CN114592092A - Fluorescence quantitative RT-PCR detection kit for feline infectious peritonitis virus - Google Patents
Fluorescence quantitative RT-PCR detection kit for feline infectious peritonitis virus Download PDFInfo
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Abstract
The invention relates to a cat infectious peritonitis virus fluorescent quantitative RT-PCR detection kit, which comprises a specific primer and a TaqMan probe for cat infectious peritonitis virus fluorescent quantitative RT-PCR detection; wherein, the sequence of the forward primer is as follows: 2, the sequence of the reverse primer is as follows: 3, SEQ ID NO; the sequence of the TaqMan probe is as follows: SEQ ID NO 4. The method has good sensitivity, specificity and stability, is suitable for rapid diagnosis of the initial infection stage of the feline infectious peritonitis virus, and has important significance for reducing the spread of the feline infectious peritonitis and improving the detection efficiency of the feline infectious peritonitis.
Description
Technical Field
The invention relates to a kit for virus molecular biology detection in the field of biotechnology, in particular to a fluorescence quantitative RT-PCR detection kit for feline infectious peritonitis viruses.
Background
Feline Infectious Peritonitis (FIP) is a clinically common Infectious disease of cats caused by Feline Infectious Peritonitis Virus (FIPV), has high mortality rate and seriously harms the health of cats. FIPV belongs to Feline Coronavirus (FCoV), an enveloped single-stranded positive-strand RNA virus. The feline infectious peritonitis virus mainly encodes 4 structural proteins, namely spike protein, membrane protein, envelope protein and nucleocapsid protein. Research has shown that the Nucleocapsid Protein (N Protein) of coronavirus is the most immunogenic Protein, and the corresponding antibody is produced at the earliest, so N Protein is often used as an index for early diagnosis. Feline coronaviruses are classified into gastroenteritis type (FECV) and transmissible peritonitis type (FIPV), and FECV is present in the intestines of most healthy cats, is generally not lethal, and causes only mild intestinal symptoms in cats. FIPV primarily infects monocytes, is highly pathogenic, and can cause lethal peritonitis in cats. The diagnosis of feline infectious peritonitis is divided into clinical and laboratory diagnostics. Clinical diagnosis depends on the manifestation of clinical symptoms and the experience of a diagnostician, while the initial symptoms of cats suffering from feline infectious peritonitis are not obvious, the disease course progresses in a large difference, so that the diagnosis result is inaccurate, the optimal treatment time is usually delayed due to misdiagnosis, and the death of the cats is finally caused. Laboratory diagnostics include pathology examinations, serological tests, RT-PCR tests, and the like. Although the RT-PCR detection is low in cost and easy to operate, the sensitivity is low, the virus content in the peritoneal fluid is very low in the early stage of FIPV infection, the RT-PCR detection easily causes omission, and the FIPV cannot be accurately detected. In addition, since the amplification product needs to be analyzed by gel electrophoresis, cross contamination is easy to generate, false positive results are easy to occur, and the experiment time is relatively long. The real-time fluorescent quantitative PCR technology is widely applied to qualitative and quantitative detection and the like due to high sensitivity, high speed, strong specificity and simple and convenient operation. The real-time fluorescent quantitative PCR technology is a method of adding fluorescent groups into a PCR system, utilizing fluorescent signal accumulation to monitor the whole PCR process in real time, and finally carrying out quantitative analysis on an unknown template through a standard curve. The current commonly used real-time fluorescent quantitative PCR is SYBR Green I fluorescent quantitative PCR and Taqman probe fluorescent quantitative PCR, and SYBR Green I can combine all dsDNA double helix minor groove regions, so that the detection specificity is not as strong as that of a TaqMan hydrolysis probe method.
At present, no report of quantitative detection kit for feline infectious peritonitis virus by using Taqman probe real-time fluorescent quantitative RT-PCR technology is seen in China. Therefore, in order to further improve the detection sensitivity, realize the quantitative detection of the feline infectious peritonitis virus in a clinical sample, provide a technical means for the early diagnosis of the feline infectious peritonitis, lay a foundation for the prevention, control and purification of the feline infectious peritonitis, and manufacture the Taqman probe real-time fluorescence quantitative PCR detection kit of the feline infectious peritonitis virus has important significance and wide market prospect.
Disclosure of Invention
The invention provides a fluorescence quantitative RT-PCR detection kit for feline infectious peritonitis viruses.
The kit comprises a conserved region fragment purification product of feline infectious peritonitis virus N gene, wherein the conserved region fragment is ATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCAAAAGACGTGGTCGTTCTAACTCTCGTGGTCGGAAGAATAGTAATATACCT.
The kit comprises a specific primer and a TaqMan probe for fluorescence quantitative RT-PCR detection of the feline infectious peritonitis virus; wherein, the sequence of the forward primer is as follows: 5'-CACAGGGACAACGCGTCAA-3', the sequence of the reverse primer is: 5'-CGACCACGAGAGTTAGAACGA-3', respectively; the sequence of the TaqMan probe is as follows: 5'-TGGGGAGATGAACCTTCCAAAAGACGT-3' are provided.
The kit of the invention comprises instructions bearing the following in weight: the total volume of the fluorescent quantitative RT-PCR reaction system is 20 mu L, and the method comprises the following steps: 2XRT-PCR Buffer 10. mu.L, 25 XRT-PCR Enzyme Mix 1. mu.L, forward primer (10 pmol/. mu.L) 0.4. mu.L, reverse primer (10 pmol/. mu.L) 0.4. mu. L, TaqMan probe (10. mu. mol/. mu.L), 50 XRox 0.4. mu.L, template 1-5. mu.L, using ddH 20 supplemented the total volume to 20. mu.L.
The kit of the invention comprises instructions bearing the following in weight: the reaction conditions of the fluorescent quantitative RT-PCR are as follows: 15min at 55 ℃; at 95 ℃ for 30 s; fluorescence signal detection was performed at the end of extension of each cycle, at 95 ℃, 10s, 60 ℃, 30s, for 45 cycles.
The specific primer and the TaqMan probe provided by the invention are designed according to the conserved region of the feline infectious peritonitis virus N gene and are used for quantitatively detecting the nucleic acid copy number of the feline infectious peritonitis virus in a sample.
The TaqMan probe is fluorescently labeled, a report fluorescent group is labeled at the 5 'end of the TaqMan probe, and a quenching fluorescent group is labeled at the 3' end of the TaqMan probe.
The reporter fluorophore is FAM and the quenching fluorophore is TAMRA.
The kit also comprises the establishment of a fluorescent quantitative PCR standard curve, and the method comprises the following steps: using recombinant plasmid containing target fragment as standard substance, and diluting it into (i) 10 times of serial dilution：3.4×108copies/μL；②：3.4×107copies/μL；③：3.4×106copies/μL；④：3.4×105copies/μL；⑤：3.4×104copies/μL；⑥：3.4×103copies/μL；⑦：3.4×102copies/μL；⑧：3.4×101copies/. mu.L. And (3) carrying out real-time fluorescent quantitative RT-PCR detection by using the standard recombinant plasmid as a template to obtain a fluorescent quantitative PCR standard curve for detecting the feline infectious peritonitis virus.
The invention has the beneficial effects that:
1. the invention relates to a TaqMan probe fluorescent quantitative RT-PCR detection kit for the infectious peritonitis virus of cat, which is established for the first time in China; the characteristic test shows that: the kit is used for detecting the feline infectious peritonitis virus, the detection sensitivity is high, and the minimum nucleic acid detection amount is 3.4x101copies/. mu.L; the kit is used for detecting the feline infectious peritonitis virus, has higher specificity, and has no cross reaction with rabies virus, feline herpes virus, calicivirus, feline parvovirus, canine influenza virus and canine distemper virus; the kit is used for detecting the feline infectious peritonitis virus, has good stability, and has the intra-batch and inter-batch test variation coefficient of less than 5 percent.
2. Compared with other detection methods of the feline infectious peritonitis virus, such as virus isolation culture identification and RT-PCR, the kit has the characteristics of convenient sampling, higher sensitivity, high detection efficiency and effective pollution prevention.
3. The kit has the characteristics of simple and easy-to-use operation program, programmed operation and suitability for large-area popularization and application.
4. The kit can be used for evaluating the infection condition of the feline infectious peritonitis virus, and can also provide a basis for researches in related fields of epidemiology, pathogenic mechanism and the like of the feline infectious peritonitis virus.
The invention is further illustrated with reference to the accompanying drawings and the specific embodiments.
Drawings
FIG. 1 is a diagram of pUC57-FIPV-N plasmid.
FIG. 2 is a diagram showing the results of screening the primer probe.
FIG. 3 is a graph showing the results of screening the probe concentration.
FIG. 4 is a schematic diagram of a standard curve of fluorescent quantitative RT-PCR in the example.
FIG. 5 is a graph of fluorescence signals from the fluorescent quantitative RT-PCR sensitive assay in the example. Wherein, the concentration of the standard substance in the amplification curve from left to right is 3.4 multiplied by 108copies/μL、3.4×107copies/μL、3.4×106copies/μL、3.4×105copies/μL、3.4×104copies/μL、3.4×103copies/μL、3.4×102copies/μL、3.4×101copies/μL。
FIG. 6 is a graph of fluorescence signals of fluorescence quantitative RT-PCR specific detection in the example.
Detailed Description
It should be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention. The experiments used in the following examples are all conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1:
four groups of primers and TaqMan probes are designed according to the complete sequence of the N gene of the feline infectious peritonitis virus, and the four groups of primers and TaqMan probes are as follows:
F3：5’-AGAAGCCTGAGGAATTGTCTGT-3’(SEQ ID NO:8)
R3：5’-TCGTAACCTCATCAATCATCTCA-3’(SEQ ID NO:9)
P3：5’-(FAM)CACAGATGTGTTTGATGACACACAGGT-3’(TAMRA)(SEQ ID NO:10)
F4：5’-CCAACAACACTTGGCACTCG-3’(SEQ ID NO:11)
R4：5’-CCAGCTTCTTCAGCAGACCA-3’(SEQ ID NO:12)
P4：5’-(FAM)CGTTGCCAATGGTAACGCTGCC-3’(TAMRA)(SEQ ID NO:13)
F5：5’-TTGTCAGAGGCCAGCGTAAG-3’(SEQ ID NO:14)
R5：5’-TTCCACGAGTGCCAAGTGTT-3’(SEQ ID NO:15)
P5：5’-(FAM)GGGTTGCAAGGGATGGAGCCA-3’(TAMRA)(SEQ ID NO:16)
F6：5’-CGGTCTAACTCTCGTGGTCG-3’(SEQ ID NO:17)
R6：5’-AAGTTCCTTACGCTGGCCTC-3’(SEQ ID NO:18)
P6：5’-(FAM)ACAGACAGGTGCGTTACCGCA-3’(TAMRA)(SEQ ID NO:19)
through fluorescent quantitative RT-PCR detection, the four groups of primers and the TaqMan probe detect a large number of clinical positive samples infected by the feline infectious peritonitis virus, the full coverage of the detected samples cannot be achieved, and no good expected result exists.
Example 2:
searching an N gene conserved region of the feline infectious peritonitis virus; design of synthetic primers and TaqMan probes
According to the N gene sequence of feline infectious peritonitis virus published by GenBank (AB086903.1, FJ917524.1, A22378.1, FJ917535.1, AB0866881.1, FJ943764.1, FJ917533.1, FJ917532.1, FJ917530.1, FJ917529.1), the conserved region is analyzed, and the specific conserved sequence of the N gene of feline infectious peritonitis virus is obtained (SEQ ID NO: 1):
ATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCAAAAGACGTGGTCGTTCTAACTCTCGTGGTCGGAAGAATAGTAATATACCT。
and (3) designing primers aiming at the conserved sequence to obtain two pairs of specific primers and two Taqman probes, wherein the fluorescent reporter group at the 5 'end of the probe is FAM, and the fluorescent quencher group at the 3' end of the probe is TAMRA.
The specific detection primers and the Taqman probe are synthesized by Baoriji medical technology Limited.
The primers and the probes are positioned in a conserved region of the feline infectious peritonitis virus N gene, and the target amplification fragment is 96 bp. The primer and probe sequences were as follows:
F1：5’-CACAGGGACAACGCGTCAA-3’；(SEQ ID NO:2)
R1：5’-CGACCACGAGAGTTAGAACGA-3’；(SEQ ID NO:3)
P1：5’-FAM-TGGGGAGATGAACCTTCCAAAAGACGT-TAMRA-3’(SEQ ID NO:4)
F2：5’-ATGGCCACACAGGGACAAC-3’；(SEQ ID NO:5)
R2：5’-ACGAGAGTTAGAACGACCACG-3’；(SEQ ID NO:6)
P2：5’-FAM-GCGTCAACTGGGGAGATGAACCT-TAMRA-3’(SEQ ID NO:7)
example 3:
construction and preparation of quantitative Standard plasmids
1. Construction of quantitative Standard plasmid
The gene containing the amplified fragment of interest was cloned into a plasmid vector pUC57 by using DNA recombination technique based on the designed PCR primers, and DNA sequencing was performed. The constructed recombinant plasmid was designated as pUC57-FIPV-N (FIG. 1) as a quantitative standard plasmid for detecting feline infectious peritonitis virus. The recombinant plasmid was synthesized by the Oncorhynchus organism Co.
2. Preparation of quantitative Standard plasmid
The standard plasmid pUC57-FIPV-N was extracted and purified by alkaline lysis, the concentration was measured by a trace nucleic acid protein analyzer, and the copy number was calculated by the following formula (copy number 6.02X 10)23X DNA concentration/mass MW, MW ═ DNA base number x 330), calculated to a standard plasmid copy number of 3.4x1011copies/. mu.L, stored at-20 ℃.
Example 4:
establishment and optimization of TaqMan probe fluorescent quantitative RT-PCR method
1. Establishment of absolute quantitative standard curve
(1) Standard plasmid preparation
The standard plasmid pUC57-FIPV-N was extracted and purified by alkaline lysis, the concentration was measured by a trace nucleic acid protein analyzer, and the copy number was calculated by the following formula (copy number 6.02X 10)23X DNA concentration/mass MW, MW ═ DNA base number x 330), calculated to a standard plasmid copy number of 3.4x1011copies/. mu.L, stored at-20 ℃.
(2) Quantitative RT-PCR reaction condition optimization
Comparing the experimental results of different PCR primers, TaqMan probes and the like in the reaction system, selecting the reaction system (figure 2 and figure 3) which has high reaction sensitivity, low background fluorescence signal, typical S-shaped amplification curve and reaction efficiency close to 1. The fluorescent quantitative PCR instrument used in the invention is an ABI Stepone Plus read-Time PCR System. The fluorescent quantitative RT-PCR detection program (Table 1) was set up to detect the fluorescent signal at the end of the second step of each cycle of the amplification stage.
TABLE 1 fluorescent quantitative PCR reaction conditions
TABLE 2 screening of reaction systems
| Reagent | Selection of concentration |
| Quantitative RT-PCR primer (upstream and downstream) | 10 pmol/. mu.L each |
| TaqMan probe | 2μmol/μL，5μmol/μL，10μmol/μL |
TABLE 3 optimized fluorescent quantitative PCR reaction System (20. mu.L reaction System)
(3) Establishment of fluorescent quantitative RT-PCR standard curve
A standard plasmid pUC57-FIPV-N containing the target fragment was serially diluted 10-fold with sterilized double distilled water to give: 3.4 is prepared108copies/μL；②：3.4×107copies/μL；③：3.4×106copies/μL；④：3.4×105copies/μL；⑤：3.4×104copies/μL；⑥：3.4×103copies/μL；⑦：3.4×102copies/μL；⑧：3.4×101copies/. mu.L. Parallel experiments were repeated 3 times for each dilution.
Adopting an optimized reaction system to carry out reaction, wherein the reaction conditions are as follows: 15min at 55 ℃; 95 ℃ and 30S; 95 ℃, 10S, 60 ℃, 30S, for a total of 45 cycles. Fluorescence signals were collected at the end of the extension phase of each cycle for real-time detection.
After the detection is finished, a standard curve (shown in figure 4) with the logarithm of the initial template number as an X axis and the CT value as a Y axis is automatically generated by computer software.
Analysis of the standard curve of FIG. 4 shows that the 8 dilution points of the template are all on the same line, indicating that the detection method is at 3.4X101-3.4×108A good linear relationship in the copies/interaction range. Regression analysis showed that R2The amplification efficiency of the fluorescent quantitative RT-PCR reaction was 100.5% at 0.9989. The detection range can reach 8 orders of magnitude (figure 5), and the sensitivity can reach 3.4 multiplied by 101copis/. mu.L. It can be seen that the sensitivity of the primer and the TaqMan probe designed by the invention is high.
2. Specificity analysis of fluorescent quantitative RT-PCR
The real-time quantitative RT-PCR detection method established by the invention is utilized to respectively detect 1 part of cat ascites, rabies virus vaccine strain (RV), feline herpes virus vaccine strain (FHV), calicivirus vaccine strain (FCV), feline parvovirus vaccine strain (FPV), Canine Influenza Virus (CIV) and canine distemper virus vaccine strain (CDV) which are detected to be positive to the feline infectious peritonitis virus by RT-PCR and sequencing, and the specificity of the method is evaluated.
The results showed that only positive feline ascites reacted positively and that all other viruses were negative (FIG. 6). The result shows that the established fluorescence quantitative RT-PCR detection method for the N gene TaqMan probe of the feline infectious peritonitis virus has good amplification effect and strong specificity.
3. Stability of fluorescent quantitative RT-PCR
Choose 3.4x108copies/μL、3.4x106copies/μL、3.4x104copies/μL、3.4x102Taking the standard plasmids with copies/mu L of 4 concentrations as templates, detecting according to the established fluorescent quantitative RT-PCR detection method, repeating the parallel test for 3 times for each sample, dividing into three batches, taking the CT average value as the test result, calculating the CT Standard Deviation (SD) and the CT variation Coefficient (CV) through statistics, and evaluating the stability of the established method. The stability test results (table 4) show that the variation coefficient of the Ct value is less than 5% in both batches and between batches, which indicates that the detection method constructed by the invention has good stability.
TABLE 4 fluorescent quantitative RT-PCR stability test results
4. Determination of detection result and quantification of virus
(1) Positive control CTValue less than 35, negative control no CTValues and no typical sigmoidal amplification curve. The positive template is plasmid quantitative standard pUC57-FIPV-N, and the negative control is sterile double distilled water;
(2) if CTThe value is less than 40, and a typical S-shaped amplification curve is presented, the positive result is judged, and the FIP virus nucleic acid is contained in the detection sample;
(3) if CTIf the value is more than 40 or no amplification signal exists, judging that the result is negative, and indicating that no FIP virus nucleic acid exists in the detection sample;
(4) if CTAnd if the value is between 40 and 45, determining the sample as a suspicious result, repeatedly detecting the sample once, and if the detection result is still within the range, determining the sample as a negative result.
(5) And (5) quantifying the virus of the positive sample, and calculating the virus content according to the established quantitative standard curve.
The calculation formula is as follows: y is 10(-3.3083X+38.445)
Y is the copy number of the virus cDNA in the sample; x: sample CTThe value is obtained.
The foregoing is only a few specific embodiments of the present invention (which are not exhaustive because the present invention includes numerical related matters), and the specific matters or common sense known in the art are not described herein in much detail. It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent substitution or equivalent transformation for those skilled in the art are within the protection scope of the present invention. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
<110> Kunming customs technology center
Fluorescent quantitative RT-PCR detection kit for cat infectious peritonitis virus
<160>19
<210>1
<211>96
<212>DNA
<213> Artificial sequence
<400>1
ATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCAAAAGACGTGGTCGTTCTAACTCTCGTGGTCGGAAGAATAGTAATATACCT
<210>2
<211>19
<212>DNA
<213> Artificial sequence
<400>2
CACAGGGACAACGCGTCAA
<210>3
<211>21
<212>DNA
<213> Artificial sequence
<400>3
CGACCACGAGAGTTAGAACGA
<210>4
<211>27
<212>DNA
<213> Artificial sequence
<400>4
TGGGGAGATGAACCTTCCAAAAGACGT
<210>5
<211>19
<212>DNA
<213> Artificial sequence
<400>5
ATGGCCACACAGGGACAAC
<210>6
<211>21
<212>DNA
<213> Artificial sequence
<400>6
ACGAGAGTTAGAACGACCACG
<210>7
<211>23
<212>DNA
<213> Artificial sequence
<400>7
GCGTCAACTGGGGAGATGAACCT
<210>8
<211>22
<212>DNA
<213> Artificial sequence
<400>8
AGAAGCCTGAGGAATTGTCTGT
<210>9
<211>23
<212>DNA
<213> Artificial sequence
<400>9
TCGTAACCTCATCAATCATCTCA
<210>10
<211>27
<212>DNA
<213> Artificial sequence
<400>10
CACAGATGTGTTTGATGACACACAGGT
<210>11
<211>20
<212>DNA
<213> Artificial sequence
<400>11
CCAACAACACTTGGCACTCG
<210>12
<211>20
<212>DNA
<213> Artificial sequence
<400>12
CCAGCTTCTTCAGCAGACCA
<210>13
<211>22
<212>DNA
<213> Artificial sequence
<400>13
CGTTGCCAATGGTAACGCTGCC
<210>14
<211>20
<212>DNA
<213> Artificial sequence
<400>14
TTGTCAGAGGCCAGCGTAAG
<210>15
<211>20
<212>DNA
<213> Artificial sequence
<400>15
TTCCACGAGTGCCAAGTGTT
<210>16
<211>21
<212>DNA
<213> Artificial sequence
<400>16
GGGTTGCAAGGGATGGAGCCA
<210>17
<211>20
<212>DNA
<213> Artificial sequence
<400>17
CGGTCTAACTCTCGTGGTCG
<210>18
<211>20
<212>DNA
<213> Artificial sequence
<400>18
AAGTTCCTTACGCTGGCCTC
<210>19
<211>21
<212>DNA
<213> Artificial sequence
<400>19
ACAGACAGGTGCGTTACCGCA
Claims (4)
1. The fluorescence quantitative RT-PCR detection kit for the feline infectious peritonitis virus is characterized by comprising a conserved domain fragment purification product of an N gene of the feline infectious peritonitis virus, wherein the conserved domain fragment is ATGGCCACACAGGGACAACGCGTCAACTGGGGAGATGAACCTTCCAAAA GACGTGGTCGTTCTAACTCTCGTGGTCGGAAGAATAGTAATATACCT.
2. The cat infectious peritonitis virus fluorescent quantitative RT-PCR detection kit is characterized by comprising a specific primer and a TaqMan probe for detecting the cat infectious peritonitis virus fluorescent quantitative RT-PCR; wherein, the sequence of the forward primer is as follows: 5'-CACAGGGACAACGCGTCAA-3', the sequence of the reverse primer is: 5'-CGACCACGAGAGTTAGAACGA-3', respectively; the sequence of the TaqMan probe is as follows: 5'-TGGGGAGATGAACCTTCCAAAAGACGT-3' are provided.
3. The kit of claim 2, wherein the kit comprises instructions that carry out the following: the total volume of the fluorescent quantitative RT-PCR reaction system is 20 mu L, and the method comprises the following steps: 2xRT-PCR Buffer 10. mu.L, 25 xRT-PCR Enzyme Mix 1. mu.L, forward primer (10 pmol/. mu.L) 0.4. mu.L, reverse primer (10 pmol/. mu.L) 0.4. mu. L, TaqMan probe (10. mu. mol/. mu.L), 50 xRox 0.4. mu.L, template 1-5. mu.L, using ddH20 supplemented the total volume to 20. mu.L.
4. The kit of claim 2, wherein the kit comprises instructions that carry out the following: the reaction conditions of the fluorescent quantitative RT-PCR are as follows: 15min at 55 ℃; at 95 ℃ for 30 s; fluorescence signal detection was performed at the end of extension of each cycle, at 95 ℃, 10s, 60 ℃, 30s, for 45 cycles.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002066686A1 (en) * | 2001-02-19 | 2002-08-29 | Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. | Feline infectious peritonitis viruses (fipv) diagnosis |
| CN107586884A (en) * | 2017-10-25 | 2018-01-16 | 东北农业大学 | A kind of RT PCR primer groups for feline infectious peritonitis virus detection, kit and its application containing the primer sets |
| CN110724768A (en) * | 2019-11-20 | 2020-01-24 | 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) | Composition, kit and method for detecting feline infectious peritonitis virus |
| CN111893212A (en) * | 2020-06-17 | 2020-11-06 | 安徽农业大学 | Real-time fluorescence quantitative PCR (polymerase chain reaction) primer group and kit for feline infectious peritonitis virus |
-
2022
- 2022-03-23 CN CN202210295257.9A patent/CN114592092A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002066686A1 (en) * | 2001-02-19 | 2002-08-29 | Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. | Feline infectious peritonitis viruses (fipv) diagnosis |
| CN107586884A (en) * | 2017-10-25 | 2018-01-16 | 东北农业大学 | A kind of RT PCR primer groups for feline infectious peritonitis virus detection, kit and its application containing the primer sets |
| CN110724768A (en) * | 2019-11-20 | 2020-01-24 | 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) | Composition, kit and method for detecting feline infectious peritonitis virus |
| CN111893212A (en) * | 2020-06-17 | 2020-11-06 | 安徽农业大学 | Real-time fluorescence quantitative PCR (polymerase chain reaction) primer group and kit for feline infectious peritonitis virus |
Non-Patent Citations (2)
| Title |
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| SANDRA FELTEN等: "Diagnosis of Feline Infectious Peritonitis: A Review of the Current Literature", 《VIRUSES》 * |
| 邓宇: "《动物疫病分子诊断技术》", 30 April 2014, 四川大学出版社 * |
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