CN116970738A - Preparation method of nfo-RPA detection kit for identifying PCV2 and PCV3 - Google Patents
Preparation method of nfo-RPA detection kit for identifying PCV2 and PCV3 Download PDFInfo
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Abstract
The application discloses a preparation method of an nfo-RPA detection kit for distinguishing PCV2 and PCV3, which comprises the following steps of: screening out gene fragments of PCV2 and PCV3 through bioinformatics analysis, amplifying corresponding gene fragments from PCV2 and PCV3 genomes, and connecting the corresponding gene fragments to pUC57 plasmids to construct positive plasmids; establishing an nfo-RPA reaction system: according to the gene fragment sequences of PCV2 and PCV3, respectively designing an nfo-RPA enzyme amplification primer, mixing the primer and the probe with the nfo-RPA enzyme to establish a reaction system, wherein the detection specificity of the detection kit is high, the reaction result can be presented by a colloidal gold test strip within 5min, and the coincidence rate of the detection result and the PCR detection result is high; the constructed nfo-RPA detection kit for distinguishing PCV2 from PCV3 is suitable for clinical rapid detection.
Description
Technical Field
The application relates to a preparation method of a detection kit, in particular to a preparation method of an nfo-RPA detection kit for identifying PCV2 and PCV3, belonging to the field of biological genetic engineering.
Background
Porcine Circovirus (PCV) is a small, non-enveloped, circular DNA virus, four different PCVs have been identified in pigs, designated by consecutive numbers according to their order of discovery, porcine circovirus type 1 (PCV 1), porcine circovirus type 2 (PCV 2), porcine circovirus type 3 (PCV 3) and porcine circovirus type 4 (PCV 4). Among them, PCV1 is generally considered to be nonpathogenic, PCV2 and PCV3 are most relevant to clinical manifestations, while PCV4 has no clear disease. Porcine circovirus type 2 (PCV 2) is considered one of the viruses that has a major economic impact on the pig industry, whereas porcine circovirus type 3 (PCV 3) has been found to be associated with swine dermatitis and nephrotic syndrome (PDNS) -like diseases, both of which are susceptible to clinical co-infection. PCV is ubiquitous in the world pig farm, and uninfected pig farms are rarely found, causing serious damage to the world pig industry and causing great economic loss to the world pig industry.
The rapid diagnostic tool for PCV detection plays an important role in disease control and eradication, and how to rapidly and effectively detect PCV infection is of great importance to pig farm effective prevention and control of the disease.
Disclosure of Invention
In order to solve the problems in the prior art, the application provides a preparation method of an nfo-RPA detection kit for identifying PCV2 and PCV3, which has the technical characteristics of high detection specificity, high detection result coincidence rate, suitability for clinical quick detection and the like.
In order to achieve the above purpose, the present application is realized by the following technical scheme:
the application discloses a preparation method of an nfo-RPA detection kit for identifying PCV2 and PCV3, which comprises the following steps:
1) Preparation of positive plasmid: screening out gene fragments of PCV2 and PCV3 through bioinformatics analysis, amplifying corresponding gene fragments from PCV3 and PCV2 genomes, and connecting the corresponding gene fragments to pUC57 plasmids to construct positive plasmids;
2) Establishing an nfo-RPA reaction system: respectively designing nfo-RPA enzyme amplification primers according to gene fragment sequences of PCV2 and PCV3, and mixing the primers and probes with the nfo-RPA enzyme to establish a reaction system;
3) And determining the optimal detection time and temperature of the nfo-RPA reaction system.
Preferably, the positive plasmid is prepared specifically by:
the amplified PCV2 and PCV3 gene fragments are subjected to double enzyme digestion by utilizing a molecular biological method and then are connected into a pUC57 vector, and are transferred into Escherichia coli Top competent bacteria for amplification, and the recombinant vectors obtained after screening and identification are named as PCV2 and PCV3 positive plasmids.
Preferably, the PCV3 gene fragment sequence is as shown in NO. 1:
no.1PCV3 gene fragment:
ATACCACTTTTTCTCCCTACAGACCTCCGTGGATCCGGGTTCCGAGGTGCCGGGTAATACTAGCCCGGCACCAAAATGAGACACAGAGCTATATTCAGAAGAAGACCCCGCCCAAGGAGACGACGACGCCACAGAAGGCGCTATGCCAGAAGAAGACTATTCATTAGGAGGCCCACAGCTGGCACATACTACACAAAGAAATACTCCACCATGAACGTCATATCCGTTGGAACCCCTCAGAATAACAAGCCCTGGCACGCCAACCACTTCATTACCCGCCTAAACGAATGGGAGACTGCAATTACCTTTGAATATTATAAGATACTAAAGATGAAAGTTACACTCAGCCCTGTAATTTCTCCGGCTCAGCAAACAAAAACTATGTTCGGGCACACAGCCATAGATCTAGACGGCGCCTGGACCACAAACACTTGGCTCCAAGACGACCCTTATGCGGAAAGTTCCACTCGTAAAGTTATGACTTCTAAAAAAAAACACAGCCGTTACTTCACCCCCAAACCACTTCTGGCGGGAACTACCAGCGCTCACCCAGGACAAAGCCTCTTCTTTTTCTCCAGACCCACCCCATGGCTCAACACATATGACCCCACCGTTCAATGGGGAGCACTGCTTTGGAGCATTTATGTCCCGGAAAAAACTGGAATGACAGACTTCTACGGCACCAAAGAAGTTTGGATTCGTTACAAGTCCGTTCTCTAAGTGAAAATAAATATAAATCTGACCCCTCCGGTGGCGACAAGCCGTGTCCCCTCGCGCGCGCTCCCGCTGCG。
preferably, the PCV2 gene fragment sequence is as shown in NO. 2:
no.2PCV2 gene fragment:
ACCAGCGCACTTCGGCAGCGGCAGCACCTCGGCAGCACCTCAGCAGCAACATGCCCAGCAAGAAGAATGGAAGAAGCGGACCCCAACCACATAAAAGGTGGGTGTTCACGCTGAATAATCCTTCCGAAGACGAGCGCAAGAAAATACGGGAGCTCCCAATCTCCCTATTTGATTATTTTATTGTTGGCGAGGAGGGTAATGAGGAAGGACGAACACCTCACCTCCAGGGGTTCGCTAATTTTGTGAAGAAGCAAACTTTTAATAAAGTGAAGTGGTATTTGGGTGCCCGCTGCTACATCGAGAAAGCCAAAGGAACTGATCAGCAGAATAAAGAATATTGCAGTAAAGAAGGCAACTTACTTATTGAATGTGGAGCTCCTCGATCTCAAGGACAACGGAGTGACCTGTCTACTGCTGTGAGTACCTTGTTGGAGAGCGGGAGTCTGGTGACCGTTGCAGAGCAGCACCCTGTAACGTTTGTCAGAAATTTCCGCGGGCTGGCTGAACTTTTGAAAGTGAGCGGGAAAATGCAGAAGCGTGATTGGAAGACCAATGTACACGTCATTGTGGGGCCACCTGGGTGTGGTAAAAGCAAATGGGCTGCTAATTTTGCAGACCCGGAAACCACATACTGGAAACCACCTAGAAACAAGTGGTGGGATGGTTACCATGGTGAAGAAGTGGTTGTTATTGATGACTTTTATGGCTGGCTGCCGTGGGATGATCTACTGAGACTGTGTGATCGATATCCATTGACTGTAGAGACTAAAGGTGGAACTGTACCTTTTTTGGCCCGCAGT。
preferably, the primer comprises the following:
PCV3-Forward GGCACATACTACACAAAGAAATACTCCACC；
PCV3-Reverse[DIG]ATATTCAAAGGTAATTGCAGTCTCCCATTCG；
PCV3-Probe[FITC]GTTGGAACCCCTCAGAATAACAAGCCCTGG[dSpacer]；
CACGCCAACCACTTCATTACCC[C3Spacer]；
PCV2-Forward GCTGCCGTGGGATGATCTACTGAGACTGTGT；
PCV2-Reverse[Biotin]CAGGGCATGGGGGGGAAAGGGTGACGAACTG；
PCV2-Probe[6-FAM]GGCCCGCAGTATTCTGATTACCAGCAATCA[dSpacer]GACCCCGTTGGAATGGTACTCC[C3Spacer]；
DIG represents digoxin, biotin represents Biotin, FITC represents fluorescein isothiocyanate, FAM represents a fluorescent reporter group, and Spacer represents a Spacer.
Preferably, the establishment of the nfo-RPA reaction system comprises: establishing a PCV2 nfo-RPA reaction system and a PCV3nfo-RPA reaction system;
establishing a PCV2 nfo-RPA reaction system: 29.4. Mu.L Buffer A, 2. Mu.L PCV3-specific-Forward, 2. Mu.L LPCV3-specific-Reverse, 0.6. Mu.L LPCV3-specific-Probe and a total of 13.5. Mu.L ddH were added to the nfo-RPA dry powder tube 2 O and Cap positive plasmid, 2.5 mu L Buffer B, after reacting for 9min at 39 ℃, taking 40 mu L of reaction product, cleaning and recovering, performing agarose gel electrophoresis, diluting the rest 10 mu L of product by 1/50 times, taking 10 mu L, dripping into a colloidal gold test strip sample adding area, observing the detection result of the colloidal gold test strip, and similarly, establishing a PCV2 nfo-RPA reaction system.
Preferably, the optimal detection time and temperature of the nfo-RPA reaction system are determined:
selecting dilution concentration of 1×10 8 Performing nfo-RPA amplification reaction by respectively setting time gradients of 8min, 9min, 10min, 11min and 12min on a PCV3 positive plasmid template of cobies/mu L, taking out a reaction tube after the reaction is finished, placing the reaction tube on ice to finish the reaction, observing a result in a colloidal gold test strip, and adopting the reaction time of a visible clear amplification strip as the optimal detection time;
on the basis of selecting the optimal detection time, the temperature gradient of 37 ℃ and 38 ℃ is further set, the nfo-RPA amplification reaction is carried out at 39 ℃, and the optimal reaction temperature is determined by observing the result in a colloidal gold test strip.
Preferably, the optimal detection time is 9min; the optimum reaction temperature is 39 ℃.
Preferably, the sensitivity verification of the nfo-RPA reaction system is included;
1X 10 Using PCV3 Positive plasmid Standard 9 The initial concentration of the copies/. Mu.L was diluted 10-fold, and the diluted concentration was 1X 10 under the reaction conditions of a reaction time of 9min and a reaction temperature of 39 ℃ 9 copies/μL、1×10 8 copies/μL、1×10 7 copies/μL、1×10 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 copies/μL、1×10 2 copies/μL、1×10 1 copies/μL、1×10 0 Cap positive plasmid of copies/. Mu.L is used as template, ddH 2 O is used as a negative control to carry out nfo-RPA amplification reaction, and the result is observed in a colloidal gold test strip;
1X 10 Using PCV2 Positive plasmid Standard 6 The initial concentration of the copies/. Mu.L was diluted 10-fold, and the diluted concentration was 1X 10 under the reaction conditions of a reaction time of 9min and a reaction temperature of 39 ℃ 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 copies/μL、1×10 2 copies/μL、1×10 1 copies/μL、1×10 0 copies/μL、1×10 -1 copies/μL、1×10 -2 copies/μL、1×10 -3 copies/μL、1×10 -4 copies/μL、1×10 -5 copies/μL、1×10 -6 PCV 2-positive plasmid of copies/. Mu.L is used as template, ddH 2 O was used as a negative control for the nfo-RPA amplification reaction and the results were observed in a colloidal gold test strip.
Preferably, the repeatability verification of the nfo-RPA reaction system further comprises:
repeating between groups: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 repeated tests were performed under the same conditions on 3 PCV3 positive plasmids at different concentrations of cobies/. Mu.L;
repeat within group: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 copies/. Mu.L of 3 PCV3 positive plasmids at different concentrations were replicated 3 times in the same experiment;
repeating between groups: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 repeated tests were performed under the same conditions on 3 PCV2 positive plasmids at different concentrations of cobies/. Mu.L;
repeat within group: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 copies/. Mu.L of 3 PCV2 positive plasmids at different concentrations were replicated 3 times in the same experiment.
Preferably, the specific identification of the nfo-RPA reaction system further comprises:
the genome of mycoplasma hyopneumoniae 168 strain, porcine circovirus 2, porcine parvovirus and pseudorabies virus are respectively used as templates, and ddH is used 2 O is a negative control, PCV3nfo-RPA reaction is carried out at the reaction temperature of 39 ℃ for 9min, and the result is observed in a colloidal gold test strip;
respectively taking genomes of mycoplasma hyopneumoniae 168 strain, porcine circovirus 3, porcine parvovirus and pseudorabies virus as templates, and ddH 2 O is a negative control, PCV2 nfo-RPA reaction was performed at a reaction temperature of 39℃for 9min, and the results were observed in a colloidal gold test strip.
The beneficial effects are that: the reaction system of the detection kit only needs to react for 9min at 39 ℃, the reaction repeatability is good, the detection specificity is high, the reaction result can be presented by a colloidal gold test strip within 5min, and the coincidence rate of the detection result and the PCR detection result is 84.17%; the constructed nfo-RPA detection kit for distinguishing PCV2 from PCV3 is suitable for clinical rapid detection.
Drawings
FIG. 1PCV3 positive plasmid schematic.
FIG. 2PCV2 positive plasmid schematic.
FIG. 3 is one of the established maps of the PCV3nfo-RPA reaction system of the present application.
FIG. 4 is a second diagram showing the establishment of the PCV3nfo-RPA reaction system of the present application.
FIG. 5 is one of the established maps of the PCV2 nfo-RPA reaction system of the present application.
FIG. 6 is a second diagram showing the establishment of the PCV2 nfo-RPA reaction system of the present application.
FIG. 7 is a plot of the nfo-RPA reaction time exploration of the present application.
FIG. 8 is a graph showing the reaction temperature probe of nfo-RPA of the present application.
FIG. 9 is a graph showing the sensitivity of the PCV3nfo-RPA reaction of the present application.
FIG. 10 is a graph showing the sensitivity of the PCV2 nfo-RPA reaction of the present application.
FIG. 11 is one of the within PCV3 group repeatability determinations of the present application.
FIG. 12 is a second graph of a repeat determination within PCV3 of the present application.
FIG. 13 is one of the within PCV2 set repeatability determinations of the present application.
FIG. 14 is a second graph of a repeat determination within the PCV2 group of the present application.
FIG. 15 is a graph showing the specificity of PCV3nfo-RPA reaction of the present application.
FIG. 16 is a graph showing the specificity of PCV2 nfo-RPA reaction of the present application.
FIG. 17 is a graph showing the detection results of nfo-RPA of the present application.
Detailed Description
The present application will be further described with reference to the accompanying drawings, but the present application is not limited to the following examples.
Currently, detection of PCV infection includes PCR, nested PCR, competitive PCR, and real-time fluorescent quantitative PCR (rtPCR). The recombinase polymerase amplification technique (Recombinase polymerase amplification, RPA) is an isothermal gene amplification technique for detection of infectious disease pathogenic molecules. RPA is carried out at a constant temperature, so that the RPA can be carried out in a water bath, in addition, the RPA is completed within about 30 minutes, nfo-RPA is obtained by adding nfo enzyme (escherichia coli endonuclease IV) into a reaction system, an amplified product of nfo-RPA carries a label simultaneously through a specific primer and a probe, and is combined with a lateral flow chromatography test strip, and the amplified product is developed on the test strip through the action of antigen-antibody combination, so that the clinical rapid, specific and visual detection can be realized without depending on special equipment. According to the application, two sections of genes of PCV2 and PCV3 are cloned respectively, and are respectively connected into pUC57 vectors, and DNA probes are designed by taking the gene fragment sequences as targets respectively. The primer and the probe are mixed with the nfo-RPA enzyme to establish a reaction system, the reaction system only needs to react for 9 minutes at 39 ℃, the detection specificity is high, the repeatability is good, and the reaction result can be presented by a colloidal gold test strip within 5 minutes, so that the nfo-RPA detection kit constructed by the method for identifying and detecting PCV2 and PCV3 viruses can be applied to clinical rapid detection.
1-17 show a specific example of a preparation method of an nfo-RPA detection kit for identifying PCV2 and PCV3, which includes the following steps:
1. preparation of positive plasmid
The amplified PCV2 and PCV3 gene fragments are subjected to double enzyme digestion by utilizing a molecular biological method and then are connected into a pUC57 vector, and are transferred into Escherichia coli Top competent bacteria for amplification, and the recombinant vectors obtained after screening and identification are named as PCV2 and PCV3 positive plasmids. The PCV3 gene fragment sequence is shown as NO.1, and the schematic diagram of the constructed PCV3 positive plasmid vector is shown as FIG. 1; the PCV2 gene fragment sequence is shown as NO.2, and the schematic diagram of the constructed PCV2 positive plasmid vector is shown as FIG. 2.
2. Establishment of nfo-RPA reaction System
The nfo-RPA kit was purchased from Fangan Anpu future Biotechnology Co., ltd, and 29.4. Mu.L of Buffer A, 2. Mu.L of PCV3-specific-Forward (10. Mu.M), 2. Mu.L of LPCV3-specific-Reverse (10. Mu.M), 0.6. Mu.L of PCV3-specific-Probe (10. Mu.M) and a total of 13.5. Mu.L of ddH were added to the nfo-RPA dry powder tube 2 O and Cap positive plasmid, 2.5. Mu.L Buffer B, at 39 ℃ for 9min, 40. Mu.L of reaction product is taken out, cleaned and recovered, and agarose gel electrophoresis is carried out. After the rest 10 mu L of the product is diluted by 1/50 times, 10 mu L of the product is dripped into a sample adding area of the colloidal gold test strip, and the detection result of the colloidal gold test strip is observed. ddH 2 O is a negative control, and the results are shown in FIGS. 3-4; similarly, a PCV2 nfo-RPA reaction system was established, and the results are shown in FIGS. 5-6. According to the application, nfo-RPA enzyme amplification primers are respectively designed according to gene fragment sequences of PCV3 and PCV2, and the primers and probes are mixed with nfo-RPA enzyme to establish a reaction system;
TABLE 1 primer sequences
Note that: in the table, DIG represents digoxin, biotin represents Biotin, FITC represents fluorescein isothiocyanate, FAM represents a fluorescent reporter group, and Spacer represents a Spacer (Spacer domain).
3. nfo-RPA reaction system and condition optimization
Selecting dilution concentration of 1×10 8 The reaction tube is taken out to be placed on ice to finish the reaction after the reaction is finished, and the result is observed in a colloidal gold test strip, as shown in FIG. 7, when the reaction time is 9min, clear amplified bands are visible.
Based on the experiment screening out the reaction time of 9min, the temperature gradient of 37 ℃ and 38 ℃ is further set, the nfo-RPA amplification reaction is carried out at 39 ℃, and the observation result is carried out in a colloidal gold test strip, as shown in FIG. 8, and the 39 ℃ is finally determined to be the optimal reaction temperature in combination with the convenience of practical clinical use.
4. Sensitivity verification of nfo-RPA reaction system
1X 10 Using PCV3 Positive plasmid Standard 9 The initial concentration of copies/. Mu.L was diluted 10-fold (10 9 To 10 0 ) The dilution concentration was 1X 10 under the reaction conditions of a reaction time of 9min and a reaction temperature of 39 ℃ 9 copies/μL、1×10 8 copies/μL、1×10 7 copies/μL、1×10 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 copies/μL、1×10 2 copies/μL、1×10 1 copies/μL、1×10 0 Cap positive plasmid of copies/. Mu.L is used as template, ddH 2 O is used as a negative control for carrying out an nfo-RPA amplification reaction, and the result is observed in a colloidal gold test strip, as shown in FIG. 9; 1X 10 Using PCV2 Positive plasmid Standard 6 The initial concentration of copies/. Mu.L was diluted 10-fold (10 6 To 10 -6 ) The dilution concentration was 1X 10 under the reaction conditions of a reaction time of 9min and a reaction temperature of 39 ℃ 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 copies/μL、1×10 2 copies/μL、1×10 1 copies/μL、1×10 0 copies/μL、1×10 -1 copies/μL、1×10 -2 copies/μL、1×10 -3 copies/μL、1×10 - 4 copies/μL、1×10 -5 copies/μL、1×10 -6 PCV 2-positive plasmid of copies/. Mu.L is used as template, ddH 2 O was used as a negative control for nfo-RPA amplification and the results were observed in a colloidal gold test strip as shown in FIG. 10. 5. Repeatability verification of nfo-RPA reaction system
Repeating between groups: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 replicates of 3 PCV3 positive plasmids at different concentrations were tested under the same conditions and the results are shown in FIG. 11.
Repeat within group: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 copies/. Mu.L of 3 PCV3 positive plasmids at different concentrations were replicated 3 times in the same experiment. The results are shown in FIG. 12.
Repeating between groups: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 repeated tests were performed under the same conditions on 3 PCV 2-positive plasmids at different concentrations of cobies/. Mu.L, and the results are shown in FIG. 13.
Repeat within group: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 copies/. Mu.L of 3 PCV2 positive plasmids at different concentrations were replicated 3 times in the same experiment. The results are shown in FIG. 14. 6. Specific identification of nfo-RPA reaction system
The genome of mycoplasma hyopneumoniae 168 strain, porcine circovirus 2, porcine parvovirus and pseudorabies virus are respectively used as templates, and ddH is used 2 O is a negative control, PCV3nfo-RPA reaction is carried out at a reaction time of 9min and a reaction temperature of 39 ℃ and the result is observed in a colloidal gold test strip, as shown in FIG. 15; respectively taking genomes of mycoplasma hyopneumoniae 168 strain, porcine circovirus 3, porcine parvovirus and pseudorabies virus as templates, and ddH 2 O is a negative control, PCV2 nfo-RPA reaction was performed at a reaction time of 9min and a reaction temperature of 39℃and the results were observed in a colloidal gold test strip, as shown in FIG. 16.
7. Use of nfo-RPA reaction system for clinical sample detection
Collecting 60 parts of pig farm disease samples, extracting disease genome DNA and performing nfo-RPA amplification reaction, wherein the result is judged as shown in figure 8, and if only a quality control line is colored as shown in figure 17 according to a color strip in the figure, the sample to be detected does not contain PCV2 and PCV3 viruses; if the test strip T2 detection line and the quality control line are both colored as shown in FIG. 17, the test strip T2 detection line and the quality control line indicate that the sample to be tested contains PCV2 virus; if the test strip T1 detection line and the quality control line are both colored as shown in FIG. 17, the test strip T1 detection line and the quality control line indicate that the sample to be tested contains PCV3 virus; if the test strip T1 detection line, the test strip T2 detection line and the test strip quality control line are all colored as shown in FIG. 17, the test strip T1 detection line, the test strip T2 detection line and the test strip quality control line indicate that PCV2 and PCV3 viruses are contained in the sample to be tested; if the detection line and the quality control line are not developed, the detection line and the quality control line indicate that amplification is not carried out or the test strip is invalid. Meanwhile, sample detection is carried out by using PCR, and the PCV3nfo-RPA amplification reaction result is compared with the PCR detection result, and the result is shown in Table 2.
TABLE 2 comparison of PCV3nfo-RPA with PCR detection results
In summary, the gene fragments of PCV3 and PCV2 were screened by bioinformatic analysis, and the corresponding gene fragments were amplified from the PCV3 and PCV2 genomes and ligated into pUC57 plasmid to construct a positive plasmid. And (5) respectively designing nfo-RPA enzyme amplification primers according to the sequence of the gene fragment. The primer and the probe are mixed with nfo-RPA enzyme to establish a reaction system, the reaction system only needs to react for 9min at 39 ℃, the reaction repeatability is good, the detection specificity is high, the reaction result can be presented by a colloidal gold test strip within 5min, and the coincidence rate of the detection result and the PCR detection result is 84.17%. The nfo-RPA detection kit constructed by the method for distinguishing PCV2 from PCV3 is suitable for clinical rapid detection.
No.1PCV3 gene fragment:
ATACCACTTTTTCTCCCTACAGACCTCCGTGGATCCGGGTTCCGAGGTGCCGGGTAATACTAGCCCGGCACCAAAATGAGACACAGAGCTATATTCAGAAGAAGACCCCGCCCAAGGAGACGACGACGCCACAGAAGGCGCTATGCCAGAAGAAGACTATTCATTAGGAGGCCCACAGCTGGCACATACTACACAAAGAAATACTCCACCATGAACGTCATATCCGTTGGAACCCCTCAGAATAACAAGCCCTGGCACGCCAACCACTTCATTACCCGCCTAAACGAATGGGAGACTGCAATTACCTTTGAATATTATAAGATACTAAAGATGAAAGTTACACTCAGCCCTGTAATTTCTCCGGCTCAGCAAACAAAAACTATGTTCGGGCACACAGCCATAGATCTAGACGGCGCCTGGACCACAAACACTTGGCTCCAAGACGACCCTTATGCGGAAAGTTCCACTCGTAAAGTTATGACTTCTAAAAAAAAACACAGCCGTTACTTCACCCCCAAACCACTTCTGGCGGGAACTACCAGCGCTCACCCAGGACAAAGCCTCTTCTTTTTCTCCAGACCCACCCCATGGCTCAACACATATGACCCCACCGTTCAATGGGGAGCACTGCTTTGGAGCATTTATGTCCCGGAAAAAACTGGAATGACAGACTTCTACGGCACCAAAGAAGTTTGGATTCGTTACAAGTCCGTTCTCTAAGTGAAAATAAATATAAATCTGACCCCTCCGGTGGCGACAAGCCGTGTCCCCTCGCGCGCGCTCCCGCTGCG。
no.2PCV2 gene fragment:
ACCAGCGCACTTCGGCAGCGGCAGCACCTCGGCAGCACCTCAGCAGCAACATGCCCAGCAAGAAGAATGGAAGAAGCGGACCCCAACCACATAAAAGGTGGGTGTTCACGCTGAATAATCCTTCCGAAGACGAGCGCAAGAAAATACGGGAGCTCCCAATCTCCCTATTTGATTATTTTATTGTTGGCGAGGAGGGTAATGAGGAAGGACGAACACCTCACCTCCAGGGGTTCGCTAATTTTGTGAAGAAGCAAACTTTTAATAAAGTGAAGTGGTATTTGGGTGCCCGCTGCTACATCGAGAAAGCCAAAGGAACTGATCAGCAGAATAAAGAATATTGCAGTAAAGAAGGCAACTTACTTATTGAATGTGGAGCTCCTCGATCTCAAGGACAACGGAGTGACCTGTCTACTGCTGTGAGTACCTTGTTGGAGAGCGGGAGTCTGGTGACCGTTGCAGAGCAGCACCCTGTAACGTTTGTCAGAAATTTCCGCGGGCTGGCTGAACTTTTGAAAGTGAGCGGGAAAATGCAGAAGCGTGATTGGAAGACCAATGTACACGTCATTGTGGGGCCACCTGGGTGTGGTAAAAGCAAATGGGCTGCTAATTTTGCAGACCCGGAAACCACATACTGGAAACCACCTAGAAACAAGTGGTGGGATGGTTACCATGGTGAAGAAGTGGTTGTTATTGATGACTTTTATGGCTGGCTGCCGTGGGATGATCTACTGAGACTGTGTGATCGATATCCATTGACTGTAGAGACTAAAGGTGGAACTGTACCTTTTTTGGCCCGCAGT。
finally, it should be noted that the application is not limited to the above embodiments, but that many variants are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present application.
Claims (10)
1. A preparation method of an nfo-RPA detection kit for identifying PCV2 and PCV3 is characterized by comprising the following steps:
1) Preparation of positive plasmid: screening out gene fragments of PCV2 and PCV3 through bioinformatics analysis, amplifying corresponding gene fragments from PCV2 and PCV3 genomes, and connecting the corresponding gene fragments to pUC57 plasmids to construct positive plasmids;
2) Establishing an nfo-RPA reaction system: respectively designing nfo-RPA enzyme amplification primers according to gene fragment sequences of PCV2 and PCV3, and mixing the primers and probes with the nfo-RPA enzyme to establish a reaction system;
3) And determining the optimal detection time and temperature of the nfo-RPA reaction system.
2. The method for preparing an nfo-RPA detection kit for discriminating PCV2 from PCV3 according to claim 1, wherein: the positive plasmid was prepared specifically as follows:
the amplified PCV2 and PCV3 gene fragments are subjected to double enzyme digestion by utilizing a molecular biological method and then are connected into a pUC57 vector, and are transferred into Escherichia coli Top competent bacteria for amplification, and the recombinant vectors obtained after screening and identification are named as PCV2 and PCV3 positive plasmids.
3. The method for preparing an nfo-RPA detection kit for discriminating PCV2 from PCV3 according to claim 2, wherein: the PCV3 gene fragment sequence is shown in NO. 1:
no.1PCV3 gene fragment:
ATACCACTTTTTCTCCCTACAGACCTCCGTGGATCCGGGTTCCGAGGTGCCGGGTAATACTAGCCCGGCACCAAAATGAGACACAGAGCTATATTCAGAAGAAGACCCCGCCCAAGGAGACGACGACGCCACAGAAGGCGCTATGCCAGAAGAAGACTATTCATTAGGAGGCCCACAGCTGGCACATACTACACAAAGAAATACTCCACCATGAACGTCATATCCGTTGGAACCCCTCAGAATAACAAGCCCTGGCACGCCAACCACTTCATTACCCGCCTAAACGAATGGGAGACTGCAATTACCTTTGAATATTATAAGATACTAAAGATGAAAGTTACACTCAGCCCTGTAATTTCTCCGGCTCAGCAAACAAAAACTATGTTCGGGCACACAGCCATAGATCTAGACGGCGCCTGGACCACAAACACTTGGCTCCAAGACGACCCTTATGCGGAAAGTTCCACTCGTAAAGTTATGACTTCTAAAAAAAAACACAGCCGTTACTTCACCCCCAAACCACTTCTGGCGGGAACTACCAGCGCTCACCCAGGACAAAGCCTCTTCTTTTTCTCCAGACCCACCCCATGGCTCAACACATATGACCCCACCGTTCAATGGGGAGCACTGCTTTGGAGCATTTATGTCCCGGAAAAAACTGGAATGACAGACTTCTACGGCACCAAAGAAGTTTGGATTCGTTACAAGTCCGTTCTCTAAGTGAAAATAAATATAAATCTGACCCCTCCGGTGGCGACAAGCCGTGTCCCCTCGCGCGCGCTCCCGCTGCG。
4. the method for preparing an nfo-RPA detection kit for discriminating PCV2 from PCV3 according to claim 2 or 3, wherein: the PCV2 gene fragment sequence is shown in NO. 2:
no.2PCV2 gene fragment:
ACCAGCGCACTTCGGCAGCGGCAGCACCTCGGCAGCACCTCAGCAGCAACATGCCCAGCAAGAAGAATGGAAGAAGCGGACCCCAACCACATAAAAGGTGGGTGTTCACGCTGAATAATCCTTCCGAAGACGAGCGCAAGAAAATACGGGAGCTCCCAATCTCCCTATTTGATTATTTTATTGTTGGCGAGGAGGGTAATGAGGAAGGACGAACACCTCACCTCCAGGGGTTCGCTAATTTTGTGAAGAAGCAAACTTTTAATAAAGTGAAGTGGTATTTGGGTGCCCGCTGCTACATCGAGAAAGCCAAAGGAACTGATCAGCAGAATAAAGAATATTGCAGTAAAGAAGGCAACTTACTTATTGAATGTGGAGCTCCTCGATCTCAAGGACAACGGAGTGACCTGTCTACTGCTGTGAGTACCTTGTTGGAGAGCGGGAGTCTGGTGACCGTTGCAGAGCAGCACCCTGTAACGTTTGTCAGAAATTTCCGCGGGCTGGCTGAACTTTTGAAAGTGAGCGGGAAAATGCAGAAGCGTGATTGGAAGACCAATGTACACGTCATTGTGGGGCCACCTGGGTGTGGTAAAAGCAAATGGGCTGCTAATTTTGCAGACCCGGAAACCACATACTGGAAACCACCTAGAAACAAGTGGTGGGATGGTTACCATGGTGAAGAAGTGGTTGTTATTGATGACTTTTATGGCTGGCTGCCGTGGGATGATCTACTGAGACTGTGTGATCGATATCCATTGACTGTAGAGACTAAAGGTGGAACTGTACCTTTTTTGGCCCGCAGT。
5. the method for preparing an nfo-RPA detection kit for discriminating PCV2 from PCV3 according to claim 2 or 3, wherein: the primer comprises the following components:
PCV3-Forward GGCACATACTACACAAAGAAATACTCCACC；
PCV3-Reverse[DIG]ATATTCAAAGGTAATTGCAGTCTCCCATTCG；
PCV3-Probe[FITC]GTTGGAACCCCTCAGAATAACAAGCCCTGG[dSpacer]；CACGCCAACCACTTCATTACCC[C3Spacer]；
PCV2-Forward GCTGCCGTGGGATGATCTACTGAGACTGTGT；
PCV2-Reverse[Biotin]CAGGGCATGGGGGGGAAAGGGTGACGAACTG；
PCV2-Probe
[6-FAM]GGCCCGCAGTATTCTGATTACCAGCAATCA[dSpacer]GACCCCGTTG GAATGGTACTCC[C3Spacer]；
wherein DIG represents digoxin, biotin represents Biotin, FITC represents fluorescein isothiocyanate, FAM represents a fluorescence reporter group, and Spacer represents a Spacer.
6. The method for preparing an nfo-RPA detection kit for discriminating PCV2 from PCV3 according to claim 1, wherein: determining the optimal detection time and temperature of an nfo-RPA reaction system:
selecting dilution concentration of 1×10 8 Performing nfo-RPA amplification reaction by respectively setting time gradients of 8min, 9min, 10min, 11min and 12min on a PCV3 positive plasmid template of cobies/mu L, taking out a reaction tube after the reaction is finished, placing the reaction tube on ice to finish the reaction, observing a result in a colloidal gold test strip, and adopting the reaction time of a visible clear amplification strip as the optimal detection time;
on the basis of selecting the optimal detection time, further setting a temperature gradient of 37 ℃, 38 ℃ and 39 ℃ for carrying out nfo-RPA amplification reaction, and observing the result in a colloidal gold test strip to determine the optimal reaction temperature;
the establishment of the nfo-RPA reaction system comprises the following steps: establishing a PCV2 nfo-RPA reaction system and a PCV3nfo-RPA reaction system;
establishing a PCV2 nfo-RPA reaction system: 29.4. Mu.L Buffer A, 2. Mu.L PCV3-specific-Forward, 2. Mu.L LPCV3-specific-Reverse, 0.6. Mu.L LPCV3-specific-Probe and a total of 13.5. Mu.L ddH were added to the nfo-RPA dry powder tube 2 O and Cap positive plasmid, 2.5 mu L Buffer B, after reacting for 9min at 39 ℃, taking 40 mu L of reaction product, cleaning and recovering, performing agarose gel electrophoresis, diluting the rest 10 mu L of product by 1/50 times, taking 10 mu L, dripping into a colloidal gold test strip sample adding area, observing the detection result of the colloidal gold test strip, and similarly, establishing a PCV2 nfo-RPA reaction system.
7. The method for preparing an nfo-RPA detection kit for discriminating between PCV2 and PCV3 according to claim 6, wherein: the optimal detection time is 9min; the optimum reaction temperature is 39 ℃.
8. The method for preparing an nfo-RPA detection kit for discriminating PCV2 from PCV3 according to claim 1, wherein: the sensitivity verification of the nfo-RPA reaction system is included;
1X 10 Using PCV3 Positive plasmid Standard 9 The initial concentration of the copies/. Mu.L was diluted 10-fold, and the diluted concentration was 1X 10 under the reaction conditions of a reaction time of 9min and a reaction temperature of 39 ℃ 9 copies/μL、1×10 8 copies/μL、1×10 7 copies/μL、1×10 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 copies/μL、1×10 2 copies/μL、1×10 1 copies/μL、1×10 0 Cap positive plasmid of copies/. Mu.L is used as template, ddH 2 O is used as a negative control to carry out nfo-RPA amplification reaction, and the result is observed in a colloidal gold test strip;
1X 10 Using PCV2 Positive plasmid Standard 6 The initial concentration of the copies/. Mu.L was diluted 10-fold, and the diluted concentration was 1X 10 under the reaction conditions of a reaction time of 9min and a reaction temperature of 39 ℃ 6 copies/μL、1×10 5 copies/μL、1×10 4 copies/μL、1×10 3 copies/μL、1×10 2 copies/μL、1×10 1 copies/μL、1×10 0 copies/μL、1×10 -1 copies/μL、1×10 -2 copies/μL、1×10 -3 copies/μL、1×10 -4 copies/μL、1×10 - 5 copies/μL、1×10 -6 PCV 2-positive plasmid of copies/. Mu.L is used as template, ddH 2 O was used as a negative control for the nfo-RPA amplification reaction and the results were observed in a colloidal gold test strip.
9. The method for preparing an nfo-RPA detection kit for discriminating PCV2 from PCV3 according to claim 1, wherein: the repeatability verification of the nfo-RPA reaction system also comprises the following steps:
repeating between groups: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 repeated tests were performed under the same conditions on 3 PCV3 positive plasmids at different concentrations of cobies/. Mu.L;
repeat within group: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 COPIES/. Mu.L 3 different concentrationsThe PCV3 positive plasmid was replicated 3 times in the same experiment;
repeating between groups: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 repeated tests were performed under the same conditions on 3 PCV2 positive plasmids at different concentrations of cobies/. Mu.L;
repeat within group: at 1X 10 8 copies/μL、1×10 7 copies/μL、1×10 6 3 copies/. Mu.L of 3 PCV2 positive plasmids at different concentrations were replicated 3 times in the same experiment.
10. The method for preparing an nfo-RPA detection kit for discriminating PCV2 from PCV3 according to claim 1, wherein: also included are nfo-RPA reaction system specific assays comprising:
the genome of mycoplasma hyopneumoniae 168 strain, porcine circovirus 2, porcine parvovirus and pseudorabies virus are respectively used as templates, and ddH is used 2 O is a negative control, PCV3nfo-RPA reaction is carried out at the reaction temperature of 39 ℃ for 9min, and the result is observed in a colloidal gold test strip;
respectively taking genomes of mycoplasma hyopneumoniae 168 strain, porcine circovirus 3, porcine parvovirus and pseudorabies virus as templates, and ddH 2 O is a negative control, PCV2 nfo-RPA reaction was performed at a reaction temperature of 39℃for 9min, and the results were observed in a colloidal gold test strip.
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