CN110218806A - The quick detection primer group of detection of Salmonella fimW gene novel visual LAMP and kit - Google Patents
The quick detection primer group of detection of Salmonella fimW gene novel visual LAMP and kit Download PDFInfo
- Publication number
- CN110218806A CN110218806A CN201910566483.4A CN201910566483A CN110218806A CN 110218806 A CN110218806 A CN 110218806A CN 201910566483 A CN201910566483 A CN 201910566483A CN 110218806 A CN110218806 A CN 110218806A
- Authority
- CN
- China
- Prior art keywords
- detection
- salmonella
- primer
- lamp
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of quick detection primer groups of novel visual LAMP and kit for targeting detection of Salmonella fimW gene.The present invention passes through optimization reaction condition, a kind of quick detection primer group of novel visual LAMP and kit for targeting detection of Salmonella fimW gene is invented, and its specificity and sensibility are tested, it is unexpectedly found with very strong specificity, the minimum bacterial concentration that detects is 7.3 × 101CFU/ml, detection sensitivity is 1000 times higher than PCR method, while simplifying inspection process, and the pre- bacterium solution for increasing bacterium can be detected directly as template, and testing result is without uncapping.The positive is yellow, and feminine gender is red, and color contrast is obvious, intuitive easily to differentiate.
Description
Technical field
The invention belongs to microorganism detection fields, specifically, being related to a kind of targeting the novel of detection of Salmonella fimW gene can
Depending on changing the quick detection primer group of LAMP and kit.
Background technique
Detection of Salmonella (salmonella) is a kind of acapsular leather as clinical common food-borne Zoonosis pathogen
Lan Shi feminine gender straight-bar bacterium.Detection of Salmonella has reported at least 2500 serotypes at present, and country's report has more than 300 kinds, different serotypes
The invasiveness of salmonella and pathogenicity are dramatically different, caused by disease be all mutual corresponding with host.Infect sramana
The symptoms such as the visible fever of bacterium, sepsis, nausea, loss of appetite, diarrhea.In addition to that human foods can be caused to be poisoned by contaminated food
Infection is outer, and Salmonella infection is also very common in Production of Livestock and Poultry, causes necrotic enteritis in pig production, can in poultry production
Cause white diarrhea, fowl typhoid, the diseases such as chicken paratyphoid often cause very big loss to production and living.
Detection of Salmonella fimbriae type is numerous, mainly there is I type~IV type etc., I type pilin by fimA, fimI, fimC,
9 gene codings of fimD, fimH, fimF, fimZ, fimY and fimW, wherein the regulation base of main pili subunit protein fimA
Because mainly having fimZ, fimY and fimW.The detection of Salmonella for only having I type pili to be widely distributed in each serotype in numerous fimbriae types
In, the serotype of alloytype pili distribution is limited.FimW sequence is compared on Gen-Bank, is contained as the result is shown
FimW gene is all detection of Salmonella, and does not find the sequence homologous with fimW, explanation in the genome of other enteric bacillis
FimW gene is the distinctive gene of detection of Salmonella.
Currently, being mainly to exist based on the methods of traditional bacterium separation and biochemical identification to the detection of salmonella
Cumbersome, the defects of sensitivity is low and accuracy rate is not high, it is not able to satisfy timely and effectively quality-monitoring and disease control demand.
With the development of molecular biology technology, new diagnostic techniques such as enzyme linked immunological, immunofluorescence, PCR, quantitative fluorescent PCR etc. is examined
The method for surveying etiology nucleic acid and protein and other has been widely applied in the detection of salmonella.However, these
Technology in application process or needs skilled operator, or needs expensive instrument and equipment, is not suitable in entry and exit port
Or animal doctor base is widely promoted and applied.
Ring mediated isothermal amplification (LAMP) technology as a kind of novel constant temperature nucleic acid amplification method, have it is easy to operate,
The features such as high specific, hypersensitivity.Its principle is a variety of special primers of multiple regions design for target gene, utilizes one kind
Archaeal dna polymerase (Bst DNAploymerase) with strand-displacement activity, acts on tens under constant temperature (60 DEG C -65 DEG C)
Minute, nucleic acid amplification reaction can be completed.Since it is low to instrument and equipment requirement, a water-bath or insulating box be can be achieved with instead
It answers, as a result observing by the naked eye can determine whether, it is simple and efficient, it is suitble to base's quick diagnosis, has been widely used in disease in recent years
Substance detection.
The result method of discrimination of LAMP includes electrophoresis, addition I fluorescent dye of SYBR Green or metal ion instruction at present
Agent carries out visualization differentiation, such as calcein (yellow green-is orange red), hydroxynaphthol blue (bluish violet-sky blue).However,
These reagents or the addition or chromatic aberration is smaller is not easy to differentiate of need to uncapping.Studies have shown that working as archaeal dna polymerase for a deoxidation core
When thuja acid molecule is integrated in new DNA double chain, a hydrogen ion can be generated as by-product, the increase of hydrogen ion concentration causes
The reduction of reacting system PH value is based on this, the judgement present invention employs cresol red as PH indicator for result, reaction knot
Fruit color difference easily differentiates (yellow is the positive, and red is feminine gender), without uncapping.Its high specificity, sensibility are higher than conventional PCR method
1000 times.Clinical detection shows that the kit coincidence rate is high, takes the pre- bacterium solution for increasing bacterium that can be detected directly as template, no
Missing inspection can occur.
As Chinese patent CN201711409788.1 discloses a kind of LAMP primer of quickly detection 3 type of pig circular ring virus
Group, kit and application.The primer sets include nucleotide sequence primer sets as shown in NO.1~6 SEQ ID；Kit includes
Above-mentioned primer sets, reaction reagent；The application method of the kit is as follows: preparation amplification reaction system first；Isothermal reaction institute
Obtain product and directly carry out naked eyes interpretation: positive findings are yellow, and negative findings are red, and the kit is easy to operate, at low cost
It is honest and clean, as a result it is easy to observe, is highly suitable for the on-site test of export quarantine, food hygiene and livestock-raising field, but the invention
Only for 3 type of pig circular ring virus.
The present invention has invented the quick detection primer group of detection of Salmonella novel visual LAMP and examination according to detection of Salmonella fimW gene
Agent box, the detection to salmonella.
Summary of the invention
In order to solve the above problem, the purpose of the present invention is to provide a kind of novel visuals for targeting detection of Salmonella fimW gene
The quick detection primer group of LAMP and kit.
Firstly, the present invention provides a kind of LAMP primer group for targeting detection of Salmonella fimW gene.
The present invention devises LAMP primer group according to detection of Salmonella fimW gene, as shown in table 1, while according to detection of Salmonella state
Family's examination criteria, standard No. are GB/T 28642-2012, synthesize detection of Salmonella PCR national standard detection primer, as shown in table 2；
Table 1
Table 2
The present invention also provides a kind of kits for targeting detection of Salmonella fimW gene, including above-mentioned targeting detection of Salmonella fimW base
The LAMP primer group of cause.
Answering for detection of Salmonella whether is infected in detection sample to be tested with above-mentioned primer sets or kit the invention discloses a kind of
With.
The invention also discloses a kind of methods whether detection sample to be tested infects detection of Salmonella, include the following steps:
(3) LAMP amplification is carried out to sample to be tested with primer sets or kit, obtains amplified production；
(2) result judges: naked eyes can determine whether；It is negative: if red is presented in amplified production, it was demonstrated that sample to be tested does not infect
Detection of Salmonella；It is positive: if yellow is presented in amplified production, it was demonstrated that sample to be tested has infected detection of Salmonella；
Further, step (1) LAMP amplification reaction system is 25 μ l, and wherein 12.5 μ L of LAMP Mix, FIP, BIP draw
3 μ L, F3, B3 primer of object (10 μm of ol/L) (10 μ l mol/L) 0.5 μ L, LF primer (10 μ l mol/L) 1 μ L, 2.5 μ L of template,
It is remaining to use ultrapure water polishing；
Further, LAMP Mix includes (NH4)2SO4, KCl, Tween-20, cresol red solution, KOH, dNTP, beet
Alkali, MgSO4With Bst enzyme；
Preferably, the ratio of outer primer, inner primer and ring primer is 1:6:2；
Preferably, step (1) LAMP reaction system, 62 DEG C of reaction temperature, reaction time 40min.
Beneficial effect of the present invention
(1) present invention establishes the LAMP method of detection of Salmonella for detection of Salmonella fimW gene for the first time；
(2) detection sensitivity of the invention is high, is 1000 times of Standard PCR detection, hence it is evident that be higher than the prior art；
(3) this invention simplifies inspection process, the pre- bacterium solution for increasing bacterium can be detected directly as template, not leaked
Inspection.
(4) indicator is developed the color by cresol red, and experimental result realizes that visual observation is highly suitable for out without uncapping
Port of entry or animal doctor base are widely promoted and applied.
Detailed description of the invention
Fig. 1 is the optimal reaction temperature the selection result figure of embodiment 2；
Fig. 2 is 1 LAMP specific test result figure of experimental example；
Fig. 3 is 2 PCR specific test result figure of experimental example；
Fig. 4 is 3 PCR detection method susceptibility results figure of experimental example；
Fig. 5 is 3 LAMP sensitivity tests result figure of experimental example；
Fig. 6 is 4 clinical sample LAMP testing result figure of experimental example.
Specific embodiment
In order to make those skilled in the art more fully understand the present invention program, with reference to the accompanying drawings and detailed description to this hair
It is bright to be described in further detail.
Mouse typhus standard bacteria ATCC 14028 of the invention purchased from american strain collection (ATCC), sramana's bacteria strain,
P. aeruginosa bacterial strain, riemerella anatipestifer bacterial strain, jejunum campylobacter bacteria strain, Campylobacter Coli bacterial strain are by Agricultural University Of South China
Laboratory qualification saves.
LB meat soup, LB agar, XLT4 agar basis, buffered peptone water solution (BPW) and four sulphur hydrochlorates of the invention
Brilliant green enrichment liquid basic (TTB) is purchased from Huankai Microbes Tech Co., Ltd., Guangdong；Phosphate buffer (PBS) is purchased from ancient cooking vessel state
Prosperous Bioisystech Co., Ltd；Bacterial genomes DNA Rapid extraction kit is purchased from Tiangeng biochemical technology (Beijing) limited public affairs
Department.
The extraction of 1 bacterial genomes DNA of embodiment
Take 4 plants of different serotypes detection of Salmonella, i.e. Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum and fowl typhoid
Detection of Salmonella；5 plants of non-detection of Salmonella, i.e. campylobacter jejuni, Campylobacter Coli, pseudomonas aeruginosa (2 plants) and riemerella anatipestifer
The streak inoculation on nutrient agar or blood agar plate takes single colonie to connect by respectively required CMC model to corresponding time, screening
It kind to after nutrient broth, shakes and cultivates in shaking table, then culture boiling cracking process or press bacterial genomes DNA Rapid extraction
Kit illustrates to carry out, and each bacterial strain DNA profiling is made.
The foundation of 2 detection of Salmonella LAMP detection method of embodiment
By the reaction time of gradient setting 15min~75min, optimum reacting time is determined.
Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd., with LAMP primer sterilizing ultrapure water after synthesis
10 μM are diluted to, PCR primer is diluted to 20 μM, puts 20 DEG C of ﹣ preservations.
LAMP reaction system is 25 μ l, wherein 12.5 μ l of LAMP Mix, FIP, BIP primer (10 μm of ol/L) 3 μ l, F3, B3
Primer (10 μ l mol/L) 0.5 μ l, LF primer (10 μ l mol/L) 1 μ l, 2.5 μ l of template, remaining uses ultrapure water polishing.LAMP
Mix includes (NH4)2SO4, KCl, Tween-20, cresol red solution, KOH, dNTP, glycine betaine, MgSO4With Bst enzyme.
Wherein optimal outer primer, inner primer, ring primer ratio be 1:6:2,62 DEG C of peak optimization reaction temperature, the peak optimization reaction time
40min, as a result as shown in Figure 1, in Fig. 1,1, reaction 15min (red)；2,20min (red) is reacted；3, reaction 25min is (red
Color)；4,30min (red) is reacted；5,40min (yellow) is reacted；6,45min (yellow) is reacted；7,60min (yellow) is reacted；
8,75min (yellow) is reacted, it is seen that when the reaction time is 40 minutes, can show yellow.
Experimental example 1LAMP specific test
(1) it extracts DNA profiling: taking 4 plants of different serotypes detection of Salmonella, i.e. Salmonella Pullorm, Salmonella enteritidis, mouse typhus
Detection of Salmonella and Salmonella gallinarum, 5 plants of non-detection of Salmonella, i.e. campylobacter jejuni, Campylobacter Coli, pseudomonas aeruginosa (2 plants) with
And the DNA profiling of riemerella anatipestifer.
(2) LAMP specific detection is carried out according to embodiment 2, as a result by naked eyes to color interpretation, it is seen that white diarrhea is husky
The detection of Salmonella templates such as door bacterium, Salmonella enteritidis, Salmonella typhimurtum and Salmonella gallinarum are the aobvious positive of amplification, and color is Huang
Color, other non-detection of Salmonella templates such as campylobacter jejuni, Campylobacter Coli, pseudomonas aeruginosa, riemerella anatipestifer are feminine gender,
Color is red, is as a result consistent as shown in Fig. 2, repeating result after testing.
1, negative control in Fig. 2；2, Salmonella Pullorm；3, Salmonella enteritidis；4, Salmonella typhimurtum；5, fowl typhoid is husky
Door bacterium；6, Campylobacter Coli；7, campylobacter jejuni；8-9, pseudomonas aeruginosa；10, riemerella anatipestifer.
Experimental example 2PCR specific test
Using national standard primer, PCR reaction system is 50 μ l:Premix Taq buffer, 25 μ L, and 4 μ l of template, concentration is 20 μ
Each 1 μ L of the upstream and downstream primer of mol/L, 19 μ l of deionized water.
Response procedures are as follows: 94 DEG C of initial denaturation 2min, 30 circulations: 94 DEG C of deformation 1min, 58.4 DEG C of annealing 40s, 72 DEG C are prolonged
Stretch 30s；Glue, amplified band 331bp are run after 72 DEG C of whole extension 7min.
PCR amplification result meets expection, only Salmonella Pullorm, Salmonella enteritidis, Salmonella typhimurtum and fowl typhoid
Etc. templates expanded, other templates do not expand, consistent with LAMP result, as a result as shown in Figure 3.
In Fig. 3, M:DNA Marker；1, Salmonella Pullorm；2, Salmonella enteritidis；3, Salmonella typhimurtum；4, chicken hurts
Cold detection of Salmonella；5, Campylobacter Coli；6, campylobacter jejuni；7-8, pseudomonas aeruginosa；9, riemerella anatipestifer；10, negative
Control)
Experimental example 3LAMP method is compared with PCR method sensibility
Mouse typhus standard bacteria 14028 is selected, XLT4 plate is drawn, picking single bacterium falls within 1ml LB meat soup, and 37 DEG C of shaking tables sway
4.5h counts it using dilution plate counting method, and each dilution does 3 repetitions, determines its bacterial concentration, then will
It carries out 10 times of doubling dilutions with sterile PBS, and obtaining ultimate density is respectively 7.3 × 107CFU/mL、7.3×106CFU/mL、
7.3×105CFU/mL、7.3×104CFU/mL、7.3×103CFU/mL、7.3×102CFU/mL、7.3×101CFU/mL、7.3
×100The bacterium solution of CFU/mL is used for sensitivity tests.
As a result as follows:
PCR detection method
Bacterium solution final concentration of 7.3 × 107CFU/ml、7.3×106CFU/ml、7.3×105CFU/ml、7.3×104CFU/
It is amplifiable to arrive target fragment when ml, purpose band size 331bp, illustrate the detection lower limit using national standard detection method for 7.3 ×
104CFU/ml, as a result as shown in figure 4, in Fig. 4,1-8.7.3 × 107CFU/ml-7.3×100CFU/ml, M:DNA Marker.
LAMP detection method
Bacterial concentration is 7.3 × 107CFU/ml-7.3×101When CFU/ml, the aobvious positive yellow of amplification, 7.3 ×
100CFU/ml is negative red, as a result as shown in figure 5, in Fig. 5,1, negative control (red)；2-9,7.3×107CFU/mL-
7.3×100CFU/ml, 9 are displayed in red, and LAMP product is run 1% Ago-Gel, is as a result consistent.
It repeats result after testing to be consistent, the detection lower limit using LAMP detection method is 7.3 × 101CFU/ml, with the side PCR
Method is compared, and Monitoring lower-cut improves 3 orders of magnitude.
This experimental example explanation, the sensibility for the LAMP method that the present invention establishes is 1000 times of conventional PCR method, this is sensitive
Property be apparently higher than existing level, it was demonstrated that the present invention target detection of Salmonella fimW gene establish LAMP method sensibility it is high.
The detection of 4 clinical sample of experimental example
30 parts of market samples (10 parts of duck, 10 parts of chicken, 10 parts of pig) are acquired, are carried out in advance with buffered peptone water solution (BPW)
After increasing bacterium, detected with LAMP method；It using national standard method, is first carried out increasing bacterium in advance with BPW, then be used with the method for improvement
TTB carries out selective enrichment, takes bacterium solution after selective enrichment, extracts DNA profiling, is detected using PCR method.It is passed with passing through
The result that bacterium of uniting separates identity process is standard, compares the detection coincidence rate for obtaining LAMP method and PCR method.
This experimental example result is as follows: in 30 parts of samples, traditional bacterium separation identity process detects 28 parts of positives,
2 parts of feminine genders；LAMP method detects 27 parts of positives, 1 part of feminine gender (C1, red), 2 parts of weakly positives, as a result as shown in fig. 6, weak sun
Bacterial concentration is also lower related after property increases bacterium with BPW in advance；PCR method detects 24 parts of positives, 6 parts of feminine genders, this and PCR detection
Sensibility is related, even the DNA of bacteria template extracted after TTB selective enrichment, segment template concentration is yet lower, PCR knot
Fruit can not run out of band, and Fig. 6 is clinical sample LAMP testing result, and P represents pig, and C represents chicken, and D represents duck, and top is corresponding compiles
Number.It is as shown in table 3 that clinical sample detects comparing result.
Table 3
The present invention devises a pair of of LAMP primer for detection of Salmonella pili specific gene fimW, by adjusting system, determination
Optimal reaction temperature, screens optimum reacting time, and test specificity and sensibility, detection clinical sample etc. have invented a kind of targeting
The quick detection primer group of the novel visual LAMP of detection of Salmonella fimW gene and kit.
Conventional bacteria separation identity process step is more, and time-consuming, generally requires that result could be gone out in 3-5 days；National standard detection of Salmonella
PCR detection method needs to increase bacterium, selective enrichment by pre-, extract DNA profiling and carry out PCR detection, because of PCR method
Sensibility is lower to be easy to cause missing inspection, and experimental facilities is also more expensive.And the LAMP that the present invention targets detection of Salmonella fimW gene is fast
Fast detection primer group and kit.High specificity, sensibility is 1000 times higher than PCR method, and the bacterium solution after pre- increasing bacterium can directly be made
It is detected for template.Using conventional bacteria separation identity process as standard, detection coincidence rate reaches 96.7%, as a result such as 3 institute of table
Show, the results showed that missing inspection will not occur.
The technology of current existing observation experiment result be electrophoresis carried out to reaction result at the end of LAMP reaction, or
I fluorescent dye of SYBR Green is added after the reaction was completed, these require to expose reaction product in air, and it is molten to form gas
Glue, test for contamination environment cause false positive.Also having technology is that Metal ion indicator, such as calcein are added in LAMP system
(yellow green-is orange red), hydroxynaphthol blue (bluish violet-sky blue) etc., observed by the variation of reaction color as a result, but this
All there is the disadvantages of visual color difference is little, and the reaction time is longer in a little methods.Present invention employs cresol reds as new
Type PH indicator is used for the judgement of result, and reaction result, which only passes through color difference, to be differentiated very well, without uncapping, wherein yellow
For the positive, red is feminine gender.It visualizes the characteristics of determining with Aerosol Pollution is prevented, and avoids false positive, simplifies operation stream
The advantages of journey.
The quick detection primer group of detection of Salmonella fimW gene novel visual LAMP of the present invention and kit have high specificity,
The high advantage of sensibility.Bacterium solution after pre- increasing bacterium can be detected directly as template, only need to pass through light water at 62 DEG C
The detection to detection of Salmonella can be completed in bath reaction 40min, and without uncapping, experimental result realizes visual observation, passes through naked eyes
It can determine that, substantially reduce detection time, be highly suitable for entry and exit port or animal doctor base is widely promoted and applied.
Claims (9)
1. a kind of LAMP primer group for targeting detection of Salmonella fimW gene, which is characterized in that by outer primer F3, B3, inner primer FIP,
BIP and ring primer I F composition；
The nucleotide sequence of outer primer F3 is as shown in SEQ ID NO.1；
The nucleotide sequence of outer primer B3 is as shown in SEQ ID NO.2；
The nucleotide sequence of inner primer FIP is as shown in SEQ ID NO.3；
The nucleotide sequence of inner primer BIP is as shown in SEQ ID NO.4；
The nucleotide sequence of ring primer LF is as shown in SEQ ID NO.5.
2. a kind of LAMP kit for detecting detection of Salmonella, which is characterized in that including targeting detection of Salmonella fimW described in claim 1
The LAMP primer group of gene.
3. LAMP kit as claimed in claim 2, which is characterized in that the kit further include LAMP Mix12.5 μ L and
2.5 μ L of DNA profiling；
Wherein, LAMP Mix includes (NH4)2SO4, KCl, Tween-20, cresol red solution, KOH, dNTP, glycine betaine, MgSO4With
Bst enzyme.
4. the application of primer sets as described in claim 1 or kit as claimed in claim 2 in detection detection of Salmonella.
5. a kind of method whether detection sample to be tested infects detection of Salmonella, includes the following steps:
(1) LAMP amplification is carried out to sample to be tested with primer sets or kit, obtains amplified production；
(2) result judges: naked eyes can determine whether；
It is negative: if red is presented in amplified production, it was demonstrated that sample to be tested does not infect detection of Salmonella；
It is positive: if yellow is presented in amplified production, it was demonstrated that sample to be tested has infected detection of Salmonella.
6. the method whether a kind of detection sample to be tested as claimed in claim 5 infects detection of Salmonella, LAMP amplification reaction system
For 25 μ L, wherein LAMP Mix12.5 μ L, FIP, BIP primer (10 μm of ol/L) 3 μ L, F3, B3 primer (10 μ lmol/L) 0.5 μ L,
LF primer (10 μ lmol/L) 1 μ L, 2.5 μ L of template, remaining uses ultrapure water polishing.
7. the method whether a kind of detection sample to be tested as claimed in claim 6 infects detection of Salmonella, wherein LAMP Mix includes
(NH4)2SO4, KCl, Tween-20, cresol red solution, KOH, dNTP, glycine betaine, MgSO4With Bst enzyme.
8. the method whether a kind of detection sample to be tested as claimed in claim 6 infects salmonellosis, wherein outer primer, interior draw
The ratio of object and ring primer is 1:6:2.
9. the method whether a kind of detection sample to be tested as claimed in claim 6 infects detection of Salmonella, wherein LAMP reaction system
In, reaction temperature is 62 DEG C, reaction time 40min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910566483.4A CN110218806A (en) | 2019-06-27 | 2019-06-27 | The quick detection primer group of detection of Salmonella fimW gene novel visual LAMP and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910566483.4A CN110218806A (en) | 2019-06-27 | 2019-06-27 | The quick detection primer group of detection of Salmonella fimW gene novel visual LAMP and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110218806A true CN110218806A (en) | 2019-09-10 |
Family
ID=67815138
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910566483.4A Pending CN110218806A (en) | 2019-06-27 | 2019-06-27 | The quick detection primer group of detection of Salmonella fimW gene novel visual LAMP and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110218806A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878368A (en) * | 2019-12-05 | 2020-03-13 | 华南农业大学 | Novel LAMP method, primer group and kit capable of detecting SNP |
| CN111057749A (en) * | 2019-11-22 | 2020-04-24 | 福州大学 | Visual constant-temperature amplification product detection method |
| WO2024067192A1 (en) * | 2022-09-30 | 2024-04-04 | 华南农业大学 | Linear displacement isothermal amplification method and use thereof |
| CN118086573A (en) * | 2024-04-18 | 2024-05-28 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Universal verticillium dahliae detection kit and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153327B (en) * | 2007-09-21 | 2010-11-03 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting salmonella |
| CN102424861B (en) * | 2012-01-06 | 2013-07-31 | 扬州大学 | Loop-mediated isothermal amplification method for detecting salmonella in food and reagent kit |
| CN104726575A (en) * | 2015-03-13 | 2015-06-24 | 江苏省家禽科学研究所 | LAMP kit for rapidly detecting salmonella in avian borne samples |
| CN103898222B (en) * | 2014-04-04 | 2015-09-09 | 江苏省家禽科学研究所 | A kind of Salmonellas molecular detection kit based on bcfD gene and nondiagnostic detection method thereof |
| CN106282375A (en) * | 2016-09-24 | 2017-01-04 | 中华人民共和国广州机场出入境检验检疫局 | The LAMP primer group of a kind of Salmonella typhimurium and test kit and using method |
| CN107022638A (en) * | 2017-06-05 | 2017-08-08 | 温和心 | The LAMP primer group of quick detection salmonella and its application |
| CN107881261A (en) * | 2017-12-23 | 2018-04-06 | 广东省农业科学院动物卫生研究所 | Detect LAMP primer group, kit and the application of the type of pig circular ring virus 3 |
| CN109182467A (en) * | 2018-08-24 | 2019-01-11 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection salmonella |
-
2019
- 2019-06-27 CN CN201910566483.4A patent/CN110218806A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153327B (en) * | 2007-09-21 | 2010-11-03 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting salmonella |
| CN102424861B (en) * | 2012-01-06 | 2013-07-31 | 扬州大学 | Loop-mediated isothermal amplification method for detecting salmonella in food and reagent kit |
| CN103898222B (en) * | 2014-04-04 | 2015-09-09 | 江苏省家禽科学研究所 | A kind of Salmonellas molecular detection kit based on bcfD gene and nondiagnostic detection method thereof |
| CN104726575A (en) * | 2015-03-13 | 2015-06-24 | 江苏省家禽科学研究所 | LAMP kit for rapidly detecting salmonella in avian borne samples |
| CN106282375A (en) * | 2016-09-24 | 2017-01-04 | 中华人民共和国广州机场出入境检验检疫局 | The LAMP primer group of a kind of Salmonella typhimurium and test kit and using method |
| CN107022638A (en) * | 2017-06-05 | 2017-08-08 | 温和心 | The LAMP primer group of quick detection salmonella and its application |
| CN107881261A (en) * | 2017-12-23 | 2018-04-06 | 广东省农业科学院动物卫生研究所 | Detect LAMP primer group, kit and the application of the type of pig circular ring virus 3 |
| CN109182467A (en) * | 2018-08-24 | 2019-01-11 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection salmonella |
Non-Patent Citations (6)
| Title |
|---|
| P. A. KOKKINOS等: "Loop-Mediated Isothermal Amplification (LAMP) for the Detection of Salmonella in Food", 《FOOD ANALYTICAL METHODS》 * |
| QIANRU YANG等: "Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time", 《BMC MICROBIOLOGY》 * |
| 刘志科等: "鸡白痢沙门菌套式PCR检测方法的建立及初步应用", 《动物医学进展》 * |
| 庞心怡等: "环介导等温扩增技术快速检测肉中沙门氏菌", 《中国食物与营养》 * |
| 张江英等: "针对fimW基因沙门氏菌的特异性PCR检测", 《中国预防兽医学报》 * |
| 高志强等: "沙门氏菌LAMP可视化检测方法的建立", 《中国动物检疫》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111057749A (en) * | 2019-11-22 | 2020-04-24 | 福州大学 | Visual constant-temperature amplification product detection method |
| CN110878368A (en) * | 2019-12-05 | 2020-03-13 | 华南农业大学 | Novel LAMP method, primer group and kit capable of detecting SNP |
| WO2024067192A1 (en) * | 2022-09-30 | 2024-04-04 | 华南农业大学 | Linear displacement isothermal amplification method and use thereof |
| CN118086573A (en) * | 2024-04-18 | 2024-05-28 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Universal verticillium dahliae detection kit and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110218806A (en) | The quick detection primer group of detection of Salmonella fimW gene novel visual LAMP and kit | |
| CN110229932B (en) | African swine fever virus nucleic acid extraction-free fluorescent isothermal amplification detection kit | |
| CN102102124B (en) | Multiplex fluorescence PCR (Polymerase Chain Reaction) detection kit for typhoid/paratyphoid saimonella | |
| CN108504778B (en) | Kit that is a kind of while detecting porcine circovirus 2 type and porcine pseudorabies virus and application | |
| CN103305638A (en) | Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus | |
| CN108048584A (en) | The brucellar probe of RAA Fluorometric assays and kit | |
| CN102094090B (en) | Cholera toxin virulence gene detection kit and detection method thereof | |
| CN110373485A (en) | A kind of ureaplasma urealyticum, three joint inspection kit of chlamydia trachomatis and gonococcus | |
| CN109913565A (en) | A kind of kit for detecting vibrio parahaemolytious, primer pair, probe and method | |
| CN106282375A (en) | The LAMP primer group of a kind of Salmonella typhimurium and test kit and using method | |
| CN104017889A (en) | Molecular beacon probe for rapid detection of Mycobacterium tuberculosis and method for detecting Mycobacterium tuberculosis | |
| CN101864483A (en) | Salmonella and shigella joint detection kit and detection method thereof | |
| CN107365869A (en) | The method and primer of food-borne Klebsiella Pneumoniae are detected using loop-mediated isothermal amplification technique | |
| CN106544436B (en) | A kind of method of salmonella in detection textile | |
| CN108277289A (en) | Escherichia coli O157:The dry powdered LAMP quick detection kits of H7 | |
| CN104152550B (en) | Adopt LAMP technology for detection ox without slurry method | |
| CN106591483A (en) | Method for rapidly detecting pseudomonas aeruginosa in textile | |
| CN109971873A (en) | Identify the method for Listeria Monocytogenes, Yi Shi listeria spp and listeria innocua | |
| CN110093402A (en) | A kind of high-throughput quantification detection kit of food-borne pathogens | |
| CN109554491A (en) | A kind of reagent and method identifying detection sheep Babesia U sp and Mohs Babesia | |
| CN110951899B (en) | PCR detection system, kit and detection method for detecting vibrio parahaemolyticus | |
| CN107488720A (en) | For detecting the primer sets and its detection method of Escherichia coli O 157 in feed | |
| CN104404132B (en) | A kind of SS2-LAMP detection kit of streptococcus suis 2-type and application | |
| CN101824482A (en) | Detection kit for vibrio cholerae O1 group and detection method thereof | |
| CN106367516B (en) | Detect the loop-mediated isothermal amplification kit and detection method of campylobacter jejuni |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190910 |
|
| RJ01 | Rejection of invention patent application after publication |