CN104894283A - Primer, reagent box and method for detecting salmonella and integron (int) - Google Patents
Primer, reagent box and method for detecting salmonella and integron (int) Download PDFInfo
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Abstract
The invention discloses primer, a reagent box and a method for detecting salmonella and integrin (int). The primer comprises upstream primer SLMF: TGCGTCAGGACCTCATAG, downstream primer SLMR: ATGCCTGGAGGAGTGTTT, upstream primer intF: TTTACGCTGCTGTATGGT, and downstream primer intR for detecting int: TTATTGCTGGGATTAGGC. The reagent box is filled with 10 uL of HRM reaction premix liquid, 0.2-3.4 uL of primer, 1 uL of DNA template and is filled into 20 uL by water. The detecting method includes extracting sample DNA and performing PCR amplification. After amplification, products are subjected to high-resolution melting curve analysis, and results are judged by an amplification curve. The reagent box is reasonable in component and proportion, is convenient to use and quick and accurate to detect, the method is convenient and quick to operate, and low in cost, and detecting results are accurate.
Description
Technical field
The present invention designs a kind of primer for detecting Salmonellas SLM and drug resistant gene integron int; The invention still further relates to a kind of double fluorescent PCR detection kit comprising above-mentioned primer, the invention still further relates to a kind of method adopting mentioned reagent box to detect Salmonellas and drug resistant gene integron int.
Background technology
Salmonellas, Classification system salmonella, be called for short SLM, it is the major cause causing bacterial food poisoning, when the mankind eat contaminated food, without sterilization or sterilization halfway food time, just may cause food poisoning, within 2008, Salmonellas epidemic situation was once broken out in the U.S., and major cause is relevant with edible contaminated tomato.Within 2009, the U.S. also once recalled the Minced Beef by Salmonellas and e. coli contamination several times, and some green salad goods are also once because suffering intestinal bacteria and being salmonella-pollutedly called back.Show that the U.S. is often only according to WHO statistical information and just cause the ill and about 3900 people death of 330-1230 ten thousand people owing to infecting Salmonellas etc., annual financial loss is about 65-349 hundred million dollars.
China is production of antibiotics and uses big country, about 210,000 tons of annual production microbiotic, except part is for (about 30,000 tons) except exporting, all the other all for medical treatment and agriculturally, the existing aquaculture area of China about 5,730,000 hectares, wherein about 300,000 hectares is green non-pollution aquaculture area, various microbiotic is prohibitted the use at water surface for aquaculture, hormone medicine, in about 95% remaining aquaculture area, in order to prevent the reason such as outburst and infection causing disease because organism density is excessive, blindly in water, throw in microbiotic, as terramycin, paraxin, Nifurazolidone etc., be mixed into bait or directly add in aquaculture water and be used for the treatment of or the generation of anti-bacteria disease.
Integron is the new removable Genetic elements of discovered in recent years, can catch and integrate the drug resistant gene of bacterium, in the propagation and diffusion of bacterial drug resistance, serve vital effect.According to the homology of the intergrase of coding and catch the difference of box gene ability, current integron is divided into 6 classes, I-III class integron research more, and wherein the Ith class integron is the most common, and is present in enterobacteriaceae more.Have investigator to analyze the incoherent gram negative bacilli of 163 strain from 14 hospitals of 9 countries, as a result 43% bacterial strain in be separated to integron.Integron basic structure is made up of 3 parts, two ends are one section of highly conserved sequence conserved segament respectively, and Cs, is called as 5.Cs and 3.Cs, therebetween region is called variable region variable region, and variable region is made up of the box gene of one or more external insertion.Integrase gene is positioned at 5 conserved terminal of integron, 3 conserved terminal structures are then different because integron type is different, bacterium constantly can catch drug resistant gene box by integrase gene from surrounding environment, and there is multi-drug resistant, and an integron can catch one or more box genes, oneself has found nearly hundred kinds of drug resistant gene boxes at present, covers the Antibiotics of most of Clinical practice.Therefore, rapid, the control detected for resistant organism accurately of integron have very important significance.
Although part resistant organism that conventional bacteria Resistance detection method can meet detects needs, still there are some shortcomings in these methods, and such as detection time, longer and detected result was not accurate enough etc.Therefore, along with the application development of Protocols in Molecular Biology, the qualification of a series of quick bacteria or molecular detection technology are paid close attention to, and the feature of these class methods is quick and accurate, generally within several hours, just can obtain detected result, substantially reduce detection time.For Salmonellas and int genes involved, researchist has carried out Standard PCR and fluorescence PCR method research, but conventional PCR method carries out electrophoresis after needing pcr amplification, not only complex operation, and easily cause pollution, also need the fluorochrome using possibility carcinogenic, and although probe method fluorescence PCR method is sensitive and accurate, but its testing cost is higher, and reagent validity period is short, is easy to degraded.
Summary of the invention
The object of the invention is to overcome weak point of the prior art, a kind of primer for detecting Salmonellas and drug resistant gene integron int is provided;
Another object of the present invention is to provide one and comprises above-mentioned primer, and component and reasonable ratio, easy to use, detect quick, accurately, be applicable to the test kit that double fluorescent PCR detects Salmonellas and drug resistant gene integron int;
Another object of the present invention is to provide a kind of method adopting mentioned reagent box to detect Salmonellas and int gene, and the method is easy and simple to handle, quick, and cost is low, and detected result is accurate.
In order to achieve the above object, the present invention adopts following scheme:
Detect a primer for Salmonellas and drug resistant gene integron, it is characterized in that the sequence of this primer is respectively:
The upstream primer SLMF:TGCGTCAGGACCTCATAG that SLM detects
The downstream primer SLMR:ATGCCTGGAGGAGTGTTT that SLM detects
The upstream primer intF:TTTACGCTGCTGTATGGT that int detects
The downstream primer intR:TTATTGCTGGGATTAGGC that int detects.
Double fluorescent PCR detects a test kit for Salmonellas and int gene, it is characterized in that in this test kit, 20 μ L reaction systems comprise following component:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
The preferred version of test kit of the present invention: 20 μ L reaction systems comprise following component:
Detect a method for Salmonellas and drug resistant gene integron, it is characterized in that comprising the following steps:
A, extraction sample DNA;
B, test kit as claimed in claim 2 is utilized to carry out pcr amplification to the sample DNA in steps A;
After C, amplification, high resolving power melting curve analysis is carried out to product, and in conjunction with amplification curve result of determination.
The method of detection Salmonellas described above and drug resistant gene integron, is characterized in that the response procedures of pcr amplification in step B carries out according to the following steps:
(1) 92 DEG C ~ 96 DEG C denaturation 30s;
(2) 92 DEG C ~ 95 DEG C sex change 10s ~ 30s, 54 DEG C ~ 64 DEG C annealing 10s ~ 30s, 72 DEG C of 10s ~ 30s, carry out 35 ~ 45 circulations altogether;
(3) 72 DEG C extend 10s ~ 30s.
The method of detection Salmonellas described above and drug resistant gene integron, is characterized in that the melting curve analysis of high resolving power described in step C, carries out according to the following steps:
(1) 92 DEG C ~ 96 DEG C sex change 1min;
(2) 40 DEG C of renaturation 1min;
(3) then initial melting temperature (Tm) 60 DEG C ~ 65 DEG C, start program heats up and melts to 90 DEG C ~ 95 DEG C, and in fusion processes, detects fluorescent signal in real time, 15 ~ 25 times/second.
The method of detection Salmonellas described above and drug resistant gene integron, is characterized in that the concrete steps extracting sample DNA described in steps A are as follows:
Get the centrifugal 2min of 1.5mL culture 10000rpm; Throw out adds the TE damping fluid of 500 μ L, and piping and druming makes it Eddy diffusion repeatedly, adds the Proteinase K of 30 μ L 10%SDS and 15 μ L, and mixing, in 37 DEG C of incubation 1h; Add 100 μ L 5mol/L NaCl, fully mix, then add 80ul CTAB/NaCl solution, 65 DEG C of incubation 10min again after mixing; Add isopyknic phenol or chloroform or primary isoamyl alcohol to mix, centrifugal 4-5min, proceeds to supernatant in a new pipe, adds the Virahol of 0.8 times of volume, and mixing is until DNA precipitates gently; After 70% washing with alcohol of precipitation 1mL, add TE solubilize precipitation.
The preparation of Plays product template of the present invention:
With conventional PCR method amplifying target genes.PCR primer is through 1% gel electrophoresis analysis, adopt PCR primer to reclaim test kit to reclaim PCR primer, PCR primer after purifying is connected with carrier pMD18-T, to connect product conversion to competent cell, screening obtains single bacterium colony, and picking individual colonies is to containing antibiotic liquid nutrient medium, incubated overnight, extract plasmid, with the recombinant plasmid dna extracted for template carries out PCR qualification, and order-checking qualification is carried out to recombinant plasmid.Extract the positive recombinant plasmid that checking is correct, and measure its concentration, by its 10 times of doubling dilutions.
Sensitivity and Specificity test in the present invention:
Adopt the method such as order-checking to carry out sequence verification to positive amplification product, result positive amplification Product Sequence carries out Blast when comparing, sequence all with Genbank aim sequence very high homology.The positive plasmid template DNA that 10 times are diluted is added previous reaction system, carries out sensitivity experiments.The each drug resistant gene detection sensitivity of result all lower than 0.1pg, and has fine repeatability.In good linear relationship between each drug resistant gene dilution template concentrations and Cp value, R
2all be greater than 0.95, illustrate that the method has good tolerance range and satisfactory stability.
It can be food that the present invention is detected sample, and detection method is non-diseases diagnostic method.
The present invention compared with prior art, has the following advantages:
1) reagent constituents of the present invention and reasonable ratio, easy to use, detects quick, accurately, is applicable to fluorescent PCR and detects Salmonellas and int gene;
2) detection method simplifies testing process, substantially reduces sense cycle, and detection time shortens about two days than traditional culture assays method;
3) the whole process of detection method, PCR primer, without the need to proceeding to other analytical equipment again, realizes stopped pipe operation, avoids crossed contamination, and can complete quantitative analysis simultaneously.
4) adopt detection method can realize detecting Salmonellas and int gene in same reaction system, easy and simple to handle, quick, cost is low, and extend test kit validity period, detected result is accurate simultaneously.
Accompanying drawing explanation
Fig. 1 is the amplification curve diagram only having Salmonellas to detect, Ct value: 21.86;
Fig. 2 is the HRM analysis chart only having Salmonellas to detect, Tm value: 76.49;
Fig. 3 is the amplification curve diagram only having int to detect, Ct value: 23.90;
Fig. 4 be only have int to detect HRM analysis chart, Tm value: 78.83;
Fig. 5 is the amplification curve diagram that Salmonellas and int detect simultaneously, Ct value: 20.48;
Fig. 6 is the HRM analysis chart that Salmonellas and int detect simultaneously, Tm value: 73.91+77.25.
Embodiment
Below in conjunction with embodiment, the present invention is described further:
Embodiment 1
Double fluorescent PCR of the present invention detects the primer of Salmonellas SLM gene and drug resistant gene int, and sequence is respectively:
Upstream primer SLMF:TGCGTCAGGACCTCATAG
Downstream primer SLMR:ATGCCTGGAGGAGTGTTT
Upstream primer intF:TTTACGCTGCTGTATGGT
Downstream primer intR:TTATTGCTGGGATTAGGC.
Embodiment 2
20 μ L reaction systems are prepared composed of the following components in test kit of the present invention:
Wherein:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
Embodiment 3
20 μ L reaction systems are prepared composed of the following components in test kit of the present invention:
Wherein:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
Embodiment 4
20 μ L reaction systems are prepared composed of the following components in test kit of the present invention:
Wherein:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
Embodiment 5
20 μ L reaction systems are prepared composed of the following components in test kit of the present invention:
Wherein:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
Embodiment 6
20 μ L reaction systems are prepared composed of the following components in test kit of the present invention:
Wherein:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
Embodiment 7
20 μ L reaction systems are prepared composed of the following components in test kit of the present invention:
Wherein:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
Embodiment 8
A kind of method detecting Salmonellas and drug resistant gene integron of the present invention, comprises the following steps:
A, extraction sample DNA;
B, utilize the test kit detecting Salmonellas and drug resistant gene integron, pcr amplification is carried out to the sample DNA in steps A; In wherein said test kit, 20 μ L reaction systems comprise following component:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
Wherein the response procedures of pcr amplification carries out according to the following steps:
(1) 92 DEG C ~ 96 DEG C denaturation 30s;
(2) 92 DEG C ~ 95 DEG C sex change 10s ~ 30s, 54 DEG C ~ 64 DEG C annealing 10s ~ 30s, 72 DEG C of 10s ~ 30s, carry out 35 ~ 45 circulations altogether;
(3) 72 DEG C extend 10s ~ 30s.
After C, amplification, high resolving power melting curve analysis is carried out to product, and in conjunction with amplification curve result of determination.Described high resolving power melting curve analysis, carries out according to the following steps:
(1) 92 DEG C ~ 96 DEG C sex change 1min;
(2) 40 DEG C of renaturation 1min;
(3) then initial melting temperature (Tm) 60 DEG C ~ 65 DEG C, start program heats up and melts to 90 DEG C ~ 95 DEG C, and in fusion processes, detects fluorescent signal in real time, 15 ~ 25 times/second.
Embodiment 9
Get the centrifugal 2min of 1.5mL culture 10000rpm; Throw out adds the TE damping fluid of 500 μ L, and piping and druming makes it Eddy diffusion repeatedly, adds the Proteinase K of 30 μ L 10%SDS and 15 μ L, and mixing, in 37 DEG C of incubation 1h; Add 100 μ L 5mol/L NaCl, fully mix, then add 80ul CTAB/NaCl solution, 65 DEG C of incubation 10min again after mixing; Add isopyknic phenol/chloroform/primary isoamyl alcohol mixing, centrifugal 4-5min, proceeds to supernatant in a new pipe, adds the Virahol of 0.8 times of volume, and mixing is until DNA precipitates gently; After 70% washing with alcohol of precipitation 1mL, add TE solubilize precipitation, then get 1 μ L extract and join following reaction system,
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
Carry out fluorescent PCR amplification, carry out according to the following steps:
(1) 92 DEG C ~ 96 DEG C denaturation 30s;
(2) 92 DEG C ~ 96 DEG C sex change 5s ~ 10s, 54 DEG C ~ 64 DEG C annealing 20s ~ 40s, carry out 35 ~ 45 circulations altogether.
A, to amplification after product carry out HRM analyze after result of determination.
HRM analysis is carried out to pcr amplification product, carries out according to the following steps:
(1) 92 DEG C ~ 96 DEG C sex change 1min;
(2) 40 DEG C of renaturation 1min;
(3) then initial melting temperature (Tm) 60 DEG C ~ 65 DEG C, start program heats up and melts to 92 DEG C ~ 96 DEG C, and in fusion processes, detects fluorescent signal in real time, 15 ~ 25 times/second.
The preparation of Plays DNA masterplate of the present invention:
With conventional PCR method amplification drug resistant gene goal gene SLM and int.PCR primer is through 1% gel electrophoresis analysis, adopt PCR primer to reclaim test kit to reclaim PCR primer, PCR primer after purifying is connected with carrier pMD18-T, to connect product conversion to competent cell, screening obtains single bacterium colony, and picking individual colonies is to containing antibiotic liquid nutrient medium, incubated overnight, extract plasmid, with the recombinant plasmid dna extracted for template carries out PCR qualification, and order-checking qualification is carried out to recombinant plasmid.Extract the positive recombinant plasmid that checking is correct, and measure its concentration, by its 10 times of doubling dilutions.
Interpretation of result
If fluorescent pcr amplification has obvious amplification curve, Ct value <35, and also melting curve has a specificity crest, and Tm value is 76.49 ± 0.15 DEG C, can be judged to Salmonellas and detect.
If fluorescent pcr amplification has obvious amplification curve, Ct value <35, melting curve has a specificity crest, and Tm value is 78.83 ± 0.15 DEG C, can be judged to int and detect.
If fluorescent pcr amplification has obvious amplification curve, Ct value <35, and have two crests, Tm value is respectively 73.91 ± 0.15 DEG C and 77.25 ± 0.15 DEG C, can be judged to Salmonellas and int all detects.
If fluorescent pcr amplification curve is obvious, 35<Ct value <40, repeat once, if still have obvious upcurve, then have a goal gene at least for detecting, according to aforementioned melting curve analysis figure crest, a situation arises and Tm value carries out corresponding judgement simultaneously.
If the obvious Ct value >40 of fluorescent pcr amplification curve, or without obvious amplification curve and Tm value, be judged to Salmonellas and int all detects feminine gender.
Sensitivity and Specificity test in the present invention:
Adopt the method such as order-checking to carry out sequence verification to positive amplification product, result positive amplification Product Sequence carries out Blast when comparing, sequence all with Genbank aim sequence very high homology.The positive plasmid template DNA that 10 times are diluted is added previous reaction system, carries out sensitivity experiments.The each drug resistant gene detection sensitivity of result all lower than 0.1pg, and has fine repeatability.In good linear relationship between each drug resistant gene dilution template concentrations and Cp value, R
2all be greater than 0.95, illustrate that the method has good tolerance range and satisfactory stability.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (7)
1. detect a primer of Salmonellas and drug resistant gene integron int, it is characterized in that the sequence of this primer is respectively:
Upstream primer SLMF:TGCGTCAGGACCTCATAG
Downstream primer SLMR:ATGCCTGGAGGAGTGTTT
Upstream primer intF:TTTACGCTGCTGTATGGT
Downstream primer intR:TTATTGCTGGGATTAGGC.
2. detect a test kit for Salmonellas and drug resistant gene integron, it is characterized in that in this test kit, 20 μ L reaction systems comprise following component:
The sequence of wherein said upstream primer SLMF: TGCGTCAGGACCTCATAG
The sequence of described downstream primer SLMR: ATGCCTGGAGGAGTGTTT
The sequence of described upstream primer intF: TTTACGCTGCTGTATGGT
The sequence of described downstream primer intR: TTATTGCTGGGATTAGGC.
3. a kind of test kit detecting Salmonellas and drug resistant gene integron according to claim 2, is characterized in that in this test kit, 20 μ L reaction systems comprise following component:
4. detect a method for Salmonellas and drug resistant gene integron, it is characterized in that comprising the following steps:
A, extraction sample DNA;
B, test kit as claimed in claim 2 is utilized to carry out pcr amplification to the sample DNA in steps A;
After C, amplification, high resolving power melting curve analysis is carried out to product, and in conjunction with amplification curve result of determination.
5. detect the method for Salmonellas and drug resistant gene integron according to claim 4, it is characterized in that the response procedures of pcr amplification in step B carries out according to the following steps:
(1) 92 DEG C ~ 96 DEG C denaturation 30s;
(2) 92 DEG C ~ 95 DEG C sex change 10s ~ 30s, 54 DEG C ~ 64 DEG C annealing 10s ~ 30s, 72 DEG C of 10s ~ 30s, carry out 35 ~ 45 circulations altogether;
(3) 72 DEG C extend 10s ~ 30s.
6. according to claim 4 or 5, detect the method for Salmonellas and drug resistant gene integron, it is characterized in that the melting curve analysis of high resolving power described in step C, carry out according to the following steps:
(1) 92 DEG C ~ 96 DEG C sex change 1min;
(2) 40 DEG C of renaturation 1min;
(3) then initial melting temperature (Tm) 60 DEG C ~ 65 DEG C, start program heats up and melts to 90 DEG C ~ 95 DEG C, and in fusion processes, detects fluorescent signal in real time, 15 ~ 25 times/second.
7. detect the method for Salmonellas and drug resistant gene integron int according to claim 6, it is characterized in that the concrete steps extracting sample DNA described in steps A are as follows:
Get the centrifugal 2min of 1.5mL culture 10000rpm; Throw out adds the TE damping fluid of 500 μ L, and piping and druming makes it Eddy diffusion repeatedly, adds the Proteinase K of 30 μ L 10%SDS and 15 μ L, and mixing, in 37 DEG C of incubation 1h; Add 100 μ L 5mol/L NaCl, fully mix, then add 80ul CTAB/NaCl solution, 65 DEG C of incubation 10min again after mixing; Add isopyknic phenol or chloroform or primary isoamyl alcohol to mix, centrifugal 4-5min, proceeds to supernatant in a new pipe, adds the Virahol of 0.8 times of volume, and mixing is until DNA precipitates gently; After 70% washing with alcohol of precipitation 1mL, add TE solubilize precipitation.
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| CN106086208A (en) * | 2016-07-29 | 2016-11-09 | 中山出入境检验检疫局检验检疫技术中心 | For detecting vibrio parahaemolyticus and the test kit of integron and method in food |
| CN106148534A (en) * | 2016-07-29 | 2016-11-23 | 中山出入境检验检疫局检验检疫技术中心 | For detecting shigella and the test kit of integron and method in food |
| CN106222252A (en) * | 2016-07-25 | 2016-12-14 | 中国水产科学研究院东海水产研究所 | Detect multiple PCR primer and the detection method of three kinds of integron related elements simultaneously |
| CN106244685A (en) * | 2016-07-29 | 2016-12-21 | 中山出入境检验检疫局检验检疫技术中心 | For detecting test kit and the method for Salmonella in Food and integron |
| CN106893779A (en) * | 2017-02-23 | 2017-06-27 | 浙江工商大学 | Primer and its application for carrying the salmonella RPA quick detections of Sulfonamides-resistant genes |
| CN107419014A (en) * | 2017-07-17 | 2017-12-01 | 蔡先全 | Detect pseudomonas aeruginosa and aprA primer, kit and method in water |
| CN107419016A (en) * | 2017-07-17 | 2017-12-01 | 蔡先全 | Detect the primer of pseudomonas aeruginosa and integron, kit and method in water |
| CN111549158A (en) * | 2020-06-08 | 2020-08-18 | 河北工程大学 | Method for detecting one-class integron gene intI1 in atmospheric environment |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015023616A2 (en) * | 2013-08-12 | 2015-02-19 | The Johns Hopkins University | Method for comprehensive, quantitative, and highly sensitive discrimination of nucleic acid sequences in homogeneous and heterogeneous populations |
| CN104651487A (en) * | 2014-11-05 | 2015-05-27 | 蔡先全 | Kit and multiple fluorescent PCR detection method for detecting food-borne pathogenic bacteria |
-
2015
- 2015-06-25 CN CN201510363834.3A patent/CN104894283A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015023616A2 (en) * | 2013-08-12 | 2015-02-19 | The Johns Hopkins University | Method for comprehensive, quantitative, and highly sensitive discrimination of nucleic acid sequences in homogeneous and heterogeneous populations |
| CN104651487A (en) * | 2014-11-05 | 2015-05-27 | 蔡先全 | Kit and multiple fluorescent PCR detection method for detecting food-borne pathogenic bacteria |
Non-Patent Citations (2)
| Title |
|---|
| ALIEDA VAN ESSEN-ZANDBERGEN: "Occurrence and characteristics of class 1, 2 and 3 integrons in Escherichia coli, Salmonella and Campylobacter spp. in the Netherlands", 《JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY》 * |
| PATRı´CIA ANTUNES: "Characterization of antimicrobial resistance and class 1 and 2 integrons in Salmonella enterica isolates from different sources in Portugal", 《JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106222252A (en) * | 2016-07-25 | 2016-12-14 | 中国水产科学研究院东海水产研究所 | Detect multiple PCR primer and the detection method of three kinds of integron related elements simultaneously |
| CN106048052A (en) * | 2016-07-29 | 2016-10-26 | 中山出入境检验检疫局检验检疫技术中心 | Kit and method for detecting staphylococcus aureus and integron in food |
| CN106086208A (en) * | 2016-07-29 | 2016-11-09 | 中山出入境检验检疫局检验检疫技术中心 | For detecting vibrio parahaemolyticus and the test kit of integron and method in food |
| CN106148534A (en) * | 2016-07-29 | 2016-11-23 | 中山出入境检验检疫局检验检疫技术中心 | For detecting shigella and the test kit of integron and method in food |
| CN106244685A (en) * | 2016-07-29 | 2016-12-21 | 中山出入境检验检疫局检验检疫技术中心 | For detecting test kit and the method for Salmonella in Food and integron |
| CN106893779A (en) * | 2017-02-23 | 2017-06-27 | 浙江工商大学 | Primer and its application for carrying the salmonella RPA quick detections of Sulfonamides-resistant genes |
| CN106893779B (en) * | 2017-02-23 | 2023-01-17 | 浙江工商大学 | Primer for rapid detection of salmonella RPA carrying sulfonamide resistance gene and application thereof |
| CN107419014A (en) * | 2017-07-17 | 2017-12-01 | 蔡先全 | Detect pseudomonas aeruginosa and aprA primer, kit and method in water |
| CN107419016A (en) * | 2017-07-17 | 2017-12-01 | 蔡先全 | Detect the primer of pseudomonas aeruginosa and integron, kit and method in water |
| CN111549158A (en) * | 2020-06-08 | 2020-08-18 | 河北工程大学 | Method for detecting one-class integron gene intI1 in atmospheric environment |
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