CN113736923B - Primer and kit for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of Chapparvovirus - Google Patents
Primer and kit for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of Chapparvovirus Download PDFInfo
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Abstract
The invention discloses a primer and a kit for real-time fluorescence quantitative PCR detection of Chapparvovirus cat, belonging to the technical field of molecular biology, wherein the primer comprises an upstream primer and a downstream primer, the upstream primer has a sequence shown as SEQ ID No.1, and the downstream primer has a sequence shown as SEQ ID No. 2. The SYBR Green I real-time fluorescent quantitative PCR technology established by the invention can realize high-efficiency amplification of target fragments in a short time, the whole reaction is simple and rapid, the detection rate is high, the repeatability is good, the SYBR Green I real-time fluorescent quantitative PCR technology is suitable for being used under the condition of large sample size, the popularization capability is strong, and the establishment of the invention can fill the blank of related fields.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a primer and a kit for real-time fluorescent quantitative PCR detection of Chapparvovirus cat.
Background
Parvoviridae are icosahedral, membrane-free, single-stranded DNA viruses with a genome size of about 4-6 Kb. The international committee on virus taxonomy divides the parvoviridae into three subfamilies, namely Densovirinae, Hamaparvovirinae and Parvovirinae. FCPV belongs to the genus Chaphamapivovirus in the subfamily Hamaparvovirinae. Members of the genus Chaphamaparvovirus have been found to infect a variety of vertebrates, including rats, mice, bats, dogs, pigs, chickens, turkeys, and the like. FCPV has been reported to be associated with diarrhea in cats.
At present, methods for molecular epidemiological detection of new viruses include a PCR method and a real-time fluorescent quantitative PCR method. The fluorescent quantitative PCR method is based on the common PCR, a pair of specific primers is added in an amplification reaction system, and a specific fluorescent probe is added at the same time, so that the SYBR Green I-based real-time fluorescent quantitative PCR technology has the advantages of high specificity, strong sensitivity, good repeatability and the like, has an intuitive reaction result, and is widely used for virus detection. However, there is no report of the use of SYBR Green for the detection of FCPV at present.
Disclosure of Invention
The invention aims to provide a primer and a kit for real-time fluorescent quantitative PCR detection of Chapparvovirus cat, which are used for solving the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a primer for real-time fluorescent quantitative PCR detection of Chapparvovirus cat, which comprises an upstream primer and a downstream primer, wherein the upstream primer has a sequence shown as SEQ ID No.1, and the downstream primer has a sequence shown as SEQ ID No. 2.
The invention also provides application of the primer for the real-time fluorescence quantitative PCR detection of the cat Chapparvovirus, which is to use the primer for the real-time fluorescence quantitative PCR detection of the cat Chapparvovirus.
The invention also provides a real-time fluorescence quantitative PCR kit for the cat Chapparvovirus, which comprises the primer for the real-time fluorescence quantitative PCR detection of the cat Chapparvovirus.
Further, the real-time fluorescent quantitative PCR kit also comprises DNA fluorescent dye.
Further, the DNA fluorescent dye is SYBR Green I.
In the present invention, SYBR Green I based real-time fluorescent quantitative PCR was established to detect FCPV, which will subsequently also be used to evaluate clinical samples.
The SYBR Green I real-time fluorescent quantitative PCR technology established by the invention can realize high-efficiency amplification of target fragments in a short time, the whole reaction is simple and rapid, the detection rate is high, the repeatability is good, the SYBR Green I real-time fluorescent quantitative PCR technology is suitable for being used under the condition of large sample size, the popularization capability is strong, and the establishment of the invention can fill the blank of related fields.
The fluorescence quantitative PCR is to continuously monitor the intensity of a fluorescence model in the PCR reaction process to measure the amount of a specific product in real time, and accordingly deduces and monitors the initial toxic content of a pathological material, so that the method not only has the characteristic of high amplification efficiency of the conventional PCR technology, but also has the characteristics of high specificity, high sensitivity and accuracy of the spectral technology and the like, simultaneously avoids the steps of subsequent electrophoresis and the like, and reduces the errors caused by pollution and manual operation. And tests prove that the method has high sensitivity and specificity and good repeatability.
The invention discloses the following technical effects:
(1) the primer provided by the invention is adopted to detect a sample, and the sensitivity can reach 101Copy number/. mu.L, 100 times more sensitive than conventional PCR methods.
(2) The specificity is good, the feline astrovirus, feline bocavirus type 1, feline parvovirus, feline coronavirus, feline calicivirus and feline herpesvirus are simultaneously detected in the experiment, and the method does not react with other common viral diseases of cats and has good applicability.
(3) Simple operation and short detection time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings 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 it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a standard curve of real-time fluorescent quantitative PCR method for FCPV;
FIG. 2 shows the specificity experiment result of real-time fluorescent quantitative PCR method for FCPV;
FIG. 3 shows the results of sensitivity experiments of real-time fluorescent quantitative PCR method for FCPV.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description 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. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, 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 herein 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
Example 1 validation experiment of real-time fluorescent quantitative PCR detection method for FCPV
1. Specific primers were designed against VP1 gene of FCPV (Carnivore chapparvorous 1 isolate VRI 849, complete genome sequence No. GenBank: MN 794869.1):
upstream primer (SEQ ID No. 1): 5'-GCGTATACCGTATGGGGTCA-3' are provided.
Downstream primer (SEQ ID No. 2): 5'-AGTCCCTGGGAATCTCCATC-3' are provided.
The upstream and downstream primers were synthesized by general-purpose company.
2. Strain
The FCPV used in the experiment was identified and stored in the laboratory. And the strain can also be obtained by obtaining the above sequence from NCBI and then synthesizing by ordinary biological methods commonly used in the art.
3. Extraction of viral nucleic acids
The virus genome is extracted from the collected samples according to the instructions of a Tiangen genome kit (Tiangen biotechnology limited), and then the virus genome is stored at the temperature of-20 ℃ for later use.
4. Construction of recombinant plasmids
4.1 connection
The purified DNA fragment was ligated into the pMD-19T vector using the TA cloning procedure. The ligation conditions were 16 ℃ for ligation for 3 h. The linking system is as follows: the gel recovered product was 4. mu. L, pMD-19T 1. mu.L, Solution I5. mu.L.
4.2 transformation
The ligation product was transformed into DH 5. alpha. competent cells by the following specific procedures:
(1) taking out DH5 alpha competent cells in advance, and placing the cells on ice to melt the cells fully;
(2) add 5. mu.L of ligation product to 50. mu.L of DH 5. alpha. competent cells;
(3) incubating at 4 ℃ for 30min, then carrying out water bath at 42 ℃ for 90s, and then quickly transferring ice to carry out ice bath for 150 s;
(4) adding 600 μ L of nonresistant LB culture medium preheated to 37 deg.C, and culturing at 37 deg.C with shaking table at 200rpm for 1-2 h;
(5) centrifuging at 6000rpm for 3min, discarding 500 μ L of supernatant, and reserving 100 μ L of liquid to fully resuspend bacterial precipitation;
(6) the resuspended liquid was aseptically spread on an ammonia-resistant solid medium and cultured overnight at 37 ℃.
4.3 plasmid extraction and sequencing
After overnight culture, a single colony in a solid medium was picked, placed in 5mL of LB medium with ampicillin resistance, and cultured at 37 ℃ for 12 hours with a shaker at 200 rpm. And meanwhile, carrying out PCR verification on the bacterial liquid, and carrying out plasmid extraction on the bacterial liquid identified as correct. The detailed steps for extracting plasmids are as follows:
(1) column equilibration step: adding 500 μ L of equilibrium liquid BL into the adsorption column, centrifuging at 12000rpm for 1min, and discarding the waste liquid;
(2) adding the bacterial liquid into a centrifugal tube, centrifuging at 12000rpm for 1min, and completely removing the supernatant;
(3) adding 250 mu L of solution P1 into the precipitate, and performing vortex oscillation to completely resuspend the precipitate;
(4) adding 250 μ L of solution P2 into the suspension in the previous step, and turning up and down for 6-8 times;
(5) adding 350 μ L of solution P3 into the suspension in the previous step, turning over for 6-8 times, and centrifuging at 12000rpm for 10 min;
(6) sucking out the supernatant to a filter column CS, and centrifuging at 12000rpm for 2 min;
(7) adding the centrifuged liquid into an adsorption column which finishes the column balancing step, centrifuging at 12000rpm for 45s, and discarding the waste liquid;
(8) adding 500 mu L PD into the adsorption column, centrifuging at 12000rpm for 45s, and discarding the waste liquid;
(9) add 600. mu.L PW to the adsorption column, centrifuge at 12000rpm for 45s, discard the waste:
(10) repeating the step (9);
(11) adding no liquid into the adsorption column, centrifuging at 12000rpm for 2min, and completely removing the liquid in the adsorption column;
(12) transferring the adsorption column into a new 1.5mL centrifuge tube, dropwise adding 50 mu L of eluent TB into the middle part of the adsorption column, standing at room temperature for 2min, and centrifuging at 12000rpm for 2 min;
(13) the obtained recombinant plasmid is put into the temperature of minus 20 ℃ for storage and standby, and meanwhile, part of the plasmid is extracted and sent to the biological (Shanghai) company Limited for sequencing verification.
Example 2 real-time fluorescent quantitative PCR reaction and Standard Curve plotting
The concentration of the recombinant plasmid was determined by a nucleic acid concentration meter, and the copy number of the recombinant plasmid was calculated to be 1.07X 1011copies/. mu.L. 10-fold serial dilutions of recombinant plasmids were used as templates on a CFX96 ™ real-time PCR detection System (Bio-Rad, Hercules, CA, USA)Line SYBR Green I assay. The system consisted of 10. mu.L SuperReal Premix Plus (Chinese Tiangen), 0.6. mu.L forward primer, 0.6. mu.L reverse primer, 1. mu.L template and 7.8. mu.L ddH2And (C) O. SYBR Green I conditions were 95 ℃ for 15min, then 95 ℃ for 10s and 60 ℃ for 30s, 40 cycles. The recombinant plasmid was diluted to 10 in a ten-fold gradient1And a standard curve is established, the abscissa represents the logarithm of the copy number of the plasmid, and the ordinate represents the Ct value. On the basis, a corresponding regression equation is obtained: y = -3.354x +34.612, R2The amplification efficiency was 1.000, 98.7%. The standard curve is shown in FIG. 1. And no primer dimer and non-specific amplification product appear in the amplification process, and the primer specificity of the established fluorescent quantitative PCR method is good.
Example 3 specificity, sensitivity and reproducibility assays
1. Experiment of specificity
The established method for implementing fluorescent quantitative PCR is adopted to amplify the nucleic acids of the FCPV positive standard, the feline astrovirus, the feline bocavirus type 1, the feline herpesvirus, the feline distemper virus, the feline coronavirus and the feline calicivirus, and negative control is established, the result is shown in figure 2, the negative is a straight line without change, and no specific amplification curve except the FCPV, which indicates that the method has good specificity.
2. Sensitivity test
10 of the ten-fold diluted recombinant plasmid8To 101As a template, the result of the fluorescent quantitative PCR amplification is shown in FIG. 3, and the lowest copy number detected by the method is 1.07X 101copies/μL。
3. Repeatability test
At 108，106，104，102The repetitive experiments of real-time fluorescent quantitative PCR were carried out using 4 copies of recombinant plasmids as templates, and the results are shown in Table 1. The variation coefficients are all less than 1% in the repeatability experiment, which shows that the method has good repeatability.
TABLE 1 real-time fluorescent quantitative PCR repeatability experiment
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
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<120> primer and kit for real-time fluorescent quantitative PCR detection of cat Chapparvovirus
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Claims (4)
1. The primer for real-time fluorescent quantitative PCR detection of the Chapparvovirus cat is characterized by comprising an upstream primer and a downstream primer, wherein the upstream primer has a sequence shown as SEQ ID No.1, and the downstream primer has a sequence shown as SEQ ID No. 2.
2. A real-time fluorescent quantitative PCR kit for the Chapparvovirus cat, which is characterized by comprising the primer for the real-time fluorescent quantitative PCR detection of the Chapparvovirus cat in claim 1.
3. The real-time fluorescent quantitative PCR kit of claim 2, wherein the real-time fluorescent quantitative PCR kit further comprises DNA fluorescent dye.
4. The real-time fluorescent quantitative PCR kit according to claim 3, wherein the DNA fluorescent dye is SYBR Green I.
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