CN101736088A - Rapid differential diagnosis method of riemerella anatipestifer disease and colibacillosis - Google Patents
Rapid differential diagnosis method of riemerella anatipestifer disease and colibacillosis Download PDFInfo
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- CN101736088A CN101736088A CN201010102938A CN201010102938A CN101736088A CN 101736088 A CN101736088 A CN 101736088A CN 201010102938 A CN201010102938 A CN 201010102938A CN 201010102938 A CN201010102938 A CN 201010102938A CN 101736088 A CN101736088 A CN 101736088A
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Abstract
The invention discloses a rapid differential diagnosis method of riemerella anatipestifer disease and colibacillosis. The method includes that riemerella anatipestifer 42-KD outer membrane protein gene and Escherichia coli phoA gene are selected to synthesize two pair of specificity primers used for gene amplification of the two germs, the amplified segments are respectively 809bp and 622bp, difference of the two is 187bp, and the established RA and E.coli double PCR method can rapidly, specifically and sensitively can detect and differentiate the two bacteria. The invention has the advantage that the method of the invention has lower required expense, higher efficiency and stronger specificity compared with the traditional differential diagnosis method.
Description
Technical field
The invention belongs to biology field, specifically is the rapid differential diagnosis method of a kind of riemerella anatipestifer and colibacillosis.
Background technology
Riemerella anatipestifer disease (Riemerella anatipestifer infection, RAI) be by riemerella anatipestifer (Riemerella anatipestifer, RA) contagious disease of a kind of main harm 2-3 duckling in age in week that causes, this disease is acute or the chronic septicemia form, its pathological characters is fibrinous pericarditis, serohepatitis, airsacculitis, caseous salpingitis and meningitis, usually, bring certain difficulty for the diagnosis and the treatment of disease with intestinal bacteria polyinfection; This sick sickness rate height, mortality ratio height, mortality height, anti-ly cross duck poor growth etc. and the provisions duck is already caused enormous economic loss.RA is a gram negative bacillus.
Colibacillosis (Colibacillosis) is that (E.coli is a gram negative bacillus for Escherichia coli, the E.coli) disease that causes of some pathogenic serological type strain by intestinal bacteria.Colibacillosis takes place in duckling, and its morbidity pathological characters is very similar to the riemerella anatipestifer disease, mainly is fibrinous pericarditis, serohepatitis and airsacculitis, differentiates that by clinical symptom and pathological change these two kinds of eqpidemic diseases have certain difficulty.
Two kinds of diseases of traditional detection are to separate by bacterium, and then carry out the evaluation of biochemical reaction respectively, and the diagnosis that the research of having reported has just been set up PCR at the detection of RA lacks two kinds of bacteriums are mixed fast diagnosis method.For example happy great waves etc. have carried out the isolation identification of bacterium to the polyinfection of RA and E.coli; Hu Qinghai etc. have carried out correlative study to using round pcr detection riemerella anatipestifer.These methods of using the bacterium isolation identification detect RA and E.coli polyinfection, but exist consuming time and susceptibility not strong, and single PCR at RA detects, and the polyinfection of RA and E.coli is then failed to finish simultaneously detection.
Summary of the invention
For overcoming the deficiencies in the prior art, the invention provides Mo Shi bacillus and colibacillary quick discriminating detection method in a kind of duck infection.
The technical scheme that the present invention solves the problems of the technologies described above is:
Present method applied molecular biology technology, Mo Shi bacillus 42-KD outer membrane protein gene and intestinal bacteria phoA gene had synthesized two pairs of Auele Specific Primers in the selection duck infected, simultaneously the gene of two kinds of germs is increased respectively at a reactive system, the size of its amplified production is respectively 809bp and 622bp, both differ 187bp at size, are enough to both are obviously distinguished.Therefore, the double PCR method of the RA of foundation and intestinal bacteria can be diagnosed these two kinds bacterial duck diseases respectively on the one hand, can also differentiate these two kinds of similar bacteriums on the other hand.Has quick, special, responsive advantage.
The detection method step is as follows:
1) preparation of enrichment medium
Enrichment medium is prepared by following method:
With Nacl, Tryptones, the beef soup mixing, under pH value 7.2～7.4,121 ℃ of temperature or 107.9kPa condition of high voltage, sterilization 15min; Substratum is cooled to room temperature, aseptic adding calf serum, and making it ultimate density is 10%, it is standby to use or seal the placement room temperature preservation behind the mixing.
2) preparation of sample to be checked
A. bacterium plate isolation thing:
Doubtful sick duck is carried out bacterium separate with chocolate dull and stereotyped, the bacterium colony of growing on the plate of making even cultivate 24～48 hours in the candle cylinder after carries out 10 with sterile physiological salt
-9Dilution.
B. bacterium increases the bacterium culture:
To the die of illness cerebral tissue of duck, grinding the back dilutes with 1: 5 part by weight of sterile saline, add enrichment medium 500 μ L in the dilution pathological material of disease, the concentration of dilution pathological material of disease in substratum is 20%, cultivate 4h～8h at 37 ℃ of shaking tables, 10000r/min is centrifugal with culture, removes supernatant 400 μ L, remaining pathological material of disease culture piping and druming is evenly kept extracting DNA and is used.
3) sample DNA extracting to be checked
Get bacterium plate isolation thing of sick duck to be checked or the about 50ul of sample that bacterium increases the bacterium culture, place the 1.5mL centrifuge tube, place 99 ℃ of water-bath 10～15min, the template DNA of acquisition is used for pcr amplification or puts-20 ℃ of preservations.
4) PCR detects
The A.DNA extracting
After bacterium plate isolation thing and bacterium increased 99 ℃ of water-bath 10～15min of bacterium culture and handle ,-20 ℃ of preservations were standby.
B. reaction solution configuration
Under condition of ice bath, in the PCR reaction tubes, add following each composition respectively by following reagent dosage:
---10 * Buffer:2.5 μ L, final concentration are 1 times;
---25mmol MgCl
2: 1.5 μ L, final concentration are 1.5mmol;
---5U/ μ L Taq archaeal dna polymerase: 0.3 μ L, final concentration are 0.06U/ μ L;
---25mmol/L RA Auele Specific Primer: 1 μ L, final concentration are 0.5mmol/L;
---25mmol/L E.coli Auele Specific Primer: 1 μ L, final concentration are 0.5mmol/L.
---dNTPs: concentration is the 0.5 μ L of 2.5mM;
---dna profiling: 3 μ L.
---RNase Free: complementing to cumulative volume is 25 μ L.
C. response procedures
Get the reaction solution that step B configures, instantaneous centrifugal mixing places the PCR reaction tubes in the PCR instrument, reacts by following program: 1. 95 ℃, and 5min; 2. 94 ℃, 1min; 3. 55 ℃, 50s; 4. 72 ℃, 1min; 2.～4. circulate 30 times; 5. 72 ℃, 8min; 6. 4 ℃ are finished reaction; The PCR product is directly used in electrophoresis or is stored in 4 ℃ of refrigerators in order to electrophoresis; The DNA minimal detectable concentration of RA and E.coli is 2.98pg.
5) the PCR detected result is judged
10 μ L PCR products and 2 μ l gel electrophoresis sample loading buffers are mixed, add in the sepharose sample well, the molecular weight dna Marker that sets up standard simultaneously contrast, carry out electrophoresis with the voltage stabilizing that 5V/cm glue is long, when the tetrabromophenol sulfonphthalein indicator arrives from the terminal 2cm of sepharose～3cm, stop.
Sepharose behind the electrophoresis placed on the ultraviolet transilluminator observe, with standard DNA molecular weight and positive control comparative analysis, result of determination, and with the gel imaging system preservation of taking pictures.
If use double PCR, a DNA cloning band appears in test sample on the fragment position of 809bp, be judged to be the RA positive, is designated as RA "+"; A DNA cloning band appears in test sample on the fragment position of 622bp, be judged to be the E.coli positive, is designated as E.coli "+"; Do not take out of now if test sample has DNA cloning in this position, two places, then be judged to be RA, E.coli feminine gender, be designated as RA (-), E.coli (-).
Advantage of the present invention is: the inventive method is compared with traditional differential diagnosis method, and the expense that needs is lower, efficient is higher, specificity is stronger.
Description of drawings
Fig. 1 is a primer specificity test agar sugar gel electrophoresis figure.
Among the figure:
1. standard DNA molecular weight (100bp ladder DNA Marker);
2-6. be RA amplification successively to sample intestinal bacteria (-), Salmonellas (-), pasteurellosis bacillus (-), staphylococcus (-) and riemerella anatipestifer (+);
7-11. be E.coli amplification successively to sample Salmonellas (-), pasteurellosis bacillus (-), staphylococcus (-), riemerella anatipestifer (-) and intestinal bacteria (+);
Annotate: the target gene fragment that " 809bp " obtains for amplification RA; The target gene fragment that " 622bp " obtains for amplification E.coli;
"-" expression detected result feminine gender; "+" expression detected result positive.
Fig. 2 is amplified reaction sensitivity test agarose gel electrophoresis figure.
Among the figure:
1. standard DNA molecular weight (100bp ladder DNA marker);
2. be the amplification electrophoresis result of 190ng/L with reference to strain RA (+) and E.coli (+) DNA concentration;
3. be the amplification electrophoresis result of 47.50ng/L with reference to strain RA (+) and E.coli (+) DNA concentration;
4. be the amplification electrophoresis result of 11.88ng/L with reference to strain RA (+) and E.coli (+) DNA concentration;
5. be the amplification electrophoresis result of 2.98ng/L with reference to strain RA (+) and E.coli (+) DNA concentration;
6. be the amplification electrophoresis result of 0.75ng/L with reference to strain RA (+) and E.coli (+) DNA concentration;
7. be the amplification electrophoresis result of 0.19ng/L with reference to strain RA (+) and E.coli (+) DNA concentration;
8. the amplification electrophoresis result of negative control (-).
Annotate: the target gene fragment that " 809bp " obtains for amplification RA; The target gene fragment that " 622bp " obtains for amplification E.coli;
"-" expression detected result feminine gender; "+" expression detected result positive.
Embodiment
1. the preparation of enrichment medium
Enrichment medium is prepared by following method:
——Nacl：0.5g；
---peptone: 1g;
---beef soup: 90mL (concentration is 0.5g/ml);
---calf serum: 10mL (5% concentration)
With Nacl: Tryptones, beef soup mixing, at the pH value 7.2～7.4 of standard, under 121 ℃ of temperature or the 107.9kPa condition of high voltage, sterilization 15min, substratum is cooled to room temperature, aseptic adding calf serum, and it is standby to use or seal the placement room temperature preservation behind the mixing.
Annotate: freshly prepd substratum all should be done sterility test, and tests with known positive bacteria and negative bacterium, to guarantee the quality of substratum.
2. the preparation of sample to be checked
Bacterium plate isolation thing: doubtful sick duck is carried out bacterium separate with chocolate dull and stereotyped, the growth bacterium colony carries out 10 on the plate of making even cultivate 24 ~ 48 hours in the candle cylinder after
-9Dilution.
Bacterium increases the bacterium culture: the cerebral tissue of the duck that will die of illness, grind back 1: 5 dilution proportion of sterile saline, the substratum 500 μ L (inoculum size 20% of dilution pathological material of disease in substratum) that add Tryptones meat soup+calf serum (10%) in the dilution pathological material of disease, 37 ℃ of shaking tables are cultivated 4h～8h, 10000r/min is centrifugal with culture, remove supernatant 400 μ L, remaining pathological material of disease culture piping and druming is evenly kept standby extracting DNA.
3. sample DNA extracting to be checked
Aseptic after taking sick duck to be checked to increase bacterium to cultivate tissue juice or the about 50ul of dilution of bacteria sample of separation and Culture, place 99 ℃ of water-bath 10～15min of 1.5mL centrifuge tube, the template DNA of acquisition can be directly used in pcr amplification or put-20 ℃ of preservations standby.
4.PCR detect
The DNA extracting: after bacterium plate isolation thing and bacterium increased 99 ℃ of water-bath 10～15min of bacterium culture and handle ,-20 ℃ of preservations were standby;
Reaction solution configuration: under condition of ice bath, in the PCR reaction tubes, add each composition respectively by following reagent dosage:
---10 * Buffer:2.5 μ L, final concentration are 1 times;
---25mmol MgCl
2: 1.5 μ L, final concentration are 1.5mmol;
---5U/ μ L Taq archaeal dna polymerase: 0.3 μ L, final concentration are 0.06U/ μ L;
---25mmol/L RA Auele Specific Primer: 1 μ L, final concentration are 0.5mmol/L;
---25mmol/L E.coli Auele Specific Primer: 1 μ L, final concentration are 0.5mmol/L
---dNTPs: concentration is the 0.5 μ L of 2.5mM;
---dna profiling: 3 μ L
---RNase Free: complementing to cumulative volume is 25 μ L.
Response procedures: add instantaneous centrifugal mixing behind the reagent.The PCR reaction tubes is placed in the PCR instrument, react by following program: 1. 95 ℃, 5min;
2. 94 ℃, 1min; 3. 55 ℃, 50s; 4. 72 ℃, 1min; 2.～4. circulate 30 times; 5. 72 ℃, 8min; 6. 4 ℃ are finished reaction.The PCR product is directly used in electrophoresis or is stored in 4 ℃ of refrigerators in order to electrophoresis.The DNA minimal detectable concentration of RA and E.coli is 2.98pg, 500nmol/L.
The primer sequence of the double PCR of table 1 and amplification fragment length
4.PCR detected result is judged
10 μ L PCR products and 2 μ l gel electrophoresis sample loading buffers are mixed, add in the sepharose sample well.The molecular weight dna Marker that sets up standard simultaneously contrast.Carry out electrophoresis with the voltage stabilizing that 5V/cm glue is long, when the tetrabromophenol sulfonphthalein indicator arrives from the terminal 2cm of sepharose～3cm, stop.
Sepharose behind the electrophoresis placed on the ultraviolet transilluminator observe, with standard DNA molecular weight and positive control comparative analysis, result of determination, and with the gel imaging system preservation of taking pictures.
If use double PCR, a DNA cloning band appears in test sample on the fragment position of 809bp, be judged to be the RA positive, is designated as RA "+"; A DNA cloning band appears in test sample on the fragment position of 622bp, be judged to be the E.coli positive, is designated as E.coli "+"; Do not take out of now if test sample has DNA cloning in this position, two places, then be judged to be RA, E.coli feminine gender, be designated as RA (-), E.coli (-).
Claims (1)
1. the rapid differential diagnosis method of riemerella anatipestifer and colibacillosis, it is characterized in that, select duck infect in Mo Shi bacillus 42-KD outer membrane protein gene and intestinal bacteria phoA gene synthesized the gene amplification that two pairs of Auele Specific Primers are used for two kinds of germs, its amplified fragments size is respectively 809bp and 622bp, both differ 187bp at size, and the double PCR method of the RA of foundation and E.coli can detect and differentiate simultaneously this two kinds of bacteriums quick, special, sensitively.The detection method step is as follows:
1) preparation of enrichment medium
Enrichment medium is prepared by following method:
With NaCl, Tryptones and beef soup mixing, under pH value 7.2～7.4,121 ℃ of temperature or 107.9kPa condition of high voltage, sterilization 15min; Substratum is cooled to room temperature, and it is 10% that aseptic adding calf serum makes it ultimate density, and it is standby to use or seal the placement room temperature preservation behind the mixing;
2) preparation of sample to be checked
A. bacterium plate isolation thing:
Doubtful sick duck is carried out bacterium separate with chocolate dull and stereotyped, the bacterium colony of growing on the plate of making even cultivate 24～48 hours in the candle cylinder after does 10 with stroke-physiological saline solution
-9Dilution;
B. bacterium increases the bacterium culture:
To the die of illness cerebral tissue of duck, grinding the back dilutes with 1: 5 part by weight of sterile saline, add enrichment medium 500 μ L in the dilution pathological material of disease, the ratio of dilution pathological material of disease in substratum is 20%, 37 ℃ of shakes are cultivated 4h～8h, and 10 000r/min are centrifugal with culture, remove supernatant 400 μ L, remaining culture piping and druming evenly gives over to extracting DNA and uses;
3) sample DNA extracting to be checked
Get bacterium plate isolation thing of sick duck to be checked or about 50 μ l of sample that bacterium increases the bacterium culture, in the 1.5mL centrifuge tube, put 99 ℃ of water-bath 10～15min, obtain template DNA, be used for pcr amplification or put-20 ℃ of preservations;
4) PCR detects
The A.DNA extracting
After bacterium plate isolation thing and bacterium increased 99 ℃ of water-bath 10～15min of bacterium culture and handle ,-20 ℃ of preservations were standby;
B. reaction solution configuration
Under condition of ice bath, in the PCR reaction tubes, add following each composition respectively by following reagent dosage:
---10 * Buffer:2.5 μ L (final concentration is 1 times);
---25mmol MgCl
2: 1.5 μ L (final concentration is 1.5mmol);
---5U/ μ L Taq archaeal dna polymerase: 0.3 μ L (final concentration is 0.06U/ μ L);
---25mmol/L RA upstream and downstream half-nest type primer: 1 μ L (final concentration is 0.5mmol/L);
---25mmol/L E.coli upstream and downstream half-nest type primer: 1 μ L (final concentration is 0.5mmol/L);
---dNTPs: concentration is the 0.5 μ L of 2.5mM;
---dna profiling: 3 μ L
---RNase Free distilled water: complementing to cumulative volume is 25 μ L;
C. response procedures
Get the reaction solution that steps A configures, instantaneous centrifugal mixing places the PCR reaction tubes in the PCR instrument, reacts by following program: 1. 95 ℃, and 5min; 2. 94 ℃, 1min; 3. 55 ℃, 50s; 4. 72 ℃, 1min; 2.～4. circulate 30 times; 5. 72 ℃, 8min; 6. 4 ℃ are finished reaction; The PCR product is directly used in electrophoresis or is stored in 4 ℃ of refrigerators in order to electrophoresis.The DNA minimal detectable concentration of RA and E.coli is 2.98pg;
5) judgement of PCR detected result
10 μ LPCR products and 2 μ l gel electrophoresis sample loading buffers are mixed, add in the sepharose sample well, the molecular weight dna Marker that sets up standard simultaneously contrast, carry out electrophoresis with the voltage stabilizing that 5V/cm glue is long, when the tetrabromophenol sulfonphthalein indicator arrives from the terminal 2cm of sepharose～3cm, stop;
Sepharose behind the electrophoresis placed on the ultraviolet transilluminator observe, with standard DNA molecular weight and positive control comparative analysis, result of determination, and with the gel imaging system preservation of taking pictures;
If use double PCR, a DNA cloning band appears in test sample on the fragment position of 809bp, be judged to be the RA positive, is designated as RA "+"; A DNA cloning band appears in test sample on the fragment position of 622bp, be judged to be the E.coli positive, is designated as E.coli "+"; Do not take out of now if test sample has DNA cloning in this position, two places, then be judged to be RA and E.coli feminine gender, be designated as RA (-) and E.coli (-).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181549A (en) * | 2011-04-14 | 2011-09-14 | 中国农业科学院上海兽医研究所 | Multi-PCR detection kit and detection method for duck-origin common bacteria |
| CN102409084A (en) * | 2010-09-21 | 2012-04-11 | 中国农业科学院上海兽医研究所 | Riemerella anatipestifer LAMP detection kit and its detection method |
| CN106702021A (en) * | 2016-12-28 | 2017-05-24 | 贵州省畜牧兽医研究所 | Duck plague and escherichia coli dual PCR diagnostic kit |
-
2010
- 2010-01-29 CN CN201010102938A patent/CN101736088A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409084A (en) * | 2010-09-21 | 2012-04-11 | 中国农业科学院上海兽医研究所 | Riemerella anatipestifer LAMP detection kit and its detection method |
| CN102409084B (en) * | 2010-09-21 | 2013-06-12 | 中国农业科学院上海兽医研究所 | Riemerella anatipestifer LAMP detection kit and its detection method |
| CN102181549A (en) * | 2011-04-14 | 2011-09-14 | 中国农业科学院上海兽医研究所 | Multi-PCR detection kit and detection method for duck-origin common bacteria |
| CN106702021A (en) * | 2016-12-28 | 2017-05-24 | 贵州省畜牧兽医研究所 | Duck plague and escherichia coli dual PCR diagnostic kit |
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Open date: 20100616 |