, high biological safety of tests and the like.
Detailed Description
For a more clear understanding of the technical features, objects and effects of the present invention, specific embodiments of the present invention will now be described in detail.
Preparation of fluorescent quantitative PCR detection reagent for African swine fever virus
1.1 design of primers and probes
A pair of specific primers and a Taqman probe are designed according to the VP72 gene of an African swine fever E75 strain (Genebank: FN 557520.1), the Taqman probe selects a FAM reporter gene and a BQ1 quenching gene, the length of an amplification region is 170bp, and a pair of primers with the sequences of SEQ ID NO: 2. SEQ ID NO: 3 and the probe with the sequence shown as SEQ ID No.4, and the upstream and downstream primers and the Taqman probe are synthesized by the Kingsler Biotech Co.
The upstream primer is SEQ ID NO: 2: 5 'GACCACTGGGTTGGTATT 3'
The downstream primer is SEQ ID NO: 3: 5 'TTCCGTAACTGCTCAT 3'
The probe is a nucleotide sequence shown as SEQ ID No. 4: FAM-5 'CATCGCACCCGGATCATCGG 3' -BQ 1.
Preparation of Positive plasmids
The ASFV-VP72 gene was synthesized by Kinry Biotech, Inc., and cloned directly into the cloning vector pMD 18T. The VP72 gene in the product intercepts VP72 gene 170bp of African swine fever E75 strain (Genebank: FN 557520.1) as a target fragment, and the plasmid sequence is SEQ ID NO: 1:
GACCACTGGGTTGGTATTCCTCCCGTGGCTTCAAAGCAAAGGTAATCATCATCGCACCCGGATCATCGGGGGTTTTAATCGCATTGCCTCCGTAGTGGAAGGGTATGTAAGAGCTGCAGAACTTTGATGGAAATTTATCGATAAGATTGATACCATGAGCAGTTACGGAA
the concentration was 2 ng/. mu.L.
Establishment of PCR detection kit for ASFV
2.1 Components of the detection kit
The detection kit comprises fluorescent PCR reaction solution, sample lysate, positive control, negative control and sterile physiological saline.
The fluorescent PCR reaction solution comprises: the primers and probes obtained in step 1.1 of the detailed description; (ii) a The negative control is SPF pig serum; the sample lysate was 1 × TE (pH 8.0).
Preparation of sample lysate
(1) Preparation of 1M Tris-HCl (pH 8.0): weighing 6.06g of Tris alkali, adding 40 mL of ultrapure water for dissolution, dropwise adding concentrated HCl of about 2.1 mL for adjusting the pH value to 8.0, and metering to 50 mL.
(2) Formulation of 0.5M EDTA (pH 8.0): weighing EDTA-Na2·2H2O9.306 g, 35 mL of ultrapure water, vigorously stirred, adjusted to pH 8.0 with about 1 g of strong sodium oxide particles, and made to volume of 50 mL (EDTA-Na)2·2H2O needs to be dissolved by adding strong sodium oxide to adjust pH to 8.0).
(3) Preparation of 1 XTE (pH 8.0): 1 mL of 1M Tris-HCl (pH 8.0), 2mL of 0.5M EDTA (pH 8.0), and 100 mL of ultrapure water to constant volume.
Optimization of real-time fluorescent PCR annealing extension temperature
The real-time fluorescent quantitative PCR reaction is carried out at 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃ and 62 ℃, the reaction system is 25 mu L, the reaction procedure is pre-denaturation at 95 ℃ for 3min, the temperature is 95 ℃ for 10s, the annealing extension temperature is 20s, and 40 cycles are carried out, and each reaction is repeated three times. The Ct values associated with different annealing temperatures are shown in Table 1.
TABLE 1 Ct values for different annealing temperatures
Through research on different annealing extension temperatures of PCR, 6 temperatures of 57-62 ℃ and Ct values corresponding to the points are selected to determine the annealing temperature, and finally the annealing extension temperature is determined to be 60 ℃.
Optimization of real-time fluorescent PCR primers and probe concentration
Primer concentrations of 0.4. mu. moL/L, 0.6. mu. moL/L, 0.8. mu. moL/L, and 1.0. mu. moL/L were designed, probe concentrations of 0.2. mu. moL/L, 0.3. mu. moL/L, 0.4. mu. moL/L, and 0.5. mu. moL/L were designed, each reaction was repeated three times, and Ct values were determined by a matrix method to determine the optimal primer concentration and probe concentration, and the results are shown in Table 2.
TABLE 2 optimization of primer and Probe concentrations
By researching the concentration of the real-time fluorescent quantitative PCR primer and the probe, the primer concentration is finally selected to be 0.8 mu mol/L, and the probe concentration is selected to be 0.3 mu mol/L.
Validation of PCR detection kit for ASFV
3.1 PCR Rapid detection kit sensitivity study for ASFV
3.1.1 reagents:
a positive plasmid; sterile water.
3.1.2 kit:
the real-time fluorescent PCR rapid detection kit for African swine fever virus obtained in the specific example 2 (20170801, 20170802, 20170803);
a real-time fluorescent PCR kit for African swine fever virus (OIE recommendations: Manual of diagnostic Tests and Vaccines for terrestial Animals.) manufactured by the OIE recommendations, batch OIE 20170801.
Vetmax;. African Swine Fever Virus (ASFV) qPCR detection kit, a 28809.
3.1.3 Main instrumentation: gentier 48E Rapid fluorescence PCR instrument.
3.1.4 PCR detection method
Positive plasmids were diluted in 10-fold gradient, corresponding to a dilution of 1.0 × 100-1.0×107The reaction system was set to 25. mu.L; pre-denaturation: 95 ℃, 3min, 1 cycle; and (3) PCR amplification: 95 ℃: the sensitivity of the kit is determined by performing fluorescence PCR reaction for 40 cycles at 60 ℃ for 10 s.
And (3) judging standard:
a. positive: the Ct value of the sample detection result is less than or equal to 35, and the obvious exponential increase shows that the virus is detected in the sample.
b. And (3) suspicious: the Ct value of the sample detection result is within the range of 35-38
c. Negative: the Ct value of the sample detection result is more than 38 or no Ct value.
3.1.5 results
Three-batch kit for detecting and diluting positive plasmids with different concentrations (1.0 × 10)0-1.0×106) The detection results show positive, and the Ct values are all lower than 30, 1.0 × 107The detection result is a negative sample, and the detection result is 100 percent in accordance with the detection results of a Vetmax box African Swine Fever Virus (ASFV) qPCR detection kit and an OIE kit.
TABLE 3 results of sensitivity test
Note: unde is no fluorescence signal detected
3.1.6 conclusion
The PCR kit obtained in the specific example 2 can detect 1.0 × 10 at the lowest6The positive plasmid with the dilution factor is 100 percent in accordance with the detection results of an OIE kit and a VetMAXTM African Swine Fever Virus (ASFV) qPCR detection kit.
Specificity research of PCR (polymerase chain reaction) rapid detection kit for ASFV (advanced sequencing kit)
3.2.1 kit:
the kit for the real-time fluorescent PCR rapid detection of the African swine fever virus comprises: 20170801, 20170802, 20170803;
a real-time fluorescent PCR kit for African swine fever virus (OIE recommendations: Manual of diagnostic Tests and Vaccines for terrestial Animals.) manufactured by the OIE recommendations, batch OIE 20170801.
Vetmax;. African Swine Fever Virus (ASFV) qPCR detection kit, a 28809.
3.2.2 negative quality control:
swine fever live vaccine, porcine reproductive and respiratory syndrome live vaccine, pseudorabies live vaccine, porcine parvovirus cell culture solution, porcine circovirus type 2 cell culture solution and SPF pig serum.
3.2.3 clinical samples:
17 parts of lymph node, 14 parts of spleen, 25 parts of pork and 44 parts of whole blood.
3.2.4 devices: gentier 48E Rapid fluorescence PCR instrument.
PCR detection method
The PCR reaction system was set to 25. mu.L; pre-denaturation: 95 ℃, 3min, 1 cycle; and (3) PCR amplification: 95 ℃: the fluorescence PCR reaction was performed for 40 cycles at 60 ℃ for 10 s. The negative quality control products (swine fever live vaccine extract, porcine reproductive and respiratory syndrome live vaccine extract, pseudorabies live vaccine extract, porcine parvovirus cell culture fluid, porcine circovirus type 2 cell culture fluid, SPF pig serum extract) and 100 clinical samples (17 parts of lymph nodes, 14 parts of spleen, 25 parts of pork and 44 parts of whole blood) are tested, and compared with the test results of an OIE kit and a VetMAX African Swine Fever Virus (ASFV) qPCR detection kit, the specificity of the kit obtained in the specific embodiment 2 is determined.
Criteria for determination
a. Positive: the Ct value of the sample detection result is less than or equal to 35, and the obvious exponential increase shows that the virus is detected in the sample.
b. And (3) suspicious: the Ct value of the sample detection result is within the range of 35-38
c. Negative: the Ct value of the sample detection result is more than 38 or no Ct value.
The detection results of the swine fever live vaccine, the porcine reproductive and respiratory syndrome live vaccine, the pseudorabies live vaccine, the porcine parvovirus cell culture solution, the porcine circovirus type 2 cell culture solution and the SPF porcine serum are all negative.
3.2.7 results
TABLE 4 results of specificity test
Note: unde is no fluorescence signal detected
TABLE 53 comparison of specificity test results for the kits
3.2.8 conclusion
The result of the specificity detection of the invention is compared with an OIE detection kit and a VetMAX African Swine Fever Virus (ASFV) qPCR detection kit, and the result shows that the negative quality control product and the clinical serum detection result are completely consistent.
PCR (polymerase chain reaction) rapid detection kit repeatability research aiming at ASFV (advanced sequencing kit)
The kits obtained in example 2 (three lots, lots 20170801, 20170802, 20170803) were used to verify product lot-to-lot and lot-to-lot reproducibility in this laboratory, and the samples used were positive quality controls (dilution factor: 1.0 × 10)0、1.0×101、1.0×102、1.0×103、1.0×104、1.0×105、1.0×106). The reaction system was set to 25. mu.L; pre-denaturation: 95 ℃, 3min, 1 cycle; and (3) PCR amplification: 95 ℃: 10s, 60 ℃, 20s, 40 cycles.
3.3.1 in-batch repeatability
In 20170801, 20170802 and 20170803 three batches of African swine fever virus real-time fluorescent PCR rapid detection kits, 3 boxes are randomly extracted from each batch, a positive quality control product is detected, the repeatability of the kit is judged through the intra-batch variation coefficient of the Ct value, and the result is shown in tables 6-8.
TABLE 6 repeatability test results for 20170801 lots
TABLE 720170802 repeatability test results in lots
TABLE 820170803 repeatability test results in lots
The in-batch repeatability detection results of the African swine fever virus real-time fluorescence PCR rapid detection kit are not obviously different, and the in-batch variation coefficient is not higher than 1.5%, which indicates that no difference exists in the batch.
Batch to batch repeatability
In 20170801, 20170802 and 20170803 three batches of African swine fever virus real-time fluorescent PCR rapid detection kits, 3 test strips are randomly drawn from each batch to detect positive quality control substances, and the batch repeatability of the kit is judged through Ct value batch variation coefficient. The results are shown in Table 9.
TABLE 9 repeatability test results between three laboratory product lots
The three batches of African swine fever virus real-time fluorescent PCR rapid detection kits in the laboratory have no obvious difference in batch-to-batch repeatability detection results, the batch-to-batch variation coefficient is lower than 1.5%, and no difference exists among batches.
The invention designs a pair of primers with the sequences of SEQ ID NO: 2. SEQ ID NO: 3 and a probe with a sequence shown as SEQ ID No. 4; the positive plasmid (sequence is SEQ ID NO: 1) constructed by the invention is used as positive quality control of real-time fluorescent quantitative PCR reaction; positive plasmid dilution 106Double as positive control, sample lysis solution of 1 × TE (pH 8.0), annealing temperature of 60 deg.C, primer concentration and probe concentration of 0.8 μmol/L and 0.3 μmol/L。
The kit provided by the invention has the advantages of sensitivity research, specificity research, repeatability research and coincidence rate research. Research results show that the detection method is feasible and reliable, the sensitivity and the specificity meet the requirements, the repeatability is good, and compared with an OIE kit and a VetMAXTM African Swine Fever Virus (ASFV) qPCR detection kit, the detection method meets 100%.
The real-time fluorescence PCR detection kit for African swine fever obtained by the invention is a method for monitoring the whole PCR process in real time by adding a fluorescent group in a PCR reaction system and utilizing fluorescent signal accumulation, and finally performing calculation analysis on an unknown template through software, and has the outstanding advantages of high sensitivity, strong specificity, rapid diagnosis, high flux, simple operation, good repeatability, high degree of automation, easy standardized operation, high biological safety of tests and the like.
Sequence listing
<110> Beijing Weideweikang Biotech Ltd
Fluorescent quantitative PCR detection reagent and kit for <120> African swine fever virus
<141>2019-02-15
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>170
<212>DNA
<213> African swine fever virus (African swine fever virus)
<400>1
gaccactggg ttggtattcc tcccgtggct tcaaagcaaa ggtaatcatc atcgcacccg 60
gatcatcggg ggttttaatc gcattgcctc cgtagtggaa gggtatgtaa gagctgcaga 120
actttgatgg aaatttatcg ataagattga taccatgagc agttacggaa 170
<210>2
<211>18
<212>DNA
<213> Artificial sequence (African swing river Virus)
<400>2
gaccactggg ttggtatt 18
<210>3
<211>16
<212>DNA
<213> Artificial sequence (African swing river Virus)
<400>3
ttccgtaact gctcat 16
<210>4
<211>20
<212>DNA
<213> Artificial sequence (African swing river Virus)
<400>4
catcgcaccc ggatcatcgg 20
Claims (10)
1. The fluorescent quantitative PCR detection reagent for the African swine fever virus is characterized by comprising the following primers and a probe matched with the primers: the sequences of the primer pairs are respectively SEQ ID NO: 2. SEQ ID NO: 3, and the probe nucleotide sequence is shown as SEQ ID No. 4.
2. The reagent of claim 1, wherein the 5 'end of the probe is labeled with FAM fluorescent reporter group labeled with fluorescent dye, and the 3' end of the probe is labeled with BQ1 fluorescent quencher group labeled with quenching fluorescent dye.
3. The fluorescent quantitative PCR detection reagent for African swine fever virus of claim 1, wherein the detection reagent takes 170bp of VP72 gene of African swine fever E75 strain as a target fragment, and the amplification target nucleotide sequence is SEQ ID NO: 1, and (b) is shown in the specification.
4. A fluorescence quantitative PCR detection kit for African swine fever virus, which is characterized by comprising the PCR detection reagent of claim 1.
5. The African swine fever virus fluorogenic quantitative PCR detection kit according to claim 4, wherein the detection kit further comprises a positive control.
6. The test kit of claim 5, wherein the positive control is a nucleic acid comprising SEQ ID NO: 1, or a plasmid having the sequence shown in 1.
7. The test kit as claimed in claims 5 to 6, wherein the positive control is a positive plasmid diluted by 106And (4) doubling.
8. The fluorescence quantitative PCR detection kit for African swine fever virus according to claims 4-7, wherein the detection kit further comprises a sample lysate.
9. The fluorescence quantitative PCR detection kit for African swine fever virus of claim 8, wherein the sample lysate is 1 XTE with pH 8.0.
10. The fluorescence quantitative PCR detection kit for African swine fever virus according to claim 4, wherein the primer concentration is 0.8 μmol/L and the probe concentration is 0.3 μmol/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795706A (en) * | 2021-03-30 | 2021-05-14 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757134A (en) * | 2014-01-13 | 2014-04-30 | 深圳澳东检验检测科技有限公司 | Fluorescent quantitative PCR (Polymerase Chain Reaction) detection reagent, kit and detection method for African swine fever virus (ASFV) |
| CN106957926A (en) * | 2017-04-14 | 2017-07-18 | 北京出入境检验检疫局检验检疫技术中心 | A kind of detection kit and primer and probe that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox |
| CN109136408A (en) * | 2018-09-29 | 2019-01-04 | 南京农业大学 | Detection reagent, kit and its application of African swine fever virus |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757134A (en) * | 2014-01-13 | 2014-04-30 | 深圳澳东检验检测科技有限公司 | Fluorescent quantitative PCR (Polymerase Chain Reaction) detection reagent, kit and detection method for African swine fever virus (ASFV) |
| CN106957926A (en) * | 2017-04-14 | 2017-07-18 | 北京出入境检验检疫局检验检疫技术中心 | A kind of detection kit and primer and probe that can simultaneously detect and differentiate classic swine fever, African swine fever and swine pox |
| CN109136408A (en) * | 2018-09-29 | 2019-01-04 | 南京农业大学 | Detection reagent, kit and its application of African swine fever virus |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795706A (en) * | 2021-03-30 | 2021-05-14 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit |
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