CN111926110B - African swine fever virus real-time fluorescent PCR amplification primer pair, probe primer and prepared kit - Google Patents

African swine fever virus real-time fluorescent PCR amplification primer pair, probe primer and prepared kit Download PDF

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CN111926110B
CN111926110B CN201910472848.7A CN201910472848A CN111926110B CN 111926110 B CN111926110 B CN 111926110B CN 201910472848 A CN201910472848 A CN 201910472848A CN 111926110 B CN111926110 B CN 111926110B
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田克恭
李向东
张海洋
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Luoyang Pu Tai Biotechnology Co ltd
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Abstract

The invention provides a real-time fluorescence PCR detection kit for African swine fever virus, wherein the kit comprises a pair of amplification primers and a probe primer; the upstream primer of the amplification primer is shown as SEQ.ID.No.1, and the downstream primer of the amplification primer is shown as SEQ.ID.No. 2; the probe primer is shown in SEQ.ID.No.3, and fluorescein is marked on the probe primer. The kit can specifically and highly sensitively detect the African swine fever virus, and the lower limit of the content of the detected DNA can reach 1 multiplied by 10‑8ng/mu l, and can be used for clinically and rapidly and accurately detecting the African swine fever virus.

Description

African swine fever virus real-time fluorescent PCR amplification primer pair, probe primer and prepared kit
Technical Field
The invention belongs to the characteristic field of single light pulse or light pulse sequence, and particularly relates to real-time fluorescence PCR detection, in particular to an African swine fever virus real-time fluorescence PCR detection primer, a probe primer and a kit.
Background
African Swine Fever (ASF) is a virulent, highly contagious infectious disease of pigs caused by African Swine Fever Virus (ASFV). The African swine fever virus is a single-molecule linear double-stranded DNA virus with an envelope, and the genome length of the African swine fever virus is about 170kb to 193 kb. ASF has the characteristics of high morbidity, high mortality and the like, and once the ASF occurs, the economic loss is huge; at present, an effective vaccine and a specific treatment means are lacked, so that the ASF becomes one of the most serious epidemic diseases endangering the pig industry at present. At present, the control of ASF only depends on the rapid diagnosis of laboratories, the killing of sick animals, and the adoption of effective quarantine measures and strict sanitation measures.
At present, ASF is firstly developed in China, is popular in a plurality of regions in China, and epidemic situation is continuously expanded. The ASFV lacks effective vaccine and rapid pathogen detection technology in China, and is an important problem in accurate monitoring and control of epidemic diseases. Therefore, establishing a rapid detection method for rapid identification of ASF (infectious disease) by using a novel etiology detection technology is a technical key for effectively and accurately preventing and controlling epidemic situations.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a real-time fluorescence PCR detection kit for African swine fever virus, wherein the kit comprises a pair of amplification primers and a probe primer; the upstream primer of the amplification primer is shown as SEQ.ID.No.1, and the downstream primer of the amplification primer is shown as SEQ.ID.No. 2; the probe primer is shown in SEQ.ID.No.3, and fluorescein is marked on the probe primer.
The African swine fever virus real-time fluorescent PCR detection primer pair and the probe primer can specifically detect the African swine fever virus, the sensitivity is high, and the lower limit of the content of the detected DNA can reach 1 multiplied by 10-8ng/mul, under specific conditions even up to 1X 10-9ng/μl。
In one embodiment of the present invention, in the kit of the present invention, the fluorescein is selected from the group consisting of: FAM (carboxyfluorescein), VIC, HEX, JOE, NED, TAMRA, CY3, ROX, or CY 5.
As an embodiment of the present invention, in the kit of the present invention, the kit further comprises: taq DNA polymerase, 5 Xreaction buffer, dNTPs, ddH2O; preferably, the concentration of Taq DNA polymerase is 5U/. mu.l, the concentration of 5 × reaction buffer is 5 × Tris-HCl (pH8.3), the concentration is 50mM, the concentration of dNTPs is 2.5mM, the concentration of the upstream primer is 10. mu.M, the concentration of the downstream primer is 10. mu.M, and the concentration of the probe primer is 5. mu.M.
As an embodiment of the invention, the kit further comprises an enzyme mixed solution, wherein the enzyme mixed solution is composed of Taq DNA polymerase, Tris-HCl, EDTA, DTT, glycerol and Tween-20, and the contents of the Taq DNA polymerase, the Tris-HCl, the EDTA, the DTT, the glycerol and the Tween-20 are respectively 0.5U/microliter, 25mM, 0.05mM, 40% V/V and 0.2% V/V.
As an embodiment of the invention, in the kit of the invention, the kit further comprises a PCR reaction solution, and the PCR reaction solution comprises the amplification primer pair, the probe primer, a reaction buffer solution, dNTPs and ddH2O is mixed according to the volume ratio of 6:10:5:19, wherein the volume ratio of the upstream primer, the downstream primer and the probe primer is 1:1: 1; the concentration of the amplification primer pair is 5 mu M, the concentration of the probe primer is 2 mu M, the reaction buffer is 5 xTris-HCl (pH8.3), the concentration is 50mM, and the concentration of dNTPs is 1.5 mM.
The invention combines the original multiple chemical reagent components of the kit into 2 types by establishing a multi-component trace combination process technology: enzyme reaction reagents are combined, and the proportion of each component is optimized; mixing the PCR reaction solution, and optimizing the proportion of each component; the process technology unexpectedly finds that the enzyme activity is enhanced, and the sensitivity of the kit is improved.
The real-time fluorescent PCR detection primer pair and the probe have the characteristics of strong specificity and high sensitivity, and the kit containing the primer pair is sensitive, convenient, fast, strong in specificity, high in sensitivity and good in reliability, so that whether the sample contains the African swine fever virus or not can be accurately judged in the early stage of infection by only one-time detection, a large amount of sample analysis can be simultaneously carried out, and powerful technical support is provided for monitoring, preventing and controlling the epidemic situation of the African swine fever virus.
In one embodiment of the present invention, the kit further comprises a positive control and a negative control; preferably, the positive control is a recombinant plasmid containing the African swine fever virus CD2v gene fragment amplified by the amplification primer, and the negative control is distilled water.
In an embodiment of the present invention, in the kit of the present invention, the kit further includes a reagent for extracting a DNA template of african swine fever virus from a sample to be tested.
The kit disclosed by the invention is strong in specificity and high in sensitivity, can be used for detecting various samples, and can be applied to serum samples, liver tissues, lymph nodes, aborted fetuses, spleen tissues, lung tissues and kidney tissues.
The invention also provides a real-time fluorescence PCR amplification reaction program of the African swine fever virus: 2min at 95 ℃; 40 cycles of 95 ℃ 10sec, 60 ℃ 30sec each; the second step of each cycle (60 ℃ C. for 60sec) was to collect the fluorescence signal.
As a preferred embodiment of the present invention, the concentration of the DNA template in the sample to be tested is more than or equal to 1 × 10-8ng/μl。
As an embodiment of the invention, the PCR reaction system used by the kit is 50 ul, and consists of 5 ul of enzyme mixed solution, 40 ul of PCR reaction solution and 5 ul of DNA template to be detected.
The invention also provides an amplification primer pair and a probe primer for real-time fluorescent PCR detection of the African swine fever virus, wherein an upstream primer of the amplification primer is shown as SEQ.ID.No.1, and a downstream primer of the amplification primer is shown as SEQ.ID.No. 2; the probe primer is shown in SEQ.ID.No.3, and fluorescein is marked on the probe primer.
As an embodiment of the present invention, the fluorescein is selected from the group consisting of: FAM (carboxyfluorescein), VIC, HEX, JOE, NED, TAMRA, CY3, ROX, or CY 5.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
Definition of
Introduction of fluorescein:
FAM: carboxyfluorescein, a kind of fluorescein derivative, is widely present in fluorescence labeling kits, and is also applicable to 488nm spectral lines of Argon-ion Laser, and Abs/Em ═ 492/518nm (pH ═ 9.0), has the general characteristics of fluorescein derivatives, and is stable in water.
VIC: green fluorescent protein, GFP, is a photoprotein derived from the marine organism aequorea Victoria (Aequoria Victoria).
HEX: hexachlorofluorescein, hexachlor fluorescein, is one of the fluorescein derivatives. The light source is suitable for an Argon-ion Laser excitation light source, and Abs/Em is 535/556 nm.
JOE: carboxyl-4 ',5 ' -dichloro-2 ', 7 ' -dimethoxy fluorescein, Carboxy-4',5 ' -dichoro-2 ', JOE has high fluorescence yield and weak pH sensitivity, and is suitable for marking protein.
TAMRA: the full name of Carboxytetramethylrhodamine, carboxytetramylrhodamine, is a rhodamine-based fluorescein derivative, and TAMRA is a few of those that can be used to label proteins.
ROX: 5-and 6-carboxy-X-rhodamine, calibration dyes.
Cy3 or Cy5, Modification, is a novel fluorescent molecule, and has good light stability, high water solubility and high fluorescence efficiency. Their excitation and emission spectra peaks were 548/562nm and 646/664nm, respectively. The molecular structures and molecular weights of Cy3 and Cy5 are very similar, but the spectra of both are very separated, so Cy3 and Cy5 are often used in many two-color experiments, such as in the fields of gene chips and protein chips.
NED: 2 '-chloro-5' -fluoro-7 ', 8' -benzene-1, 4-dichloro-6-carboxyfluorescein.
The detection channel corresponding to the fluorescein detection is the detection channel corresponding to the FTC-3000 instrument used in the experimental process of the invention, and the skilled person in the art can have different channels corresponding to different instruments in the process of repeating the invention.
The real-time fluorescence PCR detection refers to a method of adding a fluorescent group into a PCR reaction system, utilizing fluorescence signal accumulation to monitor the whole PCR process in real time, and finally carrying out quantitative analysis on an unknown template through a standard curve.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 amplification primers and Probe primers for African Swine fever Virus
One probe primer of a pair of amplification primers used in the invention is shown in a sequence table 1.
TABLE 1 primers and Probe primers for real-time fluorescence of African Swine fever Virus
Name (R) Sequence of SEQ ID NO.
Upstream primer 5’-GACACCACTTCCATACATGAACC-3’ 1
Downstream primer 5’-TTGTGTTGAGGGACGCATGT-3’ 2
Probe primer 5’-CCCAGAGAACCATTACTTCCTAAGCC-3’ 3
The reaction system and reaction conditions of the African swine fever virus real-time fluorescence PCR method are shown in Table 2 and Table 3 respectively.
TABLE 2 reaction System of African swine fever Virus real-time fluorescent PCR method
Figure BDA0002081270330000051
TABLE 3 reaction conditions of real-time fluorescent PCR method for African swine fever virus
Figure BDA0002081270330000061
Example 2 optimization of African swine fever virus real-time fluorescent PCR detection method
1. Reaction system of African swine fever virus real-time fluorescence PCR method
The optimization principle of the African swine fever virus real-time fluorescence PCR method is as follows: the maximum amplification efficiency and the minimum Ct value of the same sample are obtained by optimizing the following conditions.
The commercial 5U/. mu.l Taq enzyme is added with 2. mu.l generally in a 50. mu.l reaction system, the final concentration is 0.2U/. mu.l, in the embodiment, under the condition of further reducing the final concentration of the Taq enzyme, the original multiple chemical reagent components of the kit are combined into 2 types by establishing a multi-component trace combination process technology, and specific enzyme mixed solution and PCR reaction solution are prepared, so that the further optimization of the detection condition of the kit is realized, and the sensitivity and the specificity are greatly improved. Through optimization, the reaction system of the real-time fluorescence PCR method for the African swine fever virus is shown in Table 4. The enzyme mixture was added to a 50. mu.l reaction system in an amount of generally 5. mu.l, to a final concentration of 0.05U/. mu.l.
Table 4 optimized real-time fluorescence PCR reaction system for African swine fever virus
Figure BDA0002081270330000062
2. Determination of results
(1) Setting of conditions for analysis of results
And reading the detection result. The threshold value setting principle is based on the condition that the threshold value line just exceeds the highest point of the amplification curve of the normal negative control product, and the result is displayed.
(2) Quality control standard
Negative controls had no Ct values and no specific amplification curves;
the Ct value of the positive control is less than or equal to 30, and a specific amplification curve appears. Otherwise, this experiment is considered invalid.
(3) Result description and determination
Negative samples have no Ct value and no specific amplification curve, which indicates that the negative samples have no African swine fever virus.
The Ct value of the positive sample is less than or equal to 30, and a specific amplification curve appears, which indicates that the positive sample contains African swine fever virus.
The effective principle is as follows: the sample Ct value is between 30 < Ct < 37, and the test needs to be repeated. If the Ct of the repeated test result is less than 37, the sample is positive, otherwise, the sample is negative.
(4) Sample testing and results
The test amplifies Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Japanese encephalitis B virus (JEV), porcine pseudorabies virus (PRV) and Porcine Parvovirus (PPV), and only African swine fever virus obtains a specific fluorescence amplification curve.
EXAMPLE 3 comparative test of sensitivity and specificity of PCR detection kit
The African swine fever virus real-time fluorescent PCR kit 1 is assembled by components before condition optimization in example 1, and the African swine fever virus real-time fluorescent PCR kit 2 is assembled by components after condition optimization in example 2. The sensitivity and specificity of the two kits were compared.
After the African swine fever virus DNA template is continuously diluted by 10 times, the kit 1 and the kit 2 are respectively subjected to PCR sensitivity test according to the conditions before and after optimization, and simultaneously, a nucleic acid protein determinator is used for determining the quantity of all the DNA templates.
The concentrations of the DNA templates were 1X 10, respectively-10ng/μl、1×10-9ng/μl、1×10-8ng/μl、1×10-7ng/μl、1×10-6ng/μl、1×10-5ng/μl、1×10-4ng/μl、1×10-3ng/μl、1×10-2ng/μl、1×10-1ng/. mu.l. The result shows that the sensitivity of the kit 1 can reach 1 multiplied by 10 at least-7ng/mul, the sensitivity of the kit 2 can reach 1 multiplied by 10 at least-9ng/. mu.l. The primers screened by the invention have high sensitivity, and the sensitivity of the kit is further improved after the conditions are optimized.
The kit 1 and the kit 2 are used for carrying out specificity tests on porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, hog cholera virus, porcine parainfluenza virus, porcine parvovirus, porcine rotavirus, porcine transmissible gastroenteritis virus and African swine fever virus negative pig lung, lymph node and tonsil according to conditions before and after optimization respectively, and the result shows that the detection result of the African swine fever virus is negative. The experimental results prove that the kit has quite high specificity.
Example 4 suspected African Swine fever disease sample detection comparison
Tissue samples of suspected diseased pigs collected from clinical diseased pig farms in different regions, including 7 parts of pig liver, 11 parts of lung, 5 parts of kidney, 8 parts of lymph node, 8 parts of spleen and 5 parts of aborted fetus, were taken, and wild African swine fever virus was detected using kit 1 and kit 2 according to conditions before and after optimization, respectively. The results are shown in Table 5.
TABLE 5 detection of clinically suspected African Swine fever Virus infected pig samples
Figure BDA0002081270330000081
Figure BDA0002081270330000091
The result shows that 21 African swine fever positive morbid substances are detected by the kit 1 among 44 morbid substances, and the detection rate is 47.7%; the kit 2 can also detect the positive pathological materials detected by the kit 1, and the coincidence rate of the two is 100 percent; 32 African swine fever positive materials are detected by the kit 2, the detection rate is 72.7 percent, and is 25.0 percent higher than that of the kit 1; kit 2 detectionThe positive test piece and the kit 1 showed a small amount of pathogenic viruses, and the amount of DNA template was 1X 10 by the nucleic acid protein analyzer-8ng/μl～1×10-9ng/. mu.l. The detection sensitivity of the kit 2 after condition optimization is obviously higher than that of the kit 1, so that the pathological materials with lower virus content can be detected, and negative samples can not be detected.
EXAMPLE 5 clinical application of the kit
Sampling and taking 39 serum samples of pig farms in different areas without clinical symptoms, carrying out African swine fever wild virus detection by using a kit 1 according to the conditions before optimization, and carrying out African swine fever wild virus detection and rechecking by using a kit 2 according to the conditions after optimization. The results are shown in Table 6.
TABLE 6 clinical application of kit for African swine fever test results
Figure BDA0002081270330000101
Figure BDA0002081270330000111
The result shows that 10 African swine fever virus positive samples are detected by the kit 1 in 39 parts of serum, and the detection rate is 25.6%; the kit 2 can also detect the positive samples detected by the kit 1, and the coincidence rate reaches 100%; the kit 2 detects 20 African swine fever virus positive samples, the detection rate is 51.3 percent, and is 25.7 percent higher than that of the kit 1. The detection sensitivity of the kit 2 after condition optimization is obviously higher than that of the kit 1, and samples with lower virus content can be detected, but negative samples can not be detected. The kit can accurately detect suspected infection samples from different regional sources.
Example 6 early infection detection assay
Carrying out detection tests in a pig farm with sporadic outbreak of clinical symptoms of the African swine fever, killing all pigs with the clinical symptoms, isolating 198 other pigs, collecting serum every day, carrying out the detection of the African swine fever wild viruses by using a kit 1 according to the conditions before optimization, and carrying out the rechecking of the detection of the African swine fever wild viruses by using a kit 2 according to the conditions after optimization. The results are shown in Table 7.
TABLE 7 Positive time for early infection detection
Time of detection Kit 1 Kit 2 Rate of agreement
Day 0 5/198(2.5％) 55/198(27.8％) 100％
Day 1 6/193(3.1％) 50/193(25.9％) 100％
Day 2 6/187(3.2％) 44/187(23.5％) 100％
Day 3 8/181(4.4％) 38/181(21.0％) 100％
Day 4 9/173(5.2％) 30/173(17.3％) 100％
Day 5 13/164(7.9％) 21/164(12.8％) 100％
Day 6 8/151(5.3％) 8/151(5.3％) 100％
Day 7 0/143(0％) 0/143(0％) 100％
Day 8 0/143(0％) 0/143(0％) 100％
Day 9 0/143(0％) 0/143(0％) 100％
Day 10 0/143(0％) 0/143(0％) 100％
The positive pigs detected by the kit 1 and the kit 2 are subjected to killing treatment every day, the rest pigs are continuously monitored in an isolated mode, the result shows that the kit 1 detects 5 positive pigs on the 0 th day of the pigs without clinical symptoms, and the detection rate is 2.5%; the kit 2 can also detect positive pigs detected by the kit 1, and the coincidence rate is 100%; 55 pigs which are positive are detected by the kit 2 on the 0 th day without clinical symptoms, the detection rate is 27.8 percent and is 25.3 percent higher than that of the kit 1; by the 6 th day, 55 positive pigs are detected by the kit 1 in a cumulative way, and the number of the positive pigs is consistent with that of the positive pigs detected by the kit 2 on the 0 th day; the same positive pigs in the kit 1 only need to detect the same result as the kit 2 by the 6 th day.
The detection sensitivity of the kit 2 after the condition optimization is obviously higher than that of the kit 1, so that samples with lower virus content can be detected, and negative samples can not be detected; the kit 2 with optimized conditions can detect early under the condition that pigs are infected early, detect all infected pigs and treat the infected pigs as soon as possible, so that the kit can prevent and control the infection of the African swine fever virus early.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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Claims (5)

1. A real-time fluorescence PCR detection kit for African swine fever virus, wherein the kit comprises a pair of amplification primers and a probe primer; the upstream primer of the amplification primer is shown as SEQ.ID.No.1, and the downstream primer of the amplification primer is shown as SEQ.ID.No. 2; the probe primer is shown in SEQ.ID.No.3, fluorescein is marked on the probe primer, the kit further comprises an enzyme mixed solution, the enzyme mixed solution consists of Taq DNA polymerase, Tris-HCl, EDTA, DTT, glycerol and Tween-20, and the contents of the Taq DNA polymerase, the Tris-HCl, the EDTA, the DTT, the glycerol and the Tween-20 are respectively 0.5U/mul, 25mM, 0.05mM, 40% V/V and 0.2% V/V; the kit also comprises PCR reaction liquid, wherein the PCR reaction liquid consists of the pair of amplification primers, the probe primer, reaction buffer solution, dNTPs and ddH2O is mixed according to the volume ratio of 6:10:5:19, wherein the volume ratio of the upstream primer, the downstream primer and the probe primer is 1:1: 1; the concentration of the pair of amplification primers is 5 mu M, the concentration of the probe primer is 2 mu M, the reaction buffer solution is 5 xTris-HCl with the pH value of 8.3, the concentration is 50mM, and dNTPs is concentratedThe degree was 1.5 mM.
2. The kit of claim 1, wherein the fluorescein is selected from carboxyfluorescein FAM, VIC, HEX, JOE, NED, TAMRA, CY3, ROX, or CY 5.
3. The kit of claim 1, wherein the kit further comprises a positive control and a negative control.
4. The kit of claim 3, wherein the positive control is a recombinant plasmid containing the African swine fever virus CD2v gene fragment amplified by the amplification primer, and the negative control is distilled water.
5. The kit according to claim 1, wherein the kit further comprises a reagent for extracting the African swine fever virus DNA template from the sample to be tested.
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