CN112611867A - IFA antibody detection method of CSFV - Google Patents

IFA antibody detection method of CSFV Download PDF

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CN112611867A
CN112611867A CN202011459336.6A CN202011459336A CN112611867A CN 112611867 A CN112611867 A CN 112611867A CN 202011459336 A CN202011459336 A CN 202011459336A CN 112611867 A CN112611867 A CN 112611867A
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csfv
cells
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李鹏
王利平
刘兴友
金前跃
王寅彪
陈磊山
刘长忠
孙国鹏
岳锋
李红
张艳芳
齐永华
潘鹏涛
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Xinxiang University
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    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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Abstract

The invention discloses an IFA antibody detection method of CSFV, belonging to the field of biotechnology. According to the method for detecting the IFA antibody of the CSFV, the cell reaction plate can be prepared in advance and can be stored for a long time at the temperature of-20 ℃; the reaction results were observed by a fluorescence microscope. Has the advantages of strong specificity, high sensitivity, simple operation, and long shelf life. The invention utilizes the whole virus to detect the antibody, and can truly reflect the antibody level of animals.

Description

IFA antibody detection method of CSFV
Technical Field
The invention relates to the technical field of biology, in particular to an IFA antibody detection method of CSFV.
Background
Hog Cholera (classic Swine feber), early named Hog Cholera (HC), is an acute, febrile, and highly contagious disease of Swine caused by Hog Cholera Virus (CSFV), characterized by high Fever retention and generalized systemic hemorrhage. The morbidity and mortality of swine fever are high, and the swine fever is mainly divided into acute type, subacute type, chronic type, atypical swine fever or mild swine fever, which causes great economic loss to the swine industry all over the world. It is popular in different degrees around the world, and is classified as a type of animal disease in China, and is classified as an animal epidemic disease which must be reported by the world animal health Organization (OIE). Classical Swine Fever Virus (CSFV) belongs to the genus Pestivirus (Pestivirus) of the family Flaviviridae (Flaviviridae), is an RNA virus, and is infectious. Under natural conditions, pigs may also be infected with Bovine Viral Diarrhea Virus (BVDV) and ovine Border Disease Virus (BDV) of the same genus as Classical Swine Fever Virus (CSFV), but CSFV is pathogenic only to pigs. Although CSFV is not pathogenic to other animals, it can proliferate in animals such as mice, cattle and goats. At present, the main measure for preventing and treating the swine fever is vaccine immunity, and attenuated vaccines such as C-strain swine fever lapinized attenuated vaccine, French Thiverval-strain attenuated vaccine and Japanese cell attenuated vaccine researched by Chinese scholars such as Zhongtaichong and the like are successfully made out and play an important role in preventing and treating the swine fever. The vaccines used in China at present mainly comprise hog cholera lapinized virus vaccines, spleen stranguria vaccines and bovine testis vaccines. These vaccines have been able to control the prevalence of swine fever well, but due to the long-term use of the vaccines, swine fever has changed greatly in prevalence, mainly into atypical and mild swine fever. This causes difficulties in diagnosis and control of swine fever, and causes swine fever to be always popular, which troubles the swine industry in China. Based on the method, the establishment of a rapid and accurate diagnosis method and the research of the infection mechanism thereof have important significance for the comprehensive prevention and control of CSFV.
Therefore, it is an urgent problem to be solved by those skilled in the art to provide a method for detecting IFA antibody of CSFV.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting IFA (indirect immunofluorescence) antibody of CSFV.
In order to achieve the purpose, the invention adopts the following technical scheme:
a CSFV IFA antibody detection method comprises the following steps:
(1) PK15 cells were plated at 1.0X 105Inoculating the CSFV virus solution to a 96-well cell culture plate, diluting the CSFV virus solution with DMEM containing 2% fetal calf serum at a ratio of 1:10 after the cells adhere to the wall, and then adding the solution into the 96-well cell culture plate, wherein each well is 100 mu L; put in 5% CO at 37 DEG C2Culturing in an incubator for 48h, and observing the growth condition of the cells every day;
(2) after 2 days, the culture medium in the 96-well cell culture plate is discarded, the cells are washed mildly by PBST and are patted dry;
(3) adding precooled absolute ethyl alcohol, placing 50 mu L of the absolute ethyl alcohol in each hole at minus 20 ℃ for 30min, taking out, discarding the absolute ethyl alcohol, washing PBST, and patting dry;
(4) adding 5% skimmed milk powder, sealing, placing at 37 deg.C in incubator for 2 hr, and sealing at 200 μ L per hole; PBST cleaning and patting dry;
(5) adding CSFV positive antibody, diluting with PBS at a ratio of 1:400, adding 50 μ L into each well, and standing at 37 deg.C in incubator for 1 h; PBST cleaning and patting dry;
(6) adding goat anti-pig IgG-FITC, diluting with PBS at a ratio of 1:100, adding 50 μ L per well, standing at 37 deg.C for 1h, washing with PBST, adding 50 μ L per well, and observing with inverted fluorescence microscope.
Further, the preparation method of the CSFV virus solution comprises the following steps: grinding clinically collected pathological tissues, adding 1mL of PBS (phosphate buffer solution) into 1mg of tissue samples for dissolving, repeatedly freezing and thawing for 3 times, and taking supernatant; centrifuging at 12000rpm for 5min, and filtering with 0.22 μm filter to obtain CSFV virus solution.
According to the technical scheme, compared with the prior art, the invention discloses the IFA antibody detection method of CSFV, and the cell reaction plate can be prepared in advance and can be stored for a long time at-20 ℃; the reaction result can be observed by a fluorescence microscope; has the advantages of strong specificity, high sensitivity, simple operation, and long shelf life. The invention utilizes the whole virus to detect the antibody, and can truly reflect the antibody level of animals.
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, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the result of PCR amplification according to the present invention;
wherein, M, DL 2000; 1, CSFV amplification product;
FIG. 2 is a graph showing the results of detecting CSFV by IFA according to the present invention;
wherein, A is a virus cell test, and B is a non-virus normal cell control.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
Collecting blood samples and pathological tissues of the suspicious infected classical swine fever virus, centrifuging the collected blood, and collecting serum for later clinical serum detection. Grinding the pathological tissues, detecting CSFV virus and separating the virus. The cell line is PK15 cell, and CSFV positive serum is stored in a laboratory.
Example 1 isolation and characterization of CSFV Virus
After pathological tissues (lymph, liver, spleen, lung) suspected of CSFV virus were washed several times with PBS, 1mL of PBS was added to 1mg of tissue sample to dissolve it, and freeze-thawing was repeated 3 times, and the supernatant was taken, centrifuged at 12000rpm for 5min, and filtered through a 0.22 μm filter to obtain CSFV virus solution. Using a DNA extraction kit, RNA of CSFV virus was extracted, and then the RNA was reverse transcribed into cDNA. DNAMAN software is utilized to design a target gene primer of the classical swine fever virus, an amplified fragment is 531bp, and the specific primer sequence is as follows:
CSFV-F：5’-cggctagcctgcaaggaagattac-3’；SEQ ID NO.1；
CSFV-R：5’-tcatagatcttcattttccactgtggtgg-3’；SEQ ID NO.2。
using the designed primer and the extracted cDNA as a template to carry out PCR amplification, wherein the reaction system is as follows: 1 mu L of template, 0.5 mu L of CSFV-F, 0.5 mu L of CSFV-R, 12.5 mu L of Ex Taq enzyme and 10.5 mu L of double distilled water. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1min, and performing 30 cycles; then, the extension is carried out for 10min at 72 ℃, and the product is stored at 4 ℃ after the extension is finished. And subjected to agarose gel electrophoresis, and the results are shown in FIG. 1.
The results in FIG. 1 show that the size of the target fragment is about 531bp, and the sequencing result is consistent with the expected result; indicating that the pathological material contains CSFV.
EXAMPLE 2 establishment of clinical antibody detection method
The general PK15 cells were treated in a manner of 1.0X 105After the cells were attached to the 96-well cell culture plate, the CSFV virus solution isolated in example 1 was diluted 1:10 with DMEM containing 2% fetal bovine serum and then added to the 96-well cell culture plate at 100 μ L per well while a negative control was set up. Put in 5% CO at 37 DEG C2The incubator is used for 48 hours, and the growth condition of the cells is observed every day.
After 2 days, the culture medium in the 96-well cell culture plate is discarded, the cells are gently washed for 3 times by PBST, and the cells are patted dry; adding precooled absolute ethyl alcohol, placing 50 mu L of the absolute ethyl alcohol in each hole at minus 20 ℃ for 30min, taking out, discarding the absolute ethyl alcohol, washing PBST for 5 times, and patting to dry; adding 5% skimmed milk powder, sealing, placing at 37 deg.C in incubator for 2 hr, and sealing at 200 μ L per hole; PBST is washed for 5 times and is dried; adding CSFV positive antibody (CSFV positive antibody obtained by clinical separation according to the detection method of IDEXX kit), diluting with PBS according to a ratio of 1:400, adding 50 μ L into each well, and standing at 37 ℃ in incubator for 1 h; PBST was washed 5 times, patted dry, goat anti-porcine IgG-FITC was added, diluted with PBS at a ratio of 1:100, 50. mu.L of PBST was added to each well, incubated at 37 ℃ for 1 hour, PBST was washed 5 times, and finally 50. mu.L of PBST was added to each well, and observation was performed on an inverted fluorescence microscope to confirm that there was specific green fluorescence in nuclei of CSFV-infected positive PK15 cells, and the results are shown in FIG. 2.
And (4) IFA result judgment: the nucleus or cytoplasm of the CSFV-infected cell is green fluorescent, i.e. FIG. 2A (the virus-infected cell) is positive; whereas uninfected PK15 cells did not fluoresce green, i.e. figure 2B (non-vaccinated normal cells) was negative.
Example 3 IFA reaction conditions
1)CSFV TCID50Measurement of
(1) The PK15 cell suspension was plated in 96-well plates at 100. mu.L/well to achieve a cell mass of 2-3X 105Culturing for 12h per mL until the cells are completely attached to the wall;
(2) continuously diluting the CSFV virus liquid by 10 times in a penicillin bottle or a centrifuge tube-1-10-10；
(3) Inoculating the diluted virus to a 96-well plate with cells growing into a single layer, wherein each dilution is inoculated to a longitudinal row of 8 wells, and each well is inoculated with 100 mu L;
(4) leaving two longitudinal rows without virus inoculation, and setting normal cell control (100 μ L of maintenance solution per well, the maintenance solution is DMEM medium containing 2% fetal bovine serum);
(5) culturing for 48h, taking out and fixing after the cells are full, and placing at-20 ℃ for later use;
(6) detecting by using the established IFA antibody detection method, and observing and recording the number of holes for detecting whether lesion (CPE) occurs;
(7)TCID50the calculation of (b) is carried out according to the Reed-Muench two-degree method.
TABLE 1 TCID50Statistics of measurement results
Figure BDA0002830788520000051
TCID calculation by Reed-Muench two-law50The calculation method is as follows:
distance ratio (percentage above 50% rate of illness-50%)/(percentage above 50% rate of illness-percentage below 50% rate of illness) — (70-50)/(70-18.2) — 0.38
lgTCID50Distance ratio x difference between log of dilutions + log of dilutions above 50% disease rate-0.38 × (-1) + (-4) — 4.38.
TCID is obtained from the data50＝10-4.38/0.1ml＝10-5.38/ml
The meaning is as follows: diluting the virus 104.38Inoculation with 100. mu.l resulted in 50% of the cells being diseased.
2) Viral inoculation amount and fixation time
Respectively using 1000 TCIDs50100 TCIDs5010 TCIDs 501 TCID500.1 TCID50The virus of (4) was inoculated to PK15 cells and an unvaccinated control was set. And (3) detecting by using an IFA detection method and taking positive serum and negative serum as primary antibodies respectively, and determining the optimal virus inoculation amount by observing results. Inoculating CSFV virus (optimal virus inoculation amount) into PK15 cell bottles growing to about 60% -70%, spreading the cells on 4 96-well plates after the cells are full, and culturing at 37 deg.C with 5% CO2After 12, 24, 36 and 48 hours of culture in the incubator, the culture medium is taken out one by one and fixed. Then, positive serum and negative serum are respectively used as primary antibody for detection by using an IFA detection method, and the fixed time is determined by observing the result.
As a result, it was found that the number of TCIDs was 10050After the virus is inoculated with PK15 cells and cultured for 48 hours, IFA cells are prepared by fixationThe reaction plate is most preferred.
3) Serum dilution concentration, goat anti-porcine IgG-FITC working concentration
CSFV-infected pig serum is added into a medium according to the proportion of 1: 50. and (3) carrying out continuous multiple dilution at the ratio of 1:100, 1:200, 1:400 and the like, adding the diluted solution serving as a primary antibody into the prepared cell reaction plate, respectively carrying out detection by using an IFA antibody detection method, and determining the optimal serum dilution concentration according to the observation result.
Taking out the prepared cell reaction plate, adding 1: positive and negative sera were diluted 400-fold and then added to the 1:100, 1: 200. goat anti-porcine IgG-FITC diluted by 2 times at a ratio of 1:400, 1:800 and the like, and the optimal secondary antibody dilution concentration is determined by observing the experimental result.
The result shows that the serum to be detected starts from 1:50, the antibody titer can be detected by continuous 2-fold dilution on the basis of the strength of the antibody, the goat anti-pig IgG-FITC working concentration is 1:100 optimal, and the observation is carried out by an inverted fluorescence microscope.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> college of New county
<120> an IFA antibody detection method of CSFV
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 1
cggctagcct gcaaggaaga ttac 24
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 2
tcatagatct tcattttcca ctgtggtgg 29

Claims (2)

1. A method for detecting IFA antibody of CSFV is characterized in that the method comprises the following steps:
(1) PK15 cells were plated at 1.0X 105Inoculating the CSFV virus solution to a 96-well cell culture plate, diluting the CSFV virus solution with DMEM containing 2% fetal calf serum at a ratio of 1:10 after the cells adhere to the wall, and then adding the solution into the 96-well cell culture plate, wherein each well is 100 mu L; put in 5% CO at 37 DEG C2Culturing in an incubator for 48h, and observing the growth condition of the cells every day;
(2) after 2 days, the culture medium in the 96-well cell culture plate is discarded, the cells are washed mildly by PBST and are patted dry;
(3) adding precooled absolute ethyl alcohol, placing 50 mu L of the absolute ethyl alcohol in each hole at minus 20 ℃ for 30min, taking out, discarding the absolute ethyl alcohol, washing PBST, and patting dry;
(4) adding 5% skimmed milk powder, sealing, placing at 37 deg.C in incubator for 2 hr, and sealing at 200 μ L per hole; PBST cleaning and patting dry;
(5) adding CSFV positive antibody, diluting with PBS at a ratio of 1:400, adding 50 μ L into each well, and standing at 37 deg.C in incubator for 1 h; PBST cleaning and patting dry;
(6) adding goat anti-pig IgG-FITC, diluting with PBS at a ratio of 1:100, adding 50 μ L per well, standing at 37 deg.C for 1h, washing with PBST, adding 50 μ L per well, and observing with inverted fluorescence microscope.
2. The method according to claim 1, wherein the CSFV virus solution is prepared by the following steps: grinding clinically collected pathological tissues, adding 1mL of PBS (phosphate buffer solution) into 1mg of tissue samples for dissolving, repeatedly freezing and thawing for 3 times, and taking supernatant; centrifuging at 12000rpm for 5min, and filtering with 0.22 μm filter to obtain CSFV virus solution.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103698518A (en) * 2013-12-30 2014-04-02 山东滨州博莱威生物技术有限公司 Method for detecting virus content of swine fever live vaccine through indirect immunofluorescence
CN106383226A (en) * 2016-08-19 2017-02-08 金宇保灵生物药品有限公司 A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103698518A (en) * 2013-12-30 2014-04-02 山东滨州博莱威生物技术有限公司 Method for detecting virus content of swine fever live vaccine through indirect immunofluorescence
CN106383226A (en) * 2016-08-19 2017-02-08 金宇保灵生物药品有限公司 A quantitative detection method for the titer of a swine fever neutralizing antibody in swine serum and a detection kit

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
张朝红等: "猪瘟病毒4种检测方法的比较", 《西北农林科技大学学报(自然科学版)》 *
许保疆等: "单克隆抗体间接免疫荧光检测猪瘟病毒方法的建立", 《中国兽医学报》 *
金前跃: ""猪圆环病毒-Ⅱ型抗体检测免疫层析试纸的研制及单克隆抗体的筛选与鉴定"", 《中国知网》 *
高小静: ""PRRSV的IPMA和IFA临床抗体检测方法的建立及应用"", 《万方学位论文》 *

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