CN101974653A - Method for detecting canine distemper virus by using one-step RT-PCR method - Google Patents
Method for detecting canine distemper virus by using one-step RT-PCR method Download PDFInfo
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- CN101974653A CN101974653A CN2010105057224A CN201010505722A CN101974653A CN 101974653 A CN101974653 A CN 101974653A CN 2010105057224 A CN2010105057224 A CN 2010105057224A CN 201010505722 A CN201010505722 A CN 201010505722A CN 101974653 A CN101974653 A CN 101974653A
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Abstract
The invention discloses a method for detecting canine distemper virus by using a one-step reverse transcription-polymerase chain reaction (RT-PCR) method. The method comprises the following steps of: preprocessing a sample for the RT-PCR detection; extracting total ribonucleic acid (RNA); obtaining a specific amplification product by one-step RT-PCR reaction; and performing 1 percent agarose gel electrophoresis identification on the amplification product. The method of the invention has the characteristics of high flexibility, high specificity, simple and convenient operation procedure, low contamination rate and reliable detection result; the acquired sequence information is directly used for analyzing genetic derivation relation of an epidemic strain; and the method is suitable for etiological diagnosis and epidemiological research of canine distemper.
Description
Technical field
The invention belongs to field of virus detection, be specifically related to the method that a kind of utilization one one step RT-PCR method detects canine distemper virus.
Background technology
Canine distemper (Canine distemper) is to cause height contact, the acute infectious disease of dog by canine distemper virus (Canine distemper virus).CDV belongs to Paramyxoviridae Measles virus (MV) and belongs in classification, and between the Measles virus, rinderpest virus close antigen relation and common trait is arranged, and just the same with its ultra micro form and structure, and closely similar with newcastle disease and other paramyxovirus.Canine distemper virus is the extremely strong pathogenic agent of a kind of infectivity, is easy to propagate in the dog of susceptible and mink.Virus is present in multiple organs such as liver, spleen, lung, kidney, brain and lymphoglandula and the tissue, and the air by tears, nose liquid, saliva, urine and exhalation discharges virus.Canine distemper all can take place throughout the year, but with the Winter-Spring pilosity, this disease has certain periodicity, once is very popular in per 3 years, and the dog of different ages, sex and kind all can infect, but with minor pup susceptible the most.Bronchitis or pneumonia, gastro-enteritis and nervous symptoms with diphasic fever, acute coryza, conjunctivitis and appearance thereafter is feature clinically.Canine distemper is that the foster dog industry of current China, furbearer aquaculture and the conservation of wildlife already endanger one of maximum eqpidemic disease, often causes animals morbidities such as large quantities of dogs, ermine, fox, case fatality rate 30%-80%, and ferret is up to 100%.This has caused serious economy loss not only for furbearer plant (family), and seriously influences and restrict the fast development of whole industry.
According to epidemiologic data and the clinical symptom of CDV,, can make tentative diagnosis as cardinal symptoms such as two-way heat, mucous membrane catarrh, nervus centralis symptom and foot pad hyperkeratosiss; Making a definite diagnosis then needs to detect according to the laboratory, as manage that inclusion body inspection, virus separate etc., wherein the precondition diagnosed of detection specificity antibody is that the morbidity animal does not have the vaccine inoculation history, and these methods comprise agar diffusion precipitation test, ELISA and neutralization test, fluorescent-antibody technique and other immunohistochemistry techniques.Though aforesaid method has been obtained certain effect, what have is consuming time longer, and the susceptibility that has, specificity or accuracy are relatively poor, can not in time make a definite diagnosis.
Summary of the invention
The object of the invention is to provide a kind of utilization one one step RT-PCR method to detect the method for canine distemper virus, length consuming time, susceptibility and poor specificity when detecting to solve CDV, the problem that accuracy is not high.
In order to realize the object of the invention, technical scheme of the present invention adopts an one step RT-PCR method to detect canine distemper virus, and at a pair of Auele Specific Primer of N gene design of CDV among the GeneBank, sequence is as follows:
Upstream primer CDV 1:5 '-GGTCGGAGAATTTAGAATGAAC-3 ';
Downstream primer CDV 2:5 '-CCAAGAGCCGGATACATAG-3 '.
The concrete steps of present method are as follows:
1) the total RNA of canine distemper virus sample extracts, and surveys the concentration of extracting the total RNA in back;
2) the N gene fragment of one step RT-PCR method amplification CDV;
3) get amplified production and carry out the evaluation of 1% agarose gel electrophoresis.
The total RNA of the described canine distemper virus sample of step 1) extracts, and may further comprise the steps:
The pre-treatment of A, secretory product (nose, eyes, digestive tube), internal organs (heart, liver, spleen, lung, kidney) and cerebral tissue and lymphoglandula:
1. the pre-treatment of tissue sample: the sample thief diluent, (0.01mol/LPBS (pH7.2-7.4)) 10 * dilution grinds internal organs to be checked and cerebral tissue, gets 100 μ l lapping liquids, puts in the 1.5ml centrifuge tube, be stored in-20 ℃ to be checked.
2. the pre-treatment of liquid sample (nose liquid, eye conjunctival secretion, saliva, urine): when handling with the liquid sample of cotton swab collection, to gather cotton swab is inverted in the centrifuge tube, add diluted sample (0.01mol/LPBS (pH7.2-7.4)) 4 ℃ and leave standstill 2h, 4 ℃, the centrifugal 10min of 2000-3000r/min, get supernatant 200 μ l, put in the 1.5ml centrifuge tube, be stored in-20 ℃ to be checked.
B, extract total RNA:
Get sample 200 μ l to be checked in the 1.5mlEppendorf pipe, add 800 μ lTrizol, fully mixing, leave standstill 5min, add 200 μ l chloroform concuss 30s, 4 ℃, the centrifugal 10min of 12000r/min, water intaking phase 400-600 μ l adds isopyknic Virahol and puts upside down mixing gently, behind 4 ℃ of precooling 10min, 4 ℃, the centrifugal 10min of 10000r/min, supernatant discarded adds 600 μ l75 ﹪ ethanol, 4 ℃, the centrifugal 10min of 10000r/min, supernatant discarded adds 20 μ lDEPC-H
2O dissolving RNA precipitation.
Step 2) a described one step RT-PCR reaction system is:
Total rna solution 10.0 μ l
dNTPs（2.5mM） 5.0μl
10×Taq?Buffer 5.0μl
MgCL
2 10μl
Primer CDV 1(20 μ M) 2.0 μ l
Primer CDV 2(20 μ M) 1.0 μ l
Ex-Taq polysaccharase (5U/ μ l) 0.5 μ l
M-MuLV ThermoScript II (200U/ μ l) 1.0 μ l
RNasin（40U/μl） 1.0μl
DEPC-H
2O 14.5μl
Cumulative volume 50 μ l.
Step 2) a described one step RT-PCR response procedures is: 42 ℃ of reverse transcription 30min, 94 ℃ of deactivation ThermoScript II 2min, pre-94 ℃ of 2min of sex change; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min, this step is a circulation, totally 40 circulations; Last 72 ℃ are extended 10min.
The CDV genome is a minus strand linear rna virus; by 15690 based compositions; comprise 6 non-overlapped gene regions in the CDV genome; their encoding gene is followed successively by N-P-M-F-H-L series in proper order by 3 '～5 ' end; wherein N albumen is the stronger immunogenic protein of protectiveness; function with parcel and protection internal gene; the N gene is a conservative relatively structure gene between the different strains, different genotype of CDV, and the present invention has designed a pair of Auele Specific Primer according to the characteristics of N gene and has been used for an one step RT-PCR rapid detection.
Beneficial effect of the present invention: the present invention has set up the common method that can detect the animal tissues's sample and the liquid sample CDV of multiple canine distemper virus susceptible, have highly sensitive, quick, detect accurately, easy handling, pollution rate is low, cost is low advantage.
Description of drawings
Fig. 1 is 1% agarose gel electrophoresis figure of an one step RT-PCR amplified production, and the 1-8 swimming lane is followed successively by test sample from left to right; Swimming lane 9 positive control groups; Swimming lane 10 negative control groups; M is DL 2000bpmarker.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
Method therefor is ordinary method if no special instructions among the following embodiment.
The extraction of the total RNA of embodiment 1 liquid sample
1) pre-treatment of liquid sample (nose liquid, eye conjunctival secretion and saliva)
Respectively with nose liquid, eye conjunctival secretion and the saliva sample of cotton swab collection morbidity animal, the cotton swab of having gathered is inverted in the centrifuge tube, add sample diluting liquid (0.01mol/LPBS (pH7.2-7.4)) 4 ℃ and leave standstill 2h, the centrifugal 10min of 2000-3000r/min, get supernatant 200 μ l, put in the 1.5ml Eppendorf pipe, behind the clear and definite mark, put into-20 ℃ to be checked;
2) extraction of the total RNA of sample
Get sample 200 μ l to be checked in 1.5ml Eppendorf pipe, add 800 μ l Trizol, fully mixing leaves standstill 5min; Add 200 μ l analytical pure chloroform concuss 30s, 4 ℃, the centrifugal 10min of 12000r/min; Water intaking phase 400-600 μ l adds isopyknic Virahol and puts upside down mixing gently, behind 4 ℃ of precooling 10min, and 4 ℃, the centrifugal 10min of 10000 r/min; Supernatant discarded adds 600 μ l, 75 ﹪ ethanol, 4 ℃, the centrifugal 10min of 10000r/min; Supernatant discarded adds 20 μ lDEPC-H
2O dissolving RNA precipitation.
While,, carries out the extraction of total RNA simultaneously with test sample, and carries out mark not connect the poison cell culture as the negative control sample as the RT positive control sample with canine distemper virus low virulent strain cell culture.
1, the amplimer of design one one step RT-PCR
At a pair of Auele Specific Primer of N gene design of CDV among the GeneBank, sequence is as follows:
Upstream primer CDV 1:5 '-GGTCGGAGAATTTAGAATGAAC-3 ';
Downstream primer CDV 2:5 '-CCAAGAGCCGGATACATAG-3 ';
Primer is synthetic by the handsome Bioisystech Co., Ltd in Shanghai.
2, the foundation of RT-PCR reaction system
Carry 1) the total RNA precipitation of getting adds 20 μ l DEPC-H
2O makes its dissolving, gets 10 μ lRNA solution, adds 2.5mmol/LdNTPs 5.0 μ l, 10 * Taq Buffer, 5.0 μ l, MgCL
210 μ l, the primer CDV 1 2.0 μ l of 20pmol/ μ l, the primer CDV 2 1.0 μ l of 20pmol/ μ l, 5U/ μ lEx-Taq 0.5 μ l, the M-MuLV ThermoScript II 1.0 μ l of 200U/ μ l, the RNasin 1.0 μ l of 40U/ μ l, DEPC-H
2O 14.5 μ l, reaction totally be 50 μ l.
Be template with total RNA of positive control, total RNA of negative control respectively simultaneously, set up the RT-PCR reaction system, carry out mark.
Wherein an one step RT-PCR response procedures is:
42 ℃ of reverse transcription 30min, 94 ℃ of deactivation ThermoScript II 2min, pre-94 ℃ of 2min of sex change; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min, this step is a circulation, totally 40 circulations; Last 72 ℃ are extended 10min.
3, get amplified production and carry out the evaluation of 1% agarose gel electrophoresis
Get an one step RT-PCR amplified production of positive control, negative control and test set respectively, carry out 1% agarose gel electrophoresis, under ultraviolet lamp, observe amplification,
Experimental result is as shown in Figure 1: in positive controls, the 9th swimming lane bar segment size occurs for the specific band of 240bp, and in negative control group, the 10th swimming lane does not occur that a bar segment is big or small to be the specific band of 240bp;
At the 2nd swimming lane is the nasal discharge sample detection group of CDV dog, the 3rd swimming lane is the eye conjunctival secretion sample detection group of CDV dog, 7 swimming lanes are the saliva sample test set of CDV dog, bar segment size in these three swimming lanes, also all occurs and be the specific band of 240bp, illustrate that assay is positive;
1,4,5 swimming lanes are the nasal discharge sample detection group of healthy dogs, and 6 swimming lanes are the eye conjunctival secretion sample detection group of healthy dogs, and 8 swimming lanes are the saliva sample test set of healthy dogs, more than each swimming lane all do not have band and occur, illustrate that assay is negative;
To sum up, adopt this method can accurately detect canine distemper virus.
Claims (4)
1. utilization one an one step RT-PCR method detects the method for canine distemper virus (CDV), and it is characterized in that: at a pair of Auele Specific Primer of N gene design of CDV among the GenBank, sequence is as follows:
Upstream primer CDV 1:5 '-GGTCGGAGAATTTAGAATGAAC-3 ';
Downstream primer CDV 2:5 '-CCAAGAGCCGGATACATAG-3 ';
With CDV 1/ CDV 2 is primer, and the amplified production size is 240bp.
2. utilization one one step RT-PCR method according to claim 1 detects the method for canine distemper virus, and described detection method may further comprise the steps:
1) the total RNA of canine distemper virus sample extracts, and surveys the concentration of extracting the total RNA in back;
2) the N gene fragment of one step RT-PCR method amplification CDV;
3) get amplified production and carry out the evaluation of 1% agarose gel electrophoresis.
3. utilization one one step RT-PCR method according to claim 2 detects the method for canine distemper virus, it is characterized in that: step 2) a described one step RT-PCR reaction system is:
Total rna solution 10 μ l
dNTPs（2.5mM） 5.0μl
10×Taq?Buffer 5.0μl
MgCL
2 （2.5mM） 10μl
Primer CDV 1(20 μ M) 2.0 μ l
Primer CDV 2(20 μ M) 1.0 μ l
Ex-Taq polysaccharase (5U/ μ l) 0.5 μ l
M-MuLV ThermoScript II (200U/ μ l) 1.0 μ l
RNasin（40U/μl） 1.0μl
DEPC-H
2O 14.5μl
Cumulative volume 50 μ l.
4. utilization one one step RT-PCR method according to claim 2 detects the method for canine distemper virus, it is characterized in that: step 2) a described one step RT-PCR response procedures is: 42 ℃ of reverse transcription 30min, 94 ℃ of deactivation ThermoScript II 2min, pre-94 ℃ of 2min of sex change; 94 ℃ of 1min then, 52 ℃ of 1min, 72 ℃ of 1min, this step is a circulation, totally 40 circulations; Last 72 ℃ are extended 10min.
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| CN107916304A (en) * | 2017-12-29 | 2018-04-17 | 苏州点晶生物科技有限公司 | Canine distemper virus fluorescence EMA detection primers group, kit and detection method |
| CN108315482A (en) * | 2018-03-29 | 2018-07-24 | 河北出入境检验检疫局检验检疫技术中心 | Primer and probe and application thereof for canine distemper virus detection |
| CN111235312A (en) * | 2020-03-04 | 2020-06-05 | 东莞源和生物科技有限公司 | Primer, probe and detection kit for detecting canine distemper virus gene |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107916304A (en) * | 2017-12-29 | 2018-04-17 | 苏州点晶生物科技有限公司 | Canine distemper virus fluorescence EMA detection primers group, kit and detection method |
| CN108315482A (en) * | 2018-03-29 | 2018-07-24 | 河北出入境检验检疫局检验检疫技术中心 | Primer and probe and application thereof for canine distemper virus detection |
| CN111235312A (en) * | 2020-03-04 | 2020-06-05 | 东莞源和生物科技有限公司 | Primer, probe and detection kit for detecting canine distemper virus gene |
| CN111235312B (en) * | 2020-03-04 | 2023-12-19 | 东莞源和生物科技有限公司 | Primer, probe and detection kit for canine distemper virus gene detection |
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Application publication date: 20110216 |