CN113234861A - Triple fluorescence quantitative PCR detection reagent, detection method and kit for detecting African swine fever virus - Google Patents
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Abstract
The invention provides a triple fluorescence quantitative PCR detection reagent, a detection method and a kit for detecting African swine fever virus, and relates to the technical field of molecular biology. The triple fluorescence quantitative PCR detection reagent for detecting African swine fever virus comprises: (a) a first primer pair shown as SEQ ID NO. 1 and SEQ ID NO. 2; (b) a second primer pair shown as SEQ ID NO. 4 and SEQ ID NO. 5; and (c) a third primer set represented by SEQ ID NO. 7 and SEQ ID NO. 8. The detection reagent can simultaneously detect three genes of the African swine fever virus, primers of different genes are mutually free of interference, and the detection reagent has strong specificity, high sensitivity, good repeatability and capability of qualitatively and quantitatively detecting a target sequence.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a triple fluorescence quantitative PCR detection reagent, a detection method and a kit for detecting African swine fever viruses.
Background
African Swine Fever (ASF) is a Swine disease caused by acute and febrile filterable virus with high infectivity, and is characterized by short disease process, high mortality rate of 100 percent, fever clinically manifested by fever, skin cyanosis, and obvious bleeding of lymph nodes, kidney and gastrointestinal mucosa.
The African swine fever is first reported in Kenya in 1909, always exists in African countries south of Sahara, is successively released to Western Europe and Lamet in 1957, and is mostly put out in time, but still prevails in Portugal, southwest Spanish and Italy Sadingan island. Since 2007, African swine fever has occurred, spread, and prevailed in several countries around the world, particularly Russia and its surrounding areas. In 2017, 3 months, African swine fever epidemic situation occurs in Oakzke in the far east Russian area, and the epidemic situation is close to China and is only about 1000 km. 3-15 days 8 months in 2018, and 3 African swine fever epidemics are successively discovered in 3 distant areas of Shenyang in Liaoning, Zhengzhou in Henan and Jiangsu Lianhun harbor in China. And then spread to the whole country, causing great loss to the pig industry. Over two years, the methods and means for preventing and controlling African swine fever are increasingly improved, the toxicity of the African swine fever virus is weakened, but the loss caused by the attenuated African swine fever virus still exists, and besides biological safety measures, early diagnosis and early response measures are still the main means for preventing and controlling the African swine fever.
It is reported that the genes detected first are different according to different virus infection time, and most of the current commercialized fluorescent quantitative PCR detection kits for African swine fever are PCR amplification of a single gene. It is important to establish a method for rapidly, accurately and simultaneously detecting a plurality of viral genes.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a triple fluorescence quantitative PCR detection reagent, a detection method and a kit for detecting African swine fever virus, which can simultaneously detect three genes of the African swine fever virus, have no interference among primers of different genes, and have strong specificity and high sensitivity.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a triple fluorescent quantitative PCR detection reagent for detecting African swine fever virus, which comprises:
(a) a first primer pair shown as SEQ ID NO. 1 and SEQ ID NO. 2;
(b) a second primer pair shown as SEQ ID NO. 4 and SEQ ID NO. 5; and
(c) a third primer pair shown in SEQ ID NO. 7 and SEQ ID NO. 8.
In one embodiment, the detection reagent further comprises: a first specific probe shown as SEQ ID NO. 3, a second specific probe shown as SEQ ID NO. 6 and a third specific probe shown as SEQ ID NO. 9.
In one embodiment, the probe is a hydrolysis probe, and the probe is labeled with a fluorescent reporter at the 5 'end and a quencher at the 3' end.
In one embodiment, the fluorescent reporter group is selected from any one of FAM, HEX, JOE, VIC; the quenching group is selected from any one of BHQ1, BHQ2, BHQ3 and TAMRA.
In one embodiment, the 5' end of the different specific probes is labeled with a different fluorescent reporter.
In a specific embodiment, the 5' fluorescent reporter of the first specific probe is FAM; the 3' end quenching group is BHQ 1.
In a specific embodiment, the 5' end fluorescent reporter of the second specific probe is HEX; the 3' end quenching group is BHQ 1.
In a specific embodiment, the 5' fluorescent reporter of the third specific probe is CY 5; the 3' end quenching group is BHQ 1.
In the primer pair and the probe, the primer pair shown by SEQ ID NO. 1 and SEQ ID NO. 2 and the probe shown by SEQ ID NO. 3 are suitable for detecting the P72 gene; the primer pair shown in SEQ ID NO. 4 and SEQ ID NO. 5 and the probe shown in SEQ ID NO. 6 are used for detecting the CD2V gene, and the primer pair shown in SEQ ID NO. 7 and SEQ ID NO. 8 and the probe shown in SEQ ID NO. 9 are used for detecting the MGF gene.
The invention also provides application of the detection reagent in detecting African swine fever virus, and the application is a non-disease diagnosis purpose.
The invention also provides a method for detecting African swine fever virus for non-disease diagnosis, which comprises the steps of carrying out real-time fluorescence quantitative PCR by using the detection reagent;
preferably, in the PCR reaction system of the real-time fluorescent quantitative PCR, the concentration of the upstream primer and the downstream primer is 5-250 μ M, and the concentration of the probe is 5-250 μ M.
In one embodiment, the reaction conditions of the real-time fluorescent quantitative PCR are: 94-96 ℃ for 2-3 min; 94-96 ℃ for 10-12 s; at 58-60 deg.c for 30-35 sec; 40-42 cycles.
In a specific embodiment, the reaction conditions of the real-time fluorescent quantitative PCR are: denaturation at 95 deg.C for 2 min; 95 ℃ for 10 s; at 58 ℃ for 30 s; 42 cycles and fluorescence collection at 58 ℃.
In one embodiment, the method comprises the steps of:
(1) extracting DNA of a sample to be detected;
(2) preparing a PCR reaction system, and performing triple fluorescence quantitative PCR amplification to obtain a fluorescence curve and a Ct value;
(3) and (3) obtaining the corresponding gene category in the sample according to the fluorescence curve and the Ct value obtained in the step (2).
The invention also provides a triple fluorescence quantitative PCR kit for detecting African swine fever virus, which comprises the detection reagent.
In one embodiment, the kit further comprises a positive plasmid standard, PCR buffer, DNA polymerase, Mg2+Any one or more of dNTPs and nucleic acid-free water.
In a specific embodiment, the kit comprises 4x buffer, positive plasmid standard, and the aforementioned detection reagents (preferably containing all specific primers and probes).
In a specific embodiment, the kit comprises 2ml of 4 Xbuffer, 1ml of positive plasmid standard, 6. mu.L of each forward primer at 40. mu.M/L, 6. mu.L of each reverse primer at 40. mu.M/L, and 4. mu.L of each probe at 20. mu.M/L.
And (3) judging the result according to the cycle number Ct value when the fluorescence signal reaches a set threshold value, wherein the Ct value of the detection result of the sample to be detected is less than or equal to 36, and the result is positive and indicates that the African swine fever virus exists in the sample.
The invention can be used for detecting African swine fever virus in nasal swab, blood and tissue of pigs.
Has the advantages that:
(1) the detection reagent and the kit can realize the simultaneous detection of three genes P72, CD2V and MGF of the African swine fever virus in a single-tube reaction, and are favorable for clinical early diagnosis of the African swine fever virus.
(2) The primer pairs of different genes in the detection reagent have no interference with each other, the detection sensitivity is high, and the lowest detection limit of the detection reagent is 1 copy/mul.
(3) The detection reagent has strong specificity, can perform qualitative and quantitative detection on a target sequence, and has good repeatability.
(4) The invention optimizes the primer and the probe, so that the PCR reaction system is stable and the amplification efficiency is high.
(5) By adopting the plasmid of gene clone as a standard substance, the procedure of detecting a sample can be completed within 1 hour and 10 minutes, three strains can be distinguished simultaneously, and the time is shortened by about 2 hours and 30 minutes compared with the conventional single identification.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a standard graph provided by an embodiment of the present invention;
FIG. 2 is a schematic diagram showing three S-shaped amplification curves as a result of fluorescent quantitative PCR;
FIG. 3 is a diagram showing two sigmoid amplification curves as a result of fluorescent quantitative PCR;
FIG. 4 is a schematic diagram showing a sigmoidal amplification curve as a result of fluorescent quantitative PCR;
FIG. 5 is a melt curve analysis of fluorescent quantitative PCR;
FIG. 6 is a graph of a repeatability test standard according to an embodiment of the present invention;
FIG. 7 shows the amplification results of the detection reagents (primer pairs) of the present invention in a reaction system containing CSFV cDNA, PRRSV cDNA, PRV DNA, PCVII DNA;
FIG. 8 shows a technical scheme for constructing a recombinant plasmid in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all expressions using "first" and "second" in the present invention are used for distinguishing two entities with the same name but different names or different parameters, and it is understood that "first" and "second" are only used for convenience of expression and should not be construed as limiting the present invention.
Example 1
1.1 primer design
Specific primers and specific probes are designed according to P72, CD2V and MGF genes of African swine fever HLJ/18-7GD in GenBank, and are consigned to Shanghai biological engineering company for synthesis.
The specific primer and specific probe sequences are shown in the following table:
1.2DNA extraction
Separating serum, placing 200 μ l serum into clean 1.5ml centrifuge tube, and extracting nucleic acid according to the extraction kit specification, wherein the nucleic acid extraction kit is DNA nucleic acid extraction kit manufactured by Tiangen biology company, and has a product number of DP 315-T3.
1.3 recombinant plasmid construction
The procedures were carried out using the pEASY-Blunt Cloning Kit and the Trans1-T1 Kit from Beijing Alternal gold Biotech, Inc., all as described in the specification. The technical route is shown in figure 8. Cloning the synthetic genes P72, CD2V and MGF to a pMBP-C vector to obtain a positive plasmid with three genes, transferring the positive plasmid into escherichia coli for culture, and amplifying the plasmid; and extracting plasmids, calculating copy number, performing gradient dilution by 10 times, preparing a standard curve, and quantifying to obtain a positive standard substance.
1.4 fluorescent quantitative PCR (dye method fluorescent quantitative)
The plasmid concentration was determined by 10-fold concentration gradient dilution of a known concentration of standard plasmid: 108、107、106、105、104、103copies/uL, respectively, were subjected to fluorescent quantitative PCR.
The positive control sample is ASFV genome DNA standard substance (which is diluted 1000 times by sterile nuclease-free water and is provided by the national African swine fever reference laboratory); the negative control sample was sterile nuclease-free water.
The PCR reaction system is as follows:
reaction procedure: denaturation at 95 ℃ for 2min, 10s at 95 ℃ and 30s at 58 ℃ for 42 cycles. Fluorescence collection was performed at 58 ℃.
The standard curve shows a good linear relationship, R2The slope was-3.329 at 0.999, and the amplification efficiency E was 99.657%. The standard curve is shown in FIG. 1.
Experimental results show that the primers and the probes have no cross reaction and accurate detection results.
Melt chain curve analysis (see fig. 5): when the product is dissolved, a sharp single peak appears, and the dissolving temperature is about 85 ℃, which shows that the real-time fluorescent quantitative PCR established by the method is specific amplification, the primers are specific amplification, and the result is credible.
Example 2
For repeatability verification, standard plasmids with known concentrations were diluted in a 10-fold concentration gradient:
107、106、105、104、103copies/uL, three replicates per concentration, simultaneous fluorescent quantitative PCR, reaction system and procedure identical to example 1.
In the third repetition, the amplification curves of the standard substance with the same concentration are basically overlapped, which shows that the concentration gradient and the repeatability of the diluted standard substance are good, the obtained standard curve is reliable, and the repeatability verification curve is shown in figure 6.
As can be derived from the standard curve, the detection sensitivity of the fluorescent quantitative PCR of the present invention is at least 1 copy/. mu.l.
Example 3
200 parts of clinical samples of a pig farm which are diagnosed as having a disease through third-party detection are detected by using a method established by the designed primer probe and the kit, each sample is divided into two parts, and the two parts are simultaneously sent to the third party for detection.
According to the judgment standard: and (5) judging a result: the Ct value is less than or equal to 36, the amplification curve is three S-shaped specific amplification curves, the amplification curve is judged to be positive, the three genes can be amplified simultaneously (shown in figure 2), the amplification curve is two S-shaped specific amplification curves, the amplification curve is a strain lacking one gene (shown in figure 3), the amplification curve is one S-shaped specific amplification curve, the amplification curve is two strains lacking genes (shown in figure 4), the Ct value is more than 36, the specific amplification curve is judged to be suspected, the sampling detection is carried out again, the amplification curve is still suspected, the specific amplification curve is judged to be positive, and the amplification curve is judged to be negative without the Ct value. The detection result of the invention is as follows: 11 positive parts. The third party has 11 positive detection results, and the detection result is the same as that of the single PCR reagent used by the third party. The primer group, the probe or the kit can realize the purpose of sensitively, quickly and accurately detecting the genes P72, CD2V and MGF.
The difference between single-tube multiplex PCR and single-tube multiplex PCR is not large, and the delta Ct is +/-0.5.
Comparative example (specificity verification experiment):
the detection reagent (primer pair) of the invention can not amplify the amplification curve of the corresponding disease in a reaction system containing CSFV cDNA, PRRSV cDNA, PRV DNA and PCVII DNA. Only ASFV standard curves were generated from all three batches of reagents (see figure 7).
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Fujian Aaonong Biotech group GmbH; thai and county proud breeding, Inc.;
leshan AoXin Breeding Co., Ltd; xiamen Yixiang group Co Ltd
<120> triple fluorescence quantitative PCR detection reagent, detection method and reagent for detecting African swine fever virus
Box
<130> PA20038155
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<170> PatentIn version 3.3
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Claims (10)
1. A triple fluorescence quantitative PCR detection reagent for detecting African swine fever virus, which is characterized in that the detection reagent comprises:
(a) a first primer pair shown as SEQ ID NO. 1 and SEQ ID NO. 2;
(b) a second primer pair shown as SEQ ID NO. 4 and SEQ ID NO. 5; and
(c) a third primer pair shown in SEQ ID NO. 7 and SEQ ID NO. 8.
2. The detection reagent of claim 1, wherein the detection reagent further comprises: a first specific probe shown as SEQ ID NO. 3, a second specific probe shown as SEQ ID NO. 6 and a third specific probe shown as SEQ ID NO. 9.
3. The detection reagent according to claim 2, wherein the probe is a hydrolysis probe, and the probe is labeled with a fluorescent reporter group at the 5 'end and a quencher group at the 3' end.
4. The detection reagent according to claim 3, wherein the fluorescent reporter group is selected from any one of FAM, HEX, JOE and VIC; the quenching group is selected from any one of BHQ1, BHQ2, BHQ3 and TAMRA.
5. The detection reagent according to claim 4, wherein the 5' end of the different specific probes is labeled with a different fluorescent reporter group.
6. Use of a detection reagent according to any one of claims 1 to 5 for the detection of African swine fever virus for non-disease diagnostic purposes.
7. A method for detecting african swine fever virus for non-disease diagnostic purposes, comprising performing real-time fluorescent quantitative PCR using the detection reagent of any one of claims 1-4;
preferably, in the PCR reaction system of the real-time fluorescent quantitative PCR, the concentration of the upstream primer and the downstream primer is 5-250 μ M, and the concentration of the probe is 5-250 μ M.
8. The method of claim 7, wherein the reaction conditions of the real-time fluorescent quantitative PCR are: 94-96 ℃ for 2-3 min; 94-96 ℃ for 10-12 s; at 58-60 deg.c for 30-35 sec; 40-42 cycles.
9. A kit for triple fluorescent quantitative PCR for the detection of african swine fever virus, comprising the detection reagent according to any one of claims 1 to 5.
10. The kit of claim 9, wherein the kit further comprises a positive plasmid standard, PCR buffer, DNA polymerase, Mg2+Any one or more of dNTPs and nucleic acid-free water.
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Citations (4)
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|---|---|---|---|---|
| CN111074000A (en) * | 2019-11-18 | 2020-04-28 | 华南农业大学 | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain and double-gene deletion strain |
| CN111996191A (en) * | 2020-09-28 | 2020-11-27 | 山东省农业科学院畜牧兽医研究所 | Primer group and kit for simultaneously identifying African swine fever wild strain and gene deletion strain based on multiple qPCR technology |
| CN112094950A (en) * | 2020-10-15 | 2020-12-18 | 杭州博日科技股份有限公司 | Primer group, kit, method and application for detecting African swine fever virus wild strain and gene deletion strain |
| CN112375849A (en) * | 2021-01-14 | 2021-02-19 | 北京明日达科技发展有限责任公司 | African swine fever virus triple fluorescence PCR detection kit and application thereof |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111074000A (en) * | 2019-11-18 | 2020-04-28 | 华南农业大学 | Triple fluorescence quantitative PCR detection material and kit for distinguishing ASFV wild strain and double-gene deletion strain |
| CN111996191A (en) * | 2020-09-28 | 2020-11-27 | 山东省农业科学院畜牧兽医研究所 | Primer group and kit for simultaneously identifying African swine fever wild strain and gene deletion strain based on multiple qPCR technology |
| CN112094950A (en) * | 2020-10-15 | 2020-12-18 | 杭州博日科技股份有限公司 | Primer group, kit, method and application for detecting African swine fever virus wild strain and gene deletion strain |
| CN112375849A (en) * | 2021-01-14 | 2021-02-19 | 北京明日达科技发展有限责任公司 | African swine fever virus triple fluorescence PCR detection kit and application thereof |
Non-Patent Citations (2)
| Title |
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| YANXING LIN等: ""Development of a triplex real-time PCR assay for detection and differentiation of gene-deleted and wild-type African swine fever virus"", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 王彩霞等: "非洲猪瘟病毒CD2v基因实时荧光PCR检测方法的建立", 《中国兽医科学》 * |
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