CN102220424A - Rapid detection method for enterococcus, detection primer group and detection kit - Google Patents
Rapid detection method for enterococcus, detection primer group and detection kit Download PDFInfo
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Abstract
The invention discloses a rapid detection method for enterococcus, a detection primer group and a detection kit According to loop-mediated isothermal amplification (LAMP) technology, the primer group is obtained by analysis, design and artificial synthesis of the highly conservative part in 16sRNA gene sequence of enterococcus. Containing nucleotide sequences shown in SEQ No.1-4, the primer group shows high specificity to enterococcus. The rapid detection method for enterococcus is realized by performing a LAMP reaction to the enterococcus DNA in a sample with the detection primer group. Through identification of reaction products, whether enterococcus exists in the sample can be determined. In the invention, on the basis of the above detection method a rapid detection kit for enterococcus is also designed for rapid, simple, accurate and efficient detection and identification of enterococcus.
Description
Technical field
The invention belongs to the microorganism detection field, relate to a kind of faecalis method for quick and detect primer sets and detection kit.
Background technology
Faecalis (Enterococcus) belongs to Streptococcaceae, is the part of human and animal's normal intestinal flora, and is stronger to cold and hot tolerance, finds in causing the isolating mixing mycelia of abdominal cavity and pelvic infection institute usually.Previously think that faecalis is the commensalism bacterium harmless to the mankind, but research in recent years and clinical case have confirmed enterococcal pathogenic.In aerobic gram positive coccus, it is to be only second to staphylococcic important ward infection pathogenic bacterium; Faecalis also can cause community infection.Faecalis not only can cause urinary tract infections, skin soft-tissue infection, also can cause life-threatening abdominal cavity infection, septicemia, endocarditis and meningitis etc.Faecalis also can cause food poisoning, and common poisoning food is milk and milk preparation, meat product etc., and the report of the food poisoning that is caused by faecalis also increases gradually.The hygienist thinks that faecalis is similar to the eco-activity of coliform, but stronger to the external environment resistibility, and as the monitoring hygienic quality, the contamination index of environmental sanitary quality has more health significance.Simultaneously,, easily produce acquired resistance again because faecalis also is intrinsic resistance to multiple microbiotic, therefore very important to the monitoring of enterococcal pollution condition and fashion trend thereof.
According to bibliographical information, enterococcal contamination level reaches 10 in food
5During/g, can cause food poisoning, and grow the adult crowd of incomplete newborn infant and hypoimmunity for immunity system, lower faecalis contamination level just can cause food poisoning.National standard prevailing for the time being in force has been made the enterococcal requirement of limiting the quantity of (must not be defined as detect as natural mineral water) to part food.Mainly be accredited as the master with plate isolation in the food inspection process at present this bacterium is detected, operation is all comparatively complicated, and length consuming time, can not in time provide basis for estimation for accident.Along with the fast development of Protocols in Molecular Biology, (Loop – Mediated Isothermal Amplification LAMP) progressively is applied to the detection of food-borne pathogens to loop-mediated isothermal amplification technique.
The LAMP method is a kind of novel constant temperature nucleic acid amplification technology, and this technology is mainly utilized 6 specific regions on 4 kinds of different Auele Specific Primers identification target DNAs, and utilize a kind of have the active archaeal dna polymerase of strand displacement (
BstDNA), rapid amplifying nucleic acid under constant temperature has guaranteed the high specific and the high-level efficiency that increase.Because the nucleic acid amplification mechanism of LAMP uniqueness, its product can be presented LAMP and be reacted typical stepped band by stem one cyclic DNA of multiple multiple target sequence and the mixture that Cauliflower shape DNA is formed on agarose gel electrophoresis; Simultaneously, when nucleic acid generates in a large number, pyrophosphate ion of from dNTPs, separating out and the Mg in the reaction system
2+In conjunction with, produce macroscopic amplified reaction by product-white magnesium pyrophosphate precipitation; In addition, because amplified production generates in a large number, add fluorescence dye (as Syber Green I, ethidium bromide, Gel Red etc.).Therefore not only view mode is various for the LAMP amplification, and the result identifies simply very suitable high-throughout rapid detection.
In view of the LAMP technology has numerous advantages, now be used to the detection of pathogenic micro-organism, as mycobacterium, Enterobacter sakazakii, Salmonellas, streptococcus aureus etc.Now not seeing as yet has LAMP to be applied to the relevant report that faecalis detects.
Summary of the invention
The purpose of this invention is to provide a kind of enterococcal method for quick and detect primer sets and detection kit.
For achieving the above object, the technical solution used in the present invention is: the upstream primer of the outer primer of enterococcal detection primer sets has the sequence shown in SEQ No.1, and the downstream primer of its outer primer has the sequence shown in SEQ No.2; The upstream primer of its inner primer has the sequence shown in SEQ No.3, and the downstream primer of its inner primer has the sequence shown in SEQ No.4.
Use primer sets of the present invention to the method that faecalis carries out rapid detection to be: utilize described primer sets that the DNA of testing sample is carried out the LAMP reaction, reaction finishes the back and reaction solution is carried out yin and yang attribute judges.
Further, LAMP reaction of the present invention is undertaken by following first kind of scheme or second kind of scheme:
First kind of scheme: described LAMP is reflected at 59.5-62.5 ℃ of reaction 50-70min down;
Second kind of scheme: described LAMP reaction comprises following steps:
(1) reacts 50-70min down at 59.5-62.5 ℃ earlier;
(2) back is reacted 6-12min down at 80 ℃.
Further, the present invention is in the reaction solution that carries out described LAMP reaction, comprise concentration and be the upstream primer of described outer primer of 0.15-0.25 μ M and upstream primer and the downstream primer that downstream primer, concentration are the described inner primer of 0.6-1.0 μ M, concentration is the trimethyl-glycine of 0.6-0.7 M, and concentration is the Mg of 4.0-5.6mM
2+, 10 %(volumes) 10 *
BstDna polymerase buffer liquid, concentration are 0.2-0.4U/ μ L's
BstArchaeal dna polymerase, each concentration are the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and the triphosphoric acid deoxycytidylic acid of 0.6-1.0mM.
Use the quick detection kit of primer sets of the present invention comprise the mixing solutions of the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid and described primer sets, positive reference substance,
BstArchaeal dna polymerase, 10 *
BstDna polymerase buffer liquid, Mg
2+, alkali solution of beet and aseptic ultrapure water, the mol ratio of the acid of described triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid is 1:1:1:1, and described positive reference substance is enterococcal genomic dna.
Use the quick detection kit of primer sets of the present invention to comprise 2 * LAMP reaction solution, in described 2 * LAMP reaction solution, include described primer sets,
BstArchaeal dna polymerase, 2 *
BstDna polymerase buffer liquid, Mg
2+, alkali solution of beet, the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid, triphosphoric acid deoxycytidylic acid and aseptic ultrapure water mixed solution, the upstream primer of described outer primer and the degree of downstream primer are 0.3-0.5 μ M, the upstream primer of described inner primer and the concentration of downstream primer are 1.2-2.0 μ M, the concentration of trimethyl-glycine is 1.2-1.4 M, Mg
2+Concentration be 8.0-11.2mM,
BstThe concentration of archaeal dna polymerase is 0.4-0.8U/ μ L, and the concentration of the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid respectively is 1.2-2.0mM.
Compared with prior art, the invention has the beneficial effects as follows:
1. the present invention is according to the conservative part of disclosed faecalis 16sRNA gene order camber, analyzes design and obtains faecalis specificity LAMP detection primer sets of the present invention, and its high specificity can accurately detect the faecalis genomic dna.
2. owing to use primer sets of the present invention, the present invention's faecalis genomic dna purpose fragment that can rapidly and efficiently increase, whole LAMP amplification procedure can be finished in 1 hour, and amplified production yield height is accurate, rapid, easy, efficient to enterococcal detection.
3. in method for quick of the present invention, the LAMP amplified reaction can be finished under steady temperature, and the temperature fluctuation range that allows big (± 1.5 ℃), and therefore less demanding to the plant and instrument of temperature control, water bath or plate heater all can meet the demands.
4. method for quick of the present invention is highly sensitive, and amplification template faecalis genomic dna concentration only needs 10fg/ μ L.
5. method for quick result of the present invention judges easy, and can adopt different interpretation modes as a result according to the laboratory physical condition, and method applicability is stronger.
6. compare with the plating method of routine, method for quick of the present invention can shorten sense cycle greatly, reduce to detect link, and the disposal that can be accident etc. provides detected result accurately and reliably.
Description of drawings
Fig. 1 has shown the faecalis LAMP reaction product situation after centrifugal;
Fig. 2 has shown faecalis LAMP reaction product after the dyeing of SYBR Green I, the result who observes under ultraviolet lamp.
Embodiment
Below in conjunction with specific embodiment the present invention is further elaborated, but the present invention is not done any qualification.
The concrete detection step of present embodiment is:
1. the extraction of sample DNA
1.1 the faecalis reference culture is inoculated in the 10mL nutrient broth medium, cultivated 24 hours for 36 ℃ ± 1 ℃.
Extract bacterial genomes DNA in the cultured products 1.2 use QIAGEN bacterial genomes DNA extraction test kit.
2. PCR reaction
2.1 the upstream and downstream primer of synthetic faecalis outer primer, wherein, upstream primer F3 has the nucleotide sequence shown in SEQ No.1, and downstream primer B3 has the nucleotide sequence shown in SEQ No.2.
2.2 PCR reaction system
The PCR reaction system is: archaeal dna polymerase 0.05U/ μ L, dATP, dTTP, each 0.2mM of dGTP, dCTP, MgSO
42mM, 10 %(volumes) 10 * PCR damping fluid, each 0.5 μ M of upstream primer F3 and downstream primer B3, template DNA 1 μ L.
2.3 PCR reaction conditions
Use the PCR instrument, the PCR reaction conditions is: 95 ℃ of 5min, 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 5min; 4 ℃ of preservations.
2.4 the result observes
Reaction solution is directly carried out agarose gel electrophoresis, and deposition condition is electrophoresis 30min under 0.5 * TBE, 2.0% sepharose and the 150V voltage conditions, automatically imaging observations under the gel imaging system.By electrophoresis result visible with expect the consistent single bright amplified band of molecular weight (205bp).
2.5 result verification
Adopt SN/T 1933-2007 " the faecalis method of inspection in food and the water " that sample is detected, detect faecalis, consistent with above PCR detected result, this shows that upstream primer F3 and downstream primer B3 have good specificity.
The concrete detection step of present embodiment is:
1. the extraction of sample DNA
1.1 the faecalis reference culture is inoculated in the 10mL nutrient broth medium, cultivated 24 hours for 36 ℃ ± 1 ℃.
Extract bacterial genomes DNA in the cultured products 1.2 use QIAGEN bacterial genomes DNA extraction test kit.
2.LAMP rapid detection
2.1 synthetic faecalis LAMP detects primer sets, the upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1; The downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2; The upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3; The downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4.
2.2 LAMP reaction system
Use different Mg respectively
2+Concentration is set up 6 groups of LAMP reaction systems respectively.The cumulative volume of reaction system is 25 μ L, each group reaction system all comprises: each 0.2 μ M of the upstream primer F3 of outer primer and downstream primer B3, each 0.8 μ M of the upstream primer FIP of inner primer and downstream primer BIP, alkali solution of beet 0.65M, 10 %(volumes) 10 * BstDNA polymerase buffer, dATP, dTTP, each 0.8mM of dGTP, dCTP, BstDNA polysaccharase 0.3U/ μ L, template DNA 1 μ L; In this 6 group reaction system, MgSO
4Concentration adopt respectively: 2.4mM, 3.2mM, 4mM, 4.8mM, 5.6mM, 6.4mM; The residual volume of reaction system replenishes with aseptic ultrapure water.
2.3 LAMP reaction conditions
Use thermostatical water bath, reaction conditions is: at 61 ℃ of reaction 60min, the back is reaction 6min under 80 ℃ earlier.
2.4 reaction result is observed
Reaction product in each reaction tubes is carried out electrophoretic analysis, and deposition condition is electrophoresis 30min under 0.5 * TBE, 2.0% sepharose, the 150V voltage conditions.From electrophorogram as seen, work as Mg
2+When concentration is 4.0-5.6 mM, present comparatively bright scalariform electrophoretic band, as seen, in the inventive method, Mg
2+Peak optimization reaction concentration is 4.0-5.6mM.
The concrete detection step of present embodiment is:
1. the extraction of sample DNA
1.1 the faecalis reference culture is inoculated in the 10mL nutrient broth medium, cultivated 24 hours for 36 ℃ ± 1 ℃.
Extract bacterial genomes DNA in the cultured products 1.2 use QIAGEN bacterial genomes DNA extraction test kit.
2.LAMP rapid detection
2.1 synthetic faecalis LAMP detects primer sets, wherein, the upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1, the downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2, the upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3, and the downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4.
2.2 LAMP reaction system
Set up 9 reaction tubess, the reaction system cumulative volume is 25 μ L, and the composition of reaction system and concentration are: each 0.2 μ M of the upstream primer F3 of outer primer and downstream primer B3, each 0.8 μ M of the upstream primer FIP of inner primer and downstream primer BIP, MgSO
44.8mM, trimethyl-glycine 0.65M, 10 %(volumes) 10 *
BstDna polymerase buffer liquid, dATP, dTTP, each 0.8mM of dGTP, dCTP,
BstArchaeal dna polymerase 0.3U/ μ L.Each reaction tubes also need add faecalis DNA extraction liquid 1 μ L, and the concentration of template used DNA liquid is made as 10ng/ μ L, 1ng/ μ L, 100pg/ μ L, 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L, 0.1fg/ μ L respectively, in order to the sensitivity of this detection method of contrast test.
2.3 LAMP reaction conditions
Use thermostatical water bath, reaction conditions is: earlier at 61 ℃ of reaction 60min, the back is at 80 ℃ of reaction 6min.
2.4 reaction result is observed
Reaction product in each reaction tubes is carried out electrophoretic analysis, and deposition condition is electrophoresis 30min under 0.5 * TBE, 2.0% sepharose, the 150V voltage conditions.By electrophorogram as seen, by electrophoretic analysis to reaction product, when the concentration of template DNA liquid is reduced to 10fg/ μ L, still visible comparatively bright scalariform electrophoretic band, the detection sensitivity that the inventive method is described is 10fg/ μ L.
Present embodiment adopts milk powder as test sample, specifically detects step and is:
1. the extraction of sample DNA
1.1 get the 25g testing sample to 225mL faecalis broth culture, cultivated 24 hours for 36 ℃ ± 1 ℃.
Extract bacterial genomes DNA in the cultured products 1.2 use QIAGEN bacterial genomes DNA extraction test kit.
1.3 prepare e. coli dna simultaneously as negative control.
2.LAMP rapid detection
2.1 synthetic faecalis LAMP detects primer sets, wherein, the upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1, the downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2, the upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3, and the downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4.
2.2 LAMP reaction system
Set up 3 reaction tubess, wherein No. 1 and No. 2 reaction tubess be testing sample DNA reaction tubes, the reaction tubes of all the other 2 negative contrasts of reaction tubes and blank.The reaction system cumulative volume of each reaction tubes is respectively 25 μ L, and the composition of system and concentration are: the upstream primer F3 of outer primer and downstream primer B3 respectively are 0.15 μ M, and the upstream primer FIP of inner primer and downstream primer BIP respectively are 0.6 μ M, MgSO
4Be 4.0mM, trimethyl-glycine 0.6M, 10 %(volumes) 10 *
BstDna polymerase buffer liquid, dATP, dTTP, dGTP, dCTP respectively be 0.6mM,
BstArchaeal dna polymerase is 0.2U/ μ L, and need add template DNA in addition respectively is 1 μ L(1 number and No. 2 pipe adding testing sample DNA 1 μ L, and No. 2 pipe adds streptococcus aureus DNA 1 μ L, and No. 3 pipe adds aseptic ultrapure water 1 μ L); Residual volume replenishes with aseptic ultrapure water.
2.3 LAMP reaction conditions
Use thermostatical water bath, elder generation reacts 70min down at 59.5 ℃, and the back is reacted 6min down at 80 ℃.
2.4, reaction result observes
With reaction solution centrifugal 5min under the 6000rpm condition of each reaction tubes, wherein the bottom of No. 1 and No. 2 reaction tubess (being sample hose) presents obvious sediment, illustrates in the present embodiment sample to have faecalis.
Fig. 1 shows each reaction tubes centrifuged deposit situation.Under visual inspection, No. 1 and No. 2 reaction tubess bottom present obvious sediment, illustrate in the present embodiment sample to have faecalis; All the other 2 reaction tubes bottoms do not present precipitation, prove that the inventive method detection specificity is good.
2.5, result verification
According to SN/T 1933-2007 " the faecalis method of inspection in food and the water " sample is detected, detect faecalis, consistent with the LAMP detected result.
Present embodiment adopts milk powder as test sample, specifically detects step and is:
1. the extraction of sample DNA
1.1 get the 25g testing sample to 225mL faecalis broth culture, cultivated 24 hours for 36 ℃ ± 1 ℃.
Extract bacterial genomes DNA in the cultured products 1.2 use QIAGEN bacterial genomes DNA extraction test kit.
1.3 prepare e. coli dna simultaneously as negative control.
2.LAMP rapid detection
2.1 synthetic faecalis LAMP detects primer sets, the upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1; The downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2; The upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3; The downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4.
2.2 LAMP reaction system
Set up 4 reaction tubess, wherein No. 5 and No. 6 pipe is the reaction tubes of testing sample DNA, the reaction tubes of all the other 2 negative contrasts of reaction tubes and blank.The reaction system cumulative volume of each reaction tubes is 25 μ L, and the composition of each reaction system and concentration are: the upstream primer F3 of outer primer and the downstream primer B3 of outer primer respectively are that the upstream primer FIP and the downstream primer BIP of 0.2 μ M, inner primer respectively is 0.8 μ M, MgSO
44.8mM, trimethyl-glycine 0.65 M, 10 %(volumes) 10 *
BstDna polymerase buffer liquid, dATP, dTTP, dGTP, dCTP respectively are 0.8 mM,
BstArchaeal dna polymerase is 0.3U/ μ L, and template DNA is 1 μ L; Residual volume replenishes with aseptic ultrapure water.
2.3 LAMP reaction conditions
Use thermostatical water bath, react 60min down at 61 ℃ earlier, the back is at 80 ℃ of 9min of following reaction times.
2.4 reaction result is observed
Add dyestuff SYBR Green I in the reaction solution of each reaction tubes, Fig. 2 shows each reaction tubes fluorescence situation after adding SYBR Green I.Wherein, No. 5 and No. 6 reaction tubess (being the example reaction pipe) present obvious fluorescence under ultraviolet lamp, illustrate in the present embodiment sample to have faecalis.
Under ultraviolet lamp under the visual inspection, No. 5 and No. 6 pipes present obvious fluorescence, illustrate in the present embodiment sample to have faecalis.All the other 2 pipes do not present obvious fluorescence, prove that this method detection specificity is good.
2.5, result verification
According to SN/T 1933-2007 " the faecalis method of inspection in food and the water " sample is detected, detect faecalis, consistent with the LAMP detected result.
Present embodiment adopts milk powder as test sample, specifically detects step and is:
1. the extraction of sample DNA
1.1 get the 25g testing sample to 225mL faecalis broth culture, cultivated 24 hours for 36 ℃ ± 1 ℃.
Extract bacterial genomes DNA in the cultured products 1.2 use QIAGEN bacterial genomes DNA extraction test kit.
1.3 prepare e. coli dna simultaneously as negative control.
2.LAMP rapid detection
2.1 synthetic faecalis LAMP detects primer sets, wherein, the upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1, the downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2, the upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3, and the downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4.
2.2 LAMP reaction system
The reaction system cumulative volume is 25 μ L, and the composition of system and concentration are: the upstream primer F3 of outer primer and downstream primer B3 respectively be 0.25 μ M, the upstream primer FIP of inner primer and downstream primer BIP respectively are 1.0 μ M, MgSO
45.6mM, trimethyl-glycine 0.7M, 10 %(volumes) 10 *
BstDna polymerase buffer liquid, dATP, dTTP, dGTP, dCTP respectively are 1.0mM,
BstArchaeal dna polymerase is 0.4U/ μ L, and template DNA is 1 μ L; Residual volume replenishes with aseptic ultrapure water.
2.3 LAMP reaction conditions
Use thermostatical water bath, elder generation reacts 50min down at 62.5 ℃, and the back is reacted 12min down at 80 ℃.
2.4 reaction result is observed
Add dyestuff SYBR Green I in reaction solution, the example reaction pipe presents obvious fluorescence under ultraviolet lamp, illustrate in the present embodiment sample to have faecalis.
2.5 result verification
According to SN/T 1933-2007 " the faecalis method of inspection in food and the water " sample is detected, detect faecalis, consistent with the LAMP detected result.
This test kit is made up of following reagent:
1. faecalis LAMP detects primer, comprising:
The upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1;
The downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2;
The upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3;
The downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4;
2.
BstArchaeal dna polymerase;
3. positive reference substance: the genome of enterococcal reference culture;
4. blank product: aseptic ultrapure water;
5.10 *
BstDna polymerase buffer liquid;
6.MgSO
4Solution;
7. the mol ratio of the mixing solutions of triphosphoric acid guanine deoxyribonucleoside acid, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid and the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid is 1:1:1:1;
8. alkali solution of beet;
9. as a result the time, can contain developer SYBR Green I with fluorescence developing method observing response at test kit.
The present embodiment test kit is made up of following reagent:
1. faecalis 2 * LAMP detection reaction liquid, it is specifically formed and concentration is:
The equal 0.3 μ M of the upstream primer F3 of outer primer and downstream primer B3, each 1.2 μ M of the upstream primer FIP of inner primer and downstream primer BIP, MgSO
48.0mM, alkali solution of beet 1.2M, 2 *
BstDna polymerase buffer liquid, dATP, dTTP, the equal 1.2mM of dGTP, dCTP,
BstArchaeal dna polymerase 0.4U/ μ L, aseptic ultrapure water; Wherein,
The upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1;
The downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2;
The upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3;
The downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4;
2. positive reference substance: faecalis genomic dna;
3. blank product: aseptic ultrapure water;
4. developer: SYBR Green I.
Embodiment 9 faecalis LAMP quick detection kit
The test kit of present embodiment is made up of following reagent:
1. faecalis 2 * LAMP detection reaction liquid, it is specifically formed and concentration is:
The equal 0.4 μ M of the upstream primer F3 of outer primer and downstream primer B3, the upstream primer FIP of inner primer and downstream primer BIP 1.6 μ M, MgSO
49.6mM, alkali solution of beet 1.3M, 2 *
BstDna polymerase buffer liquid, dATP, dTTP, the equal 1.6mM of dGTP, dCTP,
BstArchaeal dna polymerase 0.6U/ μ L, aseptic ultrapure water; Wherein,
The upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1;
The downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2;
The upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3;
The downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4;
2. positive reference substance: faecalis genomic dna;
3. blank product: aseptic ultrapure water.
Embodiment 10 faecalis LAMP quick detection kit
The test kit of present embodiment is made up of following reagent:
1. faecalis 2 * LAMP detection reaction liquid, it is specifically formed and concentration is:
The equal 0.5 μ M of the upstream primer F3 of outer primer and downstream primer B3, the upstream primer FIP of inner primer and downstream primer BIP 2.0 μ M, MgSO
411.2mM, alkali solution of beet 1.4 M, 2 *
BstDna polymerase buffer liquid, dATP, dTTP, the equal 2.0mM of dGTP, dCTP,
BstArchaeal dna polymerase 0.8 U/ μ L, aseptic ultrapure water; Wherein,
The upstream primer F3 of outer primer has the nucleotide sequence shown in SEQ No.1;
The downstream primer B3 of outer primer has the nucleotide sequence shown in SEQ No.2;
The upstream primer FIP of inner primer has the nucleotide sequence shown in SEQ No.3;
The downstream primer BIP of inner primer has the nucleotide sequence shown in SEQ No.4;
2. positive reference substance: faecalis genomic dna;
3. blank product: aseptic ultrapure water.
＜110〉Zhejiang Province Quality Technology Supervision Detection Research Institute
＜120〉enterococcal method for quick and detection primer sets and detection kit
<160> 4
<170> PatentIn?version?3.1
<210> 1
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 1
gccgcggtaa?tacgtagg 18
<210> 2
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 2
tcgccactgg?tgttcctc 18
<210> 3
<211> 40
<212> DNA
＜213〉artificial sequence
<400> 3
gccgggggct?ttcacatcag?gtccggattt?attgggcgta 40
<210> 4
<211> 40
<212> DNA
＜213〉artificial sequence
<400> 4
accggggagg?gtcattggaa?tttcaccgct?acacatggaa 40
Claims (6)
1. enterococcal detection primer sets, it is characterized in that: the upstream primer of its outer primer outer primer has the sequence shown in SEQ No.1, and the downstream primer of its outer primer has the sequence shown in SEQ No.2; The upstream primer of its inner primer has the sequence shown in SEQ No.3, and the downstream primer of its inner primer has the sequence shown in SEQ No.4.
2. a primer sets of using claim 1 is carried out the method for rapid detection to faecalis, it is characterized in that: utilize described primer sets that the DNA of testing sample is carried out the LAMP reaction, reaction finishes the back and reaction solution is carried out yin and yang attribute judges.
3. method according to claim 2 is characterized in that: described LAMP reaction is undertaken by following first kind of scheme or second kind of scheme:
First kind of scheme: described LAMP is reflected at 59.5-62.5 ℃ of reaction 50-70min down;
Second kind of scheme: described LAMP reaction comprises following steps:
(1) reacts 50-70min down at 59.5-62.5 ℃ earlier;
(2) back is reacted 6-12min down at 80 ℃.
4. according to claim 2 or 3 described methods, it is characterized in that: in the reaction solution that carries out described LAMP reaction, comprise concentration and be the upstream primer of described outer primer of 0.15-0.25 μ M and upstream primer and the downstream primer that downstream primer, concentration are the described inner primer of 0.6-1.0 μ M, concentration is the trimethyl-glycine of 0.6-0.7 M, and concentration is the Mg of 4.0-5.6mM
2+, 10 %(volumes) 10 *
BstDna polymerase buffer liquid, concentration are 0.2-0.4U/ μ L's
BstArchaeal dna polymerase, each concentration are the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and the triphosphoric acid deoxycytidylic acid of 0.6-1.0mM.
5. quick detection kit of using the primer sets of claim 1 is characterized in that: comprise the mixing solutions of the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid and described primer sets, positive reference substance,
BstArchaeal dna polymerase, 10 *
BstDna polymerase buffer liquid, Mg
2+, alkali solution of beet and aseptic ultrapure water, the mol ratio of the acid of described triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid is 1:1:1:1, and described positive reference substance is enterococcal genomic dna.
6. quick detection kit of using the primer sets of claim 1 is characterized in that: comprise 2 * LAMP reaction solution, in described 2 * LAMP reaction solution, include described primer sets,
BstArchaeal dna polymerase, 2 *
BstDna polymerase buffer liquid, Mg
2+, alkali solution of beet, the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid, triphosphoric acid deoxycytidylic acid and aseptic ultrapure water mixed solution, the upstream primer of described outer primer and the degree of downstream primer are 0.3-0.5 μ M, the upstream primer of described inner primer and the concentration of downstream primer are 1.2-2.0 μ M, the concentration of trimethyl-glycine is 1.2-1.4 M, Mg
2+Concentration be 8.0-11.2mM,
BstThe concentration of archaeal dna polymerase is 0.4-0.8U/ μ L, and the concentration of the acid of triphosphoric acid guanine deoxyribonucleoside, triphosphoric acid adenyl-deoxyribonucleotide, triphosphoric acid thymidylic acid and triphosphoric acid deoxycytidylic acid respectively is 1.2-2.0mM.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014156A (en) * | 2012-11-30 | 2013-04-03 | 广东省微生物研究所 | Loop mediated isothermal amplification detection primer group, detection method and kit for enterococcus faecalis |
| KR101938557B1 (en) | 2017-06-16 | 2019-01-15 | 대한민국 | Primers for LAMP based detection of skeletal disease causing pathogen in chicken and its use |
| CN110699483A (en) * | 2019-11-28 | 2020-01-17 | 福建省农业科学院植物保护研究所 | LAMP (loop-mediated isothermal amplification) visualization-based primers for detecting isaria fumosorosea, detection method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040058336A1 (en) * | 2002-09-25 | 2004-03-25 | Cockerill Franklin R. | Detection of vancomycin-resistant enterococcus spp. |
| CN101570795A (en) * | 2008-05-30 | 2009-11-04 | 广州华峰生物科技有限公司 | Mycobacterium tuberculosis gene rapid diagnostic kit based on loop-mediated isothermal amplification technology and detection method thereof |
| CN101575640A (en) * | 2009-03-12 | 2009-11-11 | 华南农业大学 | Primer group for bovine tuberculosis mycobacterium detection, rapid detection method and detection kit |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040058336A1 (en) * | 2002-09-25 | 2004-03-25 | Cockerill Franklin R. | Detection of vancomycin-resistant enterococcus spp. |
| CN101570795A (en) * | 2008-05-30 | 2009-11-04 | 广州华峰生物科技有限公司 | Mycobacterium tuberculosis gene rapid diagnostic kit based on loop-mediated isothermal amplification technology and detection method thereof |
| CN101575640A (en) * | 2009-03-12 | 2009-11-11 | 华南农业大学 | Primer group for bovine tuberculosis mycobacterium detection, rapid detection method and detection kit |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014156A (en) * | 2012-11-30 | 2013-04-03 | 广东省微生物研究所 | Loop mediated isothermal amplification detection primer group, detection method and kit for enterococcus faecalis |
| KR101938557B1 (en) | 2017-06-16 | 2019-01-15 | 대한민국 | Primers for LAMP based detection of skeletal disease causing pathogen in chicken and its use |
| CN110699483A (en) * | 2019-11-28 | 2020-01-17 | 福建省农业科学院植物保护研究所 | LAMP (loop-mediated isothermal amplification) visualization-based primers for detecting isaria fumosorosea, detection method and application |
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