CN116855636A - Cat coronavirus detection primer probe composition, kit and method - Google Patents
Cat coronavirus detection primer probe composition, kit and method Download PDFInfo
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Classifications
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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
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- 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
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biomedicine, in particular to a cat coronavirus detection primer probe composition, a kit and a method, comprising the following steps: a first set of primers and probes designed based on the nucleotide sequence shown as SEQ ID NO. 7; a second set of primers and probes designed based on the nucleotide sequence shown as SEQ ID NO. 8; the nucleotide sequence shown in SEQ ID NO.7 is a target sequence corresponding to a cat coronavirus 7b gene, and the nucleotide sequence shown in SEQ ID NO.8 is a target sequence corresponding to a cat source sample housekeeping gene Rpp.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a cat coronavirus detection primer probe composition, a kit and a method.
Background
Feline coronavirus (Feline coronavirus, FCoV) is a single-stranded positive strand RNA virus with a non-segmented envelope, FCoV belonging to the order of the nidus virus, the family coronaviridae, genus coronavirus. According to statistics, 25% -40% of domestic cats are positive for coronaviruses, and in large cat houses or breeding cat houses, the positive rate reaches 80% -100%. Although there may be no clinical symptoms at all after infection, infectious peritonitis may occur in cats with very high mortality rates, which is often found in young cats under four years of age, especially in herding cat groups. At present, no effective vaccine prevention and no effective drug treatment exist for cat infectious peritonitis. Therefore, for FCoV management and control, a fast and accurate detection is critical.
There are many methods for detecting nucleic acids, such as polymerase chain reaction (Polymerase Chain Reaction, PCR), isothermal amplification reaction, etc. The fluorescent quantitative PCR method has the advantages of better sensitivity, better specificity and lower false positive and false negative rate, and becomes an ideal virus detection method. However, in the detection process, the collected sample is difficult to directly detect on site, long-distance transportation is required, so that the nucleic acid is easy to be damaged, or the nucleic acid is damaged or polluted due to the fact that the operation is not normal when the collection personnel collect the sample, or the nucleic acid is damaged due to the fact that the nucleic acid extraction process, finally, the sensitivity, the specificity, the false positive rate, the false negative rate and the like of the detection result of the fluorescent quantitative PCR method are unsatisfactory.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a primer probe composition, a kit and a method for detecting the cat coronavirus, and the primer probe composition, the kit and the method are adopted for detection, so that the sensitivity is high, the specificity is high, and the false positive rate and the false negative rate are low.
For this purpose, the invention provides the following technical scheme:
a feline coronavirus detection primer probe composition comprising: a first set of primers and probes designed based on the nucleotide sequence shown as SEQ ID NO. 7;
the second set of primers and probes was designed based on the nucleotide sequence shown as SEQ ID NO. 8.
Optionally, the first set of primers and probes comprises: the nucleotide sequence of the forward primer FCoV-F is shown as SEQ ID NO. 1; the nucleotide sequence of the reverse primer FCoV-R is shown as SEQ ID NO. 2; and/or probe FCoV-P, its nucleotide sequence is shown in SEQ ID NO. 3;
a second set of primers and probes comprising: the nucleotide sequence of the forward primer RPP-F is shown as SEQ ID NO. 4; the nucleotide sequence of the reverse primer RPP-R is shown as SEQ ID NO. 5; and/or the nucleotide sequence of the probe RPP-P is shown as SEQ ID NO. 6.
Optionally, the 5' end of the probe FCoV-P and the 5' end of the probe RPP-P are marked with fluorescent groups, and the 3' end of the probe FCoV-P and the probe RPP-P are marked with fluorescent quenching groups; the fluorescent groups of the probe FCoV-P and the probe RPP-P are different.
Optionally, the fluorophore is selected from at least 1 of FAM, VIC, CY, ROX, HEX, JOE, NED, texas Red and CY 3;
and/or the fluorescence quenching group is selected from at least 1 of BHQ-1, BHQ-2, BHQ-3 and MGB.
A kit for detecting the feline coronavirus comprises the feline coronavirus detection primer probe composition.
Optionally, also comprises DNA polymerase, dNTP, mg 2+ The PCR reagent comprises ROX dye, PCR enhancer, PCR stabilizer, PCR buffer solution, sample collection liquid, negative quality control product or positive quality control product.
Optionally, the method further comprises: the PCR enhancer is BSA;
and/or the PCR buffer contains Tris 10mM-500mM, chloride 10mM-500mM, (NH) 4 ) 2 SO 4 10mM-500mM, glycerol 1 v/v-50 v/v%, BSA 0.001mg/mL-1mg/mL, tween 20 0.1 v/v-10 v/v%, dithiothreitol 1mM-100mM, betaine 0.1M-3M, DMSO 0.1 v/v-10 v/v%, and pH 7.5-9.5;
and/or the sample collection liquid comprises sodium bicarbonate with the concentration range of 6-6.5g/L, sodium carbonate with the concentration range of 12-13g/L, tween-20 with the concentration range of 0.01-0.05v/v, cresol red with the concentration range of 0.01-0.05g/100ml, guanidine hydrochloride with the concentration range of 4-5M and the balance of sterile water without enzyme; the sample collection liquid is adopted to treat the sample, so that the step of extracting nucleic acid can be omitted;
and/or, the negative quality control product is enzyme-free sterile water;
and/or the positive quality control product is a mixed plasmid standard product respectively comprising nucleotide sequences shown as SEQ ID NO. 7-8.
Alternatively, a PCR amplification reaction system is included, in 25. Mu.L:
the volume of the PCR mixed solution is 12.5 mu L, the PCR mixed solution contains DNA polymerase, and the enzyme activity is 0.5-1U; dNTPs, the concentration of each base ranges from 200 mu M to 300 mu M; mg of 2+ The concentration range is 1.5-2mM; PCR buffer solution with pH value of 7.5-9.5;
the volume of the cat coronavirus detection primer probe composition is 7.5 mu L, the concentration of each primer is 0.1-0.5 mu M, and the concentration of each probe is 0.05-0.25 mu M;
the sample to be tested had a volume of 5. Mu.L.
The use of the feline coronavirus detection primer probe composition or the feline coronavirus detection kit in preparing a feline coronavirus detection product.
A method for detecting feline coronavirus for non-disease diagnosis comprising the steps of:
and (3) taking a sample to be detected as a template, preparing a PCR amplification reaction system by using the cat coronavirus detection primer probe composition or the cat coronavirus detection kit, and carrying out fluorescent quantitative PCR detection.
Optionally, adding the collected sample into a sample collection liquid for processing to obtain the sample to be tested; optionally, the treatment time is 0.5-3 minutes;
optionally, in the fluorescent quantitative PCR detection, the PCR amplification procedure is: reverse transcription, holding at 50℃for 300 seconds; pre-denaturation, holding at 95 ℃ for 30 seconds; denaturation at 95℃for 5 seconds, annealing at 57℃for 30 seconds, extension for 40 cycles;
and/or, the fluorescent quantitative PCR detection comprises a qualitative or quantitative detection method;
the qualitative method comprises the following steps: the channel corresponding to the fluorescent group of the probe in the first group of primers and the probe is a channel A; the channel corresponding to the fluorescent group of the probe in the second group of primers and the probe is a B channel; the judging method comprises the following steps:
if the Ct value of the sample to be tested in the A channel is less than or equal to 39 and the Ct value of the sample to be tested in the B channel is less than 39, the sample to be tested is positive for the cat coronavirus;
if the sample to be tested has no Ct value or Ct value more than 39 in the A channel and Ct value less than 39 in the B channel, the sample to be tested is negative to the feline coronavirus;
if the sample to be tested has no Ct value in the A channel and the B channel, retesting is carried out;
the quantitative detection method comprises the following steps of
Establishing a standard curve: diluting a standard substance comprising a nucleotide sequence shown as SEQ ID NO.7 to different concentrations; taking standard substances with different concentrations as templates, performing fluorescence quantitative PCR detection, and drawing a standard curve by taking the concentration of the standard substance as an abscissa and the corresponding Ct value as an ordinate after the detection is finished;
substituting the Ct value of the sample to be measured into the corresponding standard curve to obtain the quantitative result of the sample to be measured.
The technical scheme of the invention has the following advantages:
1. the invention provides a cat coronavirus detection primer probe composition, which comprises the following components: a first set of primers and probes designed based on the nucleotide sequence shown as SEQ ID NO. 7; a second set of primers and probes designed based on the nucleotide sequence shown as SEQ ID NO. 8; the nucleotide sequence shown in SEQ ID NO.7 is a target sequence corresponding to a cat coronavirus 7b gene, and the nucleotide sequence shown in SEQ ID NO.8 is a target sequence corresponding to a cat source sample housekeeping gene Rpp.
2. The invention provides a cat coronavirus detection primer probe composition, which comprises the following components: a first set of primers and probes comprising: the nucleotide sequence of the forward primer FCoV-F is shown as SEQ ID NO. 1; the nucleotide sequence of the reverse primer FCoV-R is shown as SEQ ID NO. 2; the nucleotide sequence of the probe FCoV-P is shown as SEQ ID NO. 3; a second set of primers and probes comprising: the nucleotide sequence of the forward primer RPP-F is shown as SEQ ID NO. 4; the nucleotide sequence of the reverse primer RPP-R is shown as SEQ ID NO. 5; the nucleotide sequence of the probe RPP-P is shown as SEQ ID NO. 6; according to the invention, by selecting the combination of the cat coronavirus 7b gene and the cat source sample housekeeping gene RPP, amplification primer pairs and detection probes are skillfully designed, so that mutual interference between a plurality of primer pairs and corresponding detection probes is avoided; and the combination of the primer and the probe is proved to have good specificity and high sensitivity when used for detecting the feline coronavirus.
3. The invention provides a method for detecting cat coronavirus for non-disease diagnosis, which comprises the following detection principles: the feline coronavirus 7b gene is selected as the detection target. Designing a set of primer probes according to the detection targets, wherein fluorescent groups are marked at the 5 'ends of the probes, and fluorescent quenching groups are marked at the 3' ends of the probes. When the probe remains intact, the fluorescence quenching group may significantly reduce the fluorescence emitted by the fluorescent group by Fluorescence Resonance Energy Transfer (FRET). During PCR amplification, the primer and the probe are simultaneously bound to the template, and the binding position of the probe is positioned between the upstream primer and the downstream primer. When the amplification extends to the position where the probe is combined, the DNA polymerase uses the 5 'exonuclease activity to cut off the fluorescent molecule connected with the 5' end of the probe from the probe, so that the fluorescent molecule emits fluorescence, and the endogenous internal standard is the same. The number of fluorescent molecules detected is proportional to the number of PCR products, and therefore, the number of initial nucleic acid templates can be calculated according to the fluorescence intensity in the PCR reaction system. The fluorescence quantitative PCR instrument automatically draws a real-time amplification curve according to the detected fluorescence signal, so that qualitative or quantitative detection of the cat coronavirus on the nucleic acid level is realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard curve drawn in experimental example 1 of the present invention;
FIG. 2 shows the sensitivity test results in Experimental example 1 of the present invention;
FIG. 3 is a sample nucleic acid which was clinically confirmed to be positive for feline coronavirus in experimental example 1 of the present invention using the method of the present invention; the ordinate RFU in the figure indicates the fluorescence signal intensity.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Positive plasmids were prepared by conventional methods, specifically as follows: cloning 7b gene sequence of cat coronavirus and sequence of cat source sample housekeeping gene Rpp to pUC57 vector, transforming bacterial competent cell DH5 alpha, extracting plasmid, sequencing and verifying to obtain plasmid standard of multiplex fluorescence quantitative PCR. The corresponding plasmid standards were designated pUC57-FCoV-7b, pUC57-Rpp, respectively. Measuring OD by ultraviolet spectrophotometry 260 The value was expressed as [ X (g/. Mu.L) DNA/DNA length (bp). Times.660 ]]×6.02×10 23 After converted to molar concentration =y (copies/. Mu.l)), diluted to 10 8 The copies/. Mu.L was stored at-20℃and diluted before use.
Example 1
The embodiment provides a cat coronavirus detection primer probe composition, which comprises the following specific components:
in order to ensure the accuracy of the detection result of the feline coronavirus, the nucleotide sequence shown as SEQ ID No.7 in the 7b gene in the viral genome is selected as a target sequence, a specific primer probe (SEQ ID No. 1-3) is designed for amplification, and the canine genome nucleic acid or other pathogen genome non-fluorescent amplification signal is detected, and the feline coronavirus genome fluorescent amplification signal is detected.
In order to eliminate the influences of false negative, false positive, pollution and extraction, a nucleotide sequence shown as SEQ ID No.8 in a cat-derived sample housekeeping gene Rpp is selected as a target sequence, a specific primer probe (SEQ ID No. 4-6) is designed for amplification, no fluorescent amplification signal is detected in other pathogen genome, and fluorescent amplification signal is detected in DNA of the cat RPP gene.
Namely, the cat coronavirus nucleic acid detection primer probe composition comprises:
the first group of primers and probes comprises a forward primer FCoV-F, a reverse primer FCoV-R and a probe FCoV-P, and the nucleotide sequences of the primers and the probes are respectively shown as SEQ ID No. 1-3; wherein, the 5 'end of the probe FCoV-P is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group MGB;
the second group of primers and probes comprises a forward primer RPP-F, a reverse primer RPP-R and a probe RPP-P, and the nucleotide sequences of the primers and the probes are respectively shown as SEQ ID No. 4-6; wherein, the 5 'end of the probe RPP-P is marked with a fluorescent group VIC, and the 3' end is marked with a fluorescence quenching group MGB.
Example 2
This example provides a feline coronavirus test kit comprising separately packaged components as shown in the following table:
TABLE 1
Example 3
This example provides a feline coronavirus test kit comprising separately packaged components as shown in the following table:
TABLE 2
Example 4
This example provides a feline coronavirus test kit comprising separately packaged components as shown in the following table:
TABLE 3 Table 3
Example 5
This example provides a method for feline coronavirus detection for non-disease diagnosis using the kit of example 2 comprising the steps of:
(1) Obtaining a sample to be tested:
sample type: including buccal swabs, fecal matter, anal swabs, and other suspicious specimens.
Sample collection: sample collection is carried out according to the requirements of a manual for collecting microorganism samples;
sample preservation: the specimens should be refrigerated (2 to 8deg.C or frozen (-20deg.C or lower) and detected as soon as possible within one week, specimens that can be detected can be stored at 4deg.C, specimens that cannot be detected can be stored at-20deg.C for a short period, and preserved below-70deg.C (after collection > 60 days) for a long period, whole blood is refrigerated (2 to 8deg.C) before plasma separation, and then stored at-20deg.C or below.
Sample processing: the sample is immersed in the sample treatment solution for 5 times with the kit carrying the sample collection solution, and the reaction is performed for about 1 minute. The positive quality control product and the negative quality control product are not involved in extraction and are directly used as templates.
(2) Nucleic acid detection: real-time fluorescent quantitative PCR (RT-qPCR) method for detecting nucleic acid of cat coronavirus and cat parvovirus:
1) Preparation of a reaction system: the reaction mixture was prepared according to the following table: in each PCR reaction, except for the sample to be tested, a negative and positive quality control product is added; all samples, including quality control materials, should be provided with parallel reaction tubes.
TABLE 4 Table 4
| Reaction mixture composition | Dosage of |
| Main reaction liquid | 12.5μL |
| Primer probe liquid | 7.5μL |
| Sample DNA/RNA to be tested | 5μL |
| Total volume of | 25μL |
2) Sample adding: after 5 mu L of each sample to be detected or quality control material is added into each reaction tube, the tube cover is tightly covered and placed on a fluorescent quantitative PCR detector.
3) Amplification parameter settings were set as follows:
TABLE 5
4) Instrument detection channel selection:
using a fluorescence quantitative PCR detector, selecting two channels FAM and VIC to collect real-time fluorescence signals, and setting the collection at 57 ℃, wherein the specific setting method refers to the using instruction of each instrument.
5) The quality control of the reaction system of the invention is shown in the following table:
TABLE 6
The qualitative detection result judging method is as follows:
TABLE 7
The quantitative detection method comprises the following steps of
Establishing a standard curve: diluting a standard substance comprising a nucleotide sequence shown as SEQ ID NO.7 to different concentrations; taking standard substances with different concentrations as templates, performing fluorescence quantitative PCR detection, and after the detection is finished, drawing a standard curve by taking the concentration (logarithm of copy number) of each standard substance in the standard substances as an abscissa and the corresponding Ct value as an ordinate (see experimental example);
substituting the Ct value of the sample to be measured into the corresponding standard curve to obtain the quantitative result of the sample to be measured.
Example 6
This example differs from example 5 in that the assay was performed using the kit of example 3.
Example 7
This example differs from example 5 in that the assay was performed using the kit of example 4.
Experimental example 1
Positive plasmid standards (pUC 57-FCoV-7 b) containing the nucleotide sequence shown in SEQ ID No.7 were diluted with sterile, enzyme-free water to concentrations of 1X 107, 1X 106, 1X 105, 1X 104, 1X 103, 1X 102, 1X 101, 1X 100 copies/. Mu.L, respectively. Taking standard substances with different concentrations as templates, then implementing the method of the embodiment 5, taking the concentration in the standard substances as an abscissa, taking the corresponding Ct value as an ordinate, drawing a standard curve, wherein the result is shown in the figure 1, the detection results are positive, the standard curve equation is y= -3.3629x+38.5, R is shown in the figure 1 2 =0.999, correlation coefficient R 2 And the detection effect is stable when the detection effect is larger than 0.99.
Diluting nucleic acid of cat coronavirus positive clinical sample to 1×10 with sterile enzyme-free water 6 、1×10 5 、1×10 4 、1×10 3 、1×10 2 、1×10 1 、1×10 0 The detection results of the method of example 5 of the present invention using copies/. Mu.L as a template are shown in FIG. 2, and the detection results are all amplified into an S-shaped curve, and the lowest detection limit of the feline coronavirus is 1X 10 0 copies/μL。
Experimental example 2
The clinically definite feline coronavirus and the positive quality control internal standard (pUC 57-Rpp) are mixed to be used as samples, and the detection is carried out by adopting the method of the example 5, wherein the detection result is shown in the figure 3, and is consistent with the clinical result, so that the detection accuracy of the invention is good.
Experimental example 3
20 parts of cat digestive tract suspected diseased anus swab samples are detected by using the method of the embodiment 5 and a conventional RT-PCR kit (using a Powerpol 2XPCR Mix kit provided by Wyowa Eboltag biotechnology Co., ltd., without adding an internal standard primer and a probe during use, and an amplification program is the same with the method), and the detection result shows that the FCoV positive number detected by the method is 8 and the detection rate is 40%; the FCoV positive number detected by conventional RT-PCR is 7, and the detection rate is 35%. The positive samples are consistent with the detection results of the invention through sequencing and BLAST analysis of nucleic acid sequences, which shows that the method of the invention has higher detection rate and low false positive rate and false negative rate compared with the conventional RT-PCR.
The above examples are given for clarity of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. A feline coronavirus detection primer probe composition comprising: a first set of primers and probes designed based on the nucleotide sequence shown as SEQ ID NO. 7;
the second set of primers and probes was designed based on the nucleotide sequence shown as SEQ ID NO. 8.
2. The feline coronavirus detection primer probe composition of claim 1, wherein,
a first set of primers and probes comprising: the nucleotide sequence of the forward primer FCoV-F is shown as SEQ ID NO. 1; the nucleotide sequence of the reverse primer FCoV-R is shown as SEQ ID NO. 2; and/or probe FCoV-P, its nucleotide sequence is shown in SEQ ID NO. 3;
a second set of primers and probes comprising: the nucleotide sequence of the forward primer RPP-F is shown as SEQ ID NO. 4; the nucleotide sequence of the reverse primer RPP-R is shown as SEQ ID NO. 5; and/or the nucleotide sequence of the probe RPP-P is shown as SEQ ID NO. 6.
3. The feline coronavirus detection primer probe composition according to claim 2, wherein the 5 'end of the probe FCoV-P, probe RPP-P is labeled with a fluorescent group and the 3' end is labeled with a fluorescence quenching group; the fluorescent groups of the probe FCoV-P and the probe RPP-P are different;
optionally, the fluorophore is selected from at least 1 of FAM, VIC, CY, ROX, HEX, JOE, NED, texas Red and CY 3;
optionally, the fluorescence quenching group is selected from at least 1 of BHQ-1, BHQ-2, BHQ-3, and MGB.
4. A feline coronavirus detection kit comprising the feline coronavirus detection primer probe composition of any one of claims 1-3.
5. The kit for detecting feline coronavirus according to claim 4, further comprising DNA polymerase, dntps, mg 2+ The PCR reagent comprises ROX dye, PCR enhancer, PCR stabilizer, PCR buffer solution, sample collection liquid, negative quality control product or positive quality control product.
6. The feline coronavirus detection kit as recited in claim 5, wherein,
the PCR enhancer is BSA;
and/or the PCR buffer contains Tris 10mM-500mM, chloride 10mM-500mM, (NH) 4 ) 2 SO 4 10mM-500mM, glycerol 1 v/v-50 v/v%, BSA 0.001mg/mL-1mg/mL, tween 20 0.1 v/v-10 v/v%, dithiothreitol 1mM-100mM, betaine 0.1M-3M, DMSO 0.1 v/v-10 v/v%, and pH 7.5-9.5;
and/or the sample collection liquid comprises sodium bicarbonate with the concentration range of 6-6.5g/L, sodium carbonate with the concentration range of 12-13g/L, tween-20 with the concentration range of 0.01-0.05v/v, cresol red with the concentration range of 0.01-0.05g/100ml, guanidine hydrochloride with the concentration range of 4-5M and the balance of sterile water without enzyme;
and/or, the negative quality control product is enzyme-free sterile water;
and/or the positive quality control product is a mixed plasmid standard product respectively comprising nucleotide sequences shown as SEQ ID NO. 7-8.
7. The feline coronavirus detection kit according to claim 5 or 6 comprising a PCR amplification reaction system of 25 μl:
the volume of the PCR mixed solution is 12.5 mu L, the PCR mixed solution contains DNA polymerase, and the enzyme activity is 0.5-1U; dNTPs, the concentration of each base ranges from 200 mu M to 300 mu M; mg of 2+ The concentration range is 1.5-2mM; PCR buffer solution with pH value of 7.5-9.5;
the volume of the cat coronavirus detection primer probe composition is 7.5 mu L, the concentration of each primer is 0.1-0.5 mu M, and the concentration of each probe is 0.05-0.25 mu M;
the sample to be tested had a volume of 5. Mu.L.
8. Use of the feline coronavirus detection primer probe composition of any one of claims 1-3 or the feline coronavirus detection kit of any one of claims 4-7 in the preparation of a feline coronavirus detection product.
9. A method for detecting feline coronavirus for non-disease diagnosis, comprising the steps of:
preparing a PCR amplification reaction system by using a sample to be detected as a template and using the feline coronavirus detection primer probe composition as set forth in any one of claims 1 to 3 or the feline coronavirus detection kit as set forth in any one of claims 4 to 7 to perform fluorescent quantitative PCR detection.
10. The method for detecting feline coronavirus diagnosed with non-disease according to claim 9,
adding the collected sample into a sample collection liquid for treatment to obtain the sample to be tested;
and/or, in the fluorescent quantitative PCR detection, the PCR amplification procedure is as follows: reverse transcription, holding at 50℃for 300 seconds; pre-denaturation, holding at 95 ℃ for 30 seconds; denaturation at 95℃for 5 seconds, annealing at 57℃for 30 seconds, extension for 40 cycles;
and/or, the fluorescent quantitative PCR detection comprises a qualitative or quantitative detection method;
the qualitative method comprises the following steps: the channel corresponding to the fluorescent group of the probe in the first group of primers and the probe is a channel A; the channel corresponding to the fluorescent group of the probe in the second group of primers and the probe is a B channel; the judging method comprises the following steps:
if the Ct value of the sample to be tested in the A channel is less than or equal to 39 and the Ct value of the sample to be tested in the B channel is less than 39, the sample to be tested is positive for the cat coronavirus;
if the sample to be tested has no Ct value or Ct value more than 39 in the A channel and Ct value less than 39 in the B channel, the sample to be tested is negative to the feline coronavirus;
if the sample to be tested has no Ct value in the A channel and the B channel, retesting is carried out;
the quantitative detection method comprises the following steps:
establishing a standard curve: diluting a standard substance comprising a nucleotide sequence shown as SEQ ID NO.7 to different concentrations; taking standard substances with different concentrations as templates, performing fluorescence quantitative PCR detection, and drawing a standard curve by taking the concentration of the standard substance as an abscissa and the corresponding Ct value as an ordinate after the detection is finished;
substituting the Ct value of the sample to be measured into the corresponding standard curve to obtain the quantitative result of the sample to be measured.
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