CN106434917A - LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit - Google Patents
LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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 invention discloses an LAMP primer group and an LAMP detection kit for staphylococcus aureus and a use method of the detection kit. The LAMP primer group for staphylococcus aureus comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. According to the LAMP primer group, the LAMP detection kit and the use method, loop-mediated isothermal amplification primers are designed according to the sequences of the conservative regions of the heat-resistant nuclease genes (nuc) of staphylococcus aureus, the specific regions of target genes are amplified by adopting the LAMP technology, thus the rapid detection for staphylococcus aureus is realized from the molecular level, and an effective and rapid nucleic acid screening detection method is provided for detecting staphylococcus aureus.
Description
Technical field：
The invention belongs to the molecular biology for detection field of food safety, more particularly to a kind of staphylococcus aureuses
LAMP primer group and detection kit.
Background technology：
Staphylococcus aureuses（Staphylococcus aureus）Belong to micrococcaceae staphylococcus, be a kind of gram
Positive cocci, is distributed widely in nature, such as：In empty gas and water, dust, the Excreta of ground and humans and animals, it is that the mankind are suppurated
One of modal pathogen in sexy dye and bacterial poisoning.As condition is suitable, staphylococcus aureuses meeting amount reproduction,
A certain amount of enterotoxin is produced, causes alimentary toxicosis.In recent years, the Center for Disease Control report, by staphylococcus aureuses
The infection for causing accounts for second, is only second to escherichia coli, and the alimentary toxicosis thing that China is caused by staphylococcus aureuses every year
Part has occupy the 4th, the whole world.At present, countries in the world are all classified as staphylococcus aureuses the legal detection of food hygiene
Mesh.Disease caused by staphylococcus aureuses has become global public health problem, serious harm human security and strong
Health.
The detection method that China includes resistant Staphylococcus aureus for staphylococcus aureuses at present includes traditional
Microorganism culturing and biochemical identification, immune detection and PCR detection technique etc..The cultural method of traditional detection staphylococcus aureuses
Complex operation, detection time is long, and sensitivity is relatively low；Immunological method shortens detection time, although sensitivity is higher but special
The opposite sex is poor.Staphylococcus aureuses enzyme rapidly and sensitively can be detected from food using round pcr, but because round pcr is to template
The quality of DNA has higher requirements, the equipment price such as the thermal cycle of needs and gel electrophoresiss is expensive and be difficult to be promoted in basic unit
And fast-field evaluation.
Ring mediated isothermal amplification（Loop-mediated isothermal amplification, LAMP）Technology is a kind of
Novel isothermal nucleic acid amplification method, main 6 specific regions using 4 kinds of different specific primer identification target genes, and profit
With a kind of archaeal dna polymerase with strand-displacement activity（BstDNAploymerase）, in constant temperature（65 DEG C or so）Lower quick
Amplification of nucleic acid, it is ensured that the high specific of amplification and high efficiency, can reach 10 in 1h9～1010Target sequence is copied.LAMP method
Have the advantages that simple to operate, detection is quick, sensitivity and specificity are high and with low cost, nucleic acids research, medical diagnosis on disease,
The fields such as Pathogen test are used widely.
Content of the invention：
It is an object of the invention to provide a kind of LAMP primer group of staphylococcus aureuses.Heat-resisting according to staphylococcus aureuses
Nuclease gene（nuc）Conserved region sequence designs loop-mediated isothermal amplification (LAMP) primer, using the specific of LAMP technology amplification target gene
Region, realizes the quick detection of staphylococcus aureuses from molecular level, is that staphylococcus aureuses detection provides one kind more
Effective faster nucleic acid screening detection method.
Another object of the present invention is to providing above-mentioned LAMP detection kit and using method.
To achieve these goals, the present invention is employed the following technical solutions：
A kind of LAMP primer group of staphylococcus aureuses, including a pair of outer primer, a pair of inner primer and a ring primer, its nucleoside
Acid sequence difference is as follows：
SAnuc-F3:CGCTACTAGTTGCTTAGTGTTA,
SAnuc-B3:GCTAAGCCACGTCCATAT；
SAnuc-FIP:ACTGTTGGATCTTCAGAACCACCAAGTCTAAGTAGCTCAGCAA,
SAnuc-BIP:AGCGATTGATGGTGATACGGTTCAGGACCATATTTCTCTACACC；
SAnuc-LF:TACGCCGTTATCTGTTTGTGA,
SAnuc-LB:AGGTCAACCAATGACATTCAGA.
A kind of detection kit containing above-mentioned LAMP primer group, 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 detection kit, the ratio of the concentration of the LAMP primer group is as follows：Outer primer, inner primer, ring draw
The mol ratio of thing is：1-2：4-8：2-4.
In above-mentioned detection kit, the archaeal dna polymerase is Bst archaeal dna polymerase.
In above-mentioned detection kit, the 2 × reaction buffer comprising buffer buffer, glycine betaine and dNTPs, three
Person's volume ratio is 10：8：7.
In above-mentioned detection kit, the fluorescent dye is SYTO-9 that concentration is 0.02mM.
In above-mentioned detection kit, the sealing fluid is mineral oil.
In above-mentioned detection kit, the standard positive template is staphylococcus aureuses reference culture genomic DNA；
The negative control is sterilizing ultra-pure water.
The method for staphylococcus aureuses being detected using above-mentioned LAMP kit, comprises the following steps：
（1）The extraction of measuring samples：DNA is extracted from measuring samples using water-boiling method；
（2）Reaction system and condition：25 μ l reaction systems contain：0.2 μM of 0.2 μM of SAnuc-F3, SAnuc-B3, SAnuc-
0.4 μM of 0.4 μM of 0.8 μM of 0.8 μM of FIP, SAnuc-BIP, SAnuc-LF, SAnuc-LB, 2 × reactant liquor 12.5 μ l, DNA gather
0.5 μ l of synthase 8U, SYTO-9,2 μ l of measuring samples, with ultra-pure water polishing to 25 μ l；It is 20 μ l that sealing fluid adds volume；Simultaneously
Positive control and negative control are set；It is centrifuged after the PCR pipe for having configured is mixed, 63 DEG C of 45～60min of reaction, and holds at 80 DEG C
Continuous 2min；
（3）Testing result judges：Above-mentioned reaction tube is placed in fluorescent PCR instrument（As ABI stepone, ZYD-S1）In, according to expansion
Increase curve to judge testing result.Amplification curve is in serpentine, and testing result is the positive, i.e., containing golden yellow Portugal in detection sample
Grape coccus；No serpentine amplification curve occurs, and testing result is feminine gender, i.e., detection sample does not contain staphylococcus aureuses.
Compared with prior art, the present invention has the advantages that：
（1）Specificity is good：The present invention is according to staphylococcus aureuses heat stable nuclease gene（nuc）Six special primers of design,
6 regions of target gene are carried out with specific amplified, high specificity, and with other bacterial strain no cross reactions.
（2）Rapidly and efficiently：Whole amplification only can be completed with 45～60min, and amplification yield is up to 109～1010Individual copy；
（3）Simple operation：Expensive, accurate equipment need not be used, low to instrument requirements, it is only necessary to which that a steady temperature instrument is just
Can react and detect, condition is fairly simple；
（4）Identification is simple：Yin and yang attribute can directly be judged by observing amplification curve, it is not necessary to other any point of loaded down with trivial details electrophoresis etc.
Analysis step, suitable Site Detection.
Description of the drawings：
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 1 pg/ μ l staphylococcus aureus gene group DNA profiling LAMP reaction result.
Fig. 2 is LAMP specificity experiments result schematic diagram；
Specific embodiment：
With reference to the accompanying drawings and examples the present invention is described in detail, but should not be regarded as limitation of the present invention.
Embodiment 1：A kind of design synthesis of LAMP primer of staphylococcus aureuses and the foundation of detection kit
（1）The design synthesis of LAMP primer
According to document report, using staphylococcus aureuses nuc gene as target gene, using primer-design software Primer
Explorer 4.0 is designed including 6 LAMP primer including ring primer.Primer is by giving birth to work biological engineering（Shanghai）Share is limited
Company synthesizes, and 6 primer sequences are as follows：
SEQ NO.1 :SAnuc-F3:CGCTACTAGTTGCTTAGTGTTA
SEQ NO.2 :SAnuc-B3:GCTAAGCCACGTCCATAT
SEQ NO.3 :SAnuc-FIP: ACTGTTGGATCTTCAGAACCACCAAGTCTAAGTAGCTCAGCAA
SEQ NO.4:SAnuc-BIP:
AGCGATTGATGGTGATACGGTTCAGGACCATATTTCTCTACACC
SEQ NO.5 :SAnuc-LF:TACGCCGTTATCTGTTTGTGA
SEQ NO.6 :SAnuc-LB:AGGTCAACCAATGACATTCAGA
（2）Staphylococcus aureuses LAMP detection kit except include above-mentioned LAMP primer group in addition to, also include archaeal dna polymerase, 2 ×
Reactant liquor, fluorescent dye, sealing fluid, positive control and negative control.Wherein reactant liquor comprising buffer buffer, glycine betaine and
DNTPs, each component volume ratio is 10：8：7.
（3）Detection method：
1）The extraction of measuring samples：Using water-boiling method, sample diluting liquid is boiled, supernatant is used as amplified reaction DNA profiling.
2）Constant temperature gene amplification detection reaction system and condition：25 μ l reaction systems contain：0.2 μM of SAnuc-F3,
0.4 μM of 0.4 μM of 0.8 μM of 0.8 μM of 0.2 μM of SAnuc-B3, SAnuc-FIP, SAnuc-BIP, SAnuc-LF, SAnuc-LB,
2 × reactant liquor, 12.5 μ l, 0.5 μ l of archaeal dna polymerase 8U, SYTO-9,2 μ l of measuring samples, with ultra-pure water polishing to 25 μ l.Sealing
It is 20 μ l that liquid adds volume.While arranging positive control and negative control.
It is centrifuged after the PCR pipe for having configured is mixed, 63 DEG C of 45～60min of reaction, and continues 2min at 80 DEG C.
3）Testing result judges：Above-mentioned reaction tube is placed in fluorescent PCR instrument（As ABI stepone, ZYD-S1）In, according to
Amplification curve is judging testing result.Amplification curve is in serpentine, and testing result is the positive, i.e., containing golden yellow in detection sample
Staphylococcuses；No serpentine amplification curve occurs, and testing result is feminine gender, i.e., detection sample does not contain staphylococcus aureuses.
Embodiment 2：LAMP sensitivity experiment
Staphylococcus aureuses reference culture is inoculated in nutrient broth, 36 DEG C ± 1 DEG C is cultivated 18 hours.Application Magen antibacterial
Gene DNA extracts kit extracts the DNA of bacteria in cultured products.The DNA of staphylococcus aureuses reference culture extracting is entered
10 times of gradient dilutions of row, are made with 1ng/ μ l, 100pg/ μ l, 10pg/ μ l, 1pg/ μ l, five gradient concentration DNA of 100fg/ μ l respectively
For template and negative control（Sterilizing ultra-pure water）Reaction system and condition according to embodiment 1 sets up detection method, to determine examination
The sensitivity of agent box detection method.
Referring to Fig. 1, as a result show：Staphylococcus aureus gene group DNA is carried out after 10 times of gradient dilutions, the gold of foundation
Staphylococcus aureus LAMP detection kit can in detection sample 1pg/ μ l L-form staphylococcus aureus.
Embodiment 3:LAMP specificity experiments
The LAMP detection kit of Application Example 1 is to Salmonella typhimurium, Enterobacter sakazakii, unicellular hypertrophy Listeria
Bacterium, shigella flexneri, colon bacillus and vibrio parahaemolyticus carry out specificity reality according to above-mentioned reaction system and condition
Test, testing result is as shown in Figure 2.It is positive control to arrange staphylococcus aureus gene group DNA, and sterilizing distilled water is negative right
According to.From figure 2 it can be seen that only serpentine amplification curve occur in staphylococcus aureuses, assume positive findingses, other bacterial strains
All there is not serpentine curve, assume negative findings until the response time extends to 60min.The LAMP reactant of embodiment 1 is described
Only there is specificity in system to staphylococcus aureuses.
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 and detection kit of a kind of staphylococcus aureuses and using method
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence SAnuc-F3
<400> 1
cgctactagt tgcttagtgt ta 22
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence SAnuc-B3
<400> 2
gctaagccac gtccatat 18
<210> 3
<211> 43
<212> DNA
<213>Artificial sequence SAnuc-FIP
<400> 3
actgttggat cttcagaacc accaagtcta agtagctcag caa 43
<210> 4
<211> 44
<212> DNA
<213>Artificial sequence SAnuc-BIP
<400> 4
agcgattgat ggtgatacgg ttcaggacca tatttctcta cacc 44
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence SAnuc-LF
<400> 5
tacgccgtta tctgtttgtg a 21
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence SAnuc-LB
<400> 6
aggtcaacca atgacattca ga 22
Claims (9)
1. the LAMP primer group of a kind of staphylococcus aureuses, it is characterised in that including a pair of outer primer, a pair of inner primer and
Ring primer, its nucleotide sequence difference is as follows：
SAnuc-F3:CGCTACTAGTTGCTTAGTGTTA,
SAnuc-B3:GCTAAGCCACGTCCATAT；
SAnuc-FIP:ACTGTTGGATCTTCAGAACCACCAAGTCTAAGTAGCTCAGCAA,
SAnuc-BIP:AGCGATTGATGGTGATACGGTTCAGGACCATATTTCTCTACACC；
SAnuc-LF:TACGCCGTTATCTGTTTGTGA,
SAnuc-LB:AGGTCAACCAATGACATTCAGA.
2. a kind of detection kit containing above-mentioned LAMP primer group, it is characterised in that including following material：（1）Archaeal dna polymerase；
（2）2 × reaction buffer；（3）Fluorescent dye；（4）Sealing fluid；（5）Standard positive template；（6）Negative control；（7）Right will
Seek the LAMP primer group described in 1.
3. detection kit as claimed in claim 2, it is characterised in that the ratio of the concentration of the LAMP primer group is as follows：
Outer primer, inner primer, the mol ratio of ring primer are：1-2：4-8：2-4.
4. detection kit as claimed in claim 2, it is characterised in that the archaeal dna polymerase be.
5. detection kit as claimed in claim 2, it is characterised in that the 2 × reaction buffer is buffered comprising buffer
Liquid, glycine betaine and dNTPs, three's volume ratio is 10：8：7.
6. detection kit as claimed in claim 2, it is characterised in that the fluorescent dye is 0.02mM for concentration
SYTO-9.
7. detection kit as claimed in claim 2, it is characterised in that the sealing fluid be.
8. detection kit as claimed in claim 2, it is characterised in that the standard positive template be
Reference culture genomic DNA；The negative control is sterilizing ultra-pure water.
9. usage right requires the method that LAMP kit described in 2 detects staphylococcus aureuses, it is characterised in that including following
Step：
（1）The extraction of measuring samples：DNA is extracted from measuring samples using water-boiling method；
（2）Reaction system and condition：25 μ l reaction systems contain：0.2 μM of 0.2 μM of SAnuc-F3, SAnuc-B3, SAnuc-
0.4 μM of 0.4 μM of 0.8 μM of 0.8 μM of FIP, SAnuc-BIP, SAnuc-LF, SAnuc-LB, 2 × reactant liquor 12.5 μ l, DNA gather
0.5 μ l of synthase 8U, SYTO-9,2 μ l of measuring samples, with ultra-pure water polishing to 25 μ l；It is 20 μ l that sealing fluid adds volume；Simultaneously
Positive control and negative control are set；It is centrifuged after the PCR pipe for having configured is mixed, 63 DEG C of 45～60min of reaction, and holds at 80 DEG C
Continuous 2min；
（3）Testing result judges：Above-mentioned reaction tube is placed in fluorescent PCR instrument, testing result is judged according to amplification curve；Expand
It is in serpentine to increase curve, and testing result is the positive, i.e., contain staphylococcus aureuses in detection sample；No serpentine amplification curve goes out
Existing, testing result is feminine gender, i.e., detection sample does not contain staphylococcus aureuses.
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| CN107513582A (en) * | 2017-10-23 | 2017-12-26 | 蔡慧娜 | Authentication chip, kit and the authentication method of positive blood culture |
| CN107663546A (en) * | 2017-09-20 | 2018-02-06 | 暨南大学 | Primer sets, kit and method based on intelligent constant-temperature augmentation detection technology for detection staphylococcus aureus |
| CN108277290A (en) * | 2017-12-29 | 2018-07-13 | 广东环凯微生物科技有限公司 | The dry powdered LAMP quick detection kits of staphylococcus aureus |
| CN108570511A (en) * | 2018-05-30 | 2018-09-25 | 华南理工大学 | A kind of LAMP detection primer group of staphylococcus aureus, its LAMP detection kit and its detection method |
| CN108866223A (en) * | 2017-05-15 | 2018-11-23 | 吉林出入境检验检疫局检验检疫技术中心 | It is a kind of for identifying the LAMP primer group and its application of staphylococcus aureus |
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