CN103614494A - Two-color fluorogenic quantitative PCR (Polymerase Chain Reaction) detection kit for CDV (canine distemper viruses) and CPV (canine parvo viruses) - Google Patents
Two-color fluorogenic quantitative PCR (Polymerase Chain Reaction) detection kit for CDV (canine distemper viruses) and CPV (canine parvo viruses) Download PDFInfo
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Abstract
The invention discloses a two-color fluorogenic quantitative PCR (Polymerase Chain Reaction) detection kit for CDV (canine distemper viruses) and CPV (canine parvo viruses). A detection method comprises the following steps: firstly, pre-treating a sample by using a nucleic acid extraction solution of the kit and extracting nucleic acid; then detecting the nucleic acids of the CDV and CPV simultaneously in a reaction system by utilizing the two-color fluorogenic quantitative PCR detection kit, and detecting whether the canine distemper and canine parvo viruses exist in the sample to be detected or not by one step; and quantifying a virus copy number. The detection method is high in detection efficiency and good in specificity and sensibility; the aims of clinical detection and epidemic situation control of animal epidemic diseases can be realized.
Description
Technical field
The present invention relates to a kind of double color fluorescent quantitative PCR detection kit, be specifically related to a kind of double color fluorescent quantitative PCR detection kit that simultaneously detects canine distemper and canine parvovirus, the invention belongs to biological technical field.
Background technology
Canine distemper virus (Caninedistempervirus, CDV) and canine parvovirus (Canine Parvovirus, CPV) be two of the most serious infectivity epidemic disease cause of disease of harm dog, caused epidemic disease is difficult to control without specific medicament, curative ratio is very low, healing person also has serious sequela, serious a large amount of dogs acute death that causes.
Canine distemper virus (Caninedistempervirus, CDV) is a kind of single strand RNA virus, and host is Canis animals and mustelid, mainly by air and spittle horizontal transmission.Sick dog is important contagium, and by the long-term toxin expelling of urine, pollutes surrounding environment; The dog of different ages, sex and kind all can infect.Infect CDV reveal any symptoms varied, relevant with virus virulence, envrionment conditions, host's age and immunological status, mainly can be divided into super acute, acute, symptom of digestive tract, nervous symptoms and 5 kinds of characteristic types of skin symptom, once there is characteristic symptom, poor prognosis.And the Minimal change that CDV causes is confined to respiratory tract and eye and is difficult to find, make the clinical diagnosis of hundstaupe very difficult.CDV infects that disease type complexity is various, and I diagnoses very difficult clinically, easily obscures mutually with upper respiratory tract infection, infectious canine hepatitis, canine Coronavirus Infection etc., must make a definite diagnosis according to Virus Isolation.
The laboratory diagnostic method that CDV infects mainly contains the following aspects: one, blood changes: comprise lymphopoiesis, thrombopenia and aplastic anemia; Two, in acute infection, can in the cell of blood smear (particularly lymphocyte, once in a while neutrophil leucocyte), find inclusion body; Three, the inspection of marrow extractum, also can find inclusion body in cell; Four, from conjunctival epithelial cell, also can detect canine distemper inclusion body; The detection method of other of CDV comprises: immunological method, separated with virus with IgM antibody with the IgG in enzyme linked immunosorbent assay (ELISA) mensuration serum, these detection methods need special equipment and the preparation of sample, and testing process is complicated, difficulty is large and tolerance range is low.
Canine parvovirus (Canine Parvovirus, CPV) is without cyst membrane, and the DNA virus of single negative strand, is comprised of 3 structural protein VP1, VP2 and VP3.Wherein VP2 is its protectiveness particulate antigen.This virus has 2 kinds of different types, i.e. CPV-1 type and CPV-2 type.CPV-1 type, claims again dog microvirus (MVC), can cause pup pneumonia, myocarditis and enteritis, or diaplacental infection causes that embryo absorbs and foetal death, but MVC only causes that clinical symptom appears in the pup below 40 ages in days.CPV-2 is very close with Feline Panleukopenia Virus (FPV) as a new virus for dog.Canine parvovirus has the contagious infection of height to dog, the dog at various ages all can infect.But more to the dog morbidity of 90 ages in days just to wean, the state of an illness is also more serious.Presented in the myocarditis symptom that pup has and sudden death.According to the kind of clinical onset dog, purebred dog and external dog are higher than soil species dog sickness rate.This disease all can occur throughout the year, but multiple with the winter-spring season of cold weather.In the ight soil of sick dog, toxic amount is higher.
The diagnostic method that CPV infects mainly carries out tentative diagnosis according to routine blood test detected result and this sick cardinal symptom.Routine blood test detects pcv to be increased, and wbc value is normal or on the low side, often points out virus disease.The bloody stool of sick dog excretion tomato juice sample or soy sauce belt transect fishy odor is this sick characteristic symptom, can be used as first visit foundation.In addition, the diagnosis of canine parvovirus disease adopts canine parvovirus Radioactive colloidal gold fast diagnose test paper to carry out more, and this method is easy fast, but often accuracy rate is not high.
In sum, the laboratory inspection of conventional canine distemper and canine parvovirus is all generally according to immunoreactive principle, by ELISA or golden mark method, to detect etc.Although can also detect by pcr amplification technology for above-mentioned two-strain in addition, but first need in sample, extract above-mentioned viral nucleic acid, canine distemper virus is a kind of RNA viruses, need to extract RNA, take this product carries out reverse transcription as template again, finally to sample, adopts pre-designed primer to increase; Canine parvovirus is DNA virus, can directly extract DNA and increase.If amplify corresponding fragment according to default fragment, viral existence is described, susceptibility will be higher than above-mentioned Serology test.Yet carry out canine distemper and canine parvovirus detection of nucleic acids by the method for regular-PCR, although sensitivity increases, but easily produce non-specific amplification, even can there is false positive because of the reason of operation, and the judgement of its result of regular-PCR TRAP need to carry out gel electrophoresis analysis to product, working method is simplified not.And canine distemper and canine parvovirus are often with the mode infection animal of independent or polyinfection, M & M is all higher, needs rapid detection, the effect that accomplish early diagnosis, early isolate, early control could arrive.
Multicolor fluorescence quantitative PCR technique refers to a plurality of goal gene that simultaneously increase in same fluorescent quantitative PCR test, and the fluorescent probe without wavelength that each goal gene adopts detects.Fluorescently-labeled probe has multiple, such as FAM, VIC, JOE, NED, HEX etc., and every kind of fluorescent signal difference that fluorescently-labeled probe produces in pcr amplification process.
Summary of the invention
In order to address the above problem, the present invention is by multicolor fluorescence quantitative PCR technique, provide that a kind of detection efficiency is high, specificity and susceptibility better, can detect method and the detection kit of CDV and CPV two-strain simultaneously.
The object of the present invention is to provide a kind of canine distemper and canine parvovirus double color fluorescent quantitative PCR detection kit.
The technical solution used in the present invention is:
A kind of canine distemper and canine parvovirus double color fluorescent quantitative PCR detection kit comprise following composition: viral RNA nucleic acid extraction liquid, viral DNA nucleic acid extraction liquid, ThermoScript II system, Taq enzyme system, double color fluorescent quantitative PCRMIX, positive quality control product, negative quality control product, DEPC water, the Auele Specific Primer and the double-colored probe that in double color fluorescent quantitative PCR MIX, contain 10 * double color fluorescent quantitative PCR buffer, dNTPs and canine distemper virus and canine parvovirus, sequence is respectively:
Canine distemper virus:
Upstream primer: CDV-F:5 '-GCAAGCGAGGATTGAGGGTAT-3 ' (SEQ ID NO.1);
Downstream primer: CDV-R:5 '-GGCCTATGGGACACACTCAAA-3 ' (SEQ ID NO.2);
Fluorescent probe: CDV-P:5 '-FAM-CACCGACCATCCAGACGTTGCTCCT-TAMRA-3 ' (SEQ ID NO.3)
Canine parvovirus:
Upstream primer: CPV-F:5 '-CGTCTACACAAGGGCCATTTA-3 ' (SEQ ID NO.4);
Downstream primer: CPV-R:5 '-CCATGTTGTCTACCAAATGCAT-3 ' (SEQ ID NO.5);
Fluorescent probe: CPV-P:5 '-VIC-CGCAAACAGATGAAAATCAAGCAGCA-TAMRA-3 ' (SEQ ID NO.6);
Wherein, the fluorescent probe reporter group of canine distemper virus is FAM, and the fluorescent probe reporter group of canine parvovirus is VIC, and quenching group is TAMRA.
Further, in the every 1000mL of above-mentioned viral RNA nucleic acid extraction liquid, contain: guanidinium isothiocyanate 480g, 0.1M Tris-HCl(pH6.4) 500mL, 0.2M EDTA(pH8.0) 120mL, surplus is water;
Further, above-mentioned viral DNA nucleic acid extraction liquid contains 20mM NaOH, 10mM Tris-HCl(pH8.0), concentration is that Chelex-100, the surplus of 2% (w/v) is water.
Further, above-mentioned double color fluorescent quantitative PCR MIX's is composed as follows:
10 * double color fluorescent quantitative PCR buffer, 2.5 μ L
CDV-F（10μM） 1μL
CDV-R（10μM） 1μL
CPV-F（10μM） 1μL
CPV-R（10μM） 1μL
CDV-P（10μM） 0.5μL
CPV-P（10μM） 0.5μL
dNTPs (10mM) 1μL
ddH
2O 8.5μL
Total 17μL。
Further, in above-mentioned 10 * double color fluorescent quantitative PCR buffer, contain following composition: 500mM Tris-HCl (pH8.0), 50mM MgCl
2, 250mM KCl and BSA of 5% etc.
A double color fluorescent quantitative PCR associated detecting method for canine distemper virus (CDV) and canine parvovirus (CPV), comprises the steps:
1) extract viral RNA and the DNA in testing sample;
2) take total RNA and the DNA obtaining is template, uses canine distemper virus
Upstream primer: CDV-F:5 '-GCAAGCGAGGATTGAGGGTAT-3 ' (SEQ ID NO.1);
Downstream primer: CDV-R:5 '-GGCCTATGGGACACACTCAAA-3 ' (SEQ ID NO.2);
Fluorescent probe: CDV-P:5 '-FAM-CACCGACCATCCAGACGTTGCTCCT-TAMRA-3 ' (SEQ ID NO.3) and canine parvovirus
Upstream primer: CPV-F:5 '-CGTCTACACAAGGGCCATTTA-3 ' (SEQ ID NO.4);
Downstream primer: CPV-R:5 '-CCATGTTGTCTACCAAATGCAT-3 ' (SEQ ID NO.5);
Fluorescent probe: CPV-P:5 '-VIC-CGCAAACAGATGAAAATCAAGCAGCA-TAMRA-3 ' (SEQ ID NO.6)
Carry out double color fluorescent quantitative PCR detection, wherein, the fluorescent probe reporter group of canine distemper virus and canine parvovirus is not identical;
3) interpretation of result: after reaction finishes, judge according to the fluorescence Ct value of testing sample whether testing sample is that canine distemper virus, canine parvovirus are positive.
Further, the system of double color fluorescent quantitative PCR detection reaction is 25 μ L, composed as follows: double color fluorescent quantitative PCR MIX 17 μ L, reversed transcriptive enzyme are that 1 μ L, Taq enzyme are canine distemper virus RNA and canine parvovirus DNA each 3 μ L or positive quality control product 6 μ L or negative quality control product 6 μ L of 1 μ L and extraction;
Further, consisting of of above-mentioned double color fluorescent quantitative PCR MIX:
10 * double color fluorescent quantitative PCR buffer, 25 μ L
CDV-F（10μM） 1μL
CDV-R（10μM） 1μL
CPV-F（10μM） 1μL
CPV-R（10μM） 1μL
CDV-P（10μM） 0.5μL
CPV-P（10μM） 0.5μL
dNTPs (10mM) 1μL
ddH
2O 8.5μL
Total 17μL。
Further, in above-mentioned 10 * double color fluorescent quantitative PCR buffer, contain following composition: the BSA of 500mM Tris-HCl (pH8.0), 50mM MgCl2,250mM KCl and 5% (w/v).
Further, double color fluorescent quantitative PCR reaction conditions is: 93 ℃, 2 minutes, then 93 ℃, 30 seconds, 55～58 ℃, 45 seconds, gather fluorescent signal, 40 circulations.
Beneficial effect of the present invention:
Test kit provided by the invention can detect canine distemper and canine parvovirus two-strain simultaneously, and be the joint-detection that is applied to RNA viruses and DNA virus, and can carry out detecting in real time accurately and quantitatively to the nucleic acid of pathogenic agent in sample, apply very easyly, be with a wide range of applications.
Test kit provided by the invention is owing to having introduced specificity amplification primer and fluorescent probe, the sensitivity and the specificity that detect are strengthened significantly, thereby avoided the not high problem of easily failing to pinpoint a disease in diagnosis with mistaken diagnosis of other detection method specificitys, based on above-mentioned advantage, this canine distemper and canine parvovirus detection kit are adapted at applying in animal healths at different levels supervision institute, animal epidemic prevention and control center pets hospital and various Animal diseases research institution.
Accompanying drawing explanation
Fig. 1 is the sensitivity experiments that in double color fluorescent quantitative PCR detection method, CDV detects, and is from left to right followed successively by 1.0 * 10
7, 1.0 * 10
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2the amplification of the standard substance of copy/μ L;
Fig. 2 is the sensitivity experiments that in double color fluorescent quantitative PCR detection method, CPV detects, and is from left to right followed successively by 1.0 * 10
7, 1.0 * 10
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2the amplification of the standard substance of copy/μ L;
Fig. 3 is the specificity experiment of CDV in double color fluorescent quantitative PCR detection method, as seen from the figure, CDV is positive, CPV, SIV, VSV, FMDV, JEV and negative quality control product all negative;
Fig. 4 is the specificity experiment of CPV in double color fluorescent quantitative PCR detection method, as seen from the figure, CPV is positive, CDV, SIV, VSV, FMDV, JEV and negative quality control product all negative.
Embodiment
embodiment 1:
one, the design of Auele Specific Primer and probe
According to the nucleotide sequence of the canine distemper virus retrieving from Genbank and canine parvovirus, be designed for the primer and the probe that detect canine distemper virus and canine parvovirus, its sequence is as follows:
Canine distemper virus:
Upstream primer: CDV-F:5 '-GCAAGCGAGGATTGAGGGTAT-3 ' (SEQ ID NO.1);
Downstream primer: CDV-R:5 '-GGCCTATGGGACACACTCAAA-3 ' (SEQ ID NO.2);
Fluorescent probe: CDV-P:5 '-FAM-CACCGACCATCCAGACGTTGCTCCT-TAMRA-3 ' (SEQ ID NO.3), amplified fragments 74 bp;
Canine parvovirus:
Upstream primer: CPV-F:5 '-CGTCTACACAAGGGCCATTTA-3 ' (SEQ ID NO.4);
Downstream primer: CPV-R:5 '-CCATGTTGTCTACCAAATGCAT-3 ' (SEQ ID NO.5);
Fluorescent probe: CPV-P:5 '-VIC-CGCAAACAGATGAAAATCAAGCAGCA-TAMRA-3 ' (SEQ ID NO.6), amplified fragments 116 bp.
Fluorescent probe reporter group for detection of canine distemper virus is FAM, and is VIC for the reporter group of canine parvovirus, and quenching group is TAMRA.
two, collection of specimens and pre-treatment
The applicable sample type of this test kit comprises the tissue, serum, Secretory product of dog etc.Tissue sample: get under aseptic condition and organize approximately 100～200mg left and right, insert in the clean EP pipe of 1.5mL, preserve to be checked.Serum: extract and examined animal venous blood 3～5mL with disposable sterilized injector, leave and take serum, preserve to be checked.Secretory product: gather under aseptic condition, preserve to be checked.The sample censorship as early as possible gathering, or be stored in-20 ℃.
three, viral nucleic acid extracts
Canine distemper virus RNA extracts:
1) solid tissue: get 0.5g left and right tissue and shred with glass homogenizer homogenate or scissors, add 1.5mL physiological saline to continue to grind, after homogenate, go in the EP pipe of 1.5mL, 10, the centrifugal 2min of 000rpm, get supernatant 100 μ L in the EP of 1.5mL pipe, add 200 μ L viral RNA nucleic acid extraction liquid fully to shake, standing 3min.
2) serum or virus liquid: get 100 μ L serum and add 200 μ L viral RNA nucleic acid extraction liquid fully to shake, standing 3min.Negative control group is set simultaneously, gets the negative quality control product of 100 μ L and add 200 μ L viral RNA nucleic acid extraction liquid fully to shake, standing 3min.
3) by step 1) or 2) in sample after processing add 200 μ L chloroforms, firmly shake 15s, 4 ℃ 13, the centrifugal 5min of 000rpm.
4) get supernatant and add isopyknic Virahol, 4 ℃ 13, the centrifugal 5min of 000rpm, abandons supernatant, adds 1ml 75% ethanol, fully mixes, and the centrifugal 5min of 13,000rpm, carefully sucks most of residual liquid.
5) by the uncovered 10min that is dried in air at room temperature of extraction tube, make to evaporate clean, by 20 μ L DEPC water dissolution precipitations, be canine distemper virus RNA to be detected.
Canine parvovirus DNA extraction:
1) solid tissue: get 0.5g left and right tissue and shred with glass homogenizer homogenate or scissors, add 1.5mL physiological saline to continue to grind, after homogenate, go in the EP pipe of 1.5mL, 10, the centrifugal 2min of 000rpm, gets supernatant 50 μ L in the EP of 1.5mL pipe, adds 200 μ L viral DNA nucleic acid extraction liquid fully to shake, standing 3min, supernatant is canine parvovirus DNA to be measured.
2) serum or virus liquid: get 50 μ L serum 10, the centrifugal 2min of 000rpm, abandons supernatant and add 50 μ L DNA virus extracting solutions, and 100 ℃ are boiled 10min, then 13, the centrifugal 5min of 000rpm, supernatant is canine parvovirus DNA to be measured.
four, the preparation of positive quality control product
Canine distemper virus (CDV) RNA and the canine parvovirus DNA extracting of take is respectively template, carries out respectively following steps:
Reverse transcription system:
Oligo dT Primer (50 μ M) 1 μ L, dNTP Mixture (10 mM) 1 μ L and CDV RNA 2 μ g, 65 ℃ of 5min, cold shock, then adds 5 * PrimeScript Buffer, 4 μ L(TAKARA companies), RNase Inhibitor (40 U/ μ L) 0.5 μ L, PrimeScript RTase (200 U/ μ L) 1 μ L and RNase free H
2o 4.5 μ L.
Reverse transcription condition:
30 ℃ of 10min, then 42 ℃, 30min, carries out reverse transcription and becomes cDNA.
PCR system:
10 * PCR Buffer, 5 μ L, TaKaRa Taq(5 U/ μ L) each 2.5 mM of 1 μ L, dNTP Mixture() 4 μ L, CDV or CPV upstream and downstream primer (10 μ M) each 0.5 μ L, above-mentioned cDNA 0.5 μ L or CPV DNA 0.5 μ L and RNase free H
2o 39.25 μ L.
PCR condition:
According to 94 ℃ of 3 minutes denaturations, 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 45 seconds, totally 35 circulations, 72 ℃ are extended 10 minutes.
After reaction, getting 5 μ L pcr amplification products detects with 2% sepharose, the PCR product of cutting glue purification is connected with pMD-18T carrier, recombinant plasmid pMD-18T-CDV or pMD-18T-CPV coat on culture dish, transformed competence colibacillus DH5 α cell, the positive bacterium colony extracting of picking plasmid, get plasmid 5 μ L dilutions and survey its A260nm and A280nm absorbance, calculate its concentration and be converted into absolute copy number, be diluted to 1.0 * 10
7copy/μ L, as the positive quality control product of double color fluorescent quantitative PCR.
five, double color fluorescent quantitative PCR amplification
Each test reaction system is formulated as follows, and double-colored PCR MIX 17 μ L, ThermoScript II are that 1 μ L, Taq enzyme are 1 μ L, instantaneous centrifugal, then adds RNA and each 3 μ L of DNA of detected sample; According to above-mentioned system, the positive and negative control are set equally, add positive quality control product or negative quality control product 6 μ L to increase.
Each reaction tubes is put into the reactive tank of quantitative PCR instrument, each title detecting and fluorophor kind are set, and (reporter group that detects CDV is selected FAM, detect the reporter group of CPV and select VIC, quenching group is selected TAMRA), set the instrument cycling conditions such as cycling condition: ABI PRISM 7700, ABI PRISM5700, ABI GeneAmp 7000, ABI PRISM7300/7500, MJ Opticon and be 93 ℃ 2 minutes, then 93 ℃ 30 seconds, 55 ℃ 45 seconds, 40 circulations; The uses such as LightCycler instrument cycling condition capillaceous be 93 ℃ 2 minutes, then 93 ℃ 5 seconds, 58 ℃ 45 seconds, totally 40 circulations.
six, interpretation of result and judgement
After reaction finishes, adjustment threshold value makes negative quality control product Ct value more than 40, and what FAM fluorescence Ct value was less than 35 circulations and S-type amplification curve is the CDV positive, and what VIC fluorescence Ct value was less than 35 circulations and S-type amplification curve is that CPV is positive, the two is all positive, is CDV and CPV common anode type.
seven, sensitivity experiment
Get the positive quality control product (1.0 * 10 in above-mentioned 3
7copy/μ L), dilution is 1.0 * 10 successively
7, 1.0 * 10
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2copy/μ L, carries out sensitivity experiment.
The sensitivity experiments that in double color fluorescent quantitative PCR system, CDV detects is shown in Fig. 1, and the susceptibility that in double color fluorescent quantitative PCR system, CPV detects is shown in Fig. 2 in fact.Fig. 1 is the sensitivity experiments that in two-color fluorescence PCR system, CDV detects, and is from left to right followed successively by 1.0 * 10
7, 1.0 * 10
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2the amplification of the standard substance of copy/μ L.Fig. 2 is the sensitivity experiments that in two-color fluorescence PCR system, CPV detects, and is from left to right followed successively by 1.0 * 10
7, 1.0 * 10
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2the amplification of the standard substance of copy/μ L.
Result proved test kit detection sensitivity is: 1.0 * 10
2copy/μ L, the method susceptibility of this double color fluorescent quantitative PCR is regular-PCR 200 times, accuracy is better than regular-PCR method.
eight, specificity experiment
According to above-mentioned double color fluorescent quantitative PCR detection method, with canine distemper virus, canine parvovirus, the two two positive and other virally carry out confirmatory experiment and product carried out to sequence verification as porcine influenza (SIV), pig vesicle Stomatovirus (VSV), foot and mouth disease virus (FMDV), Latex agglutination test (JEV), negative control, result confirms, the inventive method and test kit specificity are good, there is not false positive, the goodness of fit is 100%, and experimental result is shown in Fig. 3 and Fig. 4.Fig. 3 is the specificity experiment of CDV in double color fluorescent quantitative PCR detection method, as seen from the figure, CDV is positive, CPV, SIV, VSV, FMDV, JEV and negative quality control product all negative.Fig. 4 is the specificity experiment of CPV in double color fluorescent quantitative PCR detection method, as seen from the figure, CPV is positive, CDV, SIV, VSV, FMDV, JEV and negative quality control product all negative.
Test kit of the present invention confirms to have good repeatability and stability through the repeatedly different experiments that repeats.Under-20 ℃ of conditions, the validity period of test kit can reach 10 months.
Animal health institute of <110> Guangdong Academy of Agricultural Sciences
<120> canine distemper and canine parvovirus double color fluorescent quantitative PCR detection kit
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<170> PatentIn version 3.5
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Claims (3)
1. a canine distemper and canine parvovirus double color fluorescent quantitative PCR detection kit, comprise following composition: viral RNA nucleic acid extraction liquid, viral DNA nucleic acid extraction liquid, ThermoScript II system, Taq enzyme system, double color fluorescent quantitative PCR MIX, positive quality control product, negative quality control product and DEPC water, it is characterized in that: the Auele Specific Primer and the double-colored probe that in described double color fluorescent quantitative PCR MIX, contain 10 * double color fluorescent quantitative PCR buffer, dNTPs and canine distemper virus and canine parvovirus, sequence is respectively:
Canine distemper virus:
Upstream primer: CDV-F:5 '-GCAAGCGAGGATTGAGGGTAT-3 ';
Downstream primer: CDV-R:5 '-GGCCTATGGGACACACTCAAA-3 ';
Fluorescent probe:
CDV-P：5’-FAM- CACCGACCATCCAGACGTTGCTCCT-TAMRA-3’；
Canine parvovirus:
Upstream primer: CPV-F:5 '-CGTCTACACAAGGGCCATTTA-3 ';
Downstream primer: CPV-R:5 '-CCATGTTGTCTACCAAATGCAT-3 ';
Fluorescent probe:
CPV-P：5’-VIC- CGCAAACAGATGAAAATCAAGCAGCA-TAMRA-3’；
The fluorescent probe reporter group of canine distemper virus is FAM, and the fluorescent probe reporter group of canine parvovirus is VIC, and quenching group is TAMRA.
2. a kind of canine distemper according to claim 1 and canine parvovirus double color fluorescent quantitative PCR detection kit, it is characterized in that: in the every 1000mL of described viral RNA nucleic acid extraction liquid, contain: guanidinium isothiocyanate 480g, the Tris-HCl 500mL of 0.1M pH 6.4, the EDTA 120mL of 0.2M pH8.0, surplus is water.
3. a kind of canine distemper according to claim 1 and canine parvovirus double color fluorescent quantitative PCR detection kit, be characterised in that: Chelex-100, surplus that the Tris-HCl that described viral DNA nucleic acid extraction liquid contains 20mM NaOH, 10mM pH8.0, concentration are 2% (w/v) are water.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310562660.4A CN103614494B (en) | 2013-11-12 | 2013-11-12 | Two-color fluorogenic quantitative PCR (Polymerase Chain Reaction) detection kit for CDV (canine distemper viruses) and CPV (canine parvo viruses) |
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| CN109097488A (en) * | 2018-07-09 | 2018-12-28 | 北京市农林科学院 | For synchronizing nucleic acid, method and the kit of five kinds of dog diarrhea virus of detection and identification |
| CN110568204A (en) * | 2019-06-10 | 2019-12-13 | 青岛海润检测股份有限公司 | Chemiluminescence kit for one-step quantitative detection of canine distemper virus-canine parvovirus antibody |
| CN110607397A (en) * | 2019-09-03 | 2019-12-24 | 深圳市慧思基因科技有限公司 | Canine distemper virus dry-type real-time fluorescent PCR detection reagent |
| CN111500793A (en) * | 2020-06-22 | 2020-08-07 | 河北三狮生物科技有限公司 | Detection primer and kit for canine parvovirus and use method of detection primer and kit |
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