CN117051173A - Cat digestive tract virus PCR detection method and application - Google Patents
Cat digestive tract virus PCR detection method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention relates to the technical field of detection of cat digestive tract viruses, in particular to a cat digestive tract virus PCR detection method and application. The detection method is carried out by adopting a kit for detecting the viral PCR of the cat digestive tract, and the kit comprises an upstream primer, a downstream primer and a probe. The specific sequence of NS2 gene of cat parvovirus is amplified, and PCR amplified product is detected by fluorescence method, thereby realizing the detection of cat parvovirus. Compared with other detection methods, the method has high detection sensitivity.
Description
Technical Field
The invention relates to the technical field of detection of cat digestive tract viruses, in particular to a cat digestive tract virus PCR detection method and application.
Background
Feline parvovirus, also known as feline panleukopenia virus, feline pestivirus or feline infectious enteritis virus, is characterized primarily by hyperthermia, vomiting, severe leukopenia and enteritis, and is one of the major diseases that endanger felines. The feline parvovirus mainly infects mucosal epithelial cells of gastrointestinal tissues, causes pathological changes and death of the cells, and the clinical manifestations of feline panleukopenia caused by the virus are various and can be classified into an urgent type, an acute type, a subacute type and a non-dominant type. The acute type is sudden death, and no clinical symptoms exist; acute type death within 24 hours; subacute death occurs in about one week, accompanied by symptoms such as fever, vomiting, anorexia, and dehydration. Through a series of etiology studies, it has been demonstrated that feline parvovirus is naturally susceptible to infection in a variety of animals, such as felines and weasel, with minks being the most susceptible. At present, prevention against feline parvovirus is mainly vaccination, including inactivated viral vaccines and live attenuated vaccines. The inactivated virus vaccine has higher safety, but has a clinical effect which is not as good as that of the attenuated live vaccine. Vaccines against this virus are commercially available in some countries in europe, but diagnostic agents against this virus are not uniform. Therefore, it is important to establish a method for rapidly and sensitively detecting feline parvovirus.
Disclosure of Invention
The invention aims at providing a fluorescent quantitative PCR primer, a probe and a kit for detecting cat parvovirus aiming at the defects of the prior art. And synthesizing a specific probe and a primer according to the NS2 gene sequence of the feline parvovirus, amplifying the NS2 gene specific sequence of the feline parvovirus, and detecting a PCR amplification product by a fluorescence method or a test paper method, thereby realizing the sensitive detection of the feline parvovirus.
The invention realizes the aim through the following technical scheme:
in a first aspect, the invention provides a kit for viral PCR detection of a cat gut, comprising:
upstream primer F1:5'-tctgactccggacgtagtggaccttgcactggaaccg-3', as shown in SEQ ID NO. 1;
downstream primer R1:5'-ctctcaggtctgcctctatttcggaccacgtcg-3', as shown in SEQ ID NO. 2;
probe P1:5'-gactcgcttgcacgtctttgtgagtaacgc/iFAM6dT/C/THF/C/iBHQ1dT/gttggtttgattgtt (C3-spacer) -3' as shown in SEQ ID NO. 3.
Wherein FAM is a fluorescent group, THF is a tetrahydrofuran site, iBHQ1dT is a fluorescence quenching group, and (C3-spacer) is a blocking group.
Further, the kit also comprises RAA freeze-dried powder, a magnesium acetate solution, a reaction buffer solution and a standard plasmid; the RAA freeze-dried powder comprises recombinase, DNA polymerase, single-stranded DNA binding enzyme and dNTPs, wherein the standard plasmid is a vector carrying a specific sequence of a feline parvovirus NS2 gene, and the specific sequence is shown as SEQ ID NO. 7.
In a second aspect, the invention provides a reagent for viral PCR detection of the cat digestive tract, comprising an upstream primer, a downstream primer and a probe.
An upstream primer: 5'-tctgactccggacgtagtggaccttgcactggaaccg-3', as shown in SEQ ID NO. 1;
a downstream primer: 5'-FITC-ctctcaggtctgcctctatttcggaccacgtcg-3' as shown in SEQ ID NO. 8;
and (3) probe: 5'-BIOTIN-gactcgcttgcacgtctttgtgagtaacgc/c/THF/c/gttggtttgattgtt-PHO-3' as shown in SEQ ID NO. 9; wherein FITC is a fluorescent group, BIOTIN is BIOTIN, THF is a tetrahydrofuran site, and PHO is a phosphoric acid modification group.
Wherein, a Tetrahydrofuran (THF) is inserted into the probe sequence to simulate a basic site for modification, and the THF modified nucleotide can efficiently realize the extension of the DNA chain under the action of DNA polymerase.
The invention provides a kit designed according to an amplification primer pair and a probe for detecting feline digestive tract viruses, which comprises a RAA reaction system and an LFD test strip, wherein a detection line of the LFD test strip is provided with a biotin antibody and a fluorescent group antibody, and a quality control line is provided with an avidin-colloid Jin Teyi antibody.
Further, the fluorescent group antibody is a gold nanoparticle of a FAM antibody.
The primers and probes with biotin marks and 6-carboxyl fluorescence hormone FAM marks are subjected to amplification reaction with target nucleic acid, and the product is an amplicon with both biotin and FAM marks. The front end of the LFD test strip is a detection line close to the immersion area, and the rear end close to the liquid absorption area at the hand-held end is a quality control line. The detection line at the front end of the LFD test strip is provided with nano gold particles of FAM antibody, the detection line is also provided with biotin antibody, amplified products are dripped on the test strip, FAM groups on the amplicon react with the FAM antibody, after the biotin antibody on the detection line is combined with biotin on the amplicon, the detection line displays a strip, and the uncaptured products are combined with avidin-colloid Jin Teyi antibody on the quality control line to display the strip. The detection line and the quality control line are colored, the color seen by naked eyes is changed according to the selection change of the colloid Jin Lijing and the reducing agent, the detection result is not affected, and the proper colloid Jin Lijing and the reducing agent can be selected according to the requirement.
Further, in the RAA reaction system, the addition amount of the RAA reaction general dry powder is 1 mug/10-30 mug; the RAA reaction general dry powder comprises recombinase, bst DNA polymerase, SSB protein, repair enzyme and dNTPs. The skilled artisan can select the appropriate RAA-based universal reagent product for use as desired.
Further, the concentration of the upstream primer is 10. Mu.M; the concentration of the downstream primer was 10. Mu.M; the concentration of probe ZHU-PRO was 10. Mu.M.
Further, the concentration of magnesium acetate is 300mM.
Further, the reaction condition of the kit is that the reaction is carried out for 15-25 min at 37-42 ℃.
Further, the kit also comprises a positive control; the positive control is a standard plasmid carrying a feline parvovirus gene specific sequence. The synthesized feline parvovirus NS1 gene is constructed on a pUC57 vector, transferred into Top10 competent cells, positive particles are identified by screening, PCR and sequencing, and positive plasmids are extracted, thus obtaining the standard plasmids.
Preferably, the RAA reaction system comprises 25. Mu.L of RAA reaction buffer, 4. Mu.L of forward primer (10. Mu.M), 4. Mu.L of reverse primer (10. Mu.M), 1. Mu.L of 10 in 47.5. Mu.L 3 Copy/. Mu.L of standard plasmid, sterilized double distilled water 17.5. Mu.L. After mixing, the mixture was added to a reaction tube containing the general dry powder for RAA reaction, and then 2.5. Mu.L of 300mM magnesium acetate solution was added to each reaction tube. The above reaction systemMixing uniformly, and placing the mixture into a PCR reactor to react for 30 min at 36 ℃.
In a third aspect, a method for detecting a feline digestive tract virus in vitro using the kit comprising the steps of:
extracting genomic DNA from the cat gut sample using the DNA extraction reagent;
preparing an RRA reaction system: adding RRA freeze-dried powder into the reaction buffer solution, adding the forward primer, the reverse primer, the probe and the genomic DNA, adding sterile water, uniformly mixing and centrifuging, adding magnesium acetate, uniformly mixing and centrifuging;
and placing the RRA reaction system in a constant temperature device or environment with the temperature range of 20-50 ℃ for 20min, and incubating to obtain an amplification product.
Further, the method further comprises detecting the feline digestive tract virus from a fluorescent signal amplification curve generated from the amplification product. For example, detection of the fluorescent signal amplification curve can be determined based on whether the curve is S-shaped. For example, a standard curve is drawn according to the Ct values and the concentrations of the standards of different dilution levels, and the sample concentration is calculated according to the Ct values of the samples so as to realize quantitative detection.
Further, combining the RAA reaction with the LFD test strip, the method further comprising: sucking the amplified product into a new reaction tube, and diluting the amplified product by 50-300 times; LFD test strip detection: inserting the LFD test strip into the diluted reaction tube, and waiting for the strip to appear; directly reading a detection result according to the color development condition of the LFD test strip; the result comprises the color development results of a quality control line and a detection line on the LFD test strip. The results included: only one line appears on the quality control line, which indicates that the sample has no cat parvovirus nucleic acid or the copy number thereof is lower than the lowest detection limit of the kit; two lines appear, one is located in the detection line and the other is located in the quality control line, indicating the presence of feline parvoviral nucleic acid in the sample; no band appears on the quality control line, indicating that the nucleic acid test strip is invalid.
Further, the LFD test strip was observed within 10min, and after 10min the reading was not valid.
The invention provides a kit and a method for detecting cat parvovirus based on recombinase-mediated isothermal nucleic acid amplification, and provides a novel cat parvovirus nucleic acid adulteration on-site detection method which can realize visual detection of macroscopic results. Compared with other detection methods, the method has high detection sensitivity, can stably detect 5 copies/. Mu.L of standard plasmid, and can effectively detect 0.1 pg/. Mu.L of genome DNA sample.
In addition, the invention combines the isothermal nucleic acid amplification reagent mediated by the recombinase and the lateral flow immunological technology, and is the combined application of the RAA technology and the LFD technology. The whole detection process can be completed within 20-30 minutes without special equipment, and the result is accurate, so that the detection of the field sample can be satisfied, and the method is particularly suitable for the field detection of pet markets and the like.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification product for example 1 detection of standard plasmid using F1/R1 (lane 1) and F2/R2 (lane 2).
FIG. 2 shows the results of optimizing the amplification temperature of the standard plasmid for detection by F1/R1 in example 1, lanes 1 to 7 are the amplification temperatures of 36, 37, 38, 39, 40, 41 and 42℃in this order.
FIG. 3 shows the results of optimizing the concentration of the forward and reverse primers for detecting the standard plasmid by using F1/R1 in example 1, wherein lanes 1-6 are 0.1, 0.2, 0.3, 0.4, 0.6 and 0.8. Mu. Mol/L of forward and reverse primers in sequence.
FIG. 4 shows the results of optimizing the concentration of magnesium acetate in the test of example 1 using F1/R1 to detect standard plasmids, lanes 1-7 being 7, 9, 11, 13, 15, 17, 20mmol/L of magnesium acetate solution in this order.
FIG. 5 shows the result of example 1 for detecting sensitivity of the standard plasmid by F1/R1, lanes 1 to 8 being 10 in order 7 、10 6 、10 5 、10 4 、10 3 、10 2 Standard plasmid solutions at 10 and 5 copies/. Mu.L.
FIG. 6 shows the result of example 1 for detecting sensitivity of the standard plasmid by F2/R2, lanes 1 to 8 being 10 in order 7 、10 6 、10 5 、10 4 、10 3 、10 2 Standard plasmid solutions at 10 and 5 copies/. Mu.L.
FIG. 7 shows the results of detection of genomic DNA from a cat gut sample by the fluorescence RAA method of F1/R1/P1 in example 2, with fluorescence amplification curves of 50 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.5 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, 1 pg/. Mu.L and 0.1 pg/. Mu.L in this order from left to right.
FIG. 8 shows the results of detection of genomic DNA from a cat gut sample by the fluorescence RAA method using F2/R2/P2 in example 2, with fluorescence amplification curves of 50 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.5 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, 1 pg/. Mu.L and 0.1 pg/. Mu.L in this order from left to right.
FIG. 9 shows the results of example 3, in which the genomic DNA of the standard plasmid and cat gut sample at different concentrations was detected by the fluorescent RAA-LPF method, in the order of 10 from left to right 3 Detection results of standard plasmid of 10 and 5 copies/. Mu.L, 10 of control group 3 Detection results of 10 and 5 copies/. Mu.L of standard plasmid, detection results of 0.1 ng/. Mu.L and 0.01 ng/. Mu.L of feline parvoviral genomic DNA of the experimental group, detection results of 0.1 ng/. Mu.L and 0.01 ng/. Mu.L of feline parvoviral genomic DNA of the control group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The reagents not specifically and individually described in the present invention are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Example 1: establishment of basic reaction system
The embodiment establishes the cat digestive tract virus detection technology according to the recombinase-mediated isothermal amplification technology principle, and the method does not depend on a PCR instrument, has rapid reaction and simple and convenient operation, and is expected to provide a new means for diagnosis.
1. Construction of recombinant plasmid carrying specific sequence of feline parvovirus NS1 Gene (as Standard plasmid)
The synthesized feline parvovirus NS1 gene is constructed on a pUC57 vector, transferred into Top10 competent cells, positive particles are identified by screening, PCR and sequencing, and positive plasmids are extracted, thus obtaining the standard plasmids.
2. Primer and probe
Upstream primer F1:5'-tctgactccggacgtagtggaccttgcactggaaccg-3', as shown in SEQ ID NO. 1.
Downstream primer R1:5'-ctctcaggtctgcctctatttcggaccacgtcg-3', as shown in SEQ ID NO. 2.
Probe P1:5'-gactcgcttgcacgtctttgtgagtaacgc/iFAM6dT/C/THF/C/iBHQ1dT/gttggtttgattgtt (C3-spacer) -3' as shown in SEQ ID NO. 3.
The upstream primer F2:5'-cggcaagcaatcctcagagtcaagaccaacttc-3' as shown in SEQ ID NO. 4.
Downstream primer R2:5'-cgcttgcacgtctttgtgagtaacgccaaga-3', as shown in SEQ ID NO. 5.
Probe P2:5'-tggtttgattgttgatttgcagtttctgca/iFAM6dT/C/THF/C/iBHQ1dT/ggcgtatctggagtac (C3-spacer) -3' as shown in SEQ ID NO. 6.
The primers and probes are chemically synthesized.
3. RAA reaction system
RAA reaction system comprising 25. Mu.L of reaction buffer (Jiangsu Qiyan Gene organism), 2. Mu.L of forward primer (10. Mu.M), 2. Mu.L of reverse primer (10. Mu.M), and template DNA (10) was prepared in a 0.5ml PCR tube 3 Copy/. Mu.L, standard plasmid) 1. Mu.L, sterilized double distilled water 17.5. Mu.L. The mixed 47.5. Mu.L solution was added to dry powder reaction tubes (recombinase, DNA polymerase, single-stranded DNA binding enzyme and dNTPs) containing recombinase, and then 2.5. Mu.L of 280mM magnesium acetate solution was added to each reaction tube. The reaction systems are evenly mixed and put into a PCR reactor for reaction for 30 min at 37 ℃. After the reaction, the reaction tube was taken out, 50. Mu.L of phenol/chloroform (1:1) was added thereto, and the mixture was homogenized by shaking and centrifuged at 12000 r/min for 1min. 10. Mu.L of the upper layer solution was aspirated for agarose gel electrophoresis (gel concentration 1.2%) at 100V for 40 min, and the result was obtained by scanning on a gel imaging system. As shown in FIG. 1, the result is that the standard plasmid is used as a template for RAA amplification, F1/R1 and F2/R2 are used as primer pairs for RAA reaction, and the amplified products are subjected to agarose gel electrophoresis, and specific bands of clear 160 bp are visible under ultraviolet lamp, which are consistent with the expected result, but the concentration of the amplified bands of F2/R2 used as the primer pairs is lower.
4. Optimization of RAA amplification conditions
Preparing a reaction system according to the steps, and carrying out RAA gradient amplification at different temperatures of 36, 37, 38, 39, 40, 41 and 42 ℃; after the optimal temperature is determined, forward and reverse primers with final concentrations of 0.1, 0.2, 0.3, 0.4, 0.6 and 0.8 mu mol/L and magnesium acetate solutions with final concentrations of 7, 9, 11, 13, 15, 17 and 20mmol/L are sequentially added, and the total volume is supplemented by double distilled water. The effect of different temperatures, different primer concentrations and different magnesium ion concentrations on the DNA amplification efficiency was compared.
As shown in FIG. 2, the amplification effect was optimal at 36 ℃. As shown in FIG. 3, the amplification effect was best by adding forward and reverse primers at a final concentration of 0.8. Mu. Mol/L. As shown in FIG. 4, the amplification effect was best by adding magnesium acetate at a final concentration of 13 mmol/L.
5. Sensitivity evaluation by RAA method
Taking a standard plasmid containing an NS2 gene fragment as a reference plasmidTemplate evaluation RAA sensitivity was diluted in gradient to give different concentrations (10 7 、10 6 、10 5 、10 4 、10 3 、10 2 10 and 5 copies/. Mu.L), 1. Mu.L each was subjected to RAA reaction, and the reaction products were subjected to agarose gel electrophoresis, and scanned on a gel imaging system to obtain the results. As shown in FIG. 5, the standard plasmid with 5 copies/. Mu.L still can obtain obvious amplified bands, which indicates that the F1/R1 provided by the invention can amplify the standard plasmid with low concentration, and has very high sensitivity.
As a result, as shown in FIG. 6, the temperature was only 10 3 Obvious amplified bands can be obtained by using a copy/mu L standard plasmid, which shows that the F2/R2 provided by the invention can amplify the standard plasmid with low concentration, but the sensitivity is lower than that of F1/R1.
Example 2 establishment of recombinase-mediated isothermal amplification fluorescent method (fluorescent RAA method) for nucleic acid of feline digestive tract virus
1. Cat digestive tract sample genome DNA extraction
To the sample, 20. Mu.L of protease K was added, followed by 200. Mu.L of BufferAL, vortexed for 15s, and mixed well. Incubating at 70deg.C for 10min, rapidly centrifuging the test tube, adding 200 μl of absolute ethanol, centrifuging after vortexing for 15s, transferring the mixed solution to a collecting tube containing QIAamp Minispin column with a pipette, centrifuging at 8000r/min for 1min, and discarding the supernatant. QIAamp Mini spincolumn was placed in a fresh 2ml collection tube, 500. Mu.L Buffer AW1 was added, centrifuged at 8000r/min for 1min, and the supernatant was discarded. QIAamp Mini spin column was placed in a fresh 2mL collection tube, 500. Mu.L Buffer AW2 was added, centrifuged at 14000r/min for 1min, and the supernatant was discarded. QIAamp Mini spin column was placed in a fresh 2mL collection tube and centrifuged at full speed for 1min. QIAamp Mini spin column was placed in a fresh 1.5mL collection tube, 200 μLBuffer AE was added and incubated for 1min at room temperature. Centrifugation is performed at 8000r/min for 1min to elute DNA. The extracted DNA was stored at-80 ℃.
2. Fluorescent RAA reaction system
The initial concentration of the feline parvovirus genome extracted by the kit was 167.8 ng/. Mu.L, and the genomic DNA was diluted to 10 ng/. Mu.L, 1 ng/. Mu.L, 0.5 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, 1 pg/. Mu.L and 0.1 pg/. Mu.L of genomic DNA samples, each of which was amplified with 1. Mu.L of template DNA, and the sensitivity of the fluorescence RAA method was observed.
Based on the optimized RAA reaction system, the RAA reaction system is prepared in a 0.5ml PCR tube: 25. mu.L of reaction buffer, 2. Mu.L of 20. Mu. Mol/L forward primer, 2. Mu.L of 20. Mu. Mol/L reverse primer, 1. Mu.L of 20. Mu. Mol/L probe, 1. Mu.L of DNA template, double distilled water 16.5. Mu.L. The mixed 47.5. Mu.L solution was added to a dry powder reaction tube containing the recombinase. Then 2.5. Mu.L of 300mM magnesium acetate solution was added to each reaction tube. Immediately placing into an RAA-B6100 constant temperature vibration instrument for vibrating and centrifuging for 4min, mixing, and placing the reaction tube into an RAA fluorescence detector (RAA-F1620) for detection under the working condition of 36 ℃ for 30 min.
As shown in FIG. 7, the genomic DNA of the cat gut sample was detected by the fluorescence RAA method of F1/R1/P1, and thus, a genomic DNA sample of 0.1 pg/. Mu.L was efficiently detected (S-type amplification curve was developed). As shown in fig. 8. The fluorescence RAA method of F2/R2/P2 is adopted to detect the genomic DNA of the cat digestive tract sample, and only 0.5 ng/. Mu.L of genomic DNA sample can be effectively detected. The sensitivity of the fluorescence RAA method using F1/R1/P1 was higher than that of the fluorescence RAA method using F2/R2/P2.
Thus, the fluorescent quantitative PCR instrument detects the nucleic acid of the cat digestive tract virus according to the received fluorescent signal amplification curve to diagnose the disease. And (3) realizing qualitative detection according to whether an S-shaped amplification curve appears, drawing a standard curve according to the Ct values and the concentrations of the standard substances of different dilution levels, and calculating the sample concentration according to the sample Ct values to realize quantitative detection.
EXAMPLE 3 RAA-LFD
1. Primer and probe
Experimental group:
an upstream primer: 5'-tctgactccggacgtagtggaccttgcactggaaccg-3', as shown in SEQ ID NO. 1.
A downstream primer: 5'-FITC-ctctcaggtctgcctctatttcggaccacgtcg-3' as shown in SEQ ID NO. 8.
And (3) probe: 5'-BIOTIN-gactcgcttgcacgtctttgtgagtaacgc/c/THF/c/gttggtttgattgtt-PHO-3' as shown in SEQ ID NO. 9. Wherein FITC is a fluorescent group, BIOTIN is BIOTIN, THF is a tetrahydrofuran site, and PHO is a phosphoric acid modification group.
Control group:
an upstream primer: 5'-cggcaagcaatcctcagagtcaagaccaacttc-3' as shown in SEQ ID NO. 4.
A downstream primer: 5'-FITC-cgcttgcacgtctttgtgagtaacgccaaga-3' as shown in SEQ ID NO. 10.
And (3) probe: 5'-BIOTIN-tggtttgattgttgatttgcagtttctgca/c/THF/c/ggcgtatctggagtac-PHO-3' as shown in SEQ ID NO. 11. Wherein FITC is a fluorescent group, BIOTIN is BIOTIN, THF is a tetrahydrofuran site, and PHO is a phosphoric acid modification group.
The primers and probes are chemically synthesized.
2. RAA reaction
And the LFD test strip is provided with a biotin antibody and a fluorescent group antibody on a detection line, and an avidin-colloid Jin Teyi antibody on a quality control line. The primers and probes with biotin marks and 6-carboxyl fluorescence hormone FAM marks are subjected to amplification reaction with target nucleic acid, and the product is an amplicon with both biotin and FAM marks. The front end of the LFD test strip is a detection line close to the immersion area, and the rear end close to the liquid absorption area at the hand-held end is a quality control line. The detection line at the front end of the LFD test strip is provided with nano gold particles of FAM antibody, the detection line is also provided with biotin antibody, amplified products are dripped on the test strip, FAM groups on the amplicon react with the FAM antibody, after the biotin antibody on the detection line is combined with biotin on the amplicon, the detection line displays a strip, and the uncaptured products are combined with avidin-colloid Jin Teyi antibody on the quality control line to display the strip.
The RAA reaction system was calculated as 47.5. Mu.L and included 25. Mu.L RAA reaction buffer, 4. Mu.L forward primer (10. Mu.M), 4. Mu.L reverse primer (10. Mu.M), 1. Mu.L 10 3 Copy/. Mu.L of standard plasmid or cat gut sample genomic DNA, sterilized double distilled water 17.5. Mu.L. After mixing, the mixture was added to reaction tubes (recombinase, bst DNA polymerase, SSB protein, repair enzyme and dNTPs) containing a dry powder common to RAA reactions, and then 2.5. Mu.L of 300mM acetic acid was added to each reaction tubeMagnesium solution. The reaction systems are evenly mixed and put into a PCR reactor for reaction for 30 min at 36 ℃. After the RAA reaction is finished, the Eppendorf tube is opened, the amplified product is sucked into a new Eppendorf tube, marked and diluted by 50 times, and the test paper detection is immediately carried out.
3. LFD test strip operation
The LFD operation steps are as follows: inserting the end of the immersion area (marked with blue arrow upwards) of the LFD test strip into the Eppendorf tube, wherein the liquid level cannot exceed the MAX indication line of the immersion area, and keeping the test strip flat for 1-3min after the test strip is immersed completely (about 30-60s is needed) until the red strip appears. And directly reading the detection result according to the color development condition of the test strip. The method can generally generate a strip within about 1min, the result is observed within 10min, and the interpretation is invalid after 10 min.
4. Sensitivity experiment
The sensitivity of RAA-LFD method is evaluated by taking a standard plasmid containing NS2 gene fragment as a template, and different concentrations are obtained by gradient dilution (10) 3 10 and 5 copies/. Mu.L), or 0.1 ng/. Mu.L and 0.01 ng/. Mu.L of feline parvoviral genomic DNA as a template to evaluate the sensitivity of the RAA-LFD method, 1. Mu.L each was taken for RAA reaction, and the reaction products were subjected to LFD test strip operation to judge the sensitivity of the above-mentioned experimental group and control group based on the results. As a result, as shown in FIG. 9, the experimental group was able to detect 10 3 Standard plasmid at 10 and 5 copies/. Mu.L and cat parvovirus genomic DNA at 0.1 ng/. Mu.L, whereas the control group detected only 10 3 Copy/. Mu.L of standard plasmid, inability to detect feline parvoviral genomic DNA, again demonstrating that the sensitivity of the experimental group is higher than the control group.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (7)
1. A kit for viral PCR detection of a feline digestive tract comprising:
upstream primer F1:5'-tctgactccggacgtagtggaccttgcactggaaccg-3', as shown in SEQ ID NO. 1;
downstream primer R1:5'-ctctcaggtctgcctctatttcggaccacgtcg-3', as shown in SEQ ID NO. 2;
probe P1:5'-gactcgcttgcacgtctttgtgagtaacgc/iFAM6dT/C/THF/C/iBHQ1dT/gttggtttgattgtt (C3-spacer) -3' as shown in SEQ ID NO. 3;
wherein iFAM6dT is a fluorescent group, THF is a tetrahydrofuran site, iBHQ1dT is a fluorescence quenching group, and (C3-spacer) is a blocking group.
2. The kit of claim 1, further comprising RAA lyophilized powder, magnesium acetate solution, reaction buffer, and standard plasmid; the RAA freeze-dried powder comprises recombinase, DNA polymerase, single-stranded DNA binding enzyme and dNTPs, wherein the standard plasmid is a vector carrying a specific sequence of a feline parvovirus NS2 gene, and the specific sequence is shown as SEQ ID NO. 7.
3. A kit for viral PCR detection of a feline digestive tract comprising:
an upstream primer: 5'-tctgactccggacgtagtggaccttgcactggaaccg-3', as shown in SEQ ID NO. 1;
a downstream primer: 5'-FITC-ctctcaggtctgcctctatttcggaccacgtcg-3' as shown in SEQ ID NO. 8;
and (3) probe: 5'-BIOTIN-gactcgcttgcacgtctttgtgagtaacgc/c/THF/c/gttggtttgattgtt-PHO-3' as shown in SEQ ID NO. 9;
wherein FITC is a fluorescent group, BIOTIN is BIOTIN, THF is a tetrahydrofuran site, and PHO is a phosphoric acid modification group.
4. The kit of claim 3, further comprising RAA lyophilized powder, magnesium acetate solution, reaction buffer, and LFD test strip; the detection line of the LFD test strip is provided with a biotin antibody and a fluorescent group antibody, and the quality control line is provided with an avidin-colloid Jin Teyi antibody.
5. The kit of claim 4, wherein the RAA lyophilized powder comprises a recombinase, bst DNA polymerase, SSB protein, repair enzyme, and dNTPs.
6. The kit according to claim 3, further comprising a standard plasmid, which is a vector carrying a specific sequence of the feline parvovirus NS2 gene as shown in SEQ ID No. 7.
7. The kit of claim 1, 2 or 3, further comprising a DNA extraction reagent.
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