CN114350852B - Quantitative detection method for animal-derived live viruses - Google Patents
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Abstract
The invention discloses a quantitative detection method of animal-derived live viruses, which belongs to the technical field of biology, and comprises the following steps: a. treating a virus liquid sample to be tested by adopting ethidium azide bromide; b. further processing by isoelectric precipitation, collecting supernatant; c. collecting supernatant, and extracting virus nucleic acid; d. carrying out PCR detection on virus nucleic acid by adopting real-time fluorescent quantitative PCR, and calculating to obtain the content of live viruses in the virus liquid sample to be detected according to the Ct value of the amplification curve; the fluorescence quantitative PCR detection method for the animal-derived live viruses has the advantages of high sensitivity, strong specificity, high accuracy, short detection period and the like, overcomes the defects of the existing detection method, and meets the requirement of detecting the virus content in the production of veterinary biological products.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a quantitative detection method for animal-derived live viruses.
Background
In animal-derived virus test research and biological product production, quantitative detection of virus content is often required, and common detection methods comprise a chicken embryo half-number infection method, a cell half-number infection method, an animal half-number infection method, a virus plaque method, an indirect immunofluorescence method, a hemagglutination test method and the like, but the methods generally have the problems of more influencing factors, low accuracy and the like.
The real-time fluorescent quantitative PCR technology has been applied to the quantitative detection of microorganisms such as bacteria and viruses, and has strong sensitivity and good specificity. However, due to the changes of the environmental physical and chemical values, temperature and other factors of the virus, partial virus envelope protein denaturation or nucleocapsid rupture often occurs, so that the virus loses infectivity, whether the virus has infectivity or not cannot be effectively distinguished based on a fluorescence quantitative PCR method, namely, the live virus and the inactivated virus are difficult to distinguish, and the detection result is inaccurate.
The azide ethidium bromide is a photosensitive reaction material with high affinity to nucleic acid, can enter broken cells or bacteria, can be combined with DNA/RNA to form covalent bonds, inhibits PCR amplification reaction, has been applied to living bacteria detection of various food-borne pathogenic bacteria, has shown that after bacteria are inactivated, the thalli are rapidly broken to expose the nucleic acid, and after the treatment of the azide ethidium bromide, the influence of the inactivated bacteria nucleic acid on the PCR detection result can be eliminated, so that the quantitative detection of the living bacteria is realized.
In recent years, the application of ethidium azide to virus detection has been studied, however, the application of the technology has some problems. After virus inactivation, virus particles without infectivity exist for a long time, nucleic acid is wrapped by nucleocapsid protein, envelope protein and the like, and ethidium azide bromide cannot be combined with the unexposed nucleic acid, and the inactivated virus nucleic acid can influence the accuracy of PCR detection, so a real-time fluorescence quantitative PCR detection method capable of distinguishing live viruses from inactivated viruses is needed, and animal-derived live viruses are quantitatively detected.
Disclosure of Invention
The invention aims to provide a quantitative detection method for animal-derived live viruses, which solves the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a quantitative detection method of animal-derived live viruses, which comprises the following steps:
a. treating a virus liquid sample to be tested by adopting ethidium azide bromide;
b. further processing by isoelectric precipitation, collecting supernatant;
c. Collecting supernatant, and extracting virus nucleic acid;
d. And carrying out PCR detection on the virus nucleic acid by adopting real-time fluorescent quantitative PCR, and calculating to obtain the content of the live virus in the virus liquid sample to be detected according to the Ct value of the amplification curve.
The detection method combines the technology of adding the azido ethidium bromide, isoelectric point precipitation and the real-time fluorescence quantitative PCR technology, firstly removes the exposed virus nucleic acid by adopting the azido ethidium bromide, and then removes the inactivated virus of the unexposed nucleic acid by adopting isoelectric point precipitation, so that the inactivated virus and the live virus in the virus liquid to be detected can be distinguished, and the inactivated virus nucleic acid can not interfere with the fluorescence real-time quantitative PCR detection.
Further, the virus liquid to be detected is animal-derived virus liquid.
Further, the virus liquid sample to be detected is treated by adopting ethidium azide bromide, and the method specifically comprises the following steps: and adding an azide ethidium bromide solution into the virus liquid sample to be detected, uniformly mixing in a dark place, and adopting strong light irradiation treatment.
Further, adding an ethidium azide bromide solution into the virus liquid sample to be detected, so that the final concentration of the ethidium azide bromide in the virus liquid to be detected is 50-100 mu mol/mL.
Further, the light-shielding mixing comprises the steps of oscillating for 10-20 min under a light-shielding environment; the strong light irradiation treatment comprises the step of adopting a 300-500W LED lamp to irradiate strong light for 10-20 min.
Further, the isoelectric precipitation method specifically includes: slowly adding hydrochloric acid solution into the virus liquid sample to be detected which is subjected to ethidium bromide azide treatment, adjusting the pH of the solution to 4.0-5.0, and standing for 1-2 h.
Further, the concentration of the hydrochloric acid solution is 1-2 mol/L.
Further, the above detection method is suitable for detecting various animal-derived viruses, and viruses in the virus liquid sample to be detected include, but are not limited to: chicken pox virus, chicken infectious laryngotracheitis virus recombinant chicken pox virus, chicken newcastle disease virus, chicken infectious bronchitis virus, chicken infectious bursal disease virus, chicken infectious laryngotracheitis virus, chicken Marek's disease virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus, swine fever virus and porcine viral diarrhea virus.
The invention discloses the following technical effects:
1. According to the invention, ethidium azide and isoelectric precipitation technology are combined, so that the inactivated virus is effectively removed, the influence of the inactivated virus nucleic acid on the fluorescent PCR detection result is eliminated, the detection result reflects the content of the live virus, and the accurate quantitative detection of the live virus by the real-time fluorescent PCR method is realized for the first time.
2. The fluorescence quantitative PCR detection method has high sensitivity, strong specificity and high accuracy, and solves the problems of low sensitivity, poor specificity and the like in the conventional detection method.
3. The method of the invention obviously improves the detection efficiency of the content of the live virus, and shortens the detection period from 4-7 days of the conventional method to 3-4 hours.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the correspondence between the detection method and the virus plaque method according to embodiment 1 of the present invention;
Fig. 2 is a diagram showing the correspondence between the detection method and TCID 50 method in embodiment 2 of the present invention.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The kit and the reagent or instrument used in the detection method can be purchased from the market.
Example 1 quantitative detection of varicella virus
1. Design synthesis of primer and probe for detecting chicken poxvirus
The invention designs 1 pair of specific primers and probes by referring to the gene sequence of the chicken pox virus core protein 4b published by GenBank, and amplifies 127bp fragments.
FVP-1-F：CAACGGTATTACATATCTACTAA
FVP-1-R：CGTGAATAGAATAGTATAGTATCC
FVP-1-probe：FAM-ATACATCTCCGCCGTCGCAA-BHQ1
2. Specificity verification of primers and probes
The method comprises the steps of respectively taking virus liquids of fowl pox virus, newcastle disease virus, chicken infectious bursal disease virus, chicken infectious laryngotracheitis virus and chicken Marek's disease virus as templates (all the virus liquids are from Living vaccine for poultry of Reep (baoding) biological pharmaceutical industry Co., sold in the market), taking DNA-free water as blank control, taking chick embryo allantoic fluid as negative control, extracting nucleic acid by using a commercial nucleic acid extraction kit (purchased from Beijing full gold biotechnology Co., ltd.), and carrying out real-time fluorescence quantitative PCR detection by adopting the primers and probes designed as above.
The reaction system was 25. Mu.L, including 2 XPCR mix 12.5. Mu.L, amplification enzyme 0.2. Mu.L, each of the upstream and downstream primers 0.4. Mu.L, probe 0.2. Mu.L, detection sample 2. Mu.L, ddH 2 O9.3. Mu.L.
The reaction conditions are as follows: pre-denaturation at 95℃for 2min, denaturation at 95℃for 5 sec, annealing at 60℃for 20 sec, and fluorescence signal acquisition were performed for 40 cycles.
The test results are shown in Table 1, and it can be seen from Table 1 that: the primer and probe designed by the invention amplify chicken pox virus curve to be S-shaped fluorescence curve, ct value is 15.6, no fluorescence signal of newcastle disease virus, chicken infectious bursal disease virus, chicken infectious laryngotracheitis virus and chicken Marek' S disease virus is detected, and the result shows that the designed primer and probe have good specificity.
TABLE 1 results of specificity test of primers and probes
3. Optimization of use concentration of azide ethidium bromide
Sterile ultra-pure water was used to prepare 1mmol/mL of ethidium azide bromide solution, 5 parts of a fowlpox virus solution (live virus solution), 5 parts of an inactivated fowlpox virus solution A (30 min of inactivation treatment at 56 ℃) and 5 parts of an inactivated fowlpox virus solution B (chloroform extraction treatment and chloroform extraction can inactivate fowlpox virus and lose infectivity, and at the same time, the morphology of whole virus particles is maintained, and nucleic acid is not exposed). 2mL of the virus solution is taken and added with a proper amount of ethidium azide bromide solution at the same time, so that the final concentration of the ethidium azide bromide is 0 mu mol/mL,25 mu mol/mL,50 mu mol/mL,75 mu mol/mL and 100 mu mol/mL respectively.
Placing the virus liquid sample in a shaking table at room temperature and in a dark condition, and incubating and shaking for 10min at 140rpm to ensure that the azide ethidium bromide is fully combined with the exposed DNA; and (3) irradiating the treated sample with strong light of a 300W LED lamp for 10min, and continuously oscillating the cradle during the period to ensure that the azide ethidium bromide is fully exposed.
And (3) taking a treated virus liquid sample, extracting DNA according to the operation steps of the instruction book of the nucleic acid extraction kit, and detecting according to the reaction conditions of the fluorescent quantitative PCR.
The test results are shown in Table 2: when the final concentration of the azide ethidium bromide solution is more than or equal to 50 mu mol/mL, the azide ethidium bromide solution can be completely combined with the inactivated chicken pox virus solution A nucleic acid to inhibit PCR amplification reaction; however, the azide ethidium bromide is not effectively combined with the nucleic acid of the inactivated chicken pox virus liquid B which is extracted by chloroform, and the PCR amplification reaction cannot be inhibited. This experiment demonstrates that ethidium azide cannot bind to unexposed nucleic acid, affecting the accuracy of the PCR assay.
TABLE 2 ethidium azide concentration test results
4. Optimization of isoelectric precipitation conditions of chicken poxviruses
Taking a fowl pox virus liquid (live virus liquid) pretreated by azide ethidium bromide and an inactivated fowl pox virus liquid B (chloroform extraction treatment), dividing the two groups into 7 parts, and slowly adding a 1mol/L hydrochloric acid solution, wherein: 6 parts of pH value is respectively adjusted to 4.0, 4.1, 4.2, 4.3, 4.4 and 4.5, and 1 part of pH value is used as non-added contrast; after standing for 1 hour, the mixture was centrifuged at 12000rpm for 5 minutes, and the supernatant was collected, and DNA was extracted by referring to the procedure of the nucleic acid extraction kit, and detected under the reaction conditions of the fluorescent quantitative PCR.
The test results are shown in Table 3: the three groups of chicken pox virus liquid with the pH value less than 4.3 isoelectric precipitation treatment do not detect fluorescence signals; no fluorescence signal was detected in all four groups of inactivated chicken pox virus liquid B treated by isoelectric precipitation with pH < 4.4. The result shows that the isoelectric precipitation method can completely precipitate virus particles without infectivity, and eliminate interference of the virus particles on the fluorescence quantitative PCR detection result.
TABLE 3 isoelectric precipitation test results of chicken poxviruses
5. The invention establishes the corresponding relation between the detection method and the plaque method
Taking chicken pox virus liquid with the virus content of 2X 10 7 PFU/mL by a plaque method, firstly diluting 2 times, then diluting 10 times by serial times, simultaneously carrying out virus content detection by using the animal-derived live virus quantitative detection method (the final concentration of ethidium azide bromide is 50 mu mol/mL, and the pH value is 4.4), and counting the detection results of the detection method and the virus plaque method (see Table 4); and a relation diagram of the real-time fluorescence quantitative PCR method of the chicken pox virus and the logarithmic value of the plaque method is established by taking the Ct value as the abscissa and the logarithmic value of the virus content of the plaque method as the ordinate (see figure 1), and the detection method and the detection result of the plaque method are in a linear relation.
TABLE 4 statistical table of detection results of the detection method and plaque method of the present invention
Example 2 quantitative detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)
1. Design synthesis of primers and probes for PRRSV detection
The invention designs 1 pair of specific primers and probes by referring to the PRRSV ORF 6M gene sequence of the porcine reproductive and respiratory syndrome virus published by GenBank, and the amplified fragment is 118bp.
PRRSV-1-F:CCACAAAAGGTGCTTTTG
PRRSV-1-R CACAGTTCAGGAAGATCA
PRRSV-1-probe:FAM-TTCCATTACCTATACGCCAGTGATGAT-BHQ1
2. Taking a PRRSV virus liquid sample to be detected (virus liquid is from a PRRSV biological pharmaceutical industry Co., ltd., porcine reproductive and respiratory syndrome virus live vaccine (R98 strain, sold in the market)) and adding an ethidium azide bromide solution with the final concentration of 100 mu mol/mL; placing the mixture in a shaking table at room temperature and in a dark place, and incubating and oscillating for 20 minutes at 100rpm to enable the azide ethidium bromide to be fully combined with the exposed DNA; and then placing the mixture in a 500W LED lamp to irradiate for 20min, and continuously oscillating the cradle during the period to fully expose the azide ethidium bromide.
3. Taking the virus liquid sample, slowly adding 2mol/L hydrochloric acid solution, adjusting the pH value to 4.7, standing for 2 hours, centrifuging at 12000rpm for 5 minutes, taking supernatant, extracting DNA according to the operation steps of the instruction book of the nucleic acid extraction kit, diluting the nucleic acid 1000 times, and performing fluorescent quantitative PCR detection.
The reaction system was 25. Mu.L, including 2 XPCR mix 12.5. Mu.L, amplification enzyme 0.2. Mu.L, each of the upstream and downstream primers 0.4. Mu.L, probe 0.2. Mu.L, detection sample 2. Mu.L, ddH 2 O9.3. Mu.L.
The reaction conditions are as follows: reverse transcription is carried out at 50 ℃ for 10 minutes, pre-denaturation is carried out at 95 ℃ for 2 minutes, denaturation is carried out at 95 ℃ for 5 seconds, annealing is carried out at 60 ℃ for 20 seconds, fluorescent signals are collected, and 40 cycles are carried out.
The detection method and the cell half-number infection method (TCID 50) are counted, and a relation diagram (see figure 2) of the PRRSV real-time fluorescence quantitative PCR method and the TCID 50 logarithmic value is established by taking a Ct value as an abscissa and a TCID 50 content logarithmic value as an ordinate, wherein the detection method and the TCID 50 detection result are in a linear relation.
Example 3
The infectious bursal disease virus was detected as in example 2 and counted against the detection result of the chicken embryo half-maximal infection method (EID 50), and the results show that the detection method of the present invention is in a linear relationship with the detection result of the chicken embryo half-maximal infection method (EID 50).
Example 4
Porcine pseudorabies virus was detected according to the method of example 2, and the detection result of the porcine pseudorabies virus and the detection result of the method of the cell half-number infection method (TCID 50) are counted, and the result shows that the detection method of the invention has a linear relation with the detection result of the cell half-number infection method (TCID 50).
Example 5
Chicken marek's disease virus was detected as in example 2 and counted against the virus plaque method (PFU) detection results, which demonstrated that the detection method of the present invention was in a linear relationship with the virus plaque method (PFU) detection results.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (2)
1. The quantitative detection method of the animal-derived live virus is characterized in that the virus is a chicken pox virus;
the method comprises the following steps:
a. treating a virus liquid sample to be tested by adopting ethidium azide bromide;
b. Further processing by isoelectric precipitation, centrifuging 12000 rpm min, and collecting supernatant;
c. Collecting supernatant, and extracting virus nucleic acid;
d. Carrying out PCR detection on virus nucleic acid by adopting real-time fluorescent quantitative PCR, and calculating to obtain the content of live viruses in the virus liquid sample to be detected according to the Ct value of the amplification curve;
Wherein the virus liquid to be tested is animal-derived virus liquid;
The virus liquid sample to be tested is treated by adopting ethidium azide and ethidium bromide, and the method specifically comprises the following steps: adding an ethidium azide bromide solution into a virus liquid sample to be detected, enabling the final concentration of the ethidium azide bromide in the virus liquid to be detected to be 50 mu mol/mL, uniformly mixing in a dark place, and adopting strong light irradiation treatment;
the light-shielding mixing comprises the step of oscillating 10min under a light-shielding environment; the strong light irradiation treatment comprises the steps of adopting a 300W LED lamp to irradiate strong light for 10 min;
The isoelectric precipitation method specifically comprises the following steps: slowly adding a hydrochloric acid solution into a virus liquid sample to be detected which is subjected to ethidium bromide azide treatment, adjusting the pH of the solution to 4.4, and standing for 1 h;
the concentration of the hydrochloric acid solution is 1 mol/L;
The real-time fluorescent quantitative PCR uses specific primers and probes as follows:
FVP-1-F：CAACGGTATTACATATCTACTAA；
FVP-1-R：CGTGAATAGAATAGTATAGTATCC；
FVP-1-probe：FAM-ATACATCTCCGCCGTCGCAA-BHQ1；
the quantitative detection method is a quantitative detection method for the purposes of non-disease diagnosis and treatment.
2. A quantitative detection method of animal-derived live viruses is characterized in that the viruses are porcine reproductive and respiratory syndrome viruses;
the method comprises the following steps:
a. treating a virus liquid sample to be tested by adopting ethidium azide bromide;
b. Further processing by isoelectric precipitation, centrifuging 12000 rpm min, and collecting supernatant;
c. Collecting supernatant, and extracting virus nucleic acid;
d. After the nucleic acid is subjected to 1000-time dilution, detecting by adopting real-time fluorescent quantitative PCR, and calculating to obtain the content of the live virus in the virus liquid sample to be detected according to the Ct value of the amplification curve;
Wherein the virus liquid to be tested is animal-derived virus liquid;
The virus liquid sample to be tested is treated by adopting ethidium azide and ethidium bromide, and the method specifically comprises the following steps: adding an ethidium azide bromide solution into a virus liquid sample to be detected, enabling the final concentration of the ethidium azide bromide in the virus liquid to be detected to be 100 mu mol/mL, uniformly mixing in a dark place, and adopting strong light irradiation treatment;
the light-shielding mixing comprises the step of oscillating 20min under a light-shielding environment; the strong light irradiation treatment comprises the steps of adopting a 500W LED lamp to irradiate strong light 20 min;
The isoelectric precipitation method specifically comprises the following steps: slowly adding a hydrochloric acid solution into a virus liquid sample to be detected which is subjected to ethidium bromide azide treatment, adjusting the pH of the solution to 4.7, and standing for 2 h;
The concentration of the hydrochloric acid solution is 2 mol/L;
The real-time fluorescent quantitative PCR uses specific primers and probes as follows:
PRRSV-1-F：CCACAAAAGGTGCTTTTG；
PRRSV-1-R：CACAGTTCAGGAAGATCA；
PRRSV-1-probe：FAM-TTCCATTACCTATACGCCAGTGATGAT-BHQ1；
the quantitative detection method is a quantitative detection method for the purposes of non-disease diagnosis and treatment.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004339079A (en) * | 2003-05-13 | 2004-12-02 | Nippon Sekijiyuujishiya | Method for removing virus by filtration under virus aggregating condition |
| CN111235236A (en) * | 2020-02-15 | 2020-06-05 | 新疆农业大学 | Method for rapidly detecting viable bacteria of erwinia amylovora and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004339079A (en) * | 2003-05-13 | 2004-12-02 | Nippon Sekijiyuujishiya | Method for removing virus by filtration under virus aggregating condition |
| CN111235236A (en) * | 2020-02-15 | 2020-06-05 | 新疆农业大学 | Method for rapidly detecting viable bacteria of erwinia amylovora and application |
Non-Patent Citations (1)
| Title |
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| 郝民忠.动物微生物.重庆大学出版社,2007,第179页. * |
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