AU2021101666A4 - Primers, kit and detection method for detecting Feline panleukopenia virus - Google Patents
Primers, kit and detection method for detecting Feline panleukopenia virus Download PDFInfo
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- AU2021101666A4 AU2021101666A4 AU2021101666A AU2021101666A AU2021101666A4 AU 2021101666 A4 AU2021101666 A4 AU 2021101666A4 AU 2021101666 A AU2021101666 A AU 2021101666A AU 2021101666 A AU2021101666 A AU 2021101666A AU 2021101666 A4 AU2021101666 A4 AU 2021101666A4
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- 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
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Abstract
The present invention discloses a pair of primers, a kit and a detection method for
detecting Feline panleukopenia virus (FPLV), and belongs to the field of bioassay technology.
The primers comprises the following parts: the nucleotide sequences are as shown in the
upstream primer of SEQ ID NO: 1 and the downstream primer of SEQ ID NO: 2. The kit
comprises the primers mentioned. The detection reagent also includes the primers. The
detection method includes the steps of PCR amplification by the primers or the kit or the
detection reagent. The present invention adopts this pair of primers to detect FPLV by PCR
detection method, which can quickly and accurately identify the infection of FPLV in cats, and
provides a new method and basis for the early detection and prevention of FPLV.
Description
Primers, kit and detection method for detecting Feline panleukopenia virus
The invention relates to the technical field of biological detection, and relates to a
pair of primers, a kit and a detection method for detecting FPLV.
Feline panleukopenia virus (FPLV) belongs to the family Parvoviridaeand belongs
to the genus Parvoviridae. The virus particles are icosahedral and are the pathogen of
Feline panleukopenia. As early as 1928, the French researchers first discovered the
existence of the virus, and in 1935 was officially named Feline panleukopenia virus. The
virus can cause vomiting, diarrhea, severe hemorrhagic enteritis and significantly reduced
white blood cells in cats. The incidence rate and mortality rate are high, which often
presents endemic and serious harm to feline animals.
Currently, the most commonly applied methods for detecting FPLV are the
hemagglutination (HA) and hemagglutination inhibition tests and indirect
immunofluorescence. Among them, the HA test is the most economical, but its
disadvantage is that, on the one hand, it is difficult to obtain fresh red blood cells and, on
the other hand, the method is only suitable for the detection of samples from late stages of
FPLV infection and is not adapted to the requirements of early and rapid detection in
recent years. Therefore, there is an urgent need for a method that can detect this virus
early and quickly to prevent endemic FPLV and reduce infection to feline animals.
The purpose of the present invention is to provide a pair of primers, kit and detection
method for detecting FPLV to solve the problems of the prior art described above, by
which the pair of primers can accurately detect FPLV, which provides a new method and
basis for FPLV prevention.
To achieve the above purpose, the present invention provides the following
solutions:
The present invention provides a pair of primers for detecting FPLV with a
nucleotide sequence as shown in SEQ ID NO: 1 upstream primer and SEQ ID NO: 2
downstream primer.
The present invention also provides a kit for detecting FPLV comprising the primers
as described.
The present invention also provides a detection reagent for detecting FPLV
comprising the primers as described.
The invention also provides a method for detecting FPLV, which comprises the step
of performing PCR amplification by using the pair of primers or the kit or the detection
reagent.
Preferably, the amplification reaction system comprises the following components:
LA Taq 0.5 L, 10 x LA Buffer 5 L, dNTPs 8 L, 1 L each of upstream and
downstream primers, DNA 2 L, supplemented with water to 50 L.
Further, the PCR reaction conditions are: 95°C for 5min, 95°C for 1min, 56°C for
s, 72°C for 140s, 72°C for10min , 35 cycles, kept at 4°C.
The invention also provides the application of the pair of primers in preparing
products for detecting Feline panleukopenia virus.
Preferably, the product comprises the kit or the detection reagent mentioned above.
The invention discloses the following technical effects:
Compared with the traditional detection method, the pair of primers designed by the
invention adopts a PCR detection method to detect FPLV, so that the detection procedure
is simplified and the detection period is shortened, the FPLV can be accurately detected,
the virus can be accurately identified at an early stage, the epidemic of the virus can be
prevented and controlled in time, and the harm of the virus to cats can be reduced or
prevented. The detection method established by the invention can be used for early and
rapid diagnosis of FPLV, and can reflect the infection status of the virus and truly reflect
the distribution status of the virus in cats. The popularization and application of the kit
and the detection reagent provided by the invention are more conducive to quickly and
accurately identifying the infection situation of FPLV.
Fig. 1 is the result of PCR amplification of VP2 nucleic acid fragment of FPLV; M
is the standard control of 5000bp; 1 is the VP2 amplification result of FPLV; and 2 is
negative control;
Fig. 2 shows the results of virus specificity detection of FPLV; M is the standard
control of 5000 bp; 1 shows the amplification results of FPLV; 2 shows the amplification
results of Feline herpesvirus (FHV-1); and 3 shows the amplification results of Feline
infectious peritonitis virus (FIPV).
Now, the technical scheme of the present invention will be specifically explained by
way of examples, but it should not be regarded as a limitation of the present invention,
but rather as a more detailed description of certain aspects, characteristics and
embodiments of the present invention.
Unless otherwise specified, the test methods used in the following examples are all
conventional methods; Materials and reagents used, unless otherwise specified, are
commercially available reagents and materials.
Example 1 Primers design and synthesis for detection of Feline panleukopenia virus
Based on the sequence of Feline panleukopenia virus gene published by GenBank,
the highly conserved VP2 gene was selected as the amplification region by sequence
comparison and specific primers were designed, which were synthesized by TaKaRa
Biological Engineering (Dalian) Co.
The specific sequences are as follows:
Upstream primer: 5'-gccgaattcccaatgagtgatggagcagttcaaccag-3'(SEQ ID NO: 1),
Downstream primer: 5'-ccgctcgagctaggtgctagttgatatgtaataaac-3'(SEQ ID NO: 2).
Example 2 Establishment of PCR method for detection of Feline panleukopenia
virus
1. Test materials
1.1 Virus strain
The FPLV strain was preserved by Institute of Animal Sciences, Chinese Academy
of Agricultural Sciences, Beijing 100193, China.
1.2 Main reagents
The virus DNA extraction kit (DN40) was purchased from Beijing Aidlab Biological
Company. TaKaRa LA Taq and dNTPs Mixture reagents were purchased from TaKaRa
Bio (Dalian) Co., Ltd..
1.3 DNA extraction
The virus DNA was extracted according to the instructions of virus DNA extraction
kit.
1.4 Construction of PCR method
PCR reaction was established by cDNA from FVP as template. PCR reaction system
was follows: LA Taq 0.5tL, 10 x LA Buffer 5 L, dNTPs 8 L, upstream and downstream
primers 1 L each, template 2L, and water was added to 50[L.
PCR reaction conditions: 95°C for 5 min, 95°C for 1 min, 56°C for 45 s, 72°C for
140 s, 72°C for 10 min, 35 cycles, and maintained at 4°C.
The PCR products were subjected to 1% agarose gel electrophoresis analysis and the
results were observed.
1.5 Electrophoresis detection results
The results of electrophoretic detection of PCR amplification products were shown
in Figure 1, wherein M was 5000bp marker, 1 was the result of FPLV nucleic acid
fragment amplification with fragment size of 1822bp, and 2 was the negative control.
Example 3 specificity and sensitivity detection of PCR method for Feline
panleukopenia virus
3.1 Specific detection
Taking the FPLV PCR assay constructed in Example 2, PCR amplification was
performed on the nucleic acid templates of FPLV, FHV-1 and FIPV (all stored in the
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing
100193, China).
The results are shown in Figure 2. 1822bp was obtained after FPLV amplification
which was consistent with the sequencing results; FHV-1 and FIPV could not be
amplified.
3.2 Sensitivity detection
After detecting the concentration of FPLV template to be detected respectively, it
was diluted to 10-1-10-8g/niL by multiplication gradient dilution method, and the
sensitivity of this PCR method was determined.
The results showed that the highest detection sensitivity of FPLV was 10-8g/mL
under the optimized PCR reaction conditions in Example 2 of the present invention.
The above examples only describe the preferred mode of the invention, but do not
limit the scope of the invention. Without deviating from the design spirit of the invention,
all kinds of deformations and improvements made by ordinary technical personnel in this
field to the technical scheme of the invention shall fall within the scope of protection
determined by the claim of the invention..
Claims (8)
1. A pair of primers for detecting Feline panleukopenia virus (FPLV), which is
characterized by a nucleotide sequence as shown in SEQ ID NO: 1 upstream primer and
SEQ ID NO: 2 downstream primer.
2. A kit for detecting FPLV is characterized in that it comprises the primers of
claim.
3. A detection reagent for detecting FPLV, which is characterized in that it
comprises the pair of primers of claim 1.
4. A method for detecting FPLV, which is characterized in that it comprises the step
of PCR amplification by pair of primers as described in claim 1 or the kit as described in
claim 2 or the detection reagent as described in claim 3.
5. The method for detecting FPLV as claimed in claim 3, which is characterized in
that the amplification reaction system comprises the following components: LA Taq 0.5
L, 10 x LA Buffer 5 L, dNTPs 8 L,1 L each of upstream and downstream primers,
DNA 2 L, supplemented with water to 50 L.
6. The method for detecting FPLV as claimed in claim 3, characterized in that the
PCR reaction conditions are: 95°C for 5min, 95°C for 1min, 56°C for 45s, 72°C for 140s,
72°C for 10min, 35 cycles, kept at 4°C.
7. The application of the pair of primers according to claim 1 in preparing products
for detecting FPLV.
8. The application according to claim 7, which is characterized in that the product
comprises the kit or the detection reagent mentioned above
FIGURES
1/2
Sequence table
< 110 >Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, 2021101666
Beijing 100193, China
< 120 >Primers, kits and detection methods for detection of Feline panleukopenia
virus
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1 <211> 37 <212> DNA <213> Artificial Sequence
<400> 1 gccgaattcc caatgagtga tggagcagtt caaccag 37
<210> 2 <211> 36 <212> DNA <213> Artificial Sequence
<400> 2 ccgctcgagc taggtgctag ttgatatgta ataaac 36
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021101666A AU2021101666A4 (en) | 2021-03-31 | 2021-03-31 | Primers, kit and detection method for detecting Feline panleukopenia virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021101666A AU2021101666A4 (en) | 2021-03-31 | 2021-03-31 | Primers, kit and detection method for detecting Feline panleukopenia virus |
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| Publication Number | Publication Date |
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| AU2021101666A4 true AU2021101666A4 (en) | 2021-05-20 |
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| Application Number | Title | Priority Date | Filing Date |
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| AU2021101666A Ceased AU2021101666A4 (en) | 2021-03-31 | 2021-03-31 | Primers, kit and detection method for detecting Feline panleukopenia virus |
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| Country | Link |
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| AU (1) | AU2021101666A4 (en) |
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2021
- 2021-03-31 AU AU2021101666A patent/AU2021101666A4/en not_active Ceased
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