CN110607402A - Kit for detecting porcine transmissible gastroenteritis virus PLA - Google Patents
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Abstract
The invention relates to a swine transmissible gastroenteritis virus PLA detection kit. The kit comprises (1) two biotin-labeled antibodies: anti-TGEV antibody 1a2, anti-TGEV antibody 2B 1; (2) a probe; (3) connecting liquid; (4) PCR mix: universal PCR Assay, 2 Xfast Master Mix. Diluting two specific monoclonal antibodies of biotin labels aiming at the SA protein of the porcine transmissible gastroenteritis virus to working concentration, incubating and connecting the monoclonal antibodies with a probe, and finally amplifying a target single-stranded DNA by adopting real-time quantitative PCR to successfully detect the porcine transmissible gastroenteritis virus. The invention solves the defects of long time, large workload, low sensitivity, non-specificity, complex operation and the like in the prior art; has the advantages of no radioactive substance, low reagent and sample consumption, high sensitivity, no need of special equipment and simple operation.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a kit for rapidly detecting transmissible gastroenteritis virus (TGEV) by using a Proximity Ligation (PLA) technology.
Background
Porcine Transmissible Gastroenteritis (TGE) is a highly contagious disease characterized by severe vomiting and dehydration caused by porcine Transmissible gastroenteritis virus (TGEV). Pigs of different ages and breeds are susceptible to the disease, wherein the mortality rate of piglets within two weeks of age can reach 100%. TGE was first reported in 1946 by Doyle and Hutchings in the united states, in japan (1956) and in the united kingdom (1957), after which the occurrence of TGE was successively reported in europe, north america, asia, and other countries. The TGE is reported from the end of the last 50 years of the last century in China, the prevalence of the disease in China is in a remarkably rising trend in recent years, particularly, the disease is in local prevalence in winter and early spring cold seasons, and great harm is caused to the pig raising industry.
The latent period of the disease is very short, generally 12-18 hours, and piglets are usually seriously dehydrated and die after the disease. And the death rate of the old or adult pigs is extremely low, but the pigs lose weight and the feed reward is reduced, so that medicines and manpower are wasted, and huge economic loss is caused to the pig raising industry. Therefore, early detection and early treatment are important for preventing and controlling the disease.
The recessive infection of TGEV and the mixed infection with other pathogens are ubiquitous, the clinical diagnosis difficulty of TGEV is increased, the current TGEV infection diagnosis is mainly determined by laboratory diagnosis technologies including virus separation and identification, IPMA, IFA, gene chip, RT-LAMP, colloidal gold, RT-PCR and ELSA, but the virus separation and identification, IPMA, IFA and gene chip methods have the limitations of high test condition requirements, complex operation, complex process, high cost and the like, and are not beneficial to the rapid detection in basic laboratories. The RT-LAMP and colloidal gold methods have low requirements on instruments and equipment, but have high sensitivity, so false positive is easily caused, the RT-PCR is simple to operate and cannot quantify, the fluorescent RT-PCR method makes up the problems that the conventional RT-PCR cannot quantify and the like, but has high requirements on the technical levels of instruments, reagents and operators.
The Proximity Ligation Assay (PLA) technology is used as a new protein analysis tool, has the advantages of strong specificity, high sensitivity, simplicity, rapidness and the like, and is characterized in that a monoclonal antibody-nucleic acid compound is used as Proximity Ligation probes (Proximity probes), when a pair of Proximity probes simultaneously recognizes the same target protein molecule, the Proximity probes are adjacent to each other in spatial position, an amplifiable DNA label sequence is formed through Ligation reaction, and the label sequence can reflect the type and concentration of the protein to be detected. The technology converts the detection of protein into the detection of DNA nucleic acid sequence, and realizes the detection, quantification and positioning of special protein. The instrument is common, the required sample amount is small, and the operation is simple.
Disclosure of Invention
The invention aims to overcome the defects of other detection methods and develop a swine transmissible gastroenteritis virus PLA detection kit and a detection method thereof.
The technical scheme of the invention is as follows: a swine transmissible gastroenteritis virus PLA detection kit comprises the following components:
(1) biotin-labeled antibody: biotin-labeled anti-TGEV antibody 1a2, biotin-labeled anti-TGEV antibody 2B 1; the anti-TGEV antibody 1A2 is secreted by a cell strain TGEV-SA-1A2- (1-10) CGMCC No.18315 secreting anti-porcine transmissible gastroenteritis virus monoclonal antibody; the anti-TGEV antibody 2B1 is secreted by a cell strain TGEV-SA-2B1- (1-10) CGMCC No.18314 secreting anti-porcine transmissible gastroenteritis virus monoclonal antibody;
(2) and (3) probe: 200nM 3 'Prox-Oligo, 200nM 5' Prox-Oligo, probe dilution buffer, antibody dilution buffer;
(3) connecting liquid: 500 XDNA ligase, 1 Xligase dilution buffer solution and 20 Xligation reaction buffer solution;
(4)PCR mix：Universal PCR Assay、2×Fast Master Mix。
the method for detecting the transmissible gastroenteritis virus of the pig by using the kit PLA comprises the following specific steps
(1) Ligation of Biotin-labeled antibodies to oligonucleotide fragments
Biotin B marker 2B1 is named as Probe a, biotin B marker 1A2 is named as Probe B, 5 mu L of Probe a and 5 mu L of Prox-Oligo 3 'are sucked into a PCR tube and mixed evenly, 5 mu L of Probe B and 5 mu L of Prox-Oligo 5' are added into a new reaction tube to be mixed, after being mixed evenly by blowing, the incubation is carried out for 60min at room temperature, after the incubation is finished, 90 mu L of AssayProbe Storage Buffer is added into each PCR tube, and after being mixed evenly, the mixture is kept still for 20min at room temperature;
(2) negative control and probe solution preparation
mu.L of Antibody Dilution Buffer and 2. mu.L of ③ acid were added into the negative control reaction tube, after incubation for 1h at room temperature, 396. mu.L of label Dilution Buffer were added, respectively, after incubation for 30min at room temperature, the reaction tube was put on ice.
After 6.75. mu.L of Assay Probe Dilution Buffer, 0.375. mu.L of Assay Probe a, and 0.375. mu.L of Assay Probe b were mixed, 5. mu.L of the mixed Assay Probe was diluted 1:10 with 1 XPBS, diluted and placed on ice, 5. mu.L of the diluted Probe solution and 5. mu.L of the virus lysate were mixed, and incubated in a 37 ℃ water bath for 1 hour.
(3) Ligation reaction
Adding 96 μ L of connecting solution at 37 deg.C for 10min to 4 μ L of probe virus mixed solution and 4 μ L of negative control solution; 4 ℃ for 10 min.
(4) Real-time quantitative PCR: after mixing well 10. mu.L of Fast Master Mix and 1. mu.L of Universal PCR Assay, 9. mu.L of ligation product was added and qPCR was performed: cycling at 95 ℃ for 20s, then 95 ℃ for 3s and 60 ℃ for 30s, was repeated 40 times.
And (4) analyzing results: if the Ayg Ct (control) -Ayg Ct (forced prox-probe) is more than or equal to 8.5, the detection of the transmissible gastroenteritis virus is indicated.
The swine transmissible gastroenteritis virus ortho-position connection method (PLA) established by the kit optimizes the concentration of the biotin-labeled monoclonal antibody, selects the antibody concentration when the delta Ct maximum value is selected, continuously searches and optimizes the using amount of the antibody nucleic acid probe, avoids the false positive of an overhigh background value to a detection result, simultaneously considers the concentration of the probe when the delta Ct maximum value is selected, selects the optimal using amount, improves the accuracy and the sensitivity of an experiment, solves the defects of lower detection sensitivity, long detection time, possibility of false negative and the like existing in the method for detecting the swine transmissible gastroenteritis virus in the prior art, improves the detection sensitivity by 3-4 orders of magnitude compared with the conventional detection method, and provides a new method for sensitively, quickly and specifically detecting the swine transmissible gastroenteritis.
Compared with the prior art, the invention has the advantages and positive effects that: the invention establishes a porcine transmissible gastroenteritis virus PLA detection kit and a detection method thereof, and the kit combines an anti-TGEV-SA antibody marked by biotin with PLA for application. The invention adopts PLA technology, has strong specificity, can directly analyze complex biological samples, has low sample loss, higher specificity and detection sensitivity and simple, convenient and quick operation compared with the conventional PCR or RCA method. Can be used for detecting the transmissible gastroenteritis virus of swine, and is accurate and rapid.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention.
Example 1: preparing the monoclonal antibody for resisting the transmissible gastroenteritis virus of the pig.
(1) Constructing a baculovirus expression vector of the TGEV-Sa gene, and further transfecting sf9 to express to obtain rBacmid-TGEV-Sa fusion protein;
(2) and (2) immunizing Balb/c mice by using the protein expressed in the step (1), and fusing with myeloma cells after three times of immunization.
(3) Ten days after cell fusion, hybridoma cells are screened by an indirect Immunofluorescence (IFA) method, 7 hybridoma cells capable of stably secreting specific antibodies against transmissible gastroenteritis of swine virus are obtained after three times of subcloning, and are respectively named as Mab-TGEV-SA-1A2- (1-10), Mab-TGEV-SA-2H2, Mab-TGEV-SA-2B1- (1-10), Mab-TGEV-SA-3G1, Mab-TGEV-SA-4E3, Mab-TGEV-SA-5A1 and Mab-TGEV-SA-5E 3.
(4) The subtype of the seven monoclonal antibodies was identified using the SBA Clonotyping TM System/HRP antibody subclass kit. The results show that the heavy chains of the Mab-TGEV-SA-1A2- (1-10), the Mab-TGEV-SA-2B1- (1-10) and the Mab-TGEV-SA-5A1 are identified by subclasses as IgG1, the other four heavy chains are IgM, the light chains are all k chains, and the light chains have specific fluorescence only with TGEV. Wherein ascites titers of three monoclonal antibodies, namely Mab-TGEV-SA-1A2- (1-10), Mab-TGEV-SA-2B1- (1-10) and Mab-TGEV-SA-5A1, are respectively 1:12800, 1:25600 and 1: 1600.
The cell strain TGEV-SA-2B1- (1-10) is preserved in the general microorganism center of China microorganism culture preservation management committee (address: No. 3 of Xilu No.1 of Beijing Korean district, China academy of sciences, microorganism research institute) in 2019, 8, 12 months and 12 days, and is classified and named as a cell strain secreting the monoclonal antibody against the transmissible gastroenteritis virus of swine, and the preservation number is CGMCC No. 18314.
The cell strain TGEV-SA-1A2- (1-10) is preserved in the general microorganism center of China microorganism culture preservation management committee (address: No. 3 of Xilu No.1 of Beijing Korean district, China academy of sciences, microorganism research institute) in 2019, 8, 12 months and 12 days, and is classified and named as a cell strain secreting the monoclonal antibody against the porcine transmissible gastroenteritis virus, and the preservation number is CGMCC No. 18315.
Example 2: the porcine transmissible gastroenteritis virus PLA detection kit is prepared according to the following formula
(1) Biotin-labeled antibody: biotin-mAb-TGEV-SA-1A2, biotin-mAb-TGEV-SA-2B 1;
(2) and (3) probe: 200nM 3 'Prox-Oligo, 200nM 5' Prox-Oligo, Ab Dilution Buffer, Assay Probe Dilution Buffer, and Antibody Dilution Buffer; (TaqMan Protein Reagents Base Kit)
(3) Connecting liquid: 500 XDNA ligand, 1 Xligand Dilution Buffer, 20 Xligand Reaction Buffer;
(4)PCR mix：Universal PCR Assay、2×Fast Master Mix。
preparation of antibody nucleic acid Probe complexes from Assay Probe mixture: 2 μ L of 2, 2 μ L of 3 and 396 μ L of LAssay Probe Dilution Buffer.
Example 3: preparing a treating fluid for detecting a sample to be detected of the transmissible gastroenteritis virus by using a porcine transmissible gastroenteritis virus PLA detection kit: taking a sample from jejunum as an example
(1) Treatment of tissue samples
A. Scraping intestinal contents and jejunum mucosa tissue 0.5g, adding physiological saline containing double antibodies, fully grinding in a grinder for 3-5min to obtain a suspension, and placing the suspension in a 1.5mL centrifuge tube;
b.12,000 Xg, centrifuging for 15min, and sucking the supernatant;
C. filtering with 0.22 μm microporous membrane, and storing at-20 deg.C in refrigerator.
(2) And (3) detection:
A. ligation of tissue extracts to probes
Adding 2. mu.L of diluted probe solution, 2. mu.L of virus tissue extract and 96. mu.L of connecting solution into a PCR reaction tube, preparing a PCR reaction tube with 4. mu.L of fifth and 96. mu.L of connecting solution as negative control, and keeping the temperature at 37 ℃ for 10 min; 4 ℃, 10 min;
B. real-time quantitative PCR
Adding 11 μ L of PCR mix into 9 μ L of each of the two reaction tubes, circulating at 95 deg.C for 20s, then at 95 deg.C for 3s,
30s at 60 ℃ and 40 times. The Ct value of the sample group is 22.62, the Ct value of the negative control group is 33.12, the delta Ct value is 10.5 and is more than 8.5, which indicates that the sample contains the transmissible gastroenteritis virus, otherwise, indicates that the transmissible gastroenteritis virus in the sample is negative.
Example 4: method for detecting porcine transmissible gastroenteritis virus PLA sensitivity test
Diluting TCID 50-known porcine transmissible gastroenteritis virus liquid by 10 times as PLA detection test template, and making the copy number of the virus in tissue fluid contained in each microliter of the diluted liquid be 108，107，106，105，104，103，10210, 1; (Wangxian pig transmissible gastroenteritis virus TZ-10-2016 whole gene sequence analysis and indirect ELISA method establishment [ D]Yangzhou university, 2018.)
Respectively adding 2 mu L of virus diluent into a PCR reaction tube filled with 2 mu L of diluted probe solution, setting a blank control without adding virus tissue fluid, and incubating for 1h at room temperature;
adding 96 μ L of connecting liquid at 37 deg.C for 10 min; and (3) at 4 ℃ for 10min, then transferring 9 mu L of the connection product to a new PCR reaction tube, respectively adding 11 mu L of PCR mix, and carrying out qPCR reaction, wherein if the Ayg Ct (control) -Ayg Ct (forced prox-probe) is more than or equal to 8.5, the detection sensitivity of the kit is at least 1 virus copy, and the kit has higher detection sensitivity.
Example 5: accuracy test of porcine transmissible gastroenteritis virus PLA detection method
(1) Diluting TCID 50-known porcine transmissible gastroenteritis virus liquid by 10 times as PLA detection test template, so that each microliter of the diluted liquid contains 10 copies of virus gene8，107，106，105，104，103，102，10，1；
(2) Respectively taking virus liquid diluted by times in the step (1) as a template, and carrying out amplification detection on a target gene by using an established PLA method and a PCR method;
(3) as shown in the detection results in Table 1, the two detection methods have higher coincidence rate.
TABLE 1 comparison of conventional RT-PCR and PLA test results
Claims (2)
1. A swine transmissible gastroenteritis virus PLA detection kit comprises the following components:
(1) biotin-labeled antibody: biotin-labeled anti-TGEV antibody 1a2, biotin-labeled anti-TGEV antibody 2B 1; the anti-TGEV antibody 1A2 is secreted by a cell strain TGEV-SA-1A2- (1-10) CGMCC No.18315 secreting anti-porcine transmissible gastroenteritis virus monoclonal antibody; the anti-TGEV antibody 2B1 is secreted by a cell strain TGEV-SA-2B1- (1-10) CGMCC No.18314 secreting anti-porcine transmissible gastroenteritis virus monoclonal antibody;
(2) and (3) probe: 200nM 3 'Prox-Oligo, 200nM 5' Prox-Oligo, probe dilution buffer, antibody dilution buffer;
(3) connecting liquid: 500 XDNA ligase, 1 Xligase dilution buffer solution and 20 Xligation reaction buffer solution;
(4)PCR mix：Universal PCR Assay、2×Fast Master Mix。
2. the kit for detecting porcine transmissible gastroenteritis virus PLA according to claim 1, wherein the kit comprises the following components: 250. mu.L of biotin-labeled anti-TGEV antibody 1A, 150. mu.L of biotin-labeled anti-TGEV antibody 2B, 5. mu.L of each of 200nM 3 'oligonucleotide probe and 200nM 5' oligonucleotide probe, 800. mu.L of probe dilution buffer, 2. mu.L of antibody dilution buffer, 2. mu.L of 500 XDNA ligase, 198. mu.L of 1 Xligase dilution buffer, 50. mu.L of 20 Xligation reaction buffer, and 908. mu.L of ultrapure water.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104777312A (en) * | 2015-03-05 | 2015-07-15 | 江苏省农业科学院 | ELISA kit used for detecting swine transmissible gastroenteritis virus antibody |
| CN105527437A (en) * | 2015-12-17 | 2016-04-27 | 洛阳普莱柯万泰生物技术有限公司 | Detection kit and application thereof |
| CN105779649A (en) * | 2016-03-29 | 2016-07-20 | 扬州大学 | Immune PCR reagent kit for detecting avian leukemia virus |
| CN107942061A (en) * | 2017-11-29 | 2018-04-20 | 洛阳现代生物技术研究院有限公司 | A kind of test card, preparation and its detection method for detecting transmissible gastro-enteritis virus antibody |
| CN109232736A (en) * | 2017-07-10 | 2019-01-18 | 洛阳普莱柯万泰生物技术有限公司 | Specifically bind monoclonal antibody, pharmaceutical composition, kit and its application of transmissible gastro-enteritis virus |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104777312A (en) * | 2015-03-05 | 2015-07-15 | 江苏省农业科学院 | ELISA kit used for detecting swine transmissible gastroenteritis virus antibody |
| CN105527437A (en) * | 2015-12-17 | 2016-04-27 | 洛阳普莱柯万泰生物技术有限公司 | Detection kit and application thereof |
| CN105779649A (en) * | 2016-03-29 | 2016-07-20 | 扬州大学 | Immune PCR reagent kit for detecting avian leukemia virus |
| CN109232736A (en) * | 2017-07-10 | 2019-01-18 | 洛阳普莱柯万泰生物技术有限公司 | Specifically bind monoclonal antibody, pharmaceutical composition, kit and its application of transmissible gastro-enteritis virus |
| CN107942061A (en) * | 2017-11-29 | 2018-04-20 | 洛阳现代生物技术研究院有限公司 | A kind of test card, preparation and its detection method for detecting transmissible gastro-enteritis virus antibody |
Non-Patent Citations (3)
| Title |
|---|
| DEBAIZE 等: ""Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells:application to pre-B lymphocytes"", 《MOLECULAR CYTOGENETICS》, vol. 10, no. 27, 20 July 2017 (2017-07-20) * |
| 唐娜等: "邻位连接技术及其在病原检测中的应用", 《动物医学进展》, no. 07, 20 July 2011 (2011-07-20) * |
| 张霞等: "利用邻位连接技术检测花生过敏原成分", 《食品研究与开发》, no. 09, 10 May 2016 (2016-05-10) * |
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