CN106282375A - The LAMP primer group of a kind of Salmonella typhimurium and test kit and using method - Google Patents
The LAMP primer group of a kind of Salmonella typhimurium and test kit and using method Download PDFInfo
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- CN106282375A CN106282375A CN201610851242.0A CN201610851242A CN106282375A CN 106282375 A CN106282375 A CN 106282375A CN 201610851242 A CN201610851242 A CN 201610851242A CN 106282375 A CN106282375 A CN 106282375A
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- 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
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- 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
Abstract
The invention discloses the LAMP primer group of a kind of Salmonella typhimurium and test kit and using method.Including a pair outer primer, a pair inner primer and a pair ring primer.The present invention is according to Salmonella typhimurium invasin protein A(invA) gene conserved region sequential design loop-mediated isothermal amplification (LAMP) primer, application PrimerExplorer V4 Photographing On-line special primer group, utilize the specific region of LAMP technology amplification target gene, from molecular level, realize the quick detection of Salmonella typhimurium, provide effective and quick nucleic acid screening detection method for Salmonella typhimurium detection.
Description
Technical field:
The invention belongs to the molecular biology for detection field of food safety, particularly to a kind of Salmonella typhimurium LAMP
Primer sets and detection kit.
Background technology:
Salmonella (Salmonella spp.) is large numbers of to parasitize biochemical reaction and antigen construct in human and animal's intestinal
Similar gram negative bacilli, is modal infecting both domestic animals and human type pathogenic bacterium in enterobacteriaceae.The Salmonella being currently known
More than 2000 kinds, and almost all of Salmonella can cause serious food poisoning, World Health Organization (WHO) (WTO)
Salmonella is listed in the food transmission pathogen with serious harm and medium harm.Food transmission is human infection sramana
The main path of Salmonella, in China's bacterial food poisoning, having 70%-80% is by salmonellal, and causes sramana
In the food of Salmonella poisoning, about 90% is the livestock products such as meat, egg, milk.Salmonella typhimurium (Salmonella
Typhimurium) belong to Salmonella B group, be one of the serotype caused a disease of common Salmonella.
At present, Salmonella is detected mainly with sides such as traditional bacteria distribution, serotype experiment and biochemical identification
Method is main, has that complex operation, sensitivity is low and the most high defect of accuracy rate, it is impossible to meet quality-monitoring and disease timely and effectively
Sick demand for control.Along with the development of Protocols in Molecular Biology, the method such as new diagnostic techniques such as enzyme linked immunological, immunofluorescence, PCR
Detection cause of disease nucleic acid and protein and other are widely applied in the detection of Salmonella, but poor specificity and false positive
High and equipment and instrument high requirement can not be met the universal demand of field quick detection and basic unit.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology is 21 generation
A kind of Novel isothermal nucleic acid amplification method that several the Japanese scholars such as first Notomi of recording are reported, its principle is 6 for target gene
4 species-specific primers are designed in individual region, utilize a kind of archaeal dna polymerase (Bst DNAploymerase) with strand-displacement activity,
Act on dozens of minutes under constant temperature (60 DEG C-65 DEG C), nucleic acid amplification reaction can be completed.Keeping traditional PCR technique advantage
On the basis of, further increase the specificity of reaction, shorten the detection time simultaneously, there is good development prospect.
Summary of the invention:
It is an object of the invention to provide that a group-specific is good, highly sensitive and highly versatile for detecting Salmonella typhimurium
The LAMP primer group of bacterium.
The present invention is according to Salmonella typhimurium invasin protein A(invA) expansion of gene conserved region sequential design ring mediated isothermal
Increase primer, apply PrimerExplorer V4(http: //primerexplorer. jp/elamp4.0.0/index.html)
Photographing On-line special primer group, utilizes the specific region of LAMP technology amplification target gene, realizes mouse typhus husky from molecular level
The quick detection of door Salmonella, provides effective and quick nucleic acid screening detection method for Salmonella typhimurium detection.
Another object of the present invention is to provide LAMP detection kit and the using method of a kind of Salmonella typhimurium.
To achieve these goals, the present invention is by the following technical solutions:
The LAMP primer group of a kind of Salmonella typhimurium, including a pair outer primer, a pair inner primer and a pair ring primer, its core
Nucleotide sequence is the most as follows:
SainvA-F3:GGTCGTTCTACATTGACAGAA,
SainvA-B3:CGACACGTTCTGAACCTT；
SainvA-FIP:CGGCATCGGCTTCAATCAAGGGTACTGTTAATTACCACGCT,
SainvA-BIP:GTGAAATTATCGCCACGTTCGGGGTGACAATAGAGAAGACAACA；
SainvA-LF:TGGTACTGATCGATAATGCCAG,
SainvA-LB:TATTGGCGATAGCCTGGC。
The LAMP detection kit of a kind of Salmonella typhimurium, including following material: (1) archaeal dna polymerase；(2) 2 × anti-
Answer buffer；(3) fluorescent dye；(4) sealing fluid；(5) standard positive template；(6) negative control；(7) above-mentioned LAMP primer
Group.
In above-mentioned LAMP detection kit, described primer sets concentration ratio is as follows: outer primer, inner primer, ring draw
The mol ratio of thing is: 1-2:4-8:2-4.
In above-mentioned LAMP detection kit, described archaeal dna polymerase is Bst archaeal dna polymerase.
In above-mentioned LAMP detection kit, described 2 × reaction buffer comprise buffer buffer, glycine betaine and
DNTPs, the volume ratio of three is 10:8:7.
In above-mentioned LAMP detection kit, described fluorescent dye be concentration be the SYTO-9 of 0.02mM.
In above-mentioned LAMP detection kit, described sealing fluid is mineral oil.
In above-mentioned LAMP detection kit, described standard positive template is Salmonella typhimurium reference culture base
Because of group DNA；Described negative control is sterilizing ultra-pure water.
The method using above-mentioned LAMP detection kit detection Salmonella typhimurium, comprises the following steps:
(1) extraction of measuring samples: use water-boiling method to extract DNA from measuring samples；
(2) reaction system and condition: 25 μ l reaction systems contain: SainvA-F3 0.2 μM, SainvA-B3 0.2 μM,
SainvA-FIP 0.8 μM, SainvA-BIP 0.8 μM, SainvA-LF 0.4 μM, SainvA-LB 0.4 μM, 2 × reactant liquor
12.5 μ l, archaeal dna polymerase 8U, SYTO-9 0.5 μ l, measuring samples 2 μ l, with ultra-pure water polishing to 25 μ l；Sealing fluid adds volume
It is 20 μ l；Positive control and negative control are set simultaneously；
It is centrifuged after the PCR pipe mixing that will have configured, 63 DEG C of reactions 30～45min, and continues 2min at 80 DEG C；
(3) testing result judges: be placed in by above-mentioned reaction tube in fluorescent PCR instrument (such as ABI stepone, ZYD-S1), according to expansion
Increase curve and judge testing result.Amplification curve is serpentine, and testing result is positive, i.e. husky containing belonging to typhoid fever in detection sample
Door Salmonella；Amplification curve without serpentine occurs, testing result is negative, i.e. detection sample does not contains Salmonella typhimurium.
Compared with prior art, there is advantages that
(1) the LAMP primer group that the present invention uses adds 2 ring primers on the basis of conventional LAMP 4 primers of detection,
Accelerate amplification rate, and improve amplification efficiency.
(2) present invention uses SYTO-9 dyestuff as fluorescence indicator, can add, it is to avoid anti-in preparation reaction system
Open lid after should and add the Aerosol Pollution risk caused, and decrease reactions steps.
(3) result of the present invention judges simple, can judge detection sample yin and yang attribute by observing amplification curve, it is not necessary to electrophoresis etc.
Other any analytical procedures, are suitable for Site Detection.
Accompanying drawing illustrates:
Fig. 1 is LAMP sensitivity experiments result schematic diagram；Wherein, be followed successively by from left to right concentration 1ng/ μ l, 100pg/ μ l, 10
Pg/ μ l and 1pg/ μ l Salmonella typhimurium genomic DNA template LAMP reaction result.
Fig. 2 is LAMP specificity experiments result schematic diagram；
Detailed description of the invention:
With embodiment, the present invention is described in detail below in conjunction with the accompanying drawings, but should not be regarded as limitation of the present invention.
Embodiment 1: the design synthesis of the LAMP primer of a kind of Salmonella typhimurium and the foundation of detection kit
(1) the design synthesis of LAMP primer
Root according to the literature, using Salmonella typhimurium invA gene as target gene, uses primer-design software Primer
Explorer 4.0 design 6 LAMP primer including ring primer.Primer is limited by giving birth to work biological engineering (Shanghai) share
Company synthesizes, and 6 primer sequences are as follows:
SEQ NO.1:SainvA-F3:GGTCGTTCTACATTGACAGAA
SEQ NO.2:SainvA-B3:CGACACGTTCTGAACCTT
SEQ NO.3:SainvA-FIP:CGGCATCGGCTTCAATCAAGGGTACTGTTAATTACCACGC T
SEQ NO.4:SainvA-BIP:
GTGAAATTATCGCCACGTTCGGGGTGACAATAGAGAAGACAACA
SEQ NO.5:SainvA-LF:TGGTACTGATCGATAATGCCAG
SEQ NO.6:SainvA-LB:TATTGGCGATAGCCTGGC
(2) Salmonella typhimurium LAMP detection kit is in addition to including above-mentioned LAMP primer group, also include archaeal dna polymerase, 2 ×
Reactant liquor, fluorescent dye, sealing fluid, positive control and negative control.Wherein reactant liquor comprise buffer buffer, glycine betaine and
DNTPs, each component volume is than for 10:8:7.
(3) detection method:
1) extraction of measuring samples: carry out according to DNA extraction kit.
2) constant temperature gene amplification detection reaction system and condition: 25 μ l reaction systems contain: SainvA-F3 0.2 μM,
SainvA-B3 0.2 μM, SainvA-FIP 0.8 μM, SainvA-BIP 0.8 μM, SainvA-LF 0.4 μM, SainvA-LB
0.4 μM, 2 × reactant liquor 12.5 μ l, archaeal dna polymerase 8U, SYTO-9 0.5 μ l, measuring samples 2 μ l, with ultra-pure water polishing to 25 μ
l.It is 20 μ l that sealing fluid adds volume.Positive control and negative control are set simultaneously.
It is centrifuged after the PCR pipe mixing that will have configured, 63 DEG C of reactions 30～45min, and continues 2min at 80 DEG C.
3) testing result judges: be placed in by above-mentioned reaction tube in fluorescent PCR instrument (such as ABI stepone, ZYD-S1), according to
Amplification curve judges testing result.Amplification curve is serpentine, and testing result is positive, i.e. containing mouse typhus in detection sample
Salmonella；Amplification curve without serpentine occurs, testing result is negative, i.e. detection sample does not contains Salmonella typhimurium.
Embodiment 2:LAMP sensitivity experiment
Salmonella typhimurium reference culture is inoculated in nutrient broth, cultivates 18 hours for 36 DEG C ± 1 DEG C.Application Magen antibacterial
Gene DNA extracts test kit and extracts the DNA of bacteria in cultured products.The DNA that Salmonella typhimurium reference culture extracts is entered
10 times of gradient dilutions of row, respectively with 1ng/ μ l, 100pg/ μ l, 10pg/ μ l, 1pg/ μ l, five gradient concentration DNA of 100fg/ μ l and
Negative control (sterilizing ultra-pure water) sets up detection method as template according to reaction system and the condition of embodiment 1, to determine examination
The susceptiveness of agent box detection method.
Seeing Fig. 1, result shows: after Salmonella typhimurium genomic DNA carries out 10 times of gradient dilutions, the Mus of foundation
Salmonella typhi LAMP detection kit can detect the Salmonella typhimurium DNA of 1pg/ μ l in sample.
Embodiment 3:LAMP specificity experiments
The LAMP detection kit of Application Example 1 is to staphylococcus aureus, Enterobacter sakazakii, unicellular hypertrophy Listeria
Bacterium, shigella flexneri, colon bacillus and vibrio parahaemolyticus detect, and testing result is as shown in Figure 2.Arrange golden yellow
Color aureus gene group DNA is positive control, and sterilizing ultra-pure water is negative control.From figure 2 it can be seen that only mouse typhus
There is serpentine amplification curve in Salmonella, presents positive findings, and serpentine curve all do not occur in other bacterial strains, present negative findings
Until the response time extends to 60min.Illustrate that the LAMP reaction system of embodiment 1 only has specificity to Salmonella typhimurium.
SEQUENCE LISTING
<110>Guangzhou Airport Exit Inspection and Quarantine of the PRC, Ji'nan University, Guangzhou Double helix gene technology
Company limited
<120>the LAMP primer group of a kind of Salmonella typhimurium and test kit and using method
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>artificial sequence SainvA-F3
<400> 1
ggtcgttcta cattgacaga a 21
<210> 2
<211> 18
<212> DNA
<213>artificial sequence SainvA-B3
<400> 2
cgacacgttc tgaacctt 18
<210> 3
<211> 41
<212> DNA
<213>artificial sequence SainvA-FIP
<400> 3
cggcatcggc ttcaatcaag ggtactgtta attaccacgc t 41
<210> 4
<211> 44
<212> DNA
<213>artificial sequence SainvA-BIP
<400> 4
gtgaaattat cgccacgttc ggggtgacaa tagagaagac aaca 44
<210> 5
<211> 22
<212> DNA
<213>artificial sequence SainvA-LF
<400> 5
tggtactgat cgataatgcc ag 22
<210> 6
<211> 18
<212> DNA
<213>artificial sequence SainvA-LB
<400> 6
tattggcgat agcctggc 18
Claims (9)
1. the LAMP primer group of a Salmonella typhimurium, it is characterised in that include a pair outer primer, a pair inner primer and
To ring primer, its nucleotide sequence is the most as follows:
SainvA-F3:GGTCGTTCTACATTGACAGAA,
SainvA-B3:CGACACGTTCTGAACCTT；
SainvA-FIP:CGGCATCGGCTTCAATCAAGGGTACTGTTAATTACCACGCT,
SainvA-BIP:GTGAAATTATCGCCACGTTCGGGGTGACAATAGAGAAGACAACA；
SainvA-LF:TGGTACTGATCGATAATGCCAG,
SainvA-LB:TATTGGCGATAGCCTGGC。
2. the LAMP detection kit of a Salmonella typhimurium, it is characterised in that include following material: (1) archaeal dna polymerase；
(2) 2 × reaction buffers；(3) fluorescent dye；(4) sealing fluid；(5) standard positive template；(6) negative control；(7) right is wanted
Seek the LAMP primer group described in 1.
3. LAMP detection kit as claimed in claim 2, it is characterised in that described primer sets concentration ratio is as follows: outer
Primer, inner primer, the mol ratio of ring primer be: 1-2:4-8:2-4.
4. LAMP detection kit as claimed in claim 2, it is characterised in that described archaeal dna polymerase is Bst DNA polymerization
Enzyme.
5. LAMP detection kit as claimed in claim 2, it is characterised in that described 2 × reaction buffer comprises buffer
Buffer, glycine betaine and dNTPs, the volume ratio of three is 10:8:7.
6. LAMP detection kit as claimed in claim 2, it is characterised in that described fluorescent dye be concentration be 0.02mM
SYTO-9。
7. LAMP detection kit as claimed in claim 2, it is characterised in that described sealing fluid is mineral oil.
8. LAMP detection kit as claimed in claim 2, it is characterised in that described standard positive template is that mouse typhus is husky
Door Salmonella reference culture genomic DNA；Described negative control is sterilizing ultra-pure water.
9. use claim 2 LAMP detection kit detection Salmonella typhimurium method, it is characterised in that include with
Lower step:
(1) extraction of measuring samples: use water-boiling method to extract DNA from measuring samples；
(2) reaction system and condition: 25 μ l reaction systems contain: SainvA-F3 0.2 μM, SainvA-B3 0.2 μM,
SainvA-FIP 0.8 μM, SainvA-BIP 0.8 μM, SainvA-LF 0.4 μM, SainvA-LB 0.4 μM, 2 × reactant liquor
12.5 μ l, archaeal dna polymerase 8U, SYTO-9 0.5 μ l, measuring samples 2 μ l, with ultra-pure water polishing to 25 μ l；Sealing fluid adds volume
It is 20 μ l；Positive control and negative control are set simultaneously；
It is centrifuged after the PCR pipe mixing that will have configured, 63 DEG C of reactions 30～45min, and continues 2min at 80 DEG C；
(3) testing result judges: is placed in fluorescent PCR instrument by above-mentioned reaction tube, judges testing result according to amplification curve；Expand
Increasing curve is serpentine, and testing result is positive, i.e. containing belonging to salmonella typhi in detection sample；Amplification curve without serpentine goes out
Existing, testing result is negative, i.e. detection sample does not contains Salmonella typhimurium.
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| CN201610851242.0A CN106282375A (en) | 2016-09-24 | 2016-09-24 | The LAMP primer group of a kind of Salmonella typhimurium and test kit and using method |
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| CN108085402A (en) * | 2018-02-10 | 2018-05-29 | 上海实验动物研究中心 | The primer and its kit of one group of detection grinding tooth citric acid bacillus |
| CN109402271A (en) * | 2017-08-14 | 2019-03-01 | 中国科学院微生物研究所 | For detecting the primer system and kit and their application and the method for detecting salmonella of salmonella |
| CN110218806A (en) * | 2019-06-27 | 2019-09-10 | 华南农业大学 | The quick detection primer group of detection of Salmonella fimW gene novel visual LAMP and kit |
| CN112080572A (en) * | 2020-09-29 | 2020-12-15 | 青岛英赛特生物科技有限公司 | Triple PCR primer group and kit for simultaneously detecting salmonella typhimurium, salmonella enteritidis and clostridium welchii A types |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107022632A (en) * | 2017-05-19 | 2017-08-08 | 深圳职业技术学院 | A kind of Salmeterol fluticasone propionate primer sets, kit and application |
| CN109402271A (en) * | 2017-08-14 | 2019-03-01 | 中国科学院微生物研究所 | For detecting the primer system and kit and their application and the method for detecting salmonella of salmonella |
| CN108085402A (en) * | 2018-02-10 | 2018-05-29 | 上海实验动物研究中心 | The primer and its kit of one group of detection grinding tooth citric acid bacillus |
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| CN112080572A (en) * | 2020-09-29 | 2020-12-15 | 青岛英赛特生物科技有限公司 | Triple PCR primer group and kit for simultaneously detecting salmonella typhimurium, salmonella enteritidis and clostridium welchii A types |
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