CN114544815B - Quantitative detection method for goat pox virus - Google Patents
Quantitative detection method for goat pox virus Download PDFInfo
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- G—PHYSICS
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Abstract
The invention discloses a quantitative detection method of goat pox virus. The method comprises the following steps: firstly, identifying a target peak by using a virus specificity detection method; then measuring the concentration of the standard substance by using a protein tester; serial dilution is carried out on the standard substance, the absorption peak of the standard substance at 280nm is detected on a liquid chromatograph, and a linear regression equation of concentration and peak area is established; and calculating according to a linear regression equation and the peak area of the sample to be detected to obtain the content of goat pox virus in the sample to be detected. The method can be used for determining the content of goat pox virus in goat pox virus suspension culture solution, purified solution and goat pox inactivated vaccine, has the advantages of rapidness, accuracy, stability and good repeatability, can guide the production of the vaccine, and plays an important role in improving the quality of the vaccine.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a method for detecting and quantifying viruses, and in particular relates to a method for quantitatively detecting goat pox viruses.
Background
Capripoxviruses (GPV) are members of the poxviridae, capripoxvirus genus, double-stranded DNA viruses, with a linear genome of about 150kb in length, encoding 147 open reading frames. The basic chemical components of viruses are proteins, DNA and lipids. The virus particles are brick-shaped and have the size of 300nm multiplied by 270nm multiplied by 200nm. GPV can infect goats of all varieties, sexes and ages, with lambs being most susceptible and the infection rate reaching 100%. GPV infects other goats by inhalation or transmission through the medium of the skin, respiratory tract, after break-up by contact with the acne rash, and transmission between flocks is rapid, but is not generally disseminated to other flocks. The goat pox virus has higher resistance to drying, and the virus in the dried crust can survive for 3 to 6 months, and can survive for about 5 minutes when the dry heat reaches 100 ℃. Has stronger resistance to common disinfectants, but 500mL/L alcohol and 0.1g/L KMnO4 can inactivate the disinfectant within 1 h. Is sensitive to diethyl ether and chloroform. Repeated freeze thawing has no obvious inactivation effect on the water.
The detection method of the virus is fewer in China, and the current research on the detection method of the virus mainly relates to a PCR method and a TCID 50 A method of manufacturing the same. CN104152583B discloses a primer, a kit and a detection method for goat pox virus Taqman-MGB probe real-time fluorescence quantitative PCR detection. The target sequence of interest is detected using a two-step amplification method. CN103215385B discloses a kit for detecting goat pox virus. The kit at least comprises four primers of SEQ No. 1, SEQ No. 2, SEQ No. 3 and SEQ No. 4, dNTPs, tris-HC, KCl, (NH 4) 2SO4, mgSO4, tween20 and Bst enzyme, and template DNA. Both the above methods are based on nucleic acid detection, require primer design and primer synthesis, and CN104152583B requires fluorescent dye, and are not only improved in terms of environmental friendliness, but also can only be used for determining whether goat pox virus gene sequences exist in samples, and cannot accurately quantify complete viruses.
TCID 50 The method has complicated operation and long time, the virus is mixed with cells after a plurality of dilutions, and the mixture is placed in a carbon dioxide incubator for culturing for 7 days, and the TCID of the virus is judged according to the cytopathic number 50 (half of the amount of cell infection). The method has high requirements on the aseptic conditions of the environment, such as improper operation, and is easy to cause bacterial pollution, thereby leading to test failure.
Along with the research of goat pox virus related vaccines, development of a quantitative detection method for complete goat pox virus, which is simple to operate, environment-friendly, and good in repeatability and reproducibility, is urgently needed, is used for quality control of goat pox virus production, and improves the quality of goat pox virus vaccine products.
The HP-SEC (High Performance-Size Exclusion Chromatography High-efficiency size exclusion chromatography) is a chromatographic separation method combining High-efficiency liquid chromatography and size exclusion chromatography, and adopts a gel chromatographic column to realize effective separation on the High-efficiency liquid chromatography according to the size of a detected substance, so that viruses are not damaged in the separation process. The sample can be detected in half an hour by simple centrifugation, and the repeatability of multiple detection is very good.
Disclosure of Invention
The invention provides a goat pox virus detection method which is simple to operate, high in sensitivity and good in repeatability. According to the structural characteristics of goat pox virus, the invention designs an HP-SEC method adopting gel column separation, combines sample treatment, standard product preparation and detection condition optimization, establishes an efficient goat pox virus quantitative detection method, and provides a quality control standard for goat pox vaccine research and development.
The invention provides a quantitative detection method of goat pox virus, which is used for quantitatively detecting goat pox virus in a sample to be detected, and comprises the following steps:
(1) Detecting a goat pox virus sample by liquid chromatography, and carrying out specific identification on a target peak to prepare a standard substance;
(2) Measuring the antigen concentration in the standard by using a protein tester;
(3) Serial dilution is carried out on the standard substance, detection is carried out on a liquid chromatograph, and a linear regression equation with peak area as an abscissa and concentration as an ordinate is established;
(4) Carrying out centrifugal clarification on a sample to be tested;
(5) Detecting a sample to be detected by using the liquid chromatography in the step (3) to obtain a corresponding peak area;
(6) Calculating according to a linear regression equation and the peak area of goat pox antigen in the sample to obtain the goat pox virus antigen content in the sample to be detected;
wherein the chromatographic column used in the liquid chromatography described in (3) and (5) is a gel chromatographic column, and the detector is an ultraviolet detector, and the detection wavelength is 280nm.
The mobile phase of the liquid chromatography is a solution containing 10 mM-30 mM phosphate (Na) 2 HPO4 and NaH 2 PO 4) and 5-15% methanol by volume, pH7.2. The mobile phase, phosphate buffer is preferably 10mM phosphate buffer, and the volume percentage of methanol is preferably 10%.
The flow rate of the mobile phase is 1.0ml/min, and the sample injection amount is as follows: 50 μl
Preferably, the gel column of the liquid chromatography used in the steps (1), (3) and (5) is Bio Basic SEC-2000 or Sephacryl S-500.
In the step (4), the sample to be tested is selected from any one of suspension culture solution or purified solution or goat pox inactivated vaccine.
In the step (4), the sample to be measured is centrifuged by a centrifuge for clarification.
The retention time of goat pox virus under the above chromatographic conditions was 9.85min.
In one embodiment of the invention, in step (1), the peak of interest is identified using a virus-specific assay.
In the step (2), the concentration of the antigen in the standard substance is measured by a Nanodrop protein measuring instrument.
In the step (3), at least 5 serial dilutions of goat pox virus standard substances are taken, chromatographic detection is carried out on the diluted serial standard substances, a linear regression equation with peak area as an abscissa and concentration as an ordinate is established, and R 2 Should be greater than 0.99.
In the step (4), the sample to be tested is selected from any one of virus suspension culture solution or purified solution or goat pox inactivated vaccine.
In the step (4), the goat pox virus suspension culture solution or the purified solution of the sample to be detected is clarified by centrifugation for 5-10 minutes at 3000-5000 r/min. After the goat pox inactivated vaccine is required to be dissociated, the virus liquid is clarified by adopting centrifugation for 5-10 minutes at 3000-5000 r/min.
Advantageous effects of the invention
The invention adopts HP-SEC method to quantitatively analyze goat pox virus, the sample does not need complex pretreatment, the error of manual operation is very small, the detection method is simple to operate, the repeatability of repeated detection is good, the precision is high, the detection time is short, the result can be obtained within half an hour, and the problem that the content of complete virus can not be accurately and quantitatively determined in vaccine production is effectively solved. The production of the vaccine can be effectively guided by quantifying the content of the antigen in the goat pox virus, and the method has great guiding significance for improving the quality of the vaccine.
Drawings
FIG. 1 is a specific identification of goat pox virus standard;
FIG. 2 is a standard curve for capripoxvirus quantification;
FIG. 3 is a test result of a suspension culture sample;
FIG. 4 shows the results of 5 replicates of the same sample of suspension culture.
Detailed Description
It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way. Various changes and modifications may be made by one skilled in the art in light of the teachings herein without departing from the spirit and scope of the invention as defined in the appended claims.
The quantitative detection method of goat pox virus adopted by the invention mainly comprises the following steps:
1. material
1.1 standard: purified goat pox virus (manufactured by Zhongmu practice Co., ltd.)
1.2 instrumentation and reagents: liquid chromatograph: AKT pure, purchased from GE company; protein quantitative instrument: nanodrop2000C, available from Thermo; naH (NaH) 2 P0 4 、Na 2 HP0 4 Methanol was purchased from the national drug group.
2. Method of
2.1 specific identification of goat pox virus and preparation of standard: taking goat pox virus AV41 strain liquid, centrifuging for 5-10 minutes at 3000-5000 r/min, taking supernatant, filtering with a 0.45 mu m filter membrane, concentrating by 10 times with a 100KD ultrafiltration membrane, and detecting by a liquid chromatograph to obtain an absorption spectrum at 280nm. The components are collected and the components are specifically identified by a virus specific detection method. Based on the identification, the fraction with a retention time of 9.85min was determined to be capripoxvirus. And (3) sampling for multiple times, collecting the components with the retention time of 9.85min, and concentrating the components by using a 100KD ultrafiltration membrane to prepare the goat pox virus standard substance.
2.2 standard protein concentration determination: the standard concentration was determined with a protein meter (Nanodrop 2000C, available from Thermo);
2.3 liquid chromatography system: the mobile phase is a mixed solution containing 10 mM-30 mM phosphate and 5% -15% methanol; the gel chromatographic column is Thermo Bio Basic SEC-2000 or Sephacryl S-500; the flow rate is 1.0ml/min; the sample was introduced in an amount of 50. Mu.l.
2.4 establishing a linear regression equation: serial dilution is carried out on goat pox virus standard products with known concentration, liquid chromatography detection is carried out on the serial standard products after dilution, and a linear regression equation with peak area as an abscissa and concentration as an ordinate is established;
preparing a sample to be tested: centrifuging a sample to be detected for 5-10 minutes at a speed of 3000-5000 r/min, taking supernatant and detecting the sample to be detected;
detecting a sample to be detected: detecting a sample to be detected by using the same method as that of the standard sample to obtain a corresponding sample peak area;
and calculating according to a linear regression equation and the peak area of the sample to obtain the goat pox virus concentration in the sample to be detected.
Example 1
The purpose of this example was to investigate the effect of the buffer system on the detection results.
Three buffers with different concentrations, namely 10mM, 20mM and 30mM phosphate buffer, are selected, methanol with the volume ratio of 5%, 10% and 15% is respectively added, and the influence of different buffer systems on detection is examined by taking the separation degree as a parameter.
The influence of different phosphate buffer systems on the degree of separation was examined using goat pox suspension culture as the subject and the results are shown in Table 1.
TABLE 1 results of influence of buffer composition on degree of separation
As is clear from Table 1, the optimal degree of separation was obtained by adding 10% by volume of methanol to 10mM phosphate buffer, while the degree of separation was lower for the other buffer compositions.
Example 2
And establishing an antigen quantitative standard curve.
(1) Preparation of a standard:
chromatographic detection and component collection: taking goat pox virus AV41 strain suspension culture solution, centrifuging for 5-10 minutes at 3000-5000 r/min, taking supernatant, filtering with a 0.45 mu m filter membrane, concentrating by 10 times with a 100KD ultrafiltration membrane, and detecting by a liquid chromatograph to obtain an absorption spectrum at 280nm. The fractions were collected.
Identification of the virus: the collected components are specifically identified by a virus specific detection method. The method comprises the following steps: the components were mixed with the same amount of MEM nutrient solution and simultaneously with the blank (cell growth solution) at 37 ℃ for 1 hour. 6 well cell culture plates, 1ml per well, 4 wells per set, were inoculated with well grown monolayers of MDBK cells, respectively. Placing at 37deg.C, containing 5% CO 2 Is cultured in the incubator for 5 days, and cytopathic effect is observed daily. If cytopathy occurs, the complete goat pox virus particles are contained, and the target peak is determined. The results are shown in FIG. 1, where the virus group showed significant cytopathy, while the blank group showed no cytopathy, indicating that the standard was goat pox virus.
And (3) preparation of a standard substance: based on the identification, the fraction with a retention time of 9.85min was determined to be capripoxvirus. And (3) sampling for multiple times, collecting the components with the retention time of 9.85min, and concentrating the components by using a 100KD ultrafiltration membrane to prepare the goat pox virus standard substance.
(2) Establishing a standard curve:
protein quantification was performed on goat pox virus standards using a protein assay.
The accurate quantitative goat pox virus standard is diluted by a ratio of 10mM phosphate buffer to a standard solution with a concentration of 80 mug/ml, 40 mug/ml, 20 mug/ml, 10 mug/ml, 5 mug/ml. 50 μl was taken for detection, the flow rate was 1.0ml/min, and the detection wavelength was 280nm. The statistical retention time was 9.85min characteristic peak area, repeated 2 times per group, and the results are shown in table 2.
TABLE 2 detection results of different concentrated goat pox virus standards
Linear regression was performed on the concentration of goat pox virus and the characteristic peak area,the results are shown in fig. 2, where the linear relationship between capripoxvirus concentration and characteristic peak area is y=2×10 -5 x-0.0376 (y: concentration in μg/ml; x: peak area value); r is R 2 The value is 0.9997, which shows that the characteristic peak-to-peak area and concentration of goat pox virus have high linear relation, and the detection method provided by the invention can be used for quantitative detection of goat pox virus.
(3) Precision of the method
After adding standard substances (20 mug/ml) with known concentration into the virus-free culture solution and mixing, the test is immediately carried out for 5 times in parallel, and the test result is shown in figure 4; the peak area value of each detection characteristic peak was recorded, and the precision and recovery rate of the detection were calculated, and the results are shown in Table 3 below.
TABLE 3 precision and recovery of the measurements
As shown in Table 3, the samples were measured in parallel 5 times, the relative standard deviation was only 0.64%, and the precision of the method was high; the average recovery rate is 102.6%, and the recovery rate of the method is good.
Example 3
Quantitative detection of goat pox virus suspension culture solution
1. Instrument and reagent
A liquid chromatograph AKTA pure is equipped with an ultraviolet detector. Protein quantification apparatus Thermo Nanodrop2000C.
Preparing a mobile phase: take 0.2M Na 2 72ml of HPO4 aqueous solution and 0.2M NaH 2 28mL of PO4 aqueous solution is prepared into buffer solution containing 10mM phosphate by using ultrapure water to reach 2000mL, methanol with the volume ratio of 10 percent is added, pH7.2 is adopted, and the mobile phase is obtained, and the mobile phase is filtered by a 0.45 mu m filter membrane.
Sample to be measured: goat pox virus suspension culture (AV 41 strain, available from the middle-grazing industry Co., ltd.)
Standard substance: prepared from the middle-grazing practice stock, inc., from example 2.
2. Capripoxvirus assay in suspension culture
The goat pox virus cultured in suspension is quantified, the sample is clarified by centrifugation (the sample to be tested is centrifuged for 5-10 minutes at 3000-5000 r/min), and 50 μl of supernatant is taken for detection.
FIG. 3 is the results of a sample of an embodiment of the present invention.
As shown in FIG. 3, in the detection result map of the above sample, there was a characteristic peak of goat pox virus at 9.85min of retention time. The measurement was continued 3 times, and the content of goat pox virus in the suspension culture was calculated to be 21.4. Mu.g/ml by calculating the content of the measurement of 21.3. Mu.g/ml, 21.7. Mu.g/ml and 21.1. Mu.g/ml by the standard curve of example 2.
Example 4
Quantitative detection of goat pox virus inactivated vaccine
1. Instrument and reagent
A liquid chromatograph AKTA pure is equipped with an ultraviolet detector. Protein quantifying instrument Thermo Nanodrop2000C.
Preparing a mobile phase: take 0.2M Na 2 HPO 4 72ml of aqueous solution and 0.2M NaH 2 PO 4 28mL of aqueous solution was prepared by diluting with ultrapure water to 2000mL, preparing a buffer solution containing 10mM phosphate, adding 10% (volume ratio) of methanol, pH7.2, to obtain a mobile phase, and filtering with a 0.45 μm filter membrane.
Sample to be measured: goat pox virus inactivated vaccine (Medium-grazing practice Co., ltd.)
Standard substance: standard prepared in example 2.
2. Determination of goat pox virus content in inactivated vaccine
Dissociating an inactivated vaccine sample to be detected by using n-butanol, adding n-butanol into the vaccine according to the ratio of the vaccine to the n-butanol=6:1, vibrating for 1-3 minutes, standing for 30 minutes at 2-8 ℃, taking a lower clarified liquid, clarifying by adopting a centrifugal method (centrifuging for 5-10 minutes at 3000-5000 r/min), and taking 50 μl of an upper clarified liquid for detection. The detection was continued 3 times, and the content of goat pox virus in the vaccine dissociation solution was calculated to be 6.33. Mu.g/ml by calculating the content of 6.3. Mu.g/ml, 6.4. Mu.g/ml and 6.3. Mu.g/ml by the standard curve of example 2, and the content of goat pox virus in the vaccine was calculated to be 12.66. Mu.g/ml by calculating the ratio of antigen to adjuvant in the vaccine to 1:1.
Example operation and Effect
The quantitative detection method for goat pox virus provided by the embodiment adopts a liquid chromatography based on a gel chromatographic column to quantitatively detect the goat pox virus, can effectively separate the virus from impurities, and achieves the purpose of detecting the virus. The relative standard deviation of the embodiment is only 0.64%, the repeatability of the method is good, the precision is high, the quantitative requirement of the detection method can be met, and the method can be applied as a quantitative detection method of goat pox virus.
The liquid chromatography has the advantages of simple operation, short detection time and the like, so that the quantitative detection method of the embodiment is suitable for quality control in the goat pox virus industrialized production process.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (6)
1. The quantitative detection method of goat pox virus is used for quantitatively detecting goat pox virus in a sample to be detected, and is characterized in that: comprising the following steps:
(1) Detecting a goat pox virus sample by liquid chromatography, identifying a target peak, and preparing a standard substance;
(2) Determining the concentration of antigen in a standard;
(3) Serial dilution is carried out on the standard substance, detection is carried out by liquid chromatography, and a linear regression equation with peak area as an abscissa and concentration as an ordinate is established;
(4) Carrying out centrifugal clarification on a sample to be tested;
(5) Detecting a sample to be detected by using the liquid chromatography in the step (3) to obtain a corresponding peak area;
(6) Calculating according to a linear regression equation and the peak area of goat pox virus in the sample to obtain the goat pox virus content in the sample to be detected;
wherein,,
the gel chromatographic column of the liquid chromatography used in the steps (1), (3) and (5) is Bio Basic SEC-2000 or Sephacryl S-500;
the liquid chromatography used in the steps (1), (3) and (5) uses gel chromatographic columns, and the detector is an ultraviolet detector with the detection wavelength of 280nm;
the liquid chromatography used in the steps (1), (3) and (5) has a mobile phase of a mixed solution of phosphate buffer and methanol; the phosphate buffer solution in the mobile phase is 10mM phosphate buffer solution, the volume percentage of methanol is 10%, and the pH value is 7.2;
the flow rate of the mobile phase in the liquid chromatography used in the steps (3) and (5) was 1.0ml/min.
2. The method for quantitatively detecting capripoxviruses according to claim 1, characterized in that: in the step (1), a goat pox virus specific detection method is adopted to identify a target peak, namely obvious lesions occur after the virus reacts with MDBK cells, and no lesions occur in a blank control.
3. The method for quantitatively detecting capripoxviruses according to claim 1, characterized in that: in step (2), the antigen concentration in the standard is measured by a protein meter.
4. The method for quantitatively detecting capripoxviruses according to claim 1, characterized in that: and (3) and (5) adopting 50 mu l of sample injection amount of the liquid chromatography.
5. The method for quantitatively detecting capripoxviruses according to claim 1, characterized in that:
in the step (4), the sample to be tested is selected from any one of suspension culture solution or purified solution containing goat pox virus or goat pox inactivated vaccine.
6. The method for quantitatively detecting capripoxviruses according to claim 1, characterized in that:
in the step (4), the goat pox virus suspension culture solution or the purified solution of the sample to be detected is clarified by adopting centrifugation at 3000-5000 r/min for 5-10 min, and the supernatant is taken for detection; the goat pox inactivated vaccine to be tested needs to be dissociated and clarified; the dissociation condition is vaccine that n-butanol=3:1-9:1, mixing and vibrating for 1-3 minutes, standing for 30 minutes at 2-8 ℃, taking the supernatant of the lower layer, centrifuging for 5-10 minutes at 3000-5000 r/min for clarification, and taking the supernatant of the upper layer for testing;
in the step (3), serial dilution is carried out on goat pox virus standard substances with determined antigen content, chromatographic detection is carried out on the serial standard substances after dilution, a linear regression equation with peak area as an abscissa and concentration as an ordinate is established, and R 2 Should be greater than 0.99.
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| CN102257134A (en) * | 2008-02-12 | 2011-11-23 | 赛诺菲巴斯德有限公司 | Methods using ion exchange and gel filtration chromatography for poxvirus purification |
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| CN110274982A (en) * | 2019-06-27 | 2019-09-24 | 中牧实业股份有限公司 | The quantitative detecting method of rabies viruses inactivation antigen |
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| CN102257134A (en) * | 2008-02-12 | 2011-11-23 | 赛诺菲巴斯德有限公司 | Methods using ion exchange and gel filtration chromatography for poxvirus purification |
| CN102373303A (en) * | 2011-11-29 | 2012-03-14 | 重庆出入境检验检疫局检验检疫技术中心 | Gene chip for identifying capripoxvirus virus and detection method of same |
| CN110274982A (en) * | 2019-06-27 | 2019-09-24 | 中牧实业股份有限公司 | The quantitative detecting method of rabies viruses inactivation antigen |
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