CN101353699A - Polyase chain reaction detecting method of Campylobacter jejuni - Google Patents
Polyase chain reaction detecting method of Campylobacter jejuni Download PDFInfo
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- CN101353699A CN101353699A CNA2008100427872A CN200810042787A CN101353699A CN 101353699 A CN101353699 A CN 101353699A CN A2008100427872 A CNA2008100427872 A CN A2008100427872A CN 200810042787 A CN200810042787 A CN 200810042787A CN 101353699 A CN101353699 A CN 101353699A
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Abstract
The invention relates to a detection method of the polymerase chain reaction of jejunum campylobacter, comprising the steps that micro-oxygen-needed separated cultivation is carried out to the jejunum campylobacter, suspicious colony is selected to be dissolved in a buffer solution specially used for dissolving the jejunum campylobacter, supernatant is taken out to be used as the template of PCR after boiling decentralization; then, a pair of specific primers are designed according to a conservative segment in a VS1 gene sequence of the jejunum campylobacter to carry out PCR reaction, the reaction product is observed by using agarose gel electrophoresis, and positive result is obtained if a 516bp strip appears. The method of the invention detects the jejunum campylobacter directly from the suspicious colony, the process of bacteria-increasing cultivation is eliminated, and the designed specific primers improve the sensitivity and the specificity of the PCR detection method, thus solving the difficulty of long detection time of the jejunum campylobacter by the conventional method, and providing a reliable and fast detection method for the detection of the jejunum campylobacter in livestock, fowl and the products thereof.
Description
Technical field
The present invention relates to a kind of detection method of Amphixenosis's cause of disease-campylobacter jejuni, be specifically related to a kind of polymerase chain reaction (PCR) detection method of campylobacter jejuni, belong to medical biotechnology field.
Background technology
Campylobacter jejuni (Campylobacter jejuni) is a kind of of Campylobacter, be to be subjected to both at home and abroad the extensively infecting both domestic animals and human cause of disease bacterium of attention in recent ten years, mainly can cause human body acute enteritis and food poisoning, and immunity injury diseases such as the reactive arthritis that occurs together, hepatitis, auspicious Te Shi disease and Green-barre syndrome.Veterinarily, can cause that the domestic animal miscarriage is infertile, mastitis and cub fowl diarrhoea and poultry hepatitis.This bacterium enters in the environment by animal carrier or trouble patient's ight soil, and " live non-the cultivation " (the Viable but nonculturer-able that in water surrounding, can enter a kind of being called, VBNC) resting stage of state, the bacterium that is in the VBNC state can recover and have pathogenic under given conditions.The contact fowl poultry kind, or eat the main path that the poultry product, water source and the milk that are polluted are this bacterium infection human bodies.The World Health Organization has classified this disease as one of modal food source sexually transmitted disease.
Campylobacter jejuni culture condition harshness, do not grow at 25 ℃, 42 ℃ of well-growns VBNC that gets the hang of easily, make conventional separation and Culture and biochemical identification time and effort consuming, sensitivity not high, required time long (generally needing 48-72h), and be unfavorable for breaking out the quick diagnosis of epidemic situation, do not meet clinical pathogenic bacteria yet and detect desired principle fast and accurately.In recent years, PCR method has obtained tremendous development, and it has advantages such as highly sensitive, high specificity, detection be quick.Yet there is the not high defective of specificity in the domestic and international PCR method of reporting to the discriminating of campylobacter jejuni and campylobacter coli.Therefore, set up a kind of can detect campylobacter jejuni in the animals and animal product fast, succinctly, PCR method accurately, to realize, accurate detection easy, to effective control with prevent such food source sexually transmitted disease significant to pathogenic campylobacter jejuni.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of polyase chain reaction detecting method of campylobacter jejuni is provided, can detect the campylobacter jejuni in the animal product fast, succinctly, accurately.
For realizing this purpose, the present invention has adopted a kind of colony polymerase chain reaction (PCR) method, at first campylobacter jejuni is carried out little aerobic separation and Culture, obtain suspicious campylobacter jejuni, the single suspicious bacterium colony of picking is dissolved in the damping fluid (K Buffer) that is exclusively used in the dissolving campylobacter jejuni again, boil centrifugal after, get the template of supernatant as PCR, design a pair of Auele Specific Primer according to the conservative fragments in the campylobacter jejuni VS1 gene order then and carry out the PCR reaction, reaction product is observed with agarose gel electrophoresis, the 516bp band occurs and is positive findings.
Method concrete steps of the present invention are as follows:
1, suspicious bacterium colony separation and Culture: aseptic get animal tissues pack into the sterilization vessel in, as sample to be checked; Smash tissue to pieces homogenate, with the filtering with microporous membrane of 0.65um, directly streak inoculation is selecting to place little aerobic culture apparatus to cultivate for 42 ℃ on the culture medium flat plate earlier.
2, the suspicious bacterium colony of picking: the suspicious bacterium colony of picking from the bacterium colony of sample cultivation gained, wherein: the suspicious bacterium colony of I type is haemolysis not, canescence, pale brown look (light red may occur), flat, moistening, glossy, translucent looking looks like water droplet, protruding, smooth, the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The suspicious bacterium colony of II type is haemolysis not also, and is circular, glossy, translucent, and (diameter is 1～2mm), and the edge is complete, shinny often to be the single bacterium colony that disperses projection.
3, the preparation picking I type of pcr template or the suspicious bacterium colony of II type are dissolved in the damping fluid that is exclusively used in the dissolving campylobacter jejuni, boil behind the mixing, and the centrifuging and taking supernatant is as the template of PCR.The described damping fluid composition that is exclusively used in the dissolving campylobacter jejuni: K buffer 50mM KCl, 10mM Tris pH 8.0,2.5mMMgCl2,0.5%Tween 20,100ug/ml proteinase K.
4, the PCR design of primers is online with synthetic basis
Conservative fragments in the campylobacter jejuni VS1 gene order that (http://www.sanger.ac.uk/Projects/C_jejuni/) announces designs a pair of special primer: VS-F:5 ' TAC GAT TTG TTT CAT TG 3 ', VS-R:5 ' ATT TCT TTA GCA GGC ATA 3 '.
5, the PCR of suspicious bacterium colony detects and utilizes special primer to carry out pcr amplification according to ordinary method, and the dna fragmentation length that amplifies is 516bp.
6, the evaluation of pcr amplification product is got the PCR product at agarose gel electrophoresis, gets glue and places gel imaging system to take and analyze, and band occurs at the 516bp place, can determine that sample is a campylobacter jejuni.
The inventive method directly detects campylobacter jejuni from suspicious bacterium colony, saved and increased bacterium cultivation link, the primer of design has also increased the sensitivity and the specificity of PCR detection method, solve ordinary method and detected long difficult point of campylobacter jejuni time, provide fast for campylobacter jejuni in poultry, fowl and products thereof detects, succinctly, detection method accurately and reliably, suitable grass-roots unit applies.
Embodiment
Below by specific embodiment technical scheme of the present invention is described in further detail.Following examples do not constitute limitation of the invention.
Embodiment 1
1, suspicious bacterium colony separation and Culture: get chicken liver under the aseptic condition, pack into and add in the penicillin bottle of an amount of sterile saline, with the filtering with microporous membrane of 0.65um, directly streak inoculation is selected to place little aerobic culture apparatus to cultivate for 42 ℃ on the culture medium flat plate at improvement Camp-BAP earlier.
2, the suspicious bacterium colony of picking: the suspicious bacterium colony of picking from the bacterium colony of sample cultivation gained, wherein: the suspicious bacterium colony of I type is haemolysis not, canescence, pale brown look, flat, moistening, glossy, and drops is translucent, protruding, smooth, the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The suspicious bacterium colony of II type is haemolysis not also, and is circular, glossy, translucent, and (diameter is 1～2mm), and the edge is complete, shinny often to be the single bacterium colony that disperses projection.Above-mentioned bacterium colony all can be used as the suspicious bacterium colony of campylobacter jejuni and further identifies.
3, suspicious I type bacterium colony of the making picking of pcr template, be dissolved in 80ul and be exclusively used in (K buffer 50mM KCl in the damping fluid that dissolves campylobacter jejuni, 10mM Tris pH 8.0,2.5mM MgCl2,0.5%Tween 20,100ug/ml proteinase K), boiled behind the mixing 10 minutes, centrifugal then 5 minutes, get the template of 5 μ l supernatant liquors as PCR.
4, the PCR design of primers is with synthetic
According to the conservative fragments in the campylobacter jejuni VS1 gene order of online (http://www.sanger.ac.uk/Projects/C_jejuni/) announcement, with a pair of special primer of Premier 5.0 software designs.Sequence is as follows::
VS-F：5’TAC?GAT?TTG?TTT?CAT?TG 3’
VS-R：5’ATT?TCT?TTA?GCA?GGC?ATA 3’。
5, the PCR of suspicious I type bacterium colony detection amplified reaction cumulative volume is 25 μ l, the redistilled water 8.5 μ l that wherein sterilize, and PCR MasterMix 12.5 μ l (comprise DNATaq E, dNTP, buffer, Mg
+ 2), the 20pmol/L primer (VS-F:5 ' TAC GAT TTG TTT CAT TG 3 ', VS-R:5 ' ATT TCT TTA GCA GGCATA 3 ') each 1 μ l, template DNA 5 μ l. put the enterprising performing PCR reaction of DNA cloning instrument.Loop parameter is 94 ℃ of pre-sex change 5min, 94 ℃ of 30S, and 55 ℃ of 60S, 72 ℃ of 60S, totally 35 circulations, last 72 ℃ are extended 10min.
The dna fragmentation length that amplifies is 516bp.
6,1% sepharose is disposed in the evaluation of pcr amplification product according to a conventional method.Add ethidium bromide (EB) glue by 0.5mg/L.Get 5 μ l PCR products and 1 μ l, 10 * loading buffer mixing application of sample.Contrast with DL2000Marker.90V voltage electrophoresis 40min.Get glue and place gel imaging system to take and analyze, the result shows, clear band occurs at the 516bp place, though fuzzy assorted band background is arranged, can determine substantially to contain campylobacter jejuni in the sample.
Embodiment 2
1, suspicious bacterium colony separation and Culture: get the pig caecum under the aseptic condition and pack into and add in the penicillin bottle of an amount of sterile saline, elder generation is with the filtering with microporous membrane of 0.65um, directly streak inoculation is selected to place little aerobic culture apparatus to cultivate for 42 ℃ on the culture medium flat plate at improvement Camp-BAP.
2, the suspicious bacterium colony of picking: the suspicious bacterium colony of picking from the bacterium colony of sample cultivation gained, wherein: the suspicious bacterium colony of I type is haemolysis not, canescence, pale brown look (light red may occur), flat, moistening, glossy, translucent looking looks like water droplet, protruding, smooth, the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The suspicious bacterium colony of II type is haemolysis not also, and is circular, glossy, translucent, and (diameter is 1～2mm), and the edge is complete, shinny often to be the single bacterium colony that disperses projection.
3, suspicious II type bacterium colony of the making picking of pcr template, be dissolved in 80ul and be exclusively used in (K buffer 50mM KCl in the damping fluid that dissolves campylobacter jejuni, 10mM Tris pH 8.0,2.5mM MgCl2,0.5%Tween 20,100ug/ml proteinase K), boiled behind the mixing 10 minutes, centrifugal then 5 minutes, get the template of 5ul supernatant liquor as PCR.
4, the PCR design of primers is with synthetic
According to the conservative fragments in the campylobacter jejuni VS1 gene order of online (http://www.sanger.ac.uk/Projects/C_jejuni/) announcement, with a pair of special primer of Premier 5.0 software designs.Primer sequence is as follows:
VS-F：5’TAC?GAT?TTG?TTT?CAT?TG?3’
VS-R：5’ATT?TCT?TTA?GCA?GGC?ATA?3’。
5, the PCR of suspicious II type bacterium colony detection amplified reaction cumulative volume is 25 μ l, the redistilled water 8.5 μ l that wherein sterilize, and PCR MasterMix 12.5 μ l (comprise DNATaq E, dNTP, buffer, Mg
+ 2), the 20pmol/L primer (VS-F:5 ' TAC GAT TTG TTT CAT TG 3 ', VS-R:5 ' ATT TCT TTA GCA GGCATA 3 ') each 1 μ l, template DNA 5 μ l. put the enterprising performing PCR reaction of DNA cloning instrument.Loop parameter is 94 ℃ of pre-sex change 5min, 94 ℃ of 30S, and 58 ℃ of 60S, 72 ℃ of 60S, totally 35 circulations, last 72 ℃ are extended 10min.
The dna fragmentation length that amplifies is 516bp.
6,1% sepharose is disposed in the evaluation of pcr amplification product according to a conventional method.Add ethidium bromide (EB) glue by 0.5mg/L.Get 5 μ l PCR products and 1 μ l, 10 * loading buffer mixing application of sample.Contrast with DL2000Marker.90V voltage electrophoresis 40min.Get glue and place gel imaging system to take and analyze, the result shows, occurs at the 516bp place cleaning band, thereby contains campylobacter jejuni in definite sample.The temperature of withdrawing from a secret society or underworld gang in this step changes 58 ℃ into, and background does not have assorted band, shows very strong specificity.
Claims (1)
1, a kind of polyase chain reaction detecting method of campylobacter jejuni is characterized in that comprising the steps:
1) the aseptic animal tissues of getting packs in the sterilization vessel as sample to be checked, and animal tissues is smashed to pieces after the homogenate with the filtering with microporous membrane of 0.65um, and directly streak inoculation is selecting to place little aerobic culture apparatus to cultivate for 42 ℃ on the culture medium flat plate;
2) the suspicious bacterium colony of picking from the bacterium colony of sample cultivation gained, wherein: the suspicious bacterium colony of I type is haemolysis not, and canescence, pale brown look or light red are flat, moistening, glossy, translucent, protruding, smooth, and the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The suspicious bacterium colony of II type is haemolysis not, and is circular, glossy, translucent, and often being and disperseing the diameter of projection is the single bacterium colony of 1～2mm, and the edge is complete, shinny;
3) I type or the suspicious bacterium colony of II type with picking is dissolved in the damping fluid that is exclusively used in the dissolving campylobacter jejuni, boils behind the mixing, and the centrifuging and taking supernatant is as the template of polymerase chain reaction PCR; The described damping fluid composition that is exclusively used in the dissolving campylobacter jejuni: K buffer 50mM KCl, 10mM Tris pH 8.0,2.5mMMgCl2,0.5%Tween 20,100ug/ml proteinase K;
4) design a pair of special primer according to the conservative fragments in the campylobacter jejuni VS1 gene order: VS-F:5 ' TAC GAT TTG TTT CAT TG 3 ', VS-R:5 ' ATT TCT TTA GCA GGC ATA 3 ';
5) utilize special primer to carry out pcr amplification, the dna fragmentation length that amplifies is 516bp;
6) get pcr amplification product at agarose gel electrophoresis, get glue and place gel imaging system to take and analyze, band occurs, determine that promptly sample is a campylobacter jejuni at the 516bp place.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2008100427872A CN101353699A (en) | 2008-09-11 | 2008-09-11 | Polyase chain reaction detecting method of Campylobacter jejuni |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022334A (en) * | 2018-09-20 | 2018-12-18 | 河南省农业科学院畜牧兽医研究所 | Fast separating and purifying cultural method for Campylobacter spp |
| CN111020039A (en) * | 2019-12-30 | 2020-04-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Campylobacter jejuni species specific molecular target and rapid detection method thereof |
| CN111876509A (en) * | 2020-09-10 | 2020-11-03 | 广州医科大学附属第一医院（广州呼吸中心） | PCR method for detecting high-toxicity acinetobacter baumannii |
-
2008
- 2008-09-11 CN CNA2008100427872A patent/CN101353699A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022334A (en) * | 2018-09-20 | 2018-12-18 | 河南省农业科学院畜牧兽医研究所 | Fast separating and purifying cultural method for Campylobacter spp |
| CN111020039A (en) * | 2019-12-30 | 2020-04-17 | 广东省微生物研究所（广东省微生物分析检测中心） | Campylobacter jejuni species specific molecular target and rapid detection method thereof |
| CN111876509A (en) * | 2020-09-10 | 2020-11-03 | 广州医科大学附属第一医院（广州呼吸中心） | PCR method for detecting high-toxicity acinetobacter baumannii |
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Open date: 20090128 |