CN111733296A - Marek's disease virus serum 1 type fluorescent quantitative PCR kit and application thereof - Google Patents
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Abstract
The invention relates to a fluorescent quantitative PCR kit for detecting Marek's disease virus serum 1 strain and application thereof in quality control of poultry biological products. The kit contains a pair of specific primers and a corresponding Taqman probe for detecting the Marek's disease virus serotype 1 strain. The kit is used for detecting the Marek's disease virus serum 1 type, has good specificity, and has no specific curve amplified by other poultry virus biological products and production raw materials except that the Marek's disease virus serum 1 type strain can be amplified to form a specific curve; the method has high sensitivity, and the detection limit of the Marek's disease virus serum 1 type strains of different PFUs is 0.006 PFU; detecting the standard plasmid with 10 times of gradient dilution, wherein the detection limit is 32.8 copies/mu L; the invention can be used for detecting exogenous Marek's disease virus in biological products for poultry, and has important significance for detecting Marek's disease virus, controlling the quality of biological products for poultry and the like.
Description
Technical Field
The invention relates to a Marek's Disease Virus (MDV) serum 1 type fluorescent quantitative PCR kit and application thereof in quality control of biological products for poultry, belonging to the fields of biotechnology and animal virology.
Background
Marek's Disease (MD) is an acute or subacute lymphoproliferative disease of chickens, which is mainly characterized by mononuclear cell infiltration of peripheral nerves, skin, iris and other tissues and organs. The pathogenic Marek's Disease Virus (MDV) of the disease belongs to the Mardivrus genus of the alphaherpesvirus subclass, and is classified into 3 serotypes, namely, MDV serotype 1 (avian herpesvirus type 2), MDV serotype 2 (avian herpesvirus type 3) and MDV serotype 3 (turkey herpesvirus type 1), according to the serological response of the virus. The most notable property of avian herpes viruses over other herpes viruses is cell-binding, i.e., the absence of infectious virus particles (particularly MDV serotype 1 strains) free in cell culture.
Based on the biological characteristics of MDV, the existing exogenous MDV detection method mainly comprises the steps of observing allantoic membrane lesions by a chick embryo method, detecting the cytopathic lesions by a cell method, and detecting antibodies by an agar diffusion experiment by a chick detection method. However, with the increasing variety of veterinary biological products and the continuous improvement of biotechnology, the existing national standard for exogenous MDV test cannot completely meet the requirements of test and detection. The main expression is in two aspects: the method has the advantages that firstly, the method has sensitivity problems, for example, the MDV in the chicken inspection method adopts an agar diffusion experiment to carry out antibody detection, the sensitivity is low, the stability is poor, the detection time is long, and the antigen reactivity difference is large when the MDV is detected from different sources; secondly, the method has poor identification performance, and if different serotype Marek's disease viruses are polluted mutually, the exogenous Marek's disease viruses cannot be identified.
Fluorescent quantitative PCR has been widely used as a rapid and sensitive diagnostic method, and is particularly important in the aspect of replacing the detection of pathogens which are difficult or impossible to detect by the traditional method. In 2019, under the situation that African swine fever is too heavily abused, for African swine fever viruses which are difficult to separate and identify, PCR (polymerase chain reaction) and fluorescent quantitative PCR (polymerase chain reaction) detection methods for the polluted African swine fever viruses in biological products for pigs and pig-source raw and auxiliary materials are established in time in rural areas in China, and a solid technical support is provided for strictly closing the quality of the biological products for livestock and well controlling the African swine fever. In the existing biological products for poultry, no detection technology related to molecular biology exists in the national standard for detecting the exogenous MDV, so that a gene for coding MDV nucleocapsid protein UL19 is selected as a target, and a fluorescence quantitative PCR (polymerase chain reaction) detection method for MDV serous type 1 strains is established and is used for quality control of exogenous Marek's disease virus pollution of the biological products for poultry.
Disclosure of Invention
The invention provides a fluorescent quantitative PCR kit which is specific, sensitive, rapid and capable of quantitatively detecting the exogenous Marek's disease virus serum 1 strain, and is applied to the quality control of poultry biological products, aiming at overcoming the defects of low sensitivity, low identifiability, long detection time and the like of the existing detection method of the exogenous Marek's disease virus.
In order to achieve the above object, the present invention implements the following technical solutions:
1. a Marek's disease virus serum type 1 fluorescent quantitative PCR kit is characterized in that the kit also comprises a specific primer, a corresponding Taqman probe, a qPCR Buffer (2 x), an enzyme, a standard plasmid and enzyme-free water;
the specific primer and the corresponding Taqman probe have the sequences as follows:
Sequence 3Taqman probe (MDV1-UL 19-P): 5 '-HEX-agcctgccat cagcgcgtat-BHQ 1-3' 20;
the standard plasmid is obtained by connecting a target fragment aimed by the primer in gene synthesis to a pEASY-Blunt vector.
2. The kit is characterized in that a fluorescence quantitative PCR reaction system used by the kit is 20.0 mu L, and the kit comprises: 10.0. mu.L qPCR Buffer (2X), 0.4. mu.L enzyme, 0.4. mu.L forward primer, 0.4. mu.L reverse primer, 0.2. mu.L probe, 3.0. mu.L template, and 5.6. mu.L enzyme-free water;
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 3sec, annealing at 60 ℃ for 15sec, extension at 72 ℃ for 20sec, amplification for 40 cycles, and collection of fluorescence signals.
3. The kit is characterized by comprising the following steps of:
(1) extracting total DNA of a sample to be detected;
(2) the standard plasmid is made into 3.28 × 108copies/. mu.L to 3.28 × 100Taking the copies/mu L10-fold serial diluted solution as a template, and carrying out fluorescent quantitative PCR on the specific primer, the probe, the reaction system and the conditions to establish a standard curve;
(3) performing fluorescent quantitative PCR by taking the total DNA extracted in the step (1) as a template and the specific primer, the probe, the reaction system and the conditions;
(4) quantifying the virus copy number in a sample to be detected according to a standard curve;
(5) if the method is used for qualitative detection, a standard curve does not need to be constructed.
Positive significance of the invention
The invention relates to a Marek's disease virus serum 1 type fluorescent quantitative PCR kit and application thereof in quality control of poultry biological products. The invention has good specificity, the amplification curve of Marek's disease virus serum 1 type strain is good, and the poultry virus biological products and production raw materials do not have a specific amplification curve; the sensitivity is high, the method established by the invention is used for detecting CVI988 strains of different PFUs, and the detection limit is 0.006 PFU; detecting the standard plasmid with 10 times of gradient dilution, wherein the detection limit is 32.8 copies/mu L; solves the problems of low sensitivity, long detection time and poor identifiability of the existing exogenous MDV detection method, can be used for detecting exogenous Marek's disease virus in biological products for poultry, and has important significance for detecting Marek's disease virus, controlling the quality of biological products for poultry and the like.
Compared with the prior detection technology, the invention has the advantages that:
(1) the specificity is good, the amplification curve of Marek's disease virus serum 1 type strain is good, and other poultry virus biological products and production raw materials do not have a specific amplification curve;
(2) the sensitivity is high, and the detection limit of detecting Marek's disease virus serous type 1 strains (such as CVI988 strains) of different PFUs is 0.006 PFU; detecting the standard plasmid with 10 times of gradient dilution, wherein the detection limit is 32.8 copies/mu L;
(3) the detection is rapid, and can be completed within 3 h;
(4) not only can be detected quantitatively, but also can be detected qualitatively.
Drawings
FIG. 1 shows the sensitivity detection and the establishment of a standard curve by the MDV serum 1 type fluorescence quantitative PCR detection method;
in FIG. 1-A, 1, 2, 3, 4, 5, 6, 7, 8 and 9 represent 3.28 × 10 respectively8copies/. mu.L to 3.28 × 100Amplification curves of the plasmid standards were serially diluted 10-fold per copies/μ L;
FIG. 1-B is a graph of the results of a standard curve: amplification efficiency E98.8%, R20.992, and-3.351X + 38.828.
FIG. 2MDV serotype 1 fluorescent quantitative PCR detection method specificity detection scheme: 1. 2 and 3 represent amplification curves of CVI988, 814 and M5d strains respectively; 4 represents the amplification curve of other poultry virus biological products, virus seeds and production raw materials;
FIG. 3 is a graph showing sensitivity detection of MDV serotype 1 fluorescent quantitative PCR method: 1. 2, 3, 4, 5, 6, 7, 8 represent the amplification curves of 600, 60, 6, 0.6, 0.06, 0.006, 0.0006, 0.00006PFU CVI988 strain, respectively;
FIG. 4 fluorescent quantitative PCR kit for MDV serotype 1 is applied to the detection chart of live vaccine of Newcastle disease (Clone30 strain) contaminated by different PFU MDV serotype 1 CVI988 strains: 1. 2, 3, 4, 5, 6 represent amplification curves of the mixed 60, 6, 0.6, 0.06, 0.006, 0.0006PFU CVI988 strain in 100 feather NDV, respectively;
Detailed Description
1. Design of specific primers and probes for conserved regions of UL19 gene
Downloading and recording the whole gene sequences of all MDV serum 1 strains from GenBank, and performing sequence comparison by using two biological software, namely MegAlign and MEGA, so as to analyze the conserved regions of the strains. According to the sequence alignment result, 1 pair of specific primers (MDV1-UL19-F/MDV1-UL19-R) and corresponding Taqman probes (MDV1-UL19-P) are designed aiming at the UL19 gene conserved region of MDV serotype 1 CVI988 strain, the 5 'end of the probes is marked by a fluorescence reporter group HEX, and the 3' end of the probes is marked by a fluorescence quencher group BHQ 1.
Sequence 3Taqman probe (MDV1-UL 19-P): 5 '-HEX-agcctgccat cagcgcgtat-BHQ 1-3' 20;
establishment and condition optimization of MDV serum 1 type fluorescence quantitative PCR detection method
According to the instruction of a virus genome DNA/RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd., Cat #: DP315), extracting the genome of the CVI988 strain as a template, fixing the concentration of the template, comprehensively considering the Ct value and the fluorescence intensity of a sample, and determining the optimal primer, the concentration of a probe and the reaction condition by adopting a chessboard method.
Reaction system:
reaction conditions are as follows:
pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 3sec, annealing at 60 ℃ for 15sec, extension at 72 ℃ for 20sec, amplification for 40 cycles, and collection of fluorescence signals.
3, establishment of standard curve of MDV serum 1 type fluorescence quantitative PCR detection method
A target fragment aimed by the primer is obtained by genetic synthesis, the target fragment is connected to a pEASY-blunt vector (Beijing all-purpose gold biotechnology limited, Cat #: CB101-01), Trans1-T1 competence (Beijing all-purpose gold biotechnology limited, Cat #: CD501-02) is transformed, a positive single colony is obtained by ampicillin resistance screening and bacterial liquid PCR identification, and plasmids are extracted according to a plasmid extraction kit (Promega, Cat #: A2492) and used as a positive standard.
According to the formula N ═ (plasmid concentration (ng/. mu.l) × 10-9×6.03×1023) Calculating the recombinant plasmid copy number (recombinant plasmid base number × 660) the recombinant plasmid was selected from 3.28 × 108copies/. mu.L to 3.28 × 100The copies/mu L10-fold serial dilution is used as a standard positive template to establish a standard curve of the fluorescent quantitative PCR detection method. The standard curve is shown in FIGS. 1A and 1B: amplification efficiency E98.8%, R20.992, and-3.351X + 38.828.
(1) Sample processing (details of sample in Table 1)
Allantoic fluid, cells, embryo body, allantoic membrane production: dissolving the sample to 100 feather/200 mu L with a certain volume of normal saline, centrifuging at 12000r/min at 4 ℃ for 10min, and taking the supernatant for later use;
SPF chick embryo allantoic fluid: centrifuging at 12000r/min at 4 deg.C for 10min, and collecting supernatant;
chicken embryo fibroblast: taking the medium with an area of 75cm and a growth period of more than 7 days2Removing culture solution from the chicken embryo fibroblast, adding appropriate amount of normal saline, repeatedly freezing and thawing for 3 times, centrifuging at 12000r/min at 4 deg.C for 10min, and collecting supernatant;
SPF chick embryo, SPF chick embryo allantoic membrane: shearing a certain volume of SPF chick embryo body or an allantoic membrane of SPF chick embryo, adding 5 times of normal saline, grinding in a biological safety cabinet, repeatedly freezing and thawing the obtained homogenate for 3 times, centrifuging at 12000r/min at 4 ℃ for 10min, and taking the supernatant for later use;
(2) respectively taking 200 mu L of processed samples, extracting virus genomes as templates (for RNA viruses, the genomes of the viruses are reversely transcribed into cDNA and then are used as templates) according to the specifications of a virus genome DNA/RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd., Cat #: DP315), and detecting by using the established MDV serum 1 type fluorescence quantitative PCR detection method, wherein except the serum 1 type MDV strain, other samples do not detect specific amplification signals (figure 2), and the established detection method has good specificity.
TABLE 1 MDV serum 1 type fluorescence quantitative PCR detection method specificity evaluation research sample information
5, detecting sensitivity of MDV serum 1 type fluorescence quantitative PCR detection method
(1) Standard plasmid 3.28 × 108copies/. mu.L to 3.28 × 100The primers/mu L are used as a template after 10-fold serial dilution, and the detection is carried out by using the established MDV serum 1 type fluorescence quantitative PCR detection method, and the sensitivity of the established detection method can reach 32.8 primers/mu L (the result is shown in figure 1A), and the sensitivity is good.
(2) The MDV serum 1 type CVI988 strains of 600, 60, 6, 0.6, 0.06, 0.006, 0.0006 and 0.00006PFU are respectively taken, the virus genome is extracted as a template according to the instruction of a virus genome DNA/RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd., Cat #: DP315), and the established MDV serum 1 type fluorescence quantitative PCR detection method is used for detection, wherein the minimum of the established detection method can detect 0.006PFU (figure 3), and the sensitivity is good.
6, repeatability detection of MDV serum 1 type fluorescence quantitative PCR detection method
At 3.28 × 108copies/. mu.L to 3.28 × 101Taking the standard plasmid with 10-fold serial dilution of copies/mu L as a template, carrying out 3 times of repeatability detection on samples with each concentration, calculating the coefficient of variation, and analyzing the repeatability of the established fluorescence quantitative PCR detection method. The results are shown in table 2, and the variation coefficients of different template concentrations are lower than 1%, which indicates that the established fluorescence quantitative PCR detection method has better stability.
TABLE 2 repeatability test of MDV serum 1 type fluorescence quantitative PCR test method
Examples
The following examples are intended to better illustrate the technical solution of the present invention, but are not intended to limit the technical solution of the present invention.
Example 1 MDV serum 1 type fluorescent quantitative PCR kit for detecting poultry virus biological products, virus seeds and production raw materials
1. Sample processing (details of sample in Table 1)
Allantoic fluid, cells, embryo body, allantoic membrane production: dissolving the sample to 100 feather/200 mu L with a certain volume of normal saline, centrifuging at 12000r/min at 4 ℃ for 10min, and taking the supernatant for later use;
SPF chick embryo allantoic fluid: centrifuging at 12000r/min at 4 deg.C for 10min, and collecting supernatant;
chicken embryo fibroblast: taking the medium with an area of 75cm and a growth period of more than 7 days2Removing culture solution from the chicken embryo fibroblast, adding appropriate amount of normal saline, repeatedly freezing and thawing for 3 times, centrifuging at 12000r/min at 4 deg.C for 10min, and collecting supernatant;
SPF chick embryo, SPF chick embryo allantoic membrane: shearing a certain volume of SPF chick embryo body or an allantoic membrane of SPF chick embryo, adding 5 times of normal saline, grinding in a biological safety cabinet, repeatedly freezing and thawing the obtained homogenate for 3 times, centrifuging at 12000r/min at 4 ℃ for 10min, and taking the supernatant for later use;
2. respectively taking 200 mu L of processed samples, extracting virus genome as a template (for RNA virus, the genome of the RNA virus is reversely transcribed into cDNA and then is used as the template) according to the specification of a virus genome DNA/RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd., Cat #: DP315), and detecting by using the established MDV serum 1 type fluorescence quantitative PCR detection method, wherein except the serum 1 type MDV strain, other poultry virus biological products and production raw materials do not detect specific amplification signals (figure 2).
Example 2 determination of minimum detectable Limit of MDV serum type 1 fluorescent quantitative PCR kit
1. Standard plasmid 3.28 × 108copies/. mu.L to 3.28 × 100The copies/mu L10-fold serial dilution is used as a template, the detection is carried out by using the established MDV serum 1 type fluorescence quantitative PCR detection method, and the minimum detection limit of the kit is 32.8 copies/mu L (the result is shown in figure 1A).
2.MDV serum 1 type CVI988 strains of 600, 60, 6, 0.6, 0.06, 0.006, 0.0006 and 0.00006PFU are respectively taken, virus genomes are extracted as templates according to the instruction of a virus genome DNA/RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd., Cat #: DP315), and detection is carried out by the established MDV serum 1 type fluorescence quantitative PCR detection method, wherein the minimum detection limit of the kit is 0.006PFU (figure 3).
Example 3 repeatability test of MDV serum 1 type fluorescent quantitative PCR kit
At 3.28 × 108copies/. mu.L to 3.28 × 101The plasmid of the standard substance which is serially diluted by 10 times is copies/mu L and is used as a template, the repeatability of each sample with each concentration is detected for 3 times, the variation coefficient is calculated, and the repeatability of the kit is analyzed. The results are shown in table 2, and the coefficient of variation of different template concentrations is lower than 1%, which indicates that the kit has better repeatability.
Example 4 application of MDV serum 1 type fluorescent quantitative PCR kit in quality control of biological products for poultry
The chicken Newcastle disease live vaccine (Clone30 strain) is taken as an example. 1000 feathers of live vaccine of Newcastle disease (Clone30 strain) is taken, dissolved by 1mL of normal saline, diluted to 1000 feathers/1 mL, 100 muL is taken to be mixed with MDV serum 1 type CVI988 strain containing 60, 6, 0.6, 0.06, 0.006 and 0.0006PFU in the same volume, viral genome is extracted as a template according to the instruction of a viral genome DNA/RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd., Cat #: DP315), and the established fluorescence quantitative PCR detection method of the MDV serum 1 type strain is used for detecting the pollution of the CVI988 strain and detecting the virus of 0.006PFU (figure 4).
Sequence listing
<110> China institute for veterinary drug inspection
<120> Marek's disease virus serum 1 type fluorescent quantitative PCR kit and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> upstream primer (MDV1-UL19-F)
<400>1
<210>2
<211>20
<212>DNA
<213> downstream primer (MDV1-UL19-R)
<400>2
<210>3
<211>20
<212>DNA
<213> Taqman Probe (MDV1-UL19-P)
<400>3
Claims (3)
1. A Marek's disease virus serum type 1 fluorescent quantitative PCR kit is characterized in that the kit also comprises a specific primer, a corresponding Taqman probe, a qPCR Buffer (2 x), an enzyme, a standard plasmid and enzyme-free water;
the specific primer and the corresponding Taqman probe have the sequences as follows:
sequence 1 upstream primer (MDV1-UL 19-F): 5'-attcctggat gcaaacccgt-3' 20
Sequence 2 downstream primer (MDV1-UL 19-R): 5'-tattccacgt tccgtcagcc-3' 20
Sequence 3Taqman probe (MDV1-UL 19-P): 5 '-HEX-agcctgccat cagcgcgtat-BHQ 1-3' 20;
the standard plasmid is obtained by connecting a target fragment aimed by the primer in gene synthesis to a pEASY-Blunt vector.
2. The kit of claim 1, wherein the kit uses a fluorescent quantitative PCR reaction system of 20.0 μ L comprising: 10.0. mu.L qPCR Buffer (2X), 0.4. mu.L enzyme, 0.4. mu.L forward primer, 0.4. mu.L reverse primer, 0.2. mu.L probe, 3.0. mu.L template, and 5.6. mu.L enzyme-free water;
the reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 3sec, annealing at 60 ℃ for 15sec, extension at 72 ℃ for 20sec, amplification for 40 cycles, and collection of fluorescence signals.
3. The kit of claim 1, wherein the kit is used for detecting a serotype 1 strain of marek's disease virus according to the following steps:
(1) extracting total DNA of a sample to be detected;
(2) the standard plasmid is made into 3.28 × 108copies/. mu.L to 3.28 × 100Taking the copies/mu L10-fold serial diluted solution as a template, and carrying out fluorescent quantitative PCR on the specific primer, the probe, the reaction system and the conditions to establish a standard curve;
(3) performing fluorescent quantitative PCR by taking the total DNA extracted in the step (1) as a template and the specific primer, the probe, the reaction system and the conditions;
(4) quantifying the virus copy number in a sample to be detected according to a standard curve;
(5) if the method is used for qualitative detection, a standard curve does not need to be constructed.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994003628A1 (en) * | 1992-08-07 | 1994-02-17 | Syntro Corporation | Recombinant equine herpesviruses |
| CN103131795A (en) * | 2013-01-15 | 2013-06-05 | 北京华都诗华生物制品有限公司 | Primer and kit for identifying marek's disease virus serum 1-type vaccine virus |
| CN103361444A (en) * | 2013-07-24 | 2013-10-23 | 四川农业大学 | Kit for testing Marek's disease virus of chicken by using MEQ gene and detection method thereof |
| CN110699485A (en) * | 2019-08-16 | 2020-01-17 | 广州千寻生物技术有限公司 | RPA primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus |
-
2020
- 2020-08-10 CN CN202010794930.4A patent/CN111733296A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994003628A1 (en) * | 1992-08-07 | 1994-02-17 | Syntro Corporation | Recombinant equine herpesviruses |
| CN103131795A (en) * | 2013-01-15 | 2013-06-05 | 北京华都诗华生物制品有限公司 | Primer and kit for identifying marek's disease virus serum 1-type vaccine virus |
| CN103361444A (en) * | 2013-07-24 | 2013-10-23 | 四川农业大学 | Kit for testing Marek's disease virus of chicken by using MEQ gene and detection method thereof |
| CN110699485A (en) * | 2019-08-16 | 2020-01-17 | 广州千寻生物技术有限公司 | RPA primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus |
Non-Patent Citations (1)
| Title |
|---|
| 屈素洁: "荧光定量PCR检测鸡马立克氏病血清1型病毒方法的建立及应用", 《上海畜牧兽医通讯》 * |
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