CN107164472A - A kind of target gene, specific primer pair and detection method and kit for being used to detect the inferior salmonella of Dare - Google Patents
A kind of target gene, specific primer pair and detection method and kit for being used to detect the inferior salmonella of Dare Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 34
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- 238000001502 gel electrophoresis Methods 0.000 claims abstract description 14
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention provides a kind of target gene, specific primer pair and detection method and kit for being used to detect the inferior salmonella of Dare.Specific primer of the present invention is to being formed according to the inferior salmonella specific detection shot design of 5 Dares, and the stable high, high specificity of detection target spot, the detection method that the present invention is provided includes：Extract sample DNA, PCR amplifications；Detected through gel electrophoresis amplified production；Band after comparative electrophoresis.By experimental comparative analysis, the inventive method has unicity strong, and testing result is reliable, and the characteristics of result judgement is simple can be widely applied to food hygiene field.
Description
Technical field
The present invention relates to molecular biology and technical field of biological, it is related to a kind of for detecting the inferior salmonella of Dare
Target gene, specific primer pair and detection method and kit.
Background technology
The inferior salmonella of Dare (Salmonella Derby) be cause human infection main Salmonella serogroup it
One, the main host of this serological type strain is the mankind.The inferior salmonella of Dare can survive in livestock and poultry body, once the excrement of livestock and poultry
Or carry the raw meat of this germ and pollute food and processing site, in the environment of proper temperature, this bacterium will rapidly grow
Breeding, will result in food poisoning after bacterium number reaches certain amount in food, is eaten, and endanger the physical and mental health of people, sternly
Severe one even threat to life.Zheng Huaying, Zhou Dunjin etc. were in 2003《Enteron aisle caused by the inferior salmonella-polluted water source of Dare together
The popular investigation of outbreaks of infectious diseases》Middle display, the village of Wuhan City one occur successively because having drunk after non-sterile well water
Suffer from vomiting and diarrhoea, the symptoms such as heating of suffering from abdominal pain, subsequent reviewer carried out inspection by sampling analysis, and to find this accident be due to moral
Your inferior salmonella infection and cause.Thank to spring equality in 2013《The salmonella-polluted condition survey of Hefei district foodstuffs
Analysis》It is middle to find, the Salmonella of the common foodstuffs in Hefei district (raw meat, aquatic products, pot-stewed meat or fowl, dairy products, fermented bean products)
Bacterium pollution surveys shows, detects 22 plants of salmonella altogether in above food, wherein Salmonella derby accounts for 45.6%.
Clear stipulaties salmonella is essential items for inspection in 25th and 26 commands in 2002 of State Administration for Quality Supervision and Inspection and Quarantine.Mesh
Before, with the development for continuing to develop especially PCR (PCR) of molecular biology, detected and reflected by molecular engineering
Determining Salmonella serogroup becomes relatively reliable.
In the prior art, the inferior Salmeterol fluticasone propionate of Dare mainly uses traditional cultural method, whole detection process step
More, the time is longer, generally requires 4-6 working day, and the factor of interference is more, and the judgement of testing result is more complicated, gives
Food inspection brings very detrimental effect.And for the inferior salmonella of Dare PCR detect target gene research it is less and
Mainly due to not detecting target spot suitably.
The content of the invention
In view of the shortcomings of the prior art, the present invention provides a kind of target gene, specificity for being used to detect the inferior salmonella of Dare
Primer pair and detection method and kit, solve the inferior Salmeterol fluticasone propionate result judgement complexity of Dare in the prior art, detection
The technical problem of cycle length.
To realize object above, the present invention is achieved by the following technical programs：
It is a kind of to be used to detecting the target gene of the inferior salmonella of Dare, RU61_00441, RU61_00445, RU61_00447,
RU61_RS09205, RU61_RS06985, RU61_00441 have the nucleotide sequence or its spy as shown in SEQ ID NO.1
Specific fragment, RU61_00445 has the nucleotide sequence or its specific fragment as shown in SEQ ID NO.2, RU61_
00447 has the nucleotide sequence or its specific fragment as shown in SEQ ID NO.3, and RU61_RS09205 has such as SEQ
Nucleotide sequence or its specific fragment shown in ID NO.4, RU61_RS06985 have the nucleosides as shown in SEQ ID NO.5
Acid sequence or its specific fragment.
Specific primer pair for expanding the target gene, the specific primer is to being respectively： SD1、SD2、SD3、
SD11 and SD12, wherein,
SD1-f：Nucleotide sequence is as shown in SEQ ID NO.6, SD1-r：Nucleotide sequence is as shown in SEQ ID NO.7；
SD2-f：Nucleotide sequence is as shown in SEQ ID NO.8, SD2-r：Nucleotide sequence is as shown in SEQ ID NO.9；
SD3-f：Nucleotide sequence is as shown in SEQ ID NO.10, SD3-r：Nucleotide sequence such as SEQ ID NO.11 institutes
Show；
SD11-f：Nucleotide sequence is as shown in SEQ ID NO.12, SD3-11：Nucleotide sequence such as SEQ ID NO.13 institutes
Show；
SD12-f：Nucleotide sequence is as shown in SEQ ID NO.14, SD12-r：Nucleotide sequence such as SEQ ID NO.15 institutes
Show.
A kind of method for detecting the inferior salmonella of Dare, comprises the following steps：
S1, extraction sample DNA, PCR amplifications；
S2, detected through gel electrophoresis amplified production；
Band after S3, comparative electrophoresis, if having band, sample in 171bp, 164bp, 512bp, 608bp and 296bp position
Contain the inferior salmonella of Dare in product.
It is preferred that, the DNA fragmentation that 171bp is isolated position after the gel electrophoresis, its nucleotide sequence such as SEQ ID
Shown in NO.1；The DNA fragmentation that 164bp is isolated position, its nucleotide sequence is as shown in SEQ ID NO.2；512bp positions are separated
The DNA fragmentation gone out, its nucleotide sequence is as shown in SEQ ID NO.3；The DNA fragmentation that 608bp is isolated position, its nucleotides sequence
Row are as shown in SEQ ID NO.4；The DNA fragmentation that 296bp is isolated position, its nucleotide sequence is as shown in SEQ ID NO.5.
It is preferred that, in the PCR amplification procedures, 25 μ l include 2 × Master 12.5 μ l, 10 μM of μ l of primer pair 1, mould
Plate takes 1-5 μ l, mends ddH2O to 25 μ l.
It is preferred that, the response procedures of the PCR amplification procedures are：94 DEG C of pre-degeneration 10min, 94 DEG C are denatured 30s, 60 DEG C
Anneal 30s, 72 DEG C of extension 45s, 35 circulations, 72 DEG C of extension 10min.
It is preferred that, the gel electrophoresis uses agarose gel electrophoresis.
It is preferred that, measured with 25 μ lPCR reaction systems, for SD1, SD2, SD3, SD11 described in claim 2 and
SD12 primers, it is necessary to the inferior salmonella STb gene concentration of Dare >=2.685 ng/ μ l.
A kind of kit for being used to detect the inferior salmonella of Dare, the kit includes described specific primer pair.
A kind of described target gene or described specific primer pair or described kit are in the detection inferior Salmonella of Dare
Application in bacterium.
The present invention provide it is a kind of be used for detect the inferior salmonella of Dare target gene, specific primer pair and detection method and
Kit, compared with prior art advantage be：
The present invention analyzes the specific detection target gene for obtaining the inferior salmonella of Dare by genome comparison, so as to pass through
Specific primer is accurate to entering performing PCR amplification, the electrophoresis detection target gene, testing result of the present invention, and detection process is simple, inspection
The survey cycle is short, realizes the purpose of the inferior salmonella high specific quick detection of Dare；
The present invention provide detection 5 specific target genes of the inferior salmonella of Dare, corresponding PCR primer pairs, utilize with
Upper primer pair and target gene are detected that specific primer is to being set according to the inferior salmonella specific detection target spot of 5 Dares
Meter is formed, the stable high, high specificity of the specific detection target spot, passes through reasonably optimizing pcr amplification reaction system, gel electrophoresis inspection
Survey means, can quickly and accurately identify the inferior salmonella of Dare；
Can the inferior salmonella of Direct Identification Dare using the detection method of the present invention, it is not necessary to which serotype is tested, during detection
Between shorten within 12h, also, because detection process need not use the antiserum that must use in traditional detection method, can drop
Low testing cost, the present invention detects DNA fragment specific by PCR, strong with unicity, and testing result is reliable, result judgement
Simple the characteristics of, it can be widely applied for food hygiene field.
Brief description of the drawings
Fig. 1 is for specific primer of the present invention to SD1, SD2, SD3, SD11 and SD12 to different strain Evaluation on specificity gel
Electrophoresis result figure；
Fig. 2 is that specific primer of the present invention is evaluated different templates concentration sensitivity SD1, SD2, SD3, SD11 and SD12
Gel electrophoresis results figure.
No. 1 primer is SD1 specific primers pair in figure；No. 2 primers are SD2 specific primers pair；No. 3 primers are SD3 special
Specific primer pair；No. 11 primers are SD11 specific primers pair；No. 12 primers are SD12 specific primers pair；
1、ddH2O；2nd, the inferior salmonella of Dare；3rd, Salmonella choleraesuls；4th, moscow' paratyphi B；5th, the third type
Salmonella paratyphi；6th, Thompson salmonella；7th, Arizona salmonella；8th, salmonella dublin；9th, duck sramana
Salmonella；10th, the smooth salmonella in Persian；11st, salmonella aberdeen；12nd, S. infected cattles salmonella；13rd, mountain Fu Dunbao Salmonellas
Bacterium；14th, turkey salmonella；15th, Salmonella paratyphi A；16th, salmonella london；17th, salmonella typhimurium；
18th, Bacterium enteritidis；19th, cloth Randt salmonella；20th, Dakar salmonella；21st, Eastbourne salmonella；22nd, step
A Mi salmonellas；23rd, salmonella is received in Argonne；24th, the extra large moral salmonella of Bassens；25th, Meng Tewei sub- salmonella；26、
Jerusalem salmonella；27th, Bonn salmonella；28th, Boulogne Deng Lupu salmonellas；29th, moscow' saint paul；30、
Heidelberg salmonella；31st, salmonella kentucky；32nd, Bu Luokeli salmonellas；33rd, Beaune grace salmonella；
34th, prosperous hereby Butterworth salmonella；35th, Adelaide salmonella.
Embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, with reference to embodiment to the present invention
Technical scheme in embodiment is clearly and completely described, it is clear that described embodiment is that a part of the invention is implemented
Example, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creativeness
The every other embodiment obtained under the premise of work, belongs to the scope of protection of the invention.
Embodiment 1：
PCR detection method is to different strain Evaluation on specificity
1st, the screening of the inferior Salmonella serogroup specific gene of Dare
The complete of the inferior salmonella CVM40386 bacterial strains (NZ_JYYM01000031) of Dare is obtained in ncbi database
Genome sequence, utilizes each gene of the bacterium genome according to the method for Comparative genomic strategy the Blast programs of NCBA websites
Be compared, selection with the serotype homology higher (E value=0, Q μ ery cover=0%) and meanwhile with other microorganisms
The gene of homology very low (Q μ ery cover ＜ 10%) as the inferior salmonella of Dare quasi- specific gene.Pass through the party
Method obtains the quasi- specific gene of 12 inferior salmonellas of Dare altogether, then designs multipair primer for each gene, utilizes reality
The other serotypes of salmonella for testing room preservation carry out specificity verification.According to PCR results, RU61_00441, RU61_ are determined
00445th, RU61_00447, RU61_RS09205, RU61_RS06985 gene are used as the inferior Salmonella serogroup specificity of Dare
Detection target spot, gene order is respectively such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ
Shown in ID NO.5.
2nd, design of primers
By Primer5.0 software Design primers, set GC% scopes in 40-60%, primer size scope is 100-
1000bp, selects primer, 5 pairs of specific primers pair from alternative primer pair：SD1, SD2, SD3, SD11 and SD12, nucleosides
Acid sequence is respectively SD1-f：SEQ ID NO.6、 SD1-r：SEQ ID NO.7；SD2-f：SEQ ID NO.8、SD2-r：SEQ
ID NO.9； SD3-f：SEQ ID NO.10、SD3-r：SEQ ID NO.11；SD11-f：SEQ ID NO.12、 SD11-r：
SEQ ID NO.13；SD12-f：SEQ ID NO.14、SD12-r：SEQ ID NO.15；Entrust Shanghai life work biotechnology
Services Co., Ltd synthesizes.
3rd, the preparation of DNA profiling
46 plants of salmonella reference cultures such as the inferior salmonella of Dare (Salmonella derby) will be included and 18 plants non-
(bacterial strain that numbering is CICC is purchased from Chinese industrial Culture Collection to salmonella (such as table 1), and numbering is CMCC's
Bacterial strain be purchased from Chinese medicine Culture Collection, the bacterial strain for laboratory oneself preservation of no numbering, if other
Colleague for research need, this laboratory be ready provide the bacterial strain) each serotype be inoculated into respectively 30ml LB liquid training
Support in base, 37 DEG C of culture 8h.Each bacterium bacteria suspensions of 1ml are taken respectively in 1.5ml centrifuge tubes, and 12000r/min centrifuges 1min, abandoned
Supernatant.Twice, then with 100 μ l sterilizing pure water dissolvings precipitated with the 500 μ l pure water bacterial sediments sterilized.Boiled in boiling water bath
10min, 12000r/min are boiled, 1min is centrifuged, takes supernatant as pcr template.
4th, the foundation of pcr amplification reaction system
Set up PCR detection architectures：25 μ l reaction systems are included：2 × Master 12.5 μ l, 10 μM of μ l of primer pair 1, template
1 μ l are taken, ddH2O to 25 μ l is mended.
Set PCR response procedures：94 DEG C of pre-degenerations 10min, 94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extensions
45s, 30 circulations, 72 DEG C of extension 10min.According to set reaction system and response procedures respectively with above-mentioned step in PCR instrument
Each bacteria strain DNA extracted in rapid enters performing PCR amplification for template, is detected with doing gel electrophoresis.
5th, detected through gel electrophoresis
Using it is above-mentioned enter performing PCR amplification after product and as Marker DS2000DNA, ddH2O according to set order
Take 6 μ l to be added in 1% Ago-Gel respectively, run with 150V voltages after 40min, result is observed after GoldView dyeing.
PCR gel detections result such as Fig. 1, Tables 1 and 2, with including the inferior salmonella of Dare (Salmonella Derby)
37 plants of salmonella DNA inside as template, after PCR amplifications only the inferior salmonella of Dare 171bp, 164bp, 512bp,
There is specific electrophoretic band in 608bp and 296bp；More preferably to illustrate the specificity of the present invention, with the salmonella of separate sources
Totally 46 plants and 18 plants of nonsalmonella (in Tables 1 and 2, wherein " * " represents separation strains, other is reference culture) enter performing PCR expansion
Increase, it is as described in Table 1 that gel electrophoresis obtains result.Electrophoresis result occurs in 171bp, 164bp, 512bp, 608bp and 296bp position
During amplified band, it was demonstrated that be positive findings, represented with "+"；When the position does not have amplified band, it was demonstrated that be negative findings, with
"-" is represented；From table 1 it can also be seen that by this method PCR amplifications, the only inferior salmonella of Dare (Salmonella Derby)
There is positive reaction.Illustrate that the method stability that the present invention is provided is high dry straightly, high specificity is applied widely, inspection
Survey result accurate.
Embodiment 2：
PCR detection method is evaluated different templates concentration sensitivity
The screening of the inferior Salmonella serogroup specific gene of Dare, design of primers step such as embodiment 1.
The preparation of DNA profiling：The inferior salmonella single bacterium colony of picking Dare is transferred in 30ml LB fluid nutrient mediums, and 37
DEG C incubated overnight.The a small amount of extracts reagents of bacterial genomes DNA produced with Services Co., Ltd of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Box, extracts the inferior salmonella STb gene of Dare, after measured, and concentration is 265.8ng/ μ l.Make 10 times of gradient dilutions with sterilized water, altogether
7 gradients are diluted, pcr template is used as.
The foundation of pcr amplification reaction system：Set up PCR detection architectures：25 μ l reaction systems are included：2×Master 12.5
μ l, 10 μM of μ l of primer pair 1, template takes 5 μ l, mends ddH2O to 25 μ l.Design PCR response procedures：94 DEG C of pre-degeneration 10min, 94
DEG C denaturation 30s, 60 DEG C annealing 30s, 72 DEG C extension 45s, 30 circulation, 72 DEG C extension 10min.
6 gradient DNA profilings in above-mentioned steps are taken respectively to take 1 μ l to add PCR reaction systems, in progress reaction expansion in PCR instrument
Increase.6 μ lPCR amplified productions are taken into 1% Ago-Gel, are run with 150V voltages after 40min, are observed after GoldView dyeing
As a result.
SD1, SD2, SD3, SD11 and SD12 primer pair are directed to from the 1st to the 3rd swimming lane
There is band to show at 171bp, 164bp, 512bp, 608bp and 296bp, the 3rd swimming lane phase
The concentration answered is 2.658ng/ μ l, and (M is DNAladder I in Fig. 2 by electrophoresis result such as Fig. 2；
Template concentrations 2-10 be 265.85ng/ μ l, 26.59ng/ μ l, 2.69ng/ μ l, 268.59pg/ μ l,
26.86pg/ μ l, 2.69pg/ μ l, 268.59fg/ μ l, 26.86fg/ μ l and 2.69fg/ μ l) shown in.Cause
This, judges to examine for the PCR of 171bp, 164bp, 512bp, 608bp and 296bp primer
Survey sensitivity is 2.658ng/ μ l.
Bacterial strain and testing result used in the Evaluation on specificity of table 1
Bacterial strain and testing result used in the Evaluation on specificity of table 2
In summary, the present invention analyzes the specific detection target base for obtaining the inferior salmonella of Dare by genome comparison
Cause, thus expanded by specific primer to entering performing PCR, the electrophoresis detection target gene, testing result of the present invention is accurate, detection
Process is simple, and detection cycle is short, realizes the purpose of the inferior salmonella high specific quick detection of Dare；
The present invention provide detection 5 specific target genes of the inferior salmonella of Dare, corresponding PCR primer pairs, utilize with
Upper primer pair and target gene are detected that specific primer is to being set according to the inferior salmonella specific detection target spot of 5 Dares
Meter is formed, the stable high, high specificity of the specific detection target spot, passes through reasonably optimizing pcr amplification reaction system, gel electrophoresis inspection
Survey means, can quickly and accurately identify the inferior salmonella of Dare；
Can the inferior salmonella of Direct Identification Dare using the detection method of the present invention, it is not necessary to which serotype is tested, during detection
Between shorten within 12h, also, because detection process need not use the antiserum that must use in traditional detection method, can drop
Low testing cost, the present invention detects DNA fragment specific by PCR, strong with unicity, and testing result is reliable, result judgement
Simple the characteristics of.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
The present invention is described in detail, it will be understood by those within the art that：It still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these modification or
Replace, the essence of appropriate technical solution is departed from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (10)
- Be used to detecting the target gene of the inferior salmonella of Dare 1. a kind of, RU61_00441, RU61_00445, RU61_00447, RU61_RS09205, RU61_RS06985, it is characterised in that：RU61_00441 has the nucleotides as shown in SEQ ID NO.1 Sequence or its specific fragment, RU61_00445 have the nucleotide sequence or its specific fragment as shown in SEQ ID NO.2, RU61_00447 has the nucleotide sequence or its specific fragment as shown in SEQ ID NO.3, and RU61_RS09205 has such as Nucleotide sequence or its specific fragment shown in SEQ ID NO.4, RU61_RS06985 have as shown in SEQ ID NO.5 Nucleotide sequence or its specific fragment.
- 2. the specific primer pair for expanding target gene described in claim 1, it is characterised in that the specific primer is to dividing It is not：SD1, SD2, SD3, SD11, SD12, wherein,SD1-f：Nucleotide sequence is as shown in SEQ ID NO.6, SD1-r：Nucleotide sequence is as shown in SEQ ID NO.7；SD2-f：Nucleotide sequence is as shown in SEQ ID NO.8, SD2-r：Nucleotide sequence is as shown in SEQ ID NO.9；SD3-f：Nucleotide sequence is as shown in SEQ ID NO.10, SD3-r：Nucleotide sequence is as shown in SEQ ID NO.11；SD11-f：Nucleotide sequence is as shown in SEQ ID NO.12, SD11-r：Nucleotide sequence is as shown in SEQ ID NO.13；SD12-f：Nucleotide sequence is as shown in SEQ ID NO.14, SD12-r：Nucleotide sequence is as shown in SEQ ID NO.15.
- 3. a kind of method for detecting the inferior salmonella of Dare, it is characterised in that comprise the following steps：S1, extraction sample DNA, PCR amplifications；S2, detected through gel electrophoresis amplified production；Band after S3, comparative electrophoresis, if containing in having band, sample in 171bp, 164bp, 512bp, 608bp and 296bp position There is the inferior salmonella of Dare.
- 4. method according to claim 3, it is characterised in that：The DNA pieces that 171bp is isolated position after the gel electrophoresis Section, its nucleotide sequence is as shown in SEQ ID NO.1；The DNA fragmentation that 164bp is isolated position, its nucleotide sequence such as SEQ Shown in ID NO.2；The DNA fragmentation that 512bp is isolated position, its nucleotide sequence is as shown in SEQ ID NO.3；608bp positions The DNA fragmentation isolated, the DNA fragmentation that its nucleotide sequence 296bp positions as shown in SEQ ID NO.4 are isolated, its nucleosides Acid sequence is as shown in SEQ ID NO.5.
- 5. method according to claim 3, it is characterised in that：In the PCR amplification procedures, 25 μ l include 2 × The μ l of Master 12.5,10 μM of μ l of primer pair 1, template takes 1-5 μ l, mends ddH2O to 25 μ l.
- 6. method according to claim 3, it is characterised in that the response procedures of the PCR amplification procedures are：94 DEG C of pre- changes Property 10min, 94 DEG C denaturation 30s, 60 DEG C annealing 30s, 72 DEG C extension 45s, 35 circulation, 72 DEG C extension 10min.
- 7. method according to claim 3, it is characterised in that：The gel electrophoresis uses agarose gel electrophoresis.
- 8. method according to claim 3, it is characterised in that：Measured with 25 μ lPCR reaction systems, for claim 2 Described SD1, SD2, SD3, SD11 and SD12 primer is, it is necessary to the inferior salmonella STb gene concentration of Dare >=2.685ng/ μ l.
- 9. a kind of kit for being used to detect the inferior salmonella of Dare, it is characterised in that：The kit includes claim 2 institute The specific primer pair stated.
- 10. the specific primer pair described in target gene or claim 2 described in claim 1 or the examination described in claim 9 Application of the agent box in the detection inferior salmonella of Dare.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1397649A (en) * | 2002-08-19 | 2003-02-19 | 上海华冠生物芯片有限公司 | Biochip base on 16S rDNA gene for diagnosing frequently encountered pathogenic bacteria |
| AU2005203358A1 (en) * | 2004-07-28 | 2006-02-16 | Medvet Science Pty Ltd | Clonal identification of bacteria |
| WO2010133257A1 (en) * | 2009-05-22 | 2010-11-25 | Fondazione Parco Tecnologico Padano | Method for detection and identification of bacterial strains belonging to the classes escherichia coli, salmonella, campylobacter and listeria |
| CN104059977A (en) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | Salmonella serotype identification method and kit thereof |
-
2017
- 2017-05-19 CN CN201710354984.7A patent/CN107164472B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1397649A (en) * | 2002-08-19 | 2003-02-19 | 上海华冠生物芯片有限公司 | Biochip base on 16S rDNA gene for diagnosing frequently encountered pathogenic bacteria |
| AU2005203358A1 (en) * | 2004-07-28 | 2006-02-16 | Medvet Science Pty Ltd | Clonal identification of bacteria |
| WO2010133257A1 (en) * | 2009-05-22 | 2010-11-25 | Fondazione Parco Tecnologico Padano | Method for detection and identification of bacterial strains belonging to the classes escherichia coli, salmonella, campylobacter and listeria |
| CN104059977A (en) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | Salmonella serotype identification method and kit thereof |
Non-Patent Citations (6)
| Title |
|---|
| DECAI ZHANG等: "Label-free and high-sensitive detection of Salmonella using a surface plasmon resonance DNA-based biosensor", 《JOURNAL OF BIOTECHNOLOGY》 * |
| KEROUANTON,A.等: "Salmonella enterica subsp. enterica serovar Derby strain 07CR553, whole genome shotgun sequencing project GenBank: LAZB00000000.1", 《GENBANK》 * |
| RASHA M. GHARIEB等: "Non-Typhoidal Salmonella in poultry meat and diarrhoeic patients: prevalence, antibiogram, virulotyping, molecular detection and sequencing of class I integrons in multidrug resistant strains", 《GUT PATHOGENS》 * |
| 张毅等: "2010年上海市食源性沙门菌菌型分布和药敏分析以及快速检测方法的建立", 《现代生物医学进展》 * |
| 徐雪萍等: "《***共患病防治手册》", 30 April 2015, 金盾出版社 * |
| 翟立公: "沙门氏菌及其致病性血清型分子检测技术研究及在食品中的应用", 《中国博士学位论文全文数据库 基础科学辑》 * |
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