CN103472225A - Fluorescence immunoassay kit used for detecting porcine circovirus type 2 - Google Patents
Fluorescence immunoassay kit used for detecting porcine circovirus type 2 Download PDFInfo
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Abstract
The invention provides a fluorescence immunoassay kit used for detecting porcine circovirus type 2 (PCV2), which comprises a single factor serum antibody used for detecting PCV2, an FITC (fluorescein isothiocyanate) labeled goat anti-mouse IgG (immunoglobulin G), a positive control sample, stationary liquid and PBS (phosphate buffer) washing liquor, wherein the single factor serum antibody used for detecting PCV2 is prepared from a PCV2 Cap protein antigen immunized mouse with an amino acid sequence of SEQ ID NO: 1. The main ingredient in the kit used for detecting PCV2 prepared in the invention is a single factor serum antibody of mouse anti-porcine circovirus type 2 and is prepared from the antigen immunized mouse with the amino acid sequence of SEQ ID NO: 1, and the prepared antibody can generate a specific reaction with PCV2. The fluorescence immunoassay kit of porcine circovirus type 2 created on the basis of the single factor serum can be used for detecting antigen in the diseased tissues of an PCV2 infected pig on clinic and judging whether the pig is infected with PCV2, and the fluorescence immunoassay kit is strong in specificity, high in sensitivity, good in repeatability and high in detection speed.
Description
Technical field
The invention belongs to virus of domestic animal detection technique field, be specifically related to a kind of fluorescence immunoassay kit for detection of porcine circovirus 2 type.
Background technology
Within 1991, Canadian John Harding has reported pmws (Postweaning Multisystemic Wasting Syndrome, PMWS), by investigation and research extensively and profoundly, confirm that in 1998 pig circular ring virus (Porcine Circovirus, PCV) is main pathogen.Existing research shows, Porcine circovirus desease (Porcine Circovirus Disease, PCVD) be that a kind of clinical symptoms caused by the PCV2 infection is gradual becoming thin, expiratory dyspnea is rapid, anaemia, diarrhoea, jaundice, interstitial pneumonia, lymphnoditis and ephritis, main infringement weanling pig, with the PMWS occurred in recent years, pigskin inflammation and nephritic syndrome (Porcine Dermatitis and Nephropathy Syndrome, PDNS), porcine respiratory syndrome (Porcine Respiratory Disease Complex, PRDC), the congenital chatter of A2 type (Congenital Tremor, CT), pig hyperplasia and necrotizing pneumonia (Porcine Proliferative and Necrotizing Pneumonia, PNP), the diseases such as breeding difficulty have closely related.The harm of PCV2 is to make the immunologic function that infects pig to suffer damage, and causes Abwehrkraft des Koepers to descend, and often with the form appearance of subclinical infection, easily out in the cold.Because PCV2 infects, immune system is suffered damage, easily secondary or concurrent other pathogen, aggravate disease, and cause larger harm.It is worldwide popular that this disease is, and since 2000 confirm that there is this disease in China, now ubiquity in China swinery, cause sizable economic loss to China's pig industry.
Pig circular ring virus (PCV) belongs to PCV-II section, Circovirus on taxonomy, is one of animal virus of known minimum.Virion diameter 17nm, be 20 body symmetrical structures, without cyst membrane.PCV has two genotype, be PCV1, PCV2, Genome Size is about 1.76kb, contains 2 main reading frames, ORF1 gene outcome relevant to rdrp virus (Rep) wherein, the ORF2 gene outcome is to form viral capsid proteins (Cap) composition.PCV1 is found by Tischer etc. in 1974 first in the PK15 cell culture, to the pig no pathogenicity.The detection of PCV2 at present mainly adopts the methods such as PCR (PCR), enzyme linked immunosorbent assay (ELISA) antibody assay kit, in situ hybridization (ISH), immunohistochemistry (IHC) and the recurrence of this animal to carry out antidiastole.Wherein PCR and ISH need clear and definite and general nucleotide sequence to be differentiated, for experimenter's operative technique, require high and are prone to false positive; IHC is higher for the technical requirement of operator's pathological section, and trace routine is relatively loaded down with trivial details; And animal returns experiment and need to breed experimental animals and treat that morbidity observes clinical symptoms for differentiating, therefore diagnose consuming time long and cost is higher; The ELISA antibody assay kit can only detect PCV2 antibody, can't determine whether the pig body infects PCV2 is arranged.
Summary of the invention
The purpose of this invention is to provide a kind of fluorescence immunoassay kit for detection of porcine circovirus 2 type, can be the sensitiveer special PCV2 virus that detects, thus make up the deficiencies in the prior art.
Fluorescence immunoassay kit of the present invention, include single-factor serum antibody, FITC mark goat anti-mouse igg, positive control sample, immobile liquid and PBS washing lotion for detection of PCV2; The single-factor serum antibody of described detection PCV2 is with amino acid sequence, to be SEQ ID NO:1 prepared by Porcine circovirus type 2 Cap antigen immune mouse.
Fluorescence immunoassay kit of the present invention is to have following component to form:
1) for detection of the single-factor serum antibody of PCV2;
2) two is anti-: the goat anti-mouse IgG of FITC mark;
3) positive control sample: the PCV2SD strain that deposit number is CGMCC NO.7707;
4) immobile liquid: press acetone and methyl alcohol volume ratio 1:1 configuration immobile liquid;
5) PBS washing lotion: phosphate buffer, press Nacl8.0g; Kcl0.2g; Na
2hPO
43.58g; KH
2pO
40.24g be settled to 1L.
Wherein porcine circovirus 2 type (Porcine Circovirus2) strain PCV2SD strain, on May 31st, 2013, be deposited in and be positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3, China Committee for Culture Collection of Microorganisms's common micro-organisms center of Institute of Microorganism, Academia Sinica, deposit number is CGMCC NO.7707.
The above-mentioned serum antibody of the single-factor for detection of PCV2, its concrete preparation method is as follows: the Porcine circovirus type 2 Cap gene that is SEQ ID NO:2 by sequence inserts in expression vector PET-28a, is built into recombinant expression PET-ORF2; By conversion and IPTG, induce the BL21 Host Strains to express the His-Cap recombination fusion protein; Recombination fusion protein is carried out to purifying, and mixed immunity adjuvant immunity BALB/C mice, collect mice serum and make the single-factor serum antibody.
What the present invention was prepared is mouse resisting porcine circovirus 2 type single-factor serum antibodies for detection of principal ingredient in the kit of PCV2, be by amino acid sequence, to be SEQ ID NO:1 prepared by the antigen immune mouse, specific reaction can occur with PCV2 in the antibody of preparing.The porcine circovirus 2 type fluorescence immunoassay kit that this single-factor serum is fundamental construction of take can detect the antigen in PCV2 infected pigs pathological material of disease tissue clinically, determines whether and infects PCV2, and its high specificity, highly sensitive, favorable repeatability and detection speed are fast; Compare with PCR, ELISA, ISH, IHC and animal Orthogonal Rotational Regressive Tests diagnostic method in the past, utilize this kit more convenient, more accurate by indirect immunofluorescence assay (IFA) technology for detection.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail, the present invention's chemicals used or reagent, can select according to its function the product of existing other type, and be not limited only to the concrete record of embodiment.For the experimental technique of unreceipted actual conditions in embodiment, condition routinely usually, the condition described in " the molecular cloning experiment guide " write as J. Pehanorm Brooker (Sambrook) etc., or the condition of advising according to manufacturer operation.
(1) for detection of the preparation of the single-factor serum antibody (primary antibodie) of PCV2
The preparation of the Cap albumen of the PCV2 that 1, amino acid sequence is SEQ ID NO:2
According to one section sequence at PCV2ORF2 gene two ends and the polyclone restriction enzyme site of Expressing vector PET-28a, the Auele Specific Primer PCV2ORF2-F:5 ' of design PCV2ORF2-GGG GGA TCC ATG ACG TAT CCA AGG AGG CGT-3 ' (having added BamH I restriction enzyme site), PCV2ORF2-R:5 '-GGG AAG CTT TAA GGT TTT AAG TGG CTT GTC-3 ' (having added Hind III restriction enzyme site).The DNA that PCV2SD strain (CGMCC NO.7707) virus liquid of take extracts is template, and the amplified production size is the genes of interest sequence of 702bp, and primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Pcr amplification, and purify and reclaim kit recovery genes of interest with DNA, genes of interest also is connected and obtains the PET-ORF2 recombinant plasmid with carrier PET-28a double digestion simultaneously, this recombinant plasmid is proceeded to expressivity e. coli bl21 Host Strains according to a conventional method, the single clone of picking, enzyme cutting method is identified positive colony, shake bacterium, order-checking, sequencing result is shown in SEQ ID NO:2, the amino acid sequence of the albumen of its translation is SEQ ID NO:1; This sequence analyze is found to it is the gene order that PCV2 is new through BLAST, with known 4 strain PCV2 sequence amino acid identities 85.5～93.2%.
Be due to the error produced in amplification procedure in order to get rid of this new allele, carried out repeatedly increasing and checking order, prove that the Cap protein gene of the expression PCV2 amplified in positive control bacterium PCV2SD strain is new allele.
2, the Cap antigen that is SEQ ID NO:2 with amino acid sequence prepares single-factor serum antibody (primary antibodie)
The expressivity BL21 Host Strains that correct single clone's recombinant plasmid PET-ORF2 transforms by sequencing result, be inoculated in the LB bacteria culture media that 5ml contains ampicillin (100mg/L), 37 ℃ of jolting overnight incubation, take out next day in the LB nutrient culture media that the 1ml culture is inoculated in 120ml, after 2.5h is cultivated in 37 ℃ of joltings, bacterium liquid shakes to translucent half muddy state, and absorption photometry records OD
600=0.6～0.8, first get 10ml as contrast before inducing before inducing, and the jolting temperature is become to 30 ℃ add IPTG (final concentration is 1.0mmol/L) and induce, induce 4h to collect 10ml bacterium liquid, using induce unconverted pet vector bacterium liquid as blank, the bacterium liquid of Induction Transformation pet vector of usining contrasts as empty carrier.The bacterium liquid of abduction delivering is taken out, the centrifugal supernatant of abandoning, use again the resuspended thalline of 0.5ml1 * PBS, after the broken bacterium of ultrasonic treatment, add 2 * loading Buffer by 1:1 and boil 10min, the centrifuging and taking supernatant, supernatant obtains the approximately destination protein of 27～28kDa with 12%SDS-PAGE electrophoresis identification and analysis, utilizes His label purification kit to operate in accordance with regulations destination protein is carried out to purifying.By purifying, good spectrophotometric standard measure for the His-Cap destination protein (1.05mg/ml) adds the Freund's complete adjuvant of equivalent, 10 BALB/C mice of neck hypodermic injection, approximately 105 μ g/ are only for every 0.2ml(), 2 weeks rear Cap proteantigen back subcutaneous inoculations with containing incomplete Freunds adjuvant for the second time, 0.2ml/ only, immunity (method with for the second time) for the third time after 2 weeks again, wherein 4 not the BALB/C mice of immunizing antigen as negative control group.The rear 10d of immunity for the third time, the heart blood sampling is lethal, and the immune serum of separating out is put-20 ℃ of preservations.
Susceptibility and the specificity identification of the single-factor serum 3, prepared for PCV2SD strain Cap albumen
By PCV2SD separated strain virus liquid (10
6.25tCID
50/ ml) with the PK15 cell suspension just digested, press in 1:9 volume ratio combined inoculation to 24 porocyte culture plate (1ml/ hole), take out Tissue Culture Plate after maintaining 72h, abandoning supernatant, with 1 * PBS, wash one time, acetone: methyl alcohol (1:1) immobile liquid is in-20 ℃ of fixing 30min, 1 * PBS washes 3 times, use respectively 1 * PBS to press 1:50 mice serum, 1:100, 1:200, 1:500 makes four dilutabilitys and is added on the PK15 cell fixed, hatch 90min for 37 ℃, 1 * PBS washes 3 times, 37 ℃ of sheep anti-mouse iggs (Sigma company) that add again the FITC mark of 1:200 dilution are hatched 60min, 1 * PBS washes 3 times, discard cleansing solution, again each Kong Jiayi in 24 orifice plates is dripped to 50% glycerine, observation experiment result under fluorescent microscope, qualification result effect when the 1:100 dilutability is better.Evaluation is distinguished in other 4 strain PCV2:SDId01 strain (HM535640), AH05 strain (FJ644556), GXWM strain (EF675241) and SH03733 strain (GQ358998) that single-factor serum prepared for PCV2SD strain (positive control strain) Cap albumen by utilization is preserved laboratory by IiT (IFA), and result is all reacted and the specificity green fluorescence occurred with this 4 strain PCV2.The single-factor serum prepared with the Cap albumen for preparing of PCV2 with report is compared, single-factor serum antibody (primary antibodie) susceptibility and specificity height prepared by PCV2SD strain Cap albumen of the present invention all are significantly improved (p≤0.05), and this may have the difference of Porcine circovirus type 2 Cap amino acid sequence of the present invention and the albumen reported to cause.
(2) fluorescence immunoassay kit for detection of porcine circovirus 2 type builds and the sensitivity and specificity test
1, for detection of the porcine circovirus 2 type fluorescence immunoassay kit, build
1) primary antibodie: the anti-PCV2SD strain of mouse Cap albumen single-factor serum is the crucial constituent of this kit, the PCV2SD strain be separated to according to this laboratory preparation.
2) two is anti-: the goat anti-mouse IgG of FITC mark, and purchased from sigma company.
3) positive control poison: the PCV2SD strain that deposit number is CGMCC NO.7707;
4) immobile liquid: press acetone and methyl alcohol volume ratio 1:1 configuration immobile liquid, sealing is preserved, with front being put in-20 ℃ of Refrigerator stores.
5) washing lotion: phosphate buffer (PBS), press Nacl 8g; Kcl 0.2g; Na
2hPO
43.58g; KH
2pO
40.24g be settled to 1L.
2, for detection of porcine circovirus 2 type fluorescence immunoassay kit sensitivity, specificity and replica test 1) IFA and PCR contrast detection
Detect susceptibility and the specificity of the fluorescence immunoassay kit of porcine circovirus 2 type antigen in order to analyze the present invention, the tissue sample of applying respectively the doubtful PCV2 infection of 103 parts of clinical diagnosises of IFA and PCR method and Jiangsu, Zhejiang, Anhui, Shandong and Fujian Deng Di pig farm censorship has carried out contrast and has detected.IFA and PCR detect source, different regions tissue sample and the results are shown in following table.
2) replica test
Utilize this fluorescence immunoassay kit for detection of porcine circovirus 2 type to be detected 103 parts of clinical pathological material of diseases, extract sample DNA increases and verifies this kit testing result by PCR method simultaneously, except Jiangsu and Anhui tissue sample detect number positive: PCR method is than the equal many exceptions of IFA method, and all the other testing results are basically identical.Above-mentioned Jiangsu and Anhui sample are carried out to duplicate detection twice time simultaneously, the IFA testing result with come to the same thing for the first time, second and third time the PCR testing result is consistent with the IFA result, and the PCR testing result is described
Be prone to false positive results, thereby prove sensitivity, specificity and the repeatability of this detection kit testing result.The repeatability testing result sees the following form.
2) cross matching
Randomly draw the fluorescence immunoassay kit that a collection of utilization detects porcine circovirus 2 type, some cause of disease strains that laboratory is preserved are detected, result shows that pig breeding and disordered breathing syndrome virus, pig parvoviral, Latex agglutination test, CSFV, PRV detect, result fluorescence all do not occur at the fluorescence microscopy Microscopic observation, negative result, further illustrate the fluorescence immunoassay kit that the present invention detects porcine circovirus 2 type and not there will be false negative, there is good specificity.
Effect to kit of the present invention is described below:
A applies this kit can not produce false positive and false negative result.
B susceptibility and specificity are higher: 1:100 times of dilution effect of this single-factor serum working concentration of preparation is better, and accurately the sensitive known PCV2 virus to laboratory detects diagnosis.
The C clinical application effect is better: the samples such as porcine tissue that utilize this kit to cause clinical 103 parts of doubtful PCV2 are detected, with sensitivity, higher PCR method is verified simultaneously, result detects 23 parts of infection that contain PCV2, this kit testing result is consistent with second and third PCR method testing result, the sensitivity, specificity and the repeatability that have proved this kit testing result are higher, and clinical application effect is better.
Claims (4)
1. the fluorescence immunoassay kit for detection of porcine circovirus 2 type, include single-factor serum antibody, FITC mark goat anti-mouse igg, positive control sample, immobile liquid and PBS washing lotion for detection of PCV2; The single-factor serum antibody of described detection PCV2 is to be prepared by the Porcine circovirus type 2 Cap antigen immune mouse that amino acid sequence is SEQ ID NO:1 by sequence.
2. fluorescence immunoassay kit claimed in claim 1, it is constructed as follows:
1) for detection of the single-factor serum antibody of PCV2;
2) two is anti-: the goat anti-mouse IgG of FITC mark;
3) positive control sample: PCV2 Strain;
4) immobile liquid: press acetone and methyl alcohol volume ratio 1:1 configuration immobile liquid;
5) PBS washing lotion: phosphate buffer, press Nacl 8.0g; Kcl 0.2g; Na
2hPO
43.58g; KH
2pO
40.24g be settled to 1L.
3. kit as claimed in claim 1 or 2, it is characterized in that, the described serum antibody of the single-factor for detection of PCV2, its preparation method is as follows: the Porcine circovirus type 2 Cap gene that is SEQ ID NO:2 by sequence inserts in expression vector PET-28a, is built into recombinant expression PET-ORF2; By conversion and IPTG, induce the BL21 Host Strains to express the His-Cap recombination fusion protein; Recombination fusion protein is carried out to purifying, and mixed immunity adjuvant immunity BALB/C mice, collect mice serum and make the single-factor serum antibody.
4. kit as claimed in claim 2, is characterized in that described PCV2 Strain, is the deposit number PCV2SD strain that is CGMCC NO.7707.
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| CN104407136A (en) * | 2014-12-07 | 2015-03-11 | 青岛易邦生物工程有限公司 | Method for measuring content of viruses in avian encephalomyelitis live vaccine |
| CN106589119A (en) * | 2017-01-17 | 2017-04-26 | 青岛易邦生物工程有限公司 | Rabbit Bordetella bronchiseptica single-factor serum |
| CN108303541A (en) * | 2017-01-11 | 2018-07-20 | 上海鸣捷生物科技有限公司 | A kind of porcine circovirus type 2 antibody testing kit and its detection method |
| CN109913586A (en) * | 2019-03-25 | 2019-06-21 | 新乡学院 | A method of PCV-2 is detected using PCR-ELISA |
| CN112014561A (en) * | 2020-09-03 | 2020-12-01 | 扬州大学 | Application of porcine circovirus type 4 specific antigen in preparation of kit for detecting porcine circovirus type 4 antibody and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104407136A (en) * | 2014-12-07 | 2015-03-11 | 青岛易邦生物工程有限公司 | Method for measuring content of viruses in avian encephalomyelitis live vaccine |
| CN108303541A (en) * | 2017-01-11 | 2018-07-20 | 上海鸣捷生物科技有限公司 | A kind of porcine circovirus type 2 antibody testing kit and its detection method |
| CN108303541B (en) * | 2017-01-11 | 2023-07-25 | 上海鸣捷生物科技有限公司 | Porcine circovirus type 2 antibody detection kit and detection method thereof |
| CN106589119A (en) * | 2017-01-17 | 2017-04-26 | 青岛易邦生物工程有限公司 | Rabbit Bordetella bronchiseptica single-factor serum |
| CN109913586A (en) * | 2019-03-25 | 2019-06-21 | 新乡学院 | A method of PCV-2 is detected using PCR-ELISA |
| CN112014561A (en) * | 2020-09-03 | 2020-12-01 | 扬州大学 | Application of porcine circovirus type 4 specific antigen in preparation of kit for detecting porcine circovirus type 4 antibody and kit |
| CN112014561B (en) * | 2020-09-03 | 2022-04-19 | 扬州大学 | Application of porcine circovirus type 4 specific antigen in preparation of kit for detecting porcine circovirus type 4 antibody and kit |
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