CN102251058B - Dual PCR (Polymerase Chain Reaction) quick detection method for rabbit hemorrhagic disease virus and rabbit pasteurellosis - Google Patents
Dual PCR (Polymerase Chain Reaction) quick detection method for rabbit hemorrhagic disease virus and rabbit pasteurellosis Download PDFInfo
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Abstract
The invention discloses a dual PCR (Polymerase Chain Reaction) quick detection method for a rabbit hemorrhagic disease virus and rabbit pasteurellosis. In the method, two pairs of specific primers capable of amplifying a VP60 gene and a kmt gene are designed according to a rabbit hemorrhagic disease virus VP60 gene and a rabbit pasteurellosis kmt gene sequence. A specific stripe of 645 bp occurring in an electrophoretogram indicates a positive rabbit hemorrhagic disease virus; and a specific stripe of 215 bp occurring in the electrophoretogram indicates positive rabbit pasteurellosis. As proved by a research, the method can be used for detecting the rabbit hemorrhagic disease virus and the rabbit pasteurellosis in the same reaction system, has the characteristics of quickness, specificity, sensitivity and stability, can be applied to quick differential diagnosis of a rabbit hemorrhagic disease and a rabbit pasteurellosis disease, and plays an important role in effectively preventing and treating the two diseases; and the defects of the conventional diagnostic method are overcome.
Description
Technical field
The present invention relates to the detection technique of rabbit poison and germ, relate in particular to rabbit hemorrhagic disease virus, rabbit pasteurellosis bacillus double PCR method for quick.
Background technology
Rabbit hemorrhagic disease (is commonly called as the rabbit pest, Rabbit hemorrhagic disease, be called for short RHD) be by rabbit hemorrhagic disease virus (Rabbit hemorrhagic disease virus, abbreviation RHDV) a kind of deadly infectious disease that causes, its infectivity is strong, the course of disease short, M ﹠ M is all very high, and in the warren of acute outburst, M ﹠ M can reach 100%.Rabbit Bacillus pasteurii disease (Rabbit pasteurellosis) is a kind of acute infectious disease that is caused by pasteurella multocida (Pasteurellamultocida), often causes large quantities of rabbit invasions and death.These two kinds of epidemic diseases have been brought huge financial loss to rabbit keeping, have seriously restricted the development of rabbit keeping.Because rabbit hemorrhagic disease is similar with pathological change with rabbit Bacillus pasteurii disease clinical symptom, conventional diagnostic method is difficult to diagnose fast and accurately, and causes easily mistaken diagnosis to delay treatment.
The round pcr of the invention such as K.Mullis in 1985 has started a Biological Revolution, and nowadays round pcr has become a kind of important scientific method of diagnostic use.Multiplex PCR (multiplex-PCR is called for short MPCR) refers to comprise two pairs or more primer in the same reaction tubes (or reaction system), carries out at one time the PCR reaction of a plurality of amplification of nucleic acid sequences.Compare with regular-PCR, MPCR have save time, economic, efficient advantage, also can reduce crossed contamination.
Through retrieval, do not find the correlative study report of rabbit hemorrhagic disease virus, rabbit pasteurellosis bacillus double PCR method for quick.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of quick, special, responsive, stable rabbit hemorrhagic disease virus, rabbit pasteurellosis bacillus double PCR method for quick.
Adopt following technical scheme for solving the problems of the technologies described above the present invention: rabbit hemorrhagic disease virus, rabbit pasteurellosis bacillus double PCR method for quick, lungs from rabbit to be checked, spleen, liver, extract total RNA in the tissue such as lymphoglandula, and with rabbit hemorrhagic disease virus target gene specific downstream primer total RNA reverse transcription is become CDNA, become the doubtful rabbit pasteurellosis bacillus of separation and Culture the tissue from rabbit to be checked simultaneously, from nutrient solution, extract dna profiling, then with the CDNA and the dna profiling that obtain, and join for two pairs of synthetic Auele Specific Primers of rabbit hemorrhagic disease virus VP60 gene and rabbit pasteurellosis bacillus kmt gene design and to carry out pcr amplification in the same reaction system, pcr amplification product is judged according to electrophoresis result at last through agarose gel electrophoresis; Wherein, primer sequence and the extension increasing sequence of PCR reaction are respectively:
The VP60 extension increasing sequence is the base sequence of sequence table SEQ .ID.No.3,
Primer kmt P1 sequence 5 '-GATGTCGGCATGAATTTCTC-3 '
Primer kmt P2 sequence 5 '-ATTGACTGGCAGGATGTGAT-3 '
The kmt extension increasing sequence is the base sequence of sequence table SEQ .ID.No.6.
Reaction system and the amplification program of PCR reaction are respectively:
Reaction system: Mg
2+Ionic concn 2.0m mol/L; DNTP concentration 0.8mmol/L; Taq archaeal dna polymerase concentration 0.08U/L; Primer concentration 0.4 μ mol/L;
Amplification program: 94 ℃ of 4min; 94 ℃ of 30s, 57 ℃ of 40s, 72 ℃ of 1min, 28 circulations; 72 ℃ of 10min.
The present invention designs the Auele Specific Primer of two pairs of can increase VP60 gene and kmt genes according to rabbit hemorrhagic disease virus VP60 gene and rabbit pasteurellosis bacillus kmt gene order, has set up rabbit hemorrhagic disease virus, rabbit pasteurellosis bacillus double PCR method for quick.The specific band that occurs 645bp in the electrophoretogram is that rabbit hemorrhagic disease virus is positive; The specific band that 215bp occurs is that the rabbit pasteurellosis bacillus is positive.Studies show that, the present invention can detect rabbit hemorrhagic disease virus and rabbit pasteurellosis bacillus in same reaction system, have quick, special, responsive, stable characteristics, overcome the deficiency of routine diagnostic method, the rapid differential diagnosis that can be used for rabbit hemorrhagic disease and rabbit Bacillus pasteurii disease, significant to these two kinds of epidemic diseases of effective prevention and control.
Description of drawings
Fig. 1 is the PCR electrophorogram of the specific test of rabbit hemorrhagic disease virus of the present invention, rabbit pasteurellosis bacillus double PCR method for quick, among the figure: M is 2000bp Marker, the 1st, the rabbit pasteurellosis bacillus, the 2nd, rabbit hemorrhagic disease virus, the 3rd, rabbit pasteurellosis bacillus-rabbit hemorrhagic disease virus, the 4th, rabbit E.coli, the 5th, rabbit staphylococcus, the 6th, rabbit clostridieum welchii, the 7th, rabbit suis.
Fig. 2 is the PCR electrophorogram of the sensitivity test of rabbit hemorrhagic disease virus of the present invention, rabbit pasteurellosis bacillus double PCR method for quick, among the figure: M is 2000bp Marker, 1 to 6 rabbit pasteurellosis bacillus DNA concentration is respectively 620ng/ μ l, 62ng/ μ l, 6.2ng/ μ l, 0.62ng/ μ l, 0.062ng/ μ l, 0.0062ng/ μ l, and 1 to 6 rabbit hemorrhagic disease virus CDNA concentration is respectively 700ng/ μ l, 70ng/ μ l, 7ng/ μ l, 0.7ng/ μ l, 0.07ng/ μ l, 0.007ng/ μ l.
Fig. 3 is the PCR electrophorogram of the stability test of rabbit hemorrhagic disease virus of the present invention, rabbit pasteurellosis bacillus double PCR method for quick, and among the figure: M is 2000bp Marker, and 1 to 3 is three parallel test results.
Fig. 4 is the detected result figure of the clinical sample one of embodiment 1, and among the figure: M is 2000bp Marker, the 1st, and positive control, the 2nd, negative control, the 3rd, clinical sample one.
Fig. 5 is the detected result figure of the clinical sample two to six of embodiment 1, and among the figure: M is 2000bp Marker, the 1st, and positive control, the 2nd, negative control, 3 to 7 is clinical samples two to six.
Embodiment
The research of rabbit hemorrhagic disease virus, rabbit pasteurellosis bacillus double PCR method for quick
1, design of primers
Conservative region for rabbit hemorrhagic disease virus VP60 gene and rabbit pasteurellosis bacillus kmt gene, Comprehensive analysis results in conjunction with DNAstar, oligo6.69, Primer Express 2.0 and NCBI-blast biological software, design two pairs of Auele Specific Primers, primer is synthetic by the rich Deco skill company limited that steps in Beijing, primer sequence parameter such as table 1:
Table 1 Auele Specific Primer parameter list
2, CDNA and dna profiling preparation
(1) rabbit hemorrhagic disease virus CDNA template preparation
1. virus total RNA is extracted
Evenly 1: 5 by volume ratio of the tissues such as the lungs of the positive rabbit of clip artificial challenge rabbit hemorrhagic disease virus, spleen, liver, lymphoglandula and sterilization PBS (processing through DEPC) is ground and is made the pathological material of disease suspension, minute install to 1.5ml EP pipe, with the pathological material of disease suspension in 4 ℃ of centrifugal 5min of 8000rpm.Get supernatant 300 μ L and move to another new 1.5ml EP pipe, add TrizolReagent 500 μ L, acutely rocked 10 seconds, place 10min in room temperature, then add chloroform 200 μ L, mixing is placed 3min under the room temperature gently.In 4 ℃ of centrifugal 10min of 12000rpm.The careful supernatant 400 μ L that draw are to another new 1.5ml EP pipe, and adding Virahol 500 μ L, and room temperature leaves standstill 10min behind the mixing, precipitated rna.In 4 ℃ of centrifugal 10min of 12000rpm.Abandon supernatant, add 75% ethanol, 600 μ L, leniently repeatedly put upside down the EP pipe 3-5 time.In 4 ℃ of centrifugal 5min of 10000rpm.Abandon supernatant, the dry rear 30 μ L DEPC of adding process water, dissolving RNA.The RNA sample can directly carry out reverse transcription, or puts-70 ℃ and save backup.
2. viral CDNA is synthetic
1. the RNA that obtains is joined following reaction system carry out reverse transcription, specific as follows:
Reaction conditions: 42 ℃ of 1h, 95 ℃ of 5min.The viral CDNA lysate that obtains is used for the double PCR amplification or puts-20 ℃ saving backup.
(2) rabbit pasteurellosis bacillus dna profiling preparation
Get 500 μ L rabbit pasteurella multocida and cultivate bacterium liquid to 1.5ml EP pipe, add 20 μ L Proteinase Ks (20mg/mL) and 10%SDS 50 μ L, abundant mixing, 55 ℃ of water-bath 1h, every 15min shakes once.Add phenol/chloroform of 570 μ L/primary isoamyl alcohol mixed solution, mixing is in the centrifugal 10min of 12000rpm.The careful supernatant 400-500 μ L that draws is to microtrabeculae, in the centrifugal 2min of 10000rpm.Abandon supernatant, add 75% ethanol, 600 μ L, fully shake up, in the centrifugal 2min of 10000rpm.Abandoned stream is crossed liquid, and is empty from 5min in the spacious lid of 10000rpm.Take out microtrabeculae and put in the new 1.5ml EP pipe, add sterilization ultrapure water 25 μ L, leave standstill 3min, in the centrifugal 5min of 12000rpm.The DNA lysate that obtains is used for the double PCR amplification or puts-20 ℃ saving backup.
(3) preparation method of rabbit E.coli, rabbit staphylococcus, rabbit clostridieum welchii, rabbit streptococcus DNA template is with (2).
3, double PCR amplification
Get 1 μ L sample CDNA/DNA to be checked template and carry out the double PCR amplification, the reaction system cumulative volume is 25 μ L, and each concentration of component is: Mg
2+Ionic concn 2.0mmol/L; DNTP concentration 0.8mmol/L; Taq archaeal dna polymerase concentration 0.08U/L; Two couples of primer VP60P1, VP60P2 and kmt P1, kmt P2 concentration are 0.4 μ mol/L.And adding 2.5 μ L10 * Buffer, rest part is supplied with the sterilization ultrapure water.The PCR reaction conditions is: 94 ℃ of 4min; 94 ℃ of 30s, 57 ℃ of 40s, 72 ℃ of 1min, 28 circulations; 72 ℃ of 10min.
4, the evaluation of pcr amplification product
Get pcr amplification product 8 μ L, point sample is in 1% sepharose (containing 0.5 μ g/mL ethidium bromide), with 2000bpMarker as the standard molecule reference.Gel behind the electrophoresis carries out the result and judges under uv analyzer, as seen the specific band of 645bp appears in rabbit hemorrhagic disease virus, the specific band of 215bp appears in the rabbit pasteurellosis bacillus, and rabbit E.coli, rabbit staphylococcus, rabbit clostridieum welchii, rabbit suis band (Fig. 1) all do not occur in same position simultaneously.The result shows the method high specificity.
5, double PCR sensitivity test result
Record rabbit hemorrhagic disease virus CDNA concentration and rabbit pasteurellosis bacillus DNA concentration is respectively 700ng/ μ l and 620ng/ μ l with ultraviolet spectrophotometer, then doing continuously 10 times, to be diluted to CDNA concentration be 0.007ng/ μ l, and DNA concentration is 0.0062ng/ μ l.Detect with dual-PCR method, determine that the minimum concentrations of rabbit hemorrhagic disease virus CDNA is 0.07ng/ μ l; The minimum concentrations of rabbit pasteurellosis bacillus DNA is 0.062ng/ μ l (Fig. 2).The result shows that the method susceptibility is high.
6, double PCR replica test result
Adopt same procedure to carry out revision test 3 times, the result shows that 3 tests all can amplify the specific band of 645bp and 215bp, and band brightness there was no significant difference (Fig. 3).Show the method good stability.
Sick rabbit sample to clinically doubtful trouble rabbit hemorrhagic disease or rabbit Bacillus pasteurii disease or the two polyinfection detects.The preparation method of the preparation method of employed primer, rabbit hemorrhagic disease virus CDNA template, rabbit pasteurellosis bacillus dna profiling, double PCR amplification system and reaction conditions, and PCR Product Identification method is the same.The result shows that clinical sample one amplifies the specific band of 645bp (Fig. 4), is the rabbit hemorrhagic disease virus positive; Clinical sample two to six amplifies the specific band (Fig. 5) of 215bp, is the rabbit pasteurellosis bacillus positive.
Claims (3)
1. rabbit hemorrhagic disease virus, the non-diagnosis method for quick of rabbit pasteurellosis bacillus double PCR, it is characterized in that the lungs from rabbit to be checked, spleen, liver, extract total RNA in the lymph node tissue, and with rabbit hemorrhagic disease virus target gene specific downstream primer total RNA reverse transcription is become CDNA, become the doubtful rabbit pasteurellosis bacillus of separation and Culture the tissue from rabbit to be checked simultaneously, from nutrient solution, extract dna profiling, then with the CDNA and the dna profiling that obtain, and join for two pairs of synthetic Auele Specific Primers of rabbit hemorrhagic disease virus VP60 gene and rabbit pasteurellosis bacillus kmt gene design and to carry out pcr amplification in the same reaction system, pcr amplification product is judged according to electrophoresis result at last through agarose gel electrophoresis; Wherein, primer sequence and the extension increasing sequence of PCR reaction are respectively:
Primer VP60 P1 sequence 5 '-CTCCTGGCAGCATACTTTAC-3 '
Primer VP60 P2 sequence 5 '-CTCTTCGGTGGTCAATGT-3 '
The VP60 extension increasing sequence is the base sequence of sequence table SEQ .ID.No.3,
Primer kmt P1 sequence 5 '-GATGTCGGCATGAATTTCTC-3 '
Primer kmt P2 sequence 5 '-ATTGACTGGCAGGATGTGAT-3 '
The kmt extension increasing sequence is the base sequence of sequence table SEQ .ID.No.6.
2. rabbit hemorrhagic disease virus according to claim 1, the non-diagnosis method for quick of rabbit pasteurellosis bacillus double PCR is characterized in that reaction system and the amplification program of described PCR reaction is respectively:
Reaction system: Mg
2+Ionic concn 2.0mmol/L; DNTP concentration 0.8mmol/L; Taq archaeal dna polymerase concentration 0.08U/L; Primer concentration 0.4 μ mol/L;
Amplification program: 94 ℃ of 4min; 94 ℃ of 30s, 57 ℃ of 40s, 72 ℃ of 1min, 28 circulations; 72 ℃ of 10min.
3. be used for the sequence of rabbit hemorrhagic disease virus claimed in claim 1, the quick non-diagnosis detecting method of rabbit pasteurellosis bacillus double PCR, comprise primer sequence and extension increasing sequence, sequence is respectively:
Primer VP60P1 sequence 5 '-CTCCTGGCAGCATACTTTAC-3 '
Primer VP60P2 sequence 5 '-CTCTTCGGTGGTCAATGT-3 '
The VP60 extension increasing sequence is the base sequence of sequence table SEQ .ID.No.3,
Primer kmt P1 sequence 5 '-GATGTCGGCATGAATTTCTC-3 '
Primer kmt P2 sequence 5 '-ATTGACTGGCAGGATGTGAT-3 '
The kmt extension increasing sequence is the base sequence of sequence table SEQ .ID.No.6.
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| CN1811388A (en) * | 2006-02-23 | 2006-08-02 | 田夫林 | Viral hemorrhagic viruse fluorescence quantitative detecting method for rabbit |
| CN101979661A (en) * | 2010-11-17 | 2011-02-23 | 浙江省农业科学院 | Primer group and kit for multiple PCR detection of rabbit Pasteurella multocida and rabbit Bordetella bronchiseptica and detection method thereof |
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| CN1811388A (en) * | 2006-02-23 | 2006-08-02 | 田夫林 | Viral hemorrhagic viruse fluorescence quantitative detecting method for rabbit |
| CN101979661A (en) * | 2010-11-17 | 2011-02-23 | 浙江省农业科学院 | Primer group and kit for multiple PCR detection of rabbit Pasteurella multocida and rabbit Bordetella bronchiseptica and detection method thereof |
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Effective date of registration: 20201211 Address after: No.33 Hengyang South Road, Jinhu County, Huaian City, Jiangsu Province 211600 Patentee after: Jinhu agricultural and sideline products Marketing Association Address before: 530001 No. 51 fraternity North Road, the Guangxi Zhuang Autonomous Region, Nanning Patentee before: Guangxi Veterinary Research Institute |