CN108570511A - A kind of LAMP detection primer group of staphylococcus aureus, its LAMP detection kit and its detection method - Google Patents
A kind of LAMP detection primer group of staphylococcus aureus, its LAMP detection kit and its detection method Download PDFInfo
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Abstract
The present invention relates to biotechnologies, specifically, a kind of LAMP detection primer group of staphylococcus aureus is provided, it includes outer primer pair and inner primer pair, it can expand the target gene on staphylococcus aureus using LAMP technology, realize the quick detection of staphylococcus aureus for the specific region on staphylococcus aureus, meanwhile promoting the accuracy rate of staphylococcus aureus testing result.In addition, the present invention also provides a kind of LAMP detection kit and its detection method of the staphylococcus aureus including the LAMP detection primer group, the quick detection of staphylococcus aureus can be realized, there is high sensitivity, the good advantage of specificity.
Description
Technical field
The present invention relates to biotechnologies, in particular to a kind of LAMP detection primer of staphylococcus aureus
Group, its LAMP detection kit and its detection method.
Background technology
Staphylococcus aureus (Staphylococcus aureus) belongs to micrococcaceae staphylococcus, is a kind of leather
Lan Shi positive coccis, are distributed widely in nature, as empty gas and water, humans and animals excreta in, be mankind's pyogenic infection and
One of most common pathogen in bacterial poisoning, can cause local pyogenic infection, can also cause pneumonia, pseudomembranous enteritis,
Pericarditis etc., or even cause the general infections such as septicemia, pyemia.In recent years, the Center for Disease Control is reported, by golden yellow
Infection caused by staphylococcus accounts for the second of whole world infection, so the disease caused by staphylococcus aureus has become the whole world
Property public health problem, seriously endanger human security and health.And for the quick inspection of the infection of staphylococcus aureus,
It is the key that clinical infection is instructed to treat medication.
At present for the detection method that staphylococcus aureus includes drug-fast bacteria be mainly microculture and biochemical identification,
Immune detection and various PCR detections etc..Microculture and biochemical identification cultural method are the warps for examining staphylococcus aureus
The shortcomings of there is the laboratory for needing profession in allusion quotation method, cumbersome, and detection time is long.It is measured currently, mostly using round pcr
Staphylococcus aureus, but because technology has higher requirement to the quality and quantity of template, need thermal cycle and gel electrophoresis
Etc. equipment, price is relatively expensive, it is difficult to accomplish fast-field evaluation.
Invention content
The first object of the present invention is to provide a kind of LAMP detection primer group of staphylococcus aureus, utilize golden yellow
The staphylococcic specific region of color carries out LAMP, being capable of amplification that is quick, easy and accurately realizing staphylococcus aureus.
The second object of the present invention is to provide a kind of LAMP detection kit of staphylococcus aureus, utilize above-mentioned
LAMP detection primer group carries out LAMP, can effectively shorten detection time, improves the accuracy rate of staphylococcus aureus detection.
The third object of the present invention is to provide a kind of detection method of staphylococcus aureus, utilizes above-mentioned golden yellow
Staphylococcic LAMP detection primer group or its LAMP detection kit carry out the quick detection of staphylococcus aureus, have
High sensitivity, the good advantage of specificity.
The invention is realized in this way：
A kind of LAMP detection primer group of staphylococcus aureus comprising have outer primer pair and inner primer pair, outer primer
To including downstream primer shown in sequence sense primer as shown in SEQ ID No.1 and sequence SEQ ID No.2, inner primer
To including the sequence such as sense primer of SEQ ID No.3 and the sequence downstream primer as shown in SEQ ID No.4.
According to application of the above-mentioned LAMP detection primer group in expanding or detecting staphylococcus aureus.
A kind of LAMP detection kit of staphylococcus aureus comprising just like the LAMP of above-mentioned staphylococcus aureus
Detection primer group.
A kind of detection method of staphylococcus aureus comprising following steps：Using outer primer to, inner primer pair and/
Or ring primer pair is under LAMP reaction systems, amplifying target genes；
Outer primer is to including shown in sequence sense primer as shown in SEQ ID No.1 and sequence SEQ ID No.2
Downstream primer, inner primer to including sequence such as SEQ ID No.3 sense primer and sequence such as SEQ ID No.4 shown under
Swim primer；Ring primer pair includes sequence sense primer and sequence as shown in SEQ ID No.5 as shown in SEQ ID No.6
Downstream primer.
The invention has the advantages that：
The embodiment of the present invention provides a kind of LAMP detection primer group of staphylococcus aureus comprising have outer primer pair and
Inner primer pair can expand staphylococcus aureus for the specific region on staphylococcus aureus using LAMP technology
On target gene, realize the quick detection of staphylococcus aureus, meanwhile, promoted staphylococcus aureus testing result it is accurate
Rate.In addition, the present invention also provides a kind of LAMP detection kits of the staphylococcus aureus including the LAMP detection primer group
And its detection method, it can realize the quick detection of staphylococcus aureus, have high sensitivity, specificity good excellent
Point.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the LAMP reaction color variation diagrams under the different magnesium sulfate concentrations in seventh embodiment of the invention；
Fig. 2 is that the LAMP under the different magnesium sulfate concentrations in seventh embodiment of the invention reacts electrophoretogram (wherein, M:
marker DL2000；C:Negative control；1:MgSO4Concentration 2mM；2:3mM；3:4mM；4:5mM；5:6mM；6:7mM；7:8mM)；
Fig. 3 is LAMP specificity experiments result figures (wherein, the M in eighth embodiment of the invention:marker DL2000；C:
Negative control；1:Staphylococcus aureus ATCC 29213；2:Staphylococcus aureus ATCC 25923；3:Staphylococcus aureus
Bacterium ATCC 43300；4:Human fetal cardiomyocytes；5:Staphylococcus saprophyticus；6:Streptococcus pneumonia；7:Surface staphylococcus；8:Verdigris is false
Monad；9:Enterococcus faecalis)；
Fig. 4 is 29213 sensitivity experiments (1 of staphylococcus aureus ATCC in ninth embodiment of the invention:1ng/μL；2:
100pg/μL；3:10pg/μL；4:1pg/μL；5:100fg/μL；6:10fg/μL；7:1fg/μL)；
Fig. 5 is 29213 sensitivity experiment electrophoretogram (M of staphylococcus aureus ATCC in ninth embodiment of the invention:
marker DL2000；C:Negative control；1:1ng/μL；2:100pg/μL；3:10pg/μL；4:1pg/μL；5:100fg/μL；6:
10fg/μL；7:1f g/μL)；
Fig. 6 is 25923 sensitivity experiments (1 of staphylococcus aureus ATCC in ninth embodiment of the invention:1ng/μL；2:
100pg/μL；3:10pg/μL；4:1pg/μL；5:100fg/μL；6:10fg/μL；7:1fg/μL)；
Fig. 7 is 25923 sensitivity experiment electrophoretogram (M of staphylococcus aureus ATCC in ninth embodiment of the invention:
marker DL2000；C:Negative control；1:1ng/μL；2:100pg/μL；3:10pg/μL；4:1pg/μL；5:100fg/μL；6:
10fg/μL；7:1f g/μL).
Specific implementation mode
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
LAMP detection primer group to a kind of staphylococcus aureus of the embodiment of the present invention, its LAMP detection reagent below
Box and its detection method are specifically described.
LAMP, Loop-mediated Isothermal amplification, also known as loop-mediated isothermal amplification technique.
LAMP methods are characterized in designing four primers for six regions on target gene, using strand displacement type archaeal dna polymerase in constant temperature
Under the conditions of carry out amplification reaction, reaction can generate a large amount of amplified production, i.e. magnesium pyrophosphate white precipitate, observe by the naked eye white
The presence or absence of color precipitation judges that target gene whether there is.
The embodiment of the present invention uses ring mediated isothermal amplification (LAMP) technology, and for the resistance to thermonuclear of staphylococcus aureus
Phytase gene (nuc) devises LAMP detection primer group.
The LAMP detection primer group of a kind of staphylococcus aureus provided in an embodiment of the present invention comprising have outer primer pair
With inner primer pair, outer primer is to including shown in sequence sense primer as shown in SEQ ID No.1 and sequence SEQ ID No.2
Downstream primer, inner primer to including the sequence such as sense primer of SEQ ID No.3 and sequence as shown in SEQ ID No.4
Downstream primer.
Further, in some embodiments of the invention, the LAMP detection primer group of staphylococcus aureus can be with
Include ring primer pair, ring primer pair includes sequence sense primer as shown in SEQ ID No.5 and sequence such as SEQ ID
Downstream primer shown in No.6.Ring primer pair can increase LAMP amplified reactions on the basis of outer primer pair and inner primer pair
Rate, effectively improve the amplification efficiency of target gene.
The LAMP detection primer group that the embodiment of the present invention provides above-mentioned staphylococcus aureus is expanding or is detecting golden yellow
Application in staphylococcus.Specifically comprising the LAMP detection primer group of above-mentioned staphylococcus aureus is subjected to PCR reactions
Or LAMP reactions are to expand or detect staphylococcus aureus.
The embodiment of the present invention also provides a kind of LAMP detection kit of staphylococcus aureus comprising has above-mentioned golden yellow
The staphylococcic LAMP detection primer group of color.
Further, in some embodiments of the invention, LAMP detection kit include one or more of at
Point：BstDNA polymerases, 10 × reaction buffer, dyestuff, dNTPs, standard positive template and negative control.
Further, in some embodiments of the invention, 10 × reaction buffer include one or more of at
Point：10 × buffer buffer solutions, glycine betaine, dNTPs and magnesium sulfate.
Wherein, 10 × buffer buffer solutions may include having 200mM Tris-HCl, 8.8 pH, 100mM (NH4)2SO4、
500mM KCl、20mM MgSO4And 0.1%Triton X-100；Dyestuff is the hydroxynaphthol blue dyestuff HNB of concentration 3mM；Mark
Quasi- positive template is staphylococcus aureus reference culture genomic DNA；Negative control is sterilizing ultra-pure water.
In addition, the embodiment of the present invention also provides a kind of detection method of staphylococcus aureus comprising there is following steps：
Using outer primer pair and inner primer under LAMP reaction systems, amplifying target genes；
Outer primer is to including the sequence sense primer nuc-F3 as shown in SEQ ID No.1 and sequence SEQ ID No.2
Shown in downstream primer nuc-B3；
Inner primer to including sequence such as SEQ ID No.3 sense primer nuc-FIP and sequence such as SEQ ID No.4 institutes
The downstream primer nuc-BIP shown；
Further, in some embodiments of the invention, further include expanding using ring primer pair under LAMP reaction systems
Increase target gene；
Ring primer pair includes sequence sense primer and sequence as shown in SEQ ID No.5 as shown in SEQ ID No.6
Downstream primer.
Further, in some embodiments of the invention, in above-mentioned LAMP reaction systems, outer primer and inner primer
Molar ratio is 1：(4~5)；Further, the molar ratio of outer primer and ring primer is 1：(2~3).The molar ratio can increase mesh
Gene LAMP amplified reactions rate and stability.
Further, in some embodiments of the invention, in above-mentioned LAMP reaction systems, the reaction temperature of LAMP is
60 DEG C~67 DEG C, it is preferable that reaction temperature is 65 DEG C.Under the reaction temperature, the LAMP of above-mentioned staphylococcus aureus is detected
Primer sets can efficiently, rapidly amplifying target genes can obtain an amplification being more clear that is, under the reaction temperature
Or experimental result as a result.
LAMP amplified reactions：
First, LAMP reaction systems are prepared：BstDNA polymerases, 10 × buffer buffer solutions, dyestuff, dNTPs, beet
The genomic DNA of alkali, magnesium ion, staphylococcus aureus primer sets and sample to be tested is mended with sterilizing ultra-pure water to 25 μ L,
Positive control and negative control are set simultaneously, prepared solution is positioned in reaction tube or detection pipe, mixing is placed on 60
DEG C~67 DEG C at a temperature of react 30min, and interrupt reaction in 80 DEG C.
Then, above-mentioned reaction tube is placed in colorimetric card, is judged according to color change, testing result is in blue, detection
Result judgement is the positive, that is, detects in sample and contain staphylococcus aureus；If testing result is in purple, testing result is determined as
Feminine gender detects and does not contain staphylococcus aureus in sample.
The feature and performance of the present invention are described in further detail with reference to embodiments.
First embodiment
The present embodiment provides a kind of LAMP detection primer groups of staphylococcus aureus comprising has outer primer pair to draw with interior
Object pair.
For outer primer to including sense primer nuc-F3 and downstream primer nuc-B3, sequence is as follows:
nuc-F3:5’-TCGAGTTTGACAAAGGTCA-3’；
nuc-B3:5’-GTTGTCTTCGCTCCAAAT-3’；
For inner primer to including sense primer nuc-FIP and downstream primer nuc-BIP, sequence is as follows:
nuc-FIP:5’-TGACGAACTAAAGCTTCGTTTACC-CTGATAAATATGGACGTGGC-3’；
nuc-BIP:5-GGCTTGGCTAAAGTTGCTTATGT-TTTTTCGCTTGTGCTTCAC-3’；
Further, in the present embodiment, LAMP detection primer group further includes ring primer pair.Ring primer pair includes upstream
Primer nuc-LF and downstream primer nuc-LB, sequence are as follows:
nuc-LF:5’-CCATCAGCATAAATATACGCT-3’；
nuc-LB:5’-CCTAACAATACACATGAACAAC-3’。
Second embodiment
The present embodiment provides a kind of LAMP detection kits of staphylococcus aureus.
Specifically, LAMP detection kit includes reaction premixed liquid and detection pipe.Reaction premixed liquid includes BstDNA
Polymerase 8U, 10 × Buffer buffer solutions, 2.5 μ L (200mM Tris-HCl, pH 8.8,100mM (NH4)2SO4, 500mM KCl,
20mM MgSO4, 0.1%Triton X-100), 1 μ L of HNB dyestuffs, dNTPs1.6mM, glycine betaine 0.8M, magnesium sulfate 6mM, template
The LAMP detection primer group for the staphylococcus aureus that 1 μ L of DNA and first embodiment provide, in above-mentioned reaction premixed liquid
Addition sterilizing ultra-pure water is prepared into 25 μ L detection solution and is put into detection pipe.
3rd embodiment
The present embodiment provides a kind of detection methods of staphylococcus aureus.
Detection method includes the following steps for this：The outer primer provided using first embodiment is to, inner primer pair and ring primer
To under LAMP systems, amplifying target genes.
It is specific as follows：
First, the DNA of sample to be tested is extracted：It takes sample to be tested 1ml to carry out 12000rpm centrifugation 3min, and takes precipitation, to
50~75 μ L sterile waters are added in precipitation to be resuspended, then places in 95 DEG C~100 DEG C and keeps the temperature 10min, it is cooling after 12000rpm from
Heart 1min, takes supernatant, is positioned over 4 DEG C and saves backup.
Then, the detection primer group provided using first embodiment is carried out LAMP amplifications and drawn outside in LAMP reaction systems
The molar ratio of object and inner primer is 1：4；The molar ratio of outer primer and ring primer is 1：2.
Include in LAMP reaction systems：BstDNA polymerases 8U, 10 × Buffer buffer solutions, 2.5 μ L (200mM Tris-
HCl, pH 8.8,100mM (NH4)2SO4, 500mM KCl, 20mM MgSO4, 0.1%Triton X-100), 120 μ of HNB dyestuffs
M, the golden yellow grape that dNTPs1.6mM, glycine betaine 0.8M, magnesium sulfate 6mM, 1 μ L of template DNA and first embodiment provide
The LAMP detection primer group of coccus (0.4 μM of 0.4 μM of the sense primer of outer primer pair and downstream primer, draw by the upstream of inner primer pair
0.8 μM of 1.6 μM of 1.6 μM of object and downstream primer, 0.8 μM of the sense primer of ring primer pair and downstream primer), it is premixed in above-mentioned reaction
Addition sterilizing ultra-pure water is prepared into 25 μ L detection solution and is put into detection pipe in liquid.
The supernatant of the above-mentioned DNA containing sample to be tested of 1 μ L is taken, is added into detection pipe, 65 DEG C of reaction 30min.
Finally, by detection pipe under colorimetric card background observing response liquid color, it is blue then illustrate in sample containing golden yellow
Staphylococcus, purple then illustrate do not have staphylococcus aureus in sample.
Fourth embodiment
The present embodiment provides a kind of detection methods of staphylococcus aureus.The detection method is provided with 3rd embodiment
Detection method is roughly the same, difference lies in：In LAMP reaction systems, outside the LAMP detection primer group of first embodiment offer
The molar ratio of primer and inner primer is 1:5；The molar ratio of outer primer and ring primer is 1:2, i.e. 0.4 μM of outer primer, inner primer 2.0
μM and 0.8 μM of ring primer；
LAMP reaction temperatures are 66 DEG C；
A concentration of 5mM of magnesium ion (magnesium sulfate).
5th embodiment
The present embodiment provides a kind of detection methods of staphylococcus aureus.The detection method is provided with 3rd embodiment
Detection method is roughly the same, difference lies in：In LAMP reaction systems, outside the LAMP detection primer group of first embodiment offer
The molar ratio of primer and inner primer is 1:4；The molar ratio of outer primer and ring primer is 1:3, i.e. 0.4 μM of outer primer, inner primer 1.6
μM and 1.2 μM of ring primer.
LAMP reaction temperatures are 65 DEG C；
A concentration of 6mM of magnesium ion (magnesium sulfate).
Sixth embodiment
In the LAMP detection method for verifying the staphylococcus aureus that the embodiment of third~tetra- provides, LAMP detection primer group
Outer primer, inner primer and ring primer optimum molar ratio.
1. experimental method
The LAMP detection method Detection and Extraction of the staphylococcus aureus provided using the embodiment of third~five containing gold
The sample to be tested DNA of staphylococcus aureus.
Specifically, in the LAMP systems of 25 μ L, specific ingredient is 10 × Buffer 2.5 μ L, dNTPs (10mM) 4 μ l
(final concentration 1.6mM), glycine betaine (5M) 4 μ L (final concentration 0.8M), MgSO4(50mM) 3 μ L (final concentration 6mM), Bst DNA polymerizations
Enzyme 1 μ L, HNB dyestuffs (3mM) 1 μ l (120 μM of final concentration), 1 μ L, DEPC water of DNA profiling supply system.Outer setting negative control,
DNA profiling is replaced with 1 μ L DEPC water.
15 groups of primer ratios, outer primer are set：Ring primer：Inner primer ratio is respectively 1:1:2、1:1:3、1:1:4、1:
1:5、1:1:6、1:2:2、1:2:3、1:2:4、1:2:5、1:2:6、1:3:2、1:3:3、1:3:4、1:3:5、1:3:6, specifically ask
With reference to attached drawing 1.The concentration of a concentration of 0.4 μM of outer primer, ring primer and inner primer is with ratio respective change.
2. experimental result
The LAMP reaction results of the different primers ratio of 1 sixth embodiment of table
| Primer ratio | 1:1:2 | 1:1:3 | 1:1:4 | 1:1:5 | 1:1:6 |
| LAMP results | - | - | - | - | - |
| Primer ratio | 1:2:2 | 1:2:3 | 1:2:4 | 1:2:5 | 1:2:6 |
| LAMP results | - | - | + | + | - |
| Primer ratio | 1:3:2 | 1:3:3 | 1:3:4 | 1:3:5 | 1:3:6 |
| LAMP results | - | - | + | + | - |
Remarks：("-" indicates reaction into feminine gender, and "+" indicates that reaction is positive)
As shown in Table 1, when primer ratio is 1:2:4、1:2:5 and 1:3:4、1:3:When 5, it is anti-that LAMP amplifications can be generated
It answers, other primer ratios do not react, illustrate to work as outer primer：Ring primer：Inner primer is 1：(1~3)：When (2~6),
It can carry out LAMP reactions.
7th embodiment
In the LAMP detection method for verifying the staphylococcus aureus that the embodiment of third~five provides, magnesium sulfate it is optimal dense
Degree.
MgSO4For playing an important role for amplified reaction, Tai Gao or too low concentration can may all influence amplification instead
Should carry out.In the LAMP systems using the colour developing instruction of HNB hydroxynaphthol blues, MgSO4Solution concentration is bigger, the color of reaction solution
Will more inclined aubergine, when concentration is too low, reaction solution before the reaction i.e. in blue, lose reaction indicative function.While dNTPs
Concentration can also influence the color of reaction solution, and the concentration of dNTPs is higher, and reaction solution color is more blue, this LAMP systems keep dNTPs
Concentration it is constant, to MgSO4Concentration be optimized.The MgSO of signified optimization herein4Solution concentration does not include 10 × Buffer
In MgSO4。
1. experimental method
Using the LAMP detection method detection for the staphylococcus aureus that third and fourth embodiment provides.
Specifically, in the LAMP systems of 25 μ L, specific ingredient is 10 × Buffer 2.5 μ L, dNTPs (10mM) 4 μ l
(final concentration 1.6mM), 1 μ L, Bst archaeal dna polymerase of glycine betaine (5M) 4 μ l (final concentration 0.8M), Pirmer mix, 1 μ L, HNB dyes
Expect (3mM) 1 μ l (120 μM of final concentration), 1 μ L of DNA profiling.MgSO4(50mM) final concentration be respectively 2mM, 3mM, 4mM, 5mM,
6mM, 7mM, 8mM (such as table 2) are eventually adding DEPC water and supply system.In addition negative control is set, replaced with 1 μ L DEPC water
DNA profiling, MgSO4Concentration is set as 6mM.
2 LAMP of table reacts different magnesium sulfate concentrations
The color change for observing and recording the front and back reaction solution of reaction, carries out 1% agarose gel electrophoresis mirror after reaction
It is fixed.Take 5 μ L LAMP products and 2 μ L10 × Loading buffer after mixing, loading.Analysis reaction result determines sulfuric acid
Magnesium optium concentration, experimental result are as shown in Fig. 2.
2. experimental result
Before the reaction, it is different because of the concentration variation of magnesium sulfate to add the reaction solution color of HNB dyestuffs, with
Magnesium sulfate concentration increases to 8mM from 2mM, and color is gradually biased to aubergine (due to requiring attached drawing to be unable to chromatic colour, here from blue
Color change can not obviously show in figure, and word method is taken to illustrate).
After reaction, according to electrophoretogram, attached drawing 2 is please referred to, when magnesium sulfate concentration is 2mM and 3mM, magnesium sulfate concentration concentration
It is too low without occur LAMP amplified reactions, electrophoresis result do not occur LAMP reaction typical scalariform band.When magnesium sulfate is dense
After degree reaches 4mM, electrophoresis starts trapezoid-shaped strips occur, and band increases more and more clearly with concentration, illustrates magnesium sulfate
Concentration can realize the LAMP reactions of stability and high efficiency in 5~7mM.
But magnesium sulfate concentration is higher, and the discoloration effect of HNB dyestuffs can then die down (Fig. 1), therefore comprehensive LAMP is reacted and shown
Therefore magnesium sulfate concentration is set as 5mM~6mM by the sensitivity level of colour response, not only can guarantee that LAMP reactions efficiently carry out but also have bright
Aobvious colour-difference away from, and conducive to using naked eyes directly judge LAMP react generation.
8th embodiment
Verify the specificity of the LAMP detection primer group of staphylococcus aureus in first embodiment.
1. experimental method
Staphylococcus aureus (ATCC 29213) is chosen in this experiment, and staphylococcus aureus (ATCC 25923) is golden yellow
Color staphylococcus (ATCC 43300), streptococcus pneumonia (ATCC 49619), staphylococcus saprophyticus (ATCC 49453), epidermis Portugal
Grape coccus (ATCC 49134), Human fetal cardiomyocytes (ATCC 27844), enterococcus faecium (ATCC 19434), enterococcus faecalis (ATCC
19433), bacillus coli (ATCC 35150), the DNA that pseudomonas aeruginosa (ATCC 15442) is extracted are used for LAMP
Detection.
The detection method detection provided using the embodiment of third~tetra-.Specifically, in the LAMP systems of 25 μ L, specifically at
It is divided into 10 × Buffer 2.5 μ L, dNTPs (10mM) 4 μ l (final concentration 1.6mM), 4 μ L (final concentration 0.8M) of glycine betaine (5M),
MgSO41 μ L, Bst archaeal dna polymerases of (50mM) 3 μ L (final concentration 6mM), Pirmer mix 1 μ L, HNB dyestuffs (3mM) 1 μ l are (eventually
120 μM of concentration), 1 μ L of DNA profiling are eventually adding DEPC water and supply system.Outer setting negative control is replaced with 1 μ L DEPC water
DNA profiling.
It is foundation according to the HNB dyestuffs of addition, LAMP reactions occur, and then solution becomes blue, and unreacted solution and feminine gender are right
According to for purple it is constant.1% agarose gel electrophoresis identification is carried out after reaction, is as a result please referred to attached drawing 3, is occurred in electrophoretogram
Trapezoid-shaped strips are presented in the sample of LAMP reactions, and unreacted sample does not have trapezoid-shaped strips.
2. experimental result
From the figure 3, it may be seen that staphylococcus aureus (ATCC 29213), staphylococcus aureus (ATCC 25923) is golden yellow
There are LAMP amplified reactions in color staphylococcus (ATCC 43300) DNA, and apparent scalariform electrophoresis band occurs in electrophoresis result.Pneumonia
Streptococcus (ATCC 49619), staphylococcus saprophyticus (ATCC 49453), staphylococcus epidermis (ATCC 49134), people's grape ball
Bacterium (ATCC 27844), enterococcus faecalis (ATCC 19433), the DNA that pseudomonas aeruginosa (ATCC 15442) is extracted is not
LAMP reactions occur.Illustrate that the LAMP detection primer group that first embodiment provides has specificity.
9th embodiment
Verify the LAMP that the LAMP detection primer group or second embodiment of staphylococcus aureus in first embodiment provide
The sensitivity of detection kit.
1. experimental method
Using the DNA of kit extraction staphylococcus aureus ATCC 29213 and ATCC 25923, it is dense to measure DNA solution
It spends and dilutes following concentration：1ng/μL、100pg/μL、10pg/μL、1pg/μL、100fg/μL、10fg/μL、1fg/μL.Using
The detection method that third or four implementations provide carries out LAMP detections.
Specifically, in LAMP systems, specific ingredient is 10 × Buffer, 2.5 4 μ l (final concentrations of μ L, dNTPs (10mM)
1.6mM), 4 μ L (final concentration 0.8M) of glycine betaine (5M), MgSO4(50mM) 3 μ L (final concentration 6mM), Pirmer mix 1 μ L, Bst
Archaeal dna polymerase 1 μ L, HNB dyestuffs (3mM) 1 μ l (120 μM of final concentration), 1 μ L of DNA profiling are eventually adding DEPC water and supply system.
Outer setting negative control replaces DNA profiling with 1 μ L DEPC water.
After LAMP experiments, above-mentioned LAMP reaction solutions are subjected to electrophoresis experiment.Staphylococcus aureus ATCC's 29213
Sensitivity experiment is as shown in figure 4, its electrophoresis experiment is as shown in Figure 5.The sensitivity experiment of staphylococcus aureus ATCC 25923
As shown in fig. 6, its electrophoresis experiment is as shown in Figure 7.
It is foundation according to the HNB dyestuffs of addition, LAMP reactions occur, and then solution becomes blue, and unreacted solution and feminine gender are right
According to for purple it is constant.Trapezoid-shaped strips are presented in the sample that LAMP reactions occur in running gel figure, and unreacted sample does not have trapezoid-shaped strips.
2. experimental result
It is shown by reaction solution color change and electrophoretogram, staphylococcus aureus ATCC29213 and ATCC25923 exist
When being 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L amplified reaction occurs for DNA additive amounts, and color is by purple stain indigo plant, electricity after reaction
There are trapezoid-shaped strips in swimming glue figure.LAMP systems are for golden yellow grape in the LAMP detection method that the embodiment of third~tetra- provides
The DNA detections of coccus are limited to 1pg/ μ L.
In conclusion the embodiment of the present invention provides a kind of LAMP detection primer group of staphylococcus aureus comprising have
A pair of of outer primer and a pair of of inner primer can be expanded for the specific region on staphylococcus aureus using LAMP technology
Target gene on staphylococcus aureus realizes the quick detection of staphylococcus aureus, meanwhile, promote staphylococcus aureus
The accuracy rate of testing result.In addition, the present invention also provides a kind of staphylococcus aureuses including the LAMP detection primer group
LAMP detection kit and its detection method can realize the quick detection of staphylococcus aureus, have high sensitivity,
The good advantage of specificity.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>South China Science ＆ Engineering University and Community in Baiyunshan, Guangzhou pharmacy joint-stock company of group White Cloud Mountain pharmacy head factory
<120>A kind of LAMP detection primer composition of staphylococcus aureus, its LAMP detection kit and its detection side
Method
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
tcgagtttga caaaggtca 19
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
gttgtcttcg ctccaaat 18
<210> 3
<211> 44
<212> DNA
<213>Artificial sequence
<400> 3
tgacgaacta aagcttcgtt taccctgata aatatggacg tggc 44
<210> 4
<211> 42
<212> DNA
<213>Artificial sequence
<400> 4
ggcttggcta aagttgctta tgttttttcg cttgtgcttc ac 42
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<400> 5
ccatcagcat aaatatacgc t 21
<210> 6
<211> 22
<212> DNA
<213>Artificial sequence
<400> 6
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Claims (10)
1. a kind of LAMP detection primer group of staphylococcus aureus, which is characterized in that it includes outer primer pair and inner primer
It is right, the outer primer to include shown in sense primer shown in sequence such as SEQ ID No.1 and sequence SEQ ID No.2 under
Swim primer, the inner primer to including the sequence such as sense primer of SEQ ID No.3 and sequence as shown in SEQ ID No.4
Downstream primer.
2. the LAMP detection primer group of staphylococcus aureus according to claim 1, which is characterized in that further include having ring
Primer pair, the ring primer pair include sequence sense primer and sequence such as SEQ ID No.6 institutes as shown in SEQ ID No.5
The downstream primer shown.
3. application of the LAMP detection primer group as claimed in claim 1 or 2 in expanding or detecting staphylococcus aureus.
4. a kind of LAMP detection kit of staphylococcus aureus, which is characterized in that it includes just like claims 1 or 2 institute
State the LAMP detection primer group of staphylococcus aureus.
5. the LAMP detection kit of staphylococcus aureus according to claim 4, which is characterized in that its include with
A kind of lower or Multiple components：BstDNA polymerases, 10 × reaction buffer, dyestuff, dNTPs, standard positive template and feminine gender
Control.
6. a kind of detection method of staphylococcus aureus, which is characterized in that it includes the following steps：Utilize outer primer pair and interior
Primer pair is under LAMP reaction systems, amplifying target genes；
The outer primer is to including shown in sequence sense primer as shown in SEQ ID No.1 and sequence SEQ ID No.2
Downstream primer；
The inner primer to including sequence such as SEQ ID No.3 sense primer and sequence such as SEQ ID No.4 shown under
Swim primer.
7. detection method according to claim 6, which is characterized in that the detection method further includes being existed using ring primer pair
Under LAMP reaction systems, amplifying target genes；
The ring primer pair includes sequence sense primer and sequence as shown in SEQ ID No.5 as shown in SEQ ID No.6
Downstream primer.
8. detection method according to claim 7, which is characterized in that in the LAMP reaction systems, the outer primer
Molar ratio with the inner primer is 1：(4~5)；The molar ratio of the outer primer and the ring primer is 1：(2~3).
9. the detection method described according to claim 6 or 7, which is characterized in that in the LAMP reaction systems, LAMP reactions
Temperature is 60 DEG C~67 DEG C.
10. the detection method described according to claim 6 or 7, which is characterized in that in the LAMP reaction systems, include
The reaction density of magnesium ion, the magnesium ion is 5~6mM.
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| CN113249516B (en) * | 2020-02-10 | 2023-04-25 | 华南理工大学 | Primer group of 2019-nCoV and 2019-nCoV nucleic acid detection kit |
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