AU2021103978A4 - A primer set, reagent and method based on polymerase spiral reaction for detecting feline parvovirus - Google Patents
A primer set, reagent and method based on polymerase spiral reaction for detecting feline parvovirus Download PDFInfo
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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 39
- 241000701925 Feline parvovirus Species 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 15
- 239000002773 nucleotide Substances 0.000 claims abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 12
- 230000003321 amplification Effects 0.000 claims abstract description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 9
- 239000000980 acid dye Substances 0.000 claims abstract description 7
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 229960003237 betaine Drugs 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims 1
- -1 SYBR Green I nucleic acid Chemical class 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 241000725579 Feline coronavirus Species 0.000 description 4
- 241000282324 Felis Species 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 241000125945 Protoparvovirus Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 108060002716 Exonuclease Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 102000013165 exonuclease Human genes 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241001135557 Enteric coronavirus Species 0.000 description 1
- 241000701915 Feline panleukopenia virus Species 0.000 description 1
- 101150033828 NS1 gene Proteins 0.000 description 1
- 241001263478 Norovirus Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/701—Specific hybridization probes
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
Disclosed is a primer set, reagent and method based on polymerase spiral reaction for
detecting feline parvovirus; said primer set includes specific primers PSR-FPVF and
PSR-FPVR; accelerated primers include PSR-FPVF-FJ and PSR-FPVF-RJ;
nucleotide sequence of the specific primer PSR-FPVF is shown in SEQ ID NO.1;
nucleotide sequence of the specific primer PSR-FPVR is shown in SEQ ID NO.2;
nucleotide sequence of the accelerated primer PSR-FPVF-FJ is shown in SEQ ID
NO.3; nucleotide sequence of the accelerated primer PSR-FPVF-RJ is shown in SEQ
ID NO.4; PSR detection method constructed by said primer set is simple to operate,
has lower requirements for detection equipment, and greatly shortens the detection
time; said detection can be completed within 45 minutes at constant temperature
condition of 67C; after the amplification, add the final concentration of 20 X SYBR
Green I nucleic acid dye to the system; detection result can be recognized by naked
eye under visible light, which greatly improves the detection efficiency.
1
Description
[0001] This invention relates to the technical field of molecular biology, in particular
to a primer set, reagent and method based on polymerase spiral reaction for detecting
feline parvovirus.
[0002] Feline parvovirus (FPV), also known as feline pan-leukopenia virus, can
cause acute and highly contagious infectious disease in cats; symptoms caused by
FPV are very similar to those of other viral infections such as enteric coronavirus;
therefore, establishment of a fast, accurate, specific and sensitive detection method is
of great significance for the diagnosis and treatment of FPV.
[0003] At present, the most commonly used virus detection method is Polymerase
Chain Reaction (PCR), and technologies such as fluorescence quantitative PCR and
nest PCR are derived based on the principle of PCR; however, this series of PCR
technologies cannot get rid of the limitation of the thermal cycle of the reaction, and
requires complex temperature-variable instruments and skilled experimenters,
resulting in high detection costs and long detection times.
[0004] With intention to solve said problems, this invention provides a primer set,
reagent and method based on polymerase spiral reaction for detecting feline
parvovirus.
[0005] Technical plan adopted by this invention is introduced as follow:
[0006] A primer set based on polymerase spiral reaction for detecting feline
parvovirus includes specific primers PSR-FPVF and PSR-FPVR; accelerated primers
include PSR-FPVF-FJ and PSR-FPVF-RJ; nucleotide sequence of the specific primer
PSR-FPVF is shown in SEQ ID NO.1; nucleotide sequence of the specific primer
PSR-FPVR is shown in SEQ ID NO.2; nucleotide sequence of the accelerated primer
PSR-FPVF-FJ is shown in SEQ ID NO.3; nucleotide sequence of the accelerated
primer PSR-FPVF-RJ is shown in SEQ ID NO.4.
[0007] A reagent and method based on polymerase spiral reaction for detecting feline
parvovirus includes specific primers and accelerated primers.
[0008] Preferred,in an embodiment of this invention, said reagent includes as follow:
X Bst buffer, Bst X DNA polymerase, dNTP Mix,MgSO4, betaine, DNA template
and sterilized water.
[0009] Preferred,in an embodiment of this invention, said reagent includes SYBR
Green I nucleic acid dye.
[0010] Application of said reagent in the preparation of a detection product for
detecting feline parvovirus.
[0011] A method for detecting feline parvovirus based on polymerase spiral reaction,
wherein use specific primer PSR-FPVF, PSR-FPVR and accelerated primer
PSR-FPVF-FJ and PSR-FPVF-RJ for PSR constant temperature amplification
reaction to detect FPV in the sample to be tested.
[0012] Preferred, said detection method includes steps as follow:
[0013] Step 1: Extract the FPV genomic nucleic acid in the sample to be tested,
determine the concentration, and calculate the copy number;
[0014] Step 2: Use said extracted FPV genomic nucleic acid as a template to
construct PSR reaction system: 10 X Bst buffer 2.5uL, Bst X DNA polymerase
8U,OmM each dNTP Mix 1.75uL,2-14mmol/L MgSO4 1.5uL, 0.2-1.6mol/L betaine
4.8uL, specific primers PSR-FPVF and PSR-FPVR each luL, accelerated primers
PSR-FPVF-FJ and PSR-FPVF-RJ each luL, DNA template luL; make up to 25uL
with sterile water.
[0015] Step 3: PSR reaction system is subjected to a constant temperature reaction;
sterilized water is set as a negative control for each reaction.
[0016] Step 4: After the reaction is complete, add SYBR Green I nucleic acid dye for color reaction; color green means the sample to be tested is positive; color orange means the sample to be tested is negative.
[0017] Final concentration of SYBR Green I nucleic acid dye is 20 X.
[0018] Preferred,the temperature of said constant temperature reaction is 67°C.
[0019] Beneficial effects of this invention is as follow:
[0020] Said reagent in this invention uses Bst X DNA polymerase, which lacks 5'-3' and 3'-5' exonuclease activity; said polymerase has stronger strand displacement activity than the traditional Bst DNA polymerase; said polymerase has higher stability, good stress resistance, higher tolerance to non-ionic surfactants and high salt environment.
[0021] Compared with traditional PCR detection methods, method of this invention has simpler operation and low requirements on detection instruments; it only needs simple equipment such as centrifuges and water baths to directly complete amplification, reduces detection costs, and greatly shortens detection time; it can be completed within 45 minutes under the constant temperature of 67 °C ; after the amplification is completed, add SYBR Green I nucleic acid dye with final concentration of 20 X to the system; detection result can be judged by the naked eye under visible light, which greatly improves the detection efficiency.
[0022] In addition, PSR detection method of this invention has good specificity and is negative for both FCoV and FNV; sensitivity of PSR detection is 6.75X10 copies/uL, which is 10 times lower than that of conventional PCR detection.
[0023] Method of this invention is used to carry out repeated tests within groups and between groups, and results show that the detection results within and between groups are consistent, both are positive, and the repeatability is good; PSR method of this invention was used to detect 50 cat feces samples suspected of being infected with FPV; results showed that the detection results of the PSR method were basically consistent with those of the conventional PCR method, indicating that the PSR
method established in this study can be used to detect FPV clinical samples; said method is simpler with lower cost; said method has strong specificity, high sensitivity and reproducibility; results can be judged by the naked eye under visible light, which is suitable for rapid diagnosis of FPV infection in city and county pet hospitals.
[0024] FIG. 1 indicates the reaction temperature condition of PSR (A) and condition screening of Mg2+ concentration (B), betaine concentration (C) as well as PSR product digestion identification results (D) according to the embodiment of this invention.
[0025] With intention to further introduce the objectives, technical solutions, and advantages of the present invention,drawing and embodiments shall be provided to better introduce this invention; it should be understood that the specific embodiment described here are only used to explain the present invention, but not used to limit the present invention.
[0026] The FPV, FCoV, and FNV faces samples used in this embodiment of the present invention were all collected in pet hospitals in Lanzhou from the year of 2018 to 2020; viral nucleic acid extraction kit, 10000 X SYBR Green I, Bst X DNA polymerase, 10 X Bst Buffer, MgSO4, dNTP Mix (10mM each) solution were all purchased from Beijing Solarbio Technology Co., Ltd.; betaine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0027] Example 1
[0028] 1. Primer design
[0029] Use Oligo 6.0 software to design a pair of PCR primers for the conserved region of FPV NS1 gene: FPV-F and FPV-R; size of the amplified fragment is about 403bp, and a pair of PSR specific primers are designed according to the principle of PSR in the amplified fragment: PSR-FPVF, PSR-FPVR, and a pair of PSR
acceleration primers: PSR-FPV-FJ, PSR-FPV-RJ; PSR amplification product is a
series of ladder-shaped bands; all primers were synthesized by Beijing Tsingke
Biotechnology Co., Ltd; said specific primer names and sequences are shown in
Table 1.
[0030] PCR and PSR primers for FPV detection
[0031] 2.Nucleic acid extraction and copy number calculation
[0032] Use viral nucleic acid extraction kit to extract viral nucleic acid from FPV
positive samples, feline coronavirus (FCoV) positive samples and feline norovirus
(FNV) positive samples; use an ultra-micro spectrophotometer to determine the
concentration of FPV nucleic acid and calculate the number of copies; use FPV
genomic DNA as reaction template for subsequent experiments and store in
refrigerator at -80°C.
[0033] 3.Construction and optimization of PSR reaction system
[0034] Use said extracted FPV genomic nucleic acid as a template to construct PSR
reaction system: 10 X Bst buffer 2.5uL, Bst X DNA polymerase 8U, dNTP Mix
(10mM each) 1.75uL, MgSO4 (100mmol/L) 1.5uL, betaine (4.17mol/L) 4.8uL,
specific primers PSR-FPVF and PSR-FPVR each luL, accelerated primers
PSR-FPVF-FJ and PSR-FPVF-RJ each luL, DNA template luL; make up to 25uL
with sterile water;said reaction was kept at 65 °C for 90 minutes; electrophoresis
observations were performed after completion; negative control with sterile water as
a template was set up in each experiment.
[0035] With intention to optimize the reaction system:
[0036] Based on said PSR reaction system, perform the screening of the annealing
temperature (70°C, 67C, 64C, 61°C, 58C), reaction time (45min, 60min, 75min,
min), MgSO4 concentration (2 mmol/L, 4 mmol/L, 6 mmol/L, 8 mmol/L, 10
mmol/L, 12 mmol/L, 14 mmol/L) and betaine concentration (0.2 mol/L, 0.4 mol/L,
0.8mol/L, 1.2 mol/L, 1.6mol/L) to determine the optimal reaction conditions; at the
same time, experiment was carried out under the optimal conditions; after the
reaction, 10 uL of the amplified product was detected by agarose gel electrophoresis
with concentration of 1%; add SYBR Green I nucleic acid dye with final
concentration of 20 X to the rest of the amplified products; observe whether the
reaction tube has a color change.
[0037] Results as follow:
[0038] Using said extracted FPV genomic DNA as template, said PSR detection
method was optimized by using the square matrix method; primers PSR-FPVF and
PSR-FPVR were used at different temperatures (58C, 61°C, 64C, 67C, 70°C) to
perform PSR constant temperature amplification for 90 minutes; result shown in FIG.
1A indicates that when the reaction time reaches to 90 minutes and the reaction
temperature is 67C, the ladder-like strips are the clearest; perform PSR amplification
at 67C as the optimal reaction temperature; results are shown in FIG. 1B; results
show that 6mmol/L MgSO4 is the optimal reaction concentration in the established
MgSO4 concentration gradient; then use 67C, 6mmol/L MgSO 4 as the reaction
conditions for PSR amplification to optimize the betaine concentration; results are
shown in FIG. IC; results show that the concentration change of betaine has no
obvious effect on the reaction results, so the intermediate concentration of 0.8mol/L
can be taken as the best concentration of betaine.
[0039] With intention to verify that the gradient band amplified by PSR is FPV
specific target gene, we selected the only restriction site Sea I on the target gene to
digest the PSR product; results were shown in the FIG. ID; electrophoresis result
provides the only expected target gene band that can be seen at 100bp; said gene
band was collected by cutting gel and sent to the sequencing company; sequence
results show that the amplified product of this PSR method was a specific FPV gene
fragment.
[0040] SEQUENCE LISTING
<110> Gansu Agricultural University
<120> A primer set, reagent and method based on polymerase spiral reaction for
detecting feline parvovirus
<130> 1
<160> 6 <170> Patentln version 3.3
<210> 1
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 1
ctgtcagcac actttacact taagtactga ttctggttgg a 41
<210> 2
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<212> DNA
<213> Artificial Sequence
<400> 2
ctgtcagcac actttacact cctgtgctgt cgtcactgtg g 41
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 3
tgtctgtctt gatacttc 18
<210> 4
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<212> DNA
<213> Artificial Sequence
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cagcacactt tacactg 17
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<212> DNA
<213> Artificial Sequence
<400> 5
atggttggtg actctttgtt 20
<210>6
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 6
tacatttgat tgacacttcc 20
Claims (5)
1.A reagent for detecting feline parvovirus based on polymerase spiral reaction is
characterized in, wherein said reagent includes primer set; said primer set includes
specific primers PSR-FPVF and PSR-FPVR; accelerated primers include
PSR-FPVF-FJ and PSR-FPVF-RJ; nucleotide sequence of the specific primer
PSR-FPVF is shown in SEQ ID NO.1; nucleotide sequence of the specific primer
PSR-FPVR is shown in SEQ ID NO.2; nucleotide sequence of the accelerated primer
PSR-FPVF-FJ is shown in SEQ ID NO.3; nucleotide sequence of the accelerated
primer PSR-FPVF-RJ is shown in SEQ ID NO.4.
2.A reagent for detecting feline parvovirus based on polymerase spiral reaction as
claimed in claim 1 is characterized in, wherein said reagent includes 10 X Bst buffer,
Bst X DNA polymerase, dNTP Mix,MgSO4, betaine, DNA template and sterilized
water.
3.A reagent for detecting feline parvovirus based on polymerase spiral reaction as
claimed in claim 1 is characterized in, wherein said reagent includes SYBR Green I
nucleic acid dye.
4.An application of said reagent according to claims 2-3 in the preparation of a
detection product for detecting feline parvovirus.
5.A method for detecting feline parvovirus based on polymerase spiral reaction is
characterized in, wherein use specific primers PSR-FPVF, PSR-FPVR and
accelerated primers PSR-FPVF-FJ and PSR-FPVF-RJ for PSR constant temperature
amplification reaction to detect FPV in the sample to be tested.
FIG. 1 D R AW I N G
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736923A (en) * | 2021-10-15 | 2021-12-03 | 安徽农业大学 | Primer and kit for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of Chapparvovirus |
| CN116949224A (en) * | 2023-09-21 | 2023-10-27 | 上海基灵生物科技有限公司 | Multiplex PCR (polymerase chain reaction) kit for detecting pathogens in cat digestive tract and application thereof |
-
2021
- 2021-07-08 AU AU2021103978A patent/AU2021103978A4/en not_active Ceased
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736923A (en) * | 2021-10-15 | 2021-12-03 | 安徽农业大学 | Primer and kit for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of Chapparvovirus |
| CN116949224A (en) * | 2023-09-21 | 2023-10-27 | 上海基灵生物科技有限公司 | Multiplex PCR (polymerase chain reaction) kit for detecting pathogens in cat digestive tract and application thereof |
| CN116949224B (en) * | 2023-09-21 | 2023-12-15 | 上海基灵生物科技有限公司 | Multiplex PCR (polymerase chain reaction) kit for detecting pathogens in cat digestive tract and application thereof |
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