CN102417929B - Specific PCR detection method of Riemerella anatipestifer - Google Patents
Specific PCR detection method of Riemerella anatipestifer Download PDFInfo
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- CN102417929B CN102417929B CN201110369345.0A CN201110369345A CN102417929B CN 102417929 B CN102417929 B CN 102417929B CN 201110369345 A CN201110369345 A CN 201110369345A CN 102417929 B CN102417929 B CN 102417929B
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Abstract
The invention discloses a PCR detection method of Riemerella anatipestifer. The detection method comprises the following steps: 1, amplification primers are designed according to a conserved sequence of gyrB gene in a Riemerella anatipestifer genomic DNA sequence, wherein the conserved sequence is represented by SEQ ID NO:1; 2, DNA is extracted from a sample and is subjected to PCR amplification; 3, the obtained amplified product is detected through gel electrophoresis to determine whether the sample contains Riemerella anatipestifer; 5, if there is a corresponding single amplified strip in the electrophoresis result, the sample contains Riemerella anatipestifer; and 6, if there is no corresponding single amplified strip in the electrophoresis result, the sample contains no Riemerella anatipestifer. By adopting the detection method of the invention to detect Riemerella anatipestifer, the detection time is short, the cost is low, the detection result is specific, and the result determination is simple.
Description
Technical field
The present invention relates to a kind of poultry common disease field detection method, especially specific PCR detection method of Riemerella anatipestifer.
Background technology
Riemerella anatipestifer (Riemerella anatipestifer, RA) is the pathogenic bacteria of infectious serositis of duck, and this disease is harm aquatic bird aquaculture, particularly supports one of important epidemic disease of duck industry.In poultry, with the easy infection of duck, wherein the duck of many kinds such as cherry valley duck, kind duck, sheldrake, beautiful good duck all can fall ill.This disease is acute or chronic septicemia process, take the cheesy salpingitis of fibrinous pericarditis, serohepatitis, airsacculitis, meningitis and part, sacroiliitis as feature.Sickness rate is 10%-90%, and case fatality rate, up to 80%, all can occur throughout the year.Each foster duck area, the world nearly all has this disease popular, is that one of disease that duck industry is the most serious is supported in harm, and provisions duck industry has caused huge financial loss.Tradition identifies that RA detects some chemical constitution isophenous index of its cultural characters, form dyeing, physiological and biochemical property, thalline conventionally, but with phenotype index evaluation riemerella anatipestifer Shortcomings.In addition, traditional authentication method complicated operation, wastes time and energy, and is unfavorable for diagnosing in time the cause of disease, looks into cause of disease and controls spreading of the state of an illness.For these features of riemerella anatipestifer, many researchers have set up some detection techniques, as fluorescent-antibody technique, ELLSA, immunohistochemical methods etc. are reported in succession, but have in practice limitation in various degree.For detection riemerella anatipestifer that can be quick, accurate, easy, constantly make further progress in practice test take molecular biology as basic detection authentication method, it is one of detection method replacing the tool potentiality of traditional detection method, wherein, PCR with its sensitivity, special, easy, feature becomes one of important detection technique of molecular biology level fast.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of riemerella anatipestifer PCR detection method is provided.Adopt detection method of the present invention to detect riemerella anatipestifer, detection time is short, and cost is low, more has practicality, and detected result is special, and result judgement is simple.
The present invention realizes by following technical scheme:
A kind of riemerella anatipestifer PCR detection method, comprises the steps:
Step 1, according to the conserved sequence SEQ ID NO:1 design of amplification primers of gyrB gene in the genomic dna sequence of riemerella anatipestifer; Described primer is: forward primer sequence is as shown in SEQ ID NO:2; Reverse primer is as shown in SEQ ID NO:3;
Step 2, extracts sample DNA, the amplification of PCR method; PCR detection system is specially: 25 μ L reaction systems are specially, 10 × PCR damping fluid, 2.5 μ L, the Mg 2.0 μ L of 25mmol/L, the dNTP 1.0 μ L of 2.5mmol/L, Taq enzyme 0.25-1U, 5 μ M primer pair 1 μ L, template 2-5 μ L, finally mends to 25 μ L with distilled water; PCR detection reaction program: first 95 ℃ of denaturation 5min, start afterwards amplification cycles, the program of each circulation is: 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s; After 30 loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish;
Step 3, detected through gel electrophoresis amplified production, whether judgement sample contains riemerella anatipestifer; Described judgement is specially: if corresponding single amplified band appears in electrophoresis result, in interpret sample, contain riemerella anatipestifer; If there is not corresponding single amplified band, in sample, do not contain riemerella anatipestifer.
Described riemerella anatipestifer PCR detection method, in step 3, the described concrete grammar judging is: detect detected through gel electrophoresis amplified production and whether have single amplified band in 194bp position, if existed, in interpret sample, contain riemerella anatipestifer; If there is not corresponding single band, in sample, do not contain riemerella anatipestifer.
Compared with prior art, the present invention has following beneficial effect: adopt detection method of the present invention to detect riemerella anatipestifer, detection time is short, accurately, and fast.At the bottom of having avoided adopting traditional authentication method complex operation, length consuming time, recall rate, the shortcoming such as false positive is more.Need not adopt the antiserum(antisera) that must use in traditional method, reduced testing cost simultaneously.Detection target spot of the present invention has single specificity, and detected result is special, and result is judged simple.
Accompanying drawing explanation
Fig. 1 is PCR detection method specificity assessment experiment gel electrophoresis result figure in embodiment 2;
Fig. 2 is the sensitive evaluation experimental gel electrophoresis of PCR detection method result figure in embodiment 3.
Embodiment
Below in conjunction with concrete enforcement, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in following enforcement, conventionally according to for example Sambrook equimolecular clone of normal condition: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The foundation of riemerella anatipestifer PCR method
Pass through bioinformatic analysis, the gyrB gene that finds from riemerella anatipestifer genomic dna sequence that specific DNA is transcribed, copied, plays a significant role in restructuring and repair process, it is detected to target gene as riemerella anatipestifer, and gene order is as shown in SEQ ID NO:1;
The DNA sequence dna of this gene is input in primer-design software Primer Premier 5.0 and designs primer, it is 40-60% that GC% scope is set, product magnitude range is 150-300bp, from alternative primer pair, select primer, primer sequence following (primer is synthetic by Shanghai Ying Jun Technology Service Co., Ltd):
GYRB-L：5’-AGAGCGAGAAGAAAAAACCT-3’(SEQ ID NO：2)；
GYRB-R：5’-CTCCCATAAGCATAGAGAAGA-3’(SEQ ID NO：3)；
Step 2, the preparation of DNA profiling
The bacterial strain of the various serotypes of riemerella anatipestifer is seeded to respectively in the tryptic soy broth liquid nutrient medium of 50ml, increases after bacterium 12h at 37 ℃, get 1ml bacterium liquid and put into the centrifuge tube of 1.5ml, the centrifugal 10min of 3000r/min, get supernatant liquor, then at the centrifugal 5min of 12000r/min, collect thalline.With aseptic double-distilled water Eddy diffusion thalline, after centrifugal washing, add the aseptic ultrapure water of 100 μ L, in boiling water bath, boil 15 minutes, take out immediately, place 30min at-20 ℃; 37 ℃ thaw after, the centrifugal 5min of 12000r/min, get supernatant liquor place-20 ℃ for subsequent use.
Step 3 PCR detects
PCR detection system is specially: 25 μ L reaction systems are specially, 10 × PCR damping fluid, 2.5 μ L, the Mg 2.0 μ L of 25mmol/L, the dNTP 1.0 μ L of 2.5mmol/L, Taq enzyme 0,25-1U, 5 μ M primer pair 1 μ L, template solution is got 2-5 μ L, finally mends to 25 μ L with distilled water.PCR detection system Amplification is specially: first 95 ℃ of denaturation 5min, start afterwards amplification cycles, and the program of each circulation is: 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations; 72 ℃ are extended 10min, are cooled to 12 ℃, finish.
Step 4 judging criterion
Described judgement is specially: get pcr amplification product 5ul, the sepharose 2% carries out electrophoretic analysis, under ultra violet lamp, observes, if the single amplified band of corresponding 194bp appears in electrophoresis result, in interpret sample, contains riemerella anatipestifer; If there is not the single amplified band of corresponding 194bp, in sample, do not contain riemerella anatipestifer.
Result: get pcr amplification product 5ul, after 2% agarose gel electrophoresis, observe the single amplified band of about 194bp under ultra violet lamp, illustrate that set up PCR can detect riemerella anatipestifer.
Embodiment 2
The experiment of PCR Evaluation on specificity
Respectively according to implementing DNA profiling extracting method and PCR detection method in 1, the reference cultures such as intestinal bacteria, Salmonellas, streptococcus aureus, pasteurella multocida, bacillus pumilus, suis, Salmonella typhimurium, salmonella dublin, white dysentery Salmonellas, riemerella anatipestifer (as shown in table 1, bacterial strain shown in table be those skilled in the art can obtain by disclosed channel) are carried out to amplified reaction.
The detected result of specificity experiment is shown in Fig. 1, and negative control bacterial strain and strain number refer to accompanying drawing explanation.In figure: swimming lane 1-10: pasteurella multocida Pasteurella.multiocida PM966, Salmonellas Salmonella CMCC 50083-4, streptococcus aureus Staphylococcus aureus ATCC 6538, intestinal bacteria Escherichia 046, suis Streptococcosis CMCC32223 strain, bacillus pumilus Bacillus subtil CMCC63202 strain, mouse typhus Salmonella Salmonella.typhimurium 50115-13 strain, salmonella dublin Salmonella.dulin 50761 strains, white dysentery Salmonellas Salmonella.pullorum50047-2 strain, riemerella anatipestifer Riemerella anatipestiferATCC 11845 strains, swimming lane 11:ddH2O, swimming lane M:2000bp molecular weight standard.As can be known from Fig. 1, except riemerella anatipestifer reference culture ATCC 11845 strains, all the other bacteriums all do not have 194bp specific amplified band.
Table 1 Evaluation on specificity bacterial strain uses therefor and test-results
| Bacterial strain | Numbering | Bacterial strain | Result |
| Pasteurella.multiocida | PM966 | 1 | - |
| Salmonella | CMCC 50083-4 | 1 | - |
| Staphylococcus aureus | ATCC 6538 | 1 | - |
| Escherichia coli | 046 | 1 | - |
| Streptococcosis | CMCC32223 strain | 1 | - |
| Bacillus subtil | CMCC63202 strain | 1 | - |
| Salmonella.typhimurium | 50115-13 | 1 | - |
| Salmonella.dulin | 50761 | 1 | - |
| Salmonella.pullorum | 50047-2 | 1 | - |
| Riemerella anatipestifer | ATCC 11845 | 1 | + |
In table 1 ,-: PCR result is negative :+: PCR result is positive.
Embodiment 3
The sensitivity evaluation experimental of PCR
Extract the mensuration of the riemerella anatipestifer template DNA process OD260/280 obtaining with embodiment 1, the concentration of the total DNA solution of riemerella anatipestifer is 350ng/ μ l, do 10 times of gradient dilutions with sterilized water, dilute altogether 6 gradients, each gradient is got respectively 1 μ L and is added PCR reaction system, detected through gel electrophoresis amplified production is observed gel electrophoresis result in gel imaging instrument; As shown in Figure 2, in figure: swimming lane 1-6:DNA template content is respectively 350ng, 35ng, 3.5ng, 350pg, 35pg, 3.5pg and 350fg reaction; Swimming lane 7:ddH
2o; Swimming lane M:DL2000bp molecular weight standard.As shown in Figure 2, can see band clearly at the 5th article of swimming lane, the corresponding DNA concentration 35pg of institute, has good susceptibility.
Embodiment 4
The detection of the doubtful bacterial strain of riemerella anatipestifer
Utilize the specific PCR detection method of Riemerella anatipestifer that embodiment 1 sets up to detect the doubtful riemerella anatipestifer of 30 strain separating in the duck group of Sichuan periphery 20 Duo Ge large-scale cultivation factories; carry out Biolog system identification (by instrument and matched reagent specification sheets) simultaneously, the two result is compared.
Result: utilize specific PCR detection method of Riemerella anatipestifer that embodiment 1 sets up therefrom to detect the doubtful bacterial strain of the 10 strain result that is positive, the Biolog system identification result of the doubtful bacterial strain of this 10 strain is also riemerella anatipestifer (concrete authentication step refers to by instrument and matched reagent specification sheets); And other negative doubtful bacterial strain of PCR detection method, it is not riemerella anatipestifer that Biolog identifies.Prove by this enforcement, the specific PCR method of the riemerella anatipestifer of setting up has extreme high reliability.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (1)
1. the non-diagnostic assays method of riemerella anatipestifer PCR, is characterized in that, comprises the steps:
Step 1, according to the conserved sequence SEQID NO:1 design of amplification primers of gyrB gene in the genomic dna sequence of riemerella anatipestifer; Described primer is: forward primer sequence is as shown in SEQ ID NO:2; Reverse primer is as shown in SEQ ID NO:3;
Step 2, extracts sample DNA, the amplification of PCR method; PCR detection system is specially: 25 μ L reaction systems are specially, 10 × PCR damping fluid, 2.5 μ L, the Mg of 25mmol/L
2+2.0 μ L, the dNTP1.0 μ L of 2.5mmol/L, Taq enzyme 0.25-1U, 5 μ M primer pair 1 μ L, template 2-5 μ L, finally mends to 25 μ L with distilled water; PCR detection reaction program: first 95 ℃ of denaturation 5min, start afterwards amplification cycles, the program of each circulation is: 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s; After 30 loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish;
Step 3, detected through gel electrophoresis amplified production, whether judgement sample contains riemerella anatipestifer; Described judgement is specially: if corresponding single amplified band appears in electrophoresis result, in interpret sample, contain riemerella anatipestifer; If there is not corresponding single amplified band, in sample, do not contain riemerella anatipestifer; The described concrete grammar judging is: detect detected through gel electrophoresis amplified production and whether have single amplified band in 194bp position, if existed, in interpret sample, contain riemerella anatipestifer; If there is not corresponding single band, in sample, do not contain riemerella anatipestifer.
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| CN104962558A (en) * | 2015-07-06 | 2015-10-07 | 中国农业科学院兰州兽医研究所 | Specific primers for detection of riemerella anatipestifer and PCR (Polymerase Chain Reaction) detection kit |
| CN105886658B (en) * | 2016-06-28 | 2019-04-30 | 福建省农业科学院畜牧兽医研究所 | It is a kind of for detecting the multiple PCR primer of Riemerellosis Anatipestifer Major Virulence Factors |
| CN108823325B (en) * | 2018-07-27 | 2021-07-06 | 四川农业大学 | Application of riemerella anatipestifer Imp gene, PCR detection kit and method thereof |
| CN108866218B (en) * | 2018-07-27 | 2021-07-06 | 四川农业大学 | Application of Imp gene in preparation of kit for detecting riemerella anatipestifer serum 1 and method |
| CN109957622A (en) * | 2019-03-27 | 2019-07-02 | 华南农业大学 | It is a kind of detect duck infectious serositis RPA-LFD visualizing agent box and its application |
| CN111197095B (en) * | 2020-01-14 | 2023-07-07 | 温氏食品集团股份有限公司 | Screening method of vaccine strain |
| CN112626247A (en) * | 2021-01-20 | 2021-04-09 | 重庆永健生物技术有限责任公司 | Primer pair, kit and detection method for detecting riemerella anatipestifer |
| CN114874914B (en) * | 2022-01-18 | 2024-01-26 | 吉林大学 | Broad-spectrum antibacterial self-flocculating non-saccharomyces cerevisiae strain CC-P5 and application thereof |
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