CN110172499A - A kind of method and application improving food-borne pathogenic microorganism LAMP amplified reaction efficiency - Google Patents
A kind of method and application improving food-borne pathogenic microorganism LAMP amplified reaction efficiency Download PDFInfo
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Abstract
The invention discloses a kind of method and its application for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency, the optimization to LAMP amplification is realized by adding colloidal gold material into food-borne pathogenic microorganism LAMP amplification reaction system, and colloidal gold is added in LAMP amplification reaction system and carries out isothermal duplication.This method can effectively improve the sensitivity of LAMP amplified reaction, and be able to suppress the appearance of false positive results, reduce non-specific amplification, to improve the detection efficiency of food-borne pathogenic microorganism.This method can be applied to field of detection of food safety, have wide application prospect.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of raising food-borne pathogenic microorganism LAMP amplified reaction effect
The method and its application of rate.
Background technique
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) is 2000
A kind of isothermal Progress of Nucleic Acid Amplification Technologies of invention, the technology is directed to staphylococcus aureus, salmonella, Dan Zengsheng in recent years
The LAMP detection method of the food-borne pathogenic microorganisms such as listeria spp, vibrio parahemolyticus, comma bacillus is tentatively established,
Quickly to detect in food whether contain these pathogenic microorganisms.The technology is poly- dependent on a kind of DNA with strand displacement characteristic
Synthase (such as Bst enzyme) utilizes 6 different zones of a pair of of outer primer (F3/B3) and a pair of of inner primer (FIP/BIP) and target gene
Anneal makes self pairing of product form a kind of special dumbbell structure, then passes through the mutual of inner primer and dumbbell structure
It recruits pair, carries out under isothermal conditions, amplification 15min-1h can produce 109~1010Amplicon again.Since the technology is sensitive
Degree is high, if experimental situation by Aerosol Pollution, is easy to generate false positive results.In addition, due to LAMP amplified reaction process
In be related to a plurality of primer, inevitably occur between primer non-specific binding generate primer dimer, can consume in reaction system
Reaction substrate, to reduce the efficiency of reaction, reducing detection sensitivity is reduced, while being easy to cause result false positive again, is made
Obtain result erroneous judgement.Therefore, how to establish the existing more high detection sensitivity of one kind, inhibit the LAMP amplification of non-specific amplification anti-again
System is answered, is always the LAMP amplification technique field skill urgently to be solved including food-borne pathogenic microorganism etc. including test objects
Art problem.
Colloidal gold is by gold chloride (HAuCl4) reducing agent such as white phosphorus, ascorbic acid, sodium citrate, the tannic acid the effects of
Under, it can be grouped to the gold particle of a certain size (generally between 1nm-150nm), and since electrostatic interaction becomes a kind of stable
Colloidal state forms electronegative hydrophobic sol solution, becomes stable colloidal state due to electrostatic interaction, therefore referred to as colloid
Gold.In recent years, colloidal gold has fragmentary report in terms of being applied to nucleic acid amplification reaction, and Ru Lihai is wealthy to wait (2004,2005) open
Application of the colloidal gold in terms of improving PCR reaction efficiency.Zhou et al.(2013) after LAMP amplified reaction, it will
Colloidal gold is added in reaction product as color developing agent, is used to indicate amplified reaction result.Kumvongpin et al.
(2016) and Ye et al.(2018) then report colloidal gold improve high-risk human mammilla papillomavirus and rotavirus virus
With the research in terms of-actin gene LAMP amplified reaction efficiency, but there has been no by colloidal gold be applied to the micro- life of food-borne pathogenic
The report of object LAMP amplified reaction efficiency.
The present invention provides a kind of methods for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency, can be further
It improves the sensitivity of detection, inhibit non-specific amplification, can be very good to solve field of food safety in the augmentation detection side LAMP
The technical problem that face encounters.
Summary of the invention
For staphylococcus aureus, salmonella, single monocytogenes, vibrio parahemolyticus, comma bacillus etc.
Food-borne pathogenic microorganism is encountered in existing LAMP amplification technique since high sensitivity, experimental situation are easily by aerosol dirt
The problem of non-specific binding occurs between dye and a plurality of primer and is easy to produce false positive and sensitivity decrease, the object of the invention
It is to provide a kind of method for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency.
Another object of the present invention is: providing a kind of application of the above method in the detection.
The object of the invention passes through following proposal and realizes: a kind of to improve food-borne pathogenic microorganism LAMP amplified reaction efficiency
Method is added colloidal gold in food-borne pathogenic microorganism LAMP amplified reaction body based on ring mediated isothermal amplification LAMP
In system, realizes the optimization expanded to LAMP, include the following:
(1) LAMP amplification reaction system optimizes: colloidal gold is added in food-borne pathogenic microorganism LAMP amplification reaction system,
Then LAMP amplification is carried out, wherein dosage are as follows: glue is added in the food-borne pathogenic microorganism LAMP amplification reaction system of 25 μ L
The final concentration of 0.25-15nmol/L of body gold；
(2) LAMP amplified production detects: can pass through electrophoresis detection, Turbidity measurement or color developing detection.
Wherein, prepared by colloidal gold: colloid gold reagent utilizes prior art preparation.
The food-borne pathogenic microorganism LAMP amplification reaction system includes outer primer F3 and B3, inner primer FIP and BIP,
Ring primer LF and LB, Bst archaeal dna polymerase, 10 × polymerase buffer, deoxyribonucleoside triphosphate (dNTP), magnesium salts (Mg2 +), glycine betaine, colloidal gold.
The present invention provides a kind of application of the above method in detection food-borne pathogenic microorganism.
The present invention provides a kind of application of the above method in detection staphylococcus aureus.
The present invention provides a kind of application of upper the method in detection salmonella.
The present invention provides a kind of application of the above method in detection Listeria monocytogenes.
The food-borne pathogenic microorganism LAMP amplification reaction system includes outer primer F3 and B3, inner primer FIP and BIP,
Ring primer LF and LB, Bst archaeal dna polymerase, 10 × polymerase buffer, dNTP, Mg2+, glycine betaine, colloidal gold, in distilled water
It is one or more.
The present invention is realized pair by adding colloidal gold material into food-borne pathogenic microorganism LAMP amplification reaction system
The optimization of LAMP amplification provides a kind of method for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency.Using this hair
Bright detection method detects food-borne pathogenic microorganism, has the advantages that highly sensitive and high specific.
In the present invention, the food-borne pathogenic microorganism LAMP amplification reaction system includes outer primer F3 and B3, inner primer
FIP and BIP, ring primer LF and LB, Bst archaeal dna polymerase, 10 × polymerase buffer, dNTP, Mg2+, glycine betaine, colloidal gold,
One of distilled water is a variety of.
In the method for the present invention, in a specific embodiment (primer containing ring), the LAMP amplification reaction system includes outer
Primers F 3 and B3, inner primer FIP and BIP, 8U Bst archaeal dna polymerase, 1 × polymerase buffer, 1.0-1.6mmol/L
DNTP, 2-9mmol/L Mg2+, 0-1.5mol/L glycine betaine, 0.25-15nmol/L colloidal gold, including ring primer LF and/or LB；
The ratio between concentration of the outer primer and inner primer is 1:(2-8)；The ratio between concentration of the outer primer and ring primer is 1:(2-8)；
In another specific embodiment (being free of ring primer), the LAMP amplification reaction system includes outer primer F3 and B3, inner primer
FIP and BIP, 8U Bst archaeal dna polymerase, 1 × polymerase buffer, 1.0-1.6mmol/L dNTP, 2-9mmol/L Mg2+, 0-
The ratio between concentration of 1.5mol/L glycine betaine, 0.25-15nmol/L colloidal gold, the outer primer and inner primer is 1:(2-8).
Ring primer helps to improve reaction efficiency, for example, 1 × Bst DNA polymerase reaction buffer can select 1 ×
Thermopol reaction buffer includes 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10 mmol/L
(NH4)2SO4, 0.1% Triton X-100,2mM MgSO4；MgSO in 1 × Bst DNA polymerase reaction buffer4And enzyme
Magnesium ion Mg in reaction system2+Do merging treatment.
In the method for the present invention, the response procedures of the isothermal amplification reactions are 1. 60~65 DEG C of 10~90min of incubation；②80
DEG C terminate reaction 2 ~ 20min.The present invention does not limit by the way that other are suitable for response procedures and realizes detection method.
In the method for the present invention, detection method includes but is not limited to electrophoresis detection, Turbidity measurement or color developing detection (including naked eyes
Directly observe or carry out amplification curve judgement by instrument) etc..
The invention also provides application of the method in detection food-borne pathogenic microorganism, wherein the food-borne cause
Sick microorganism includes but is not limited to bacterium, virus and other food-borne microorganisms that can be caused a disease.Wherein, the bacterium, including but
It is not limited to salmonella, staphylococcus aureus, Listeria monocytogenes and other food-borne pathogenic microorganisms.
Beneficial effect of the present invention includes: to have high specificity, spirit using food-borne pathogenic microorganism detection method of the present invention
Sensitivity is high, the advantages of.Compared with current common LAMP amplification detection method, the present invention is by food-borne pathogenic microorganism LAMP
Colloidal gold material is added in amplification reaction system to realize that easy to operate, excellent is very suitable for the optimization of LAMP amplification
The mechanisms such as food production and safety detection promote the use of.Above-mentioned each optimum condition can be carried out any group based on common sense in the field
It closes, the category scope of the present invention.
This method can effectively improve the sensitivity of LAMP amplified reaction, and be able to suppress the appearance of false positive results, subtract
Few non-specific amplification, to improve the detection efficiency of food-borne pathogenic microorganism.This method can be applied to food safety
Detection field has wide application prospect.
Detailed description of the invention
Attached drawing 1 shows that 1 colloidal gold of the embodiment of the present invention shows staphylococcus aureus LAMP amplification reaction system optimization
Color testing result；
Attached drawing 2 shows that 1 colloidal gold of the embodiment of the present invention examines the electrophoresis that staphylococcus aureus LAMP amplification reaction system optimizes
Survey result；
Attached drawing 3 shows optimization of 2 colloidal gold of the embodiment of the present invention to salmonella LAMP amplification reaction system；
Attached drawing 4 shows optimization of 3 colloidal gold of the embodiment of the present invention to Listeria monocytogenes LAMP amplification reaction system.
Specific embodiment
In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and using appended claims as protection scope.Implement process of the invention,
Condition, reagent, experimental method etc. are among the general principles and common general knowledge in the art in addition to what is specifically mentioned below,
There are no special restrictions to content by the present invention.
Embodiment 1
A method of food-borne pathogenic microorganism LAMP amplified reaction efficiency is improved, is with ring mediated isothermal amplification LAMP system
Basis, comprising: outer primer F3 and B3, inner primer FIP and BIP, Bst archaeal dna polymerase, 10 × polymerase buffer, by colloidal gold
Addition realizes the optimization expanded to LAMP in staphylococcus aureus LAMP amplification reaction system, as follows:
(1) LAMP amplification reaction system is prepared, composition is as follows:
Final concentration of 0.25-15nmol/L
Wherein, primer LF, FIP, BIP, F3 and B3 is staphylococcus aureus specific LAMP amplimer, and template is golden yellow
Staphylococcus DNA derives from China General Microbiological Culture Collection Center CGMCC 1.2465, and bacterial strain uses Beijing after cultivation
The bacterial nucleic acid extracts kit of Tiangeng bio-engineering corporation extracts genomic DNA；
(2) optimization material colloids gold is added into the above system, the colloidal gold solution of 1.8 μ L is added in the system of each 25 μ L,
The corresponding control experiment of optimization material is not added simultaneously, LAMP is then carried out under the conditions of 62 DEG C and reacts 60min, 80 DEG C of terminations are anti-
Answer 2min；
(3) SYBR Green I is added in amplified production and carries out color developing detection, then carry out agarose gel electrophoresis detection:
Amplification is as depicted in figs. 1 and 2, and respectively color developing detection result and electrophoresis detection are as a result, sample 1-2: being added without this
Invention optimization material colloids gold, sample 1 are that template L-form staphylococcus aureus is added, and sample 2 is to be added without template golden yellow Portugal
The negative control of grape coccus DNA；Sample 3-4: present invention optimization material colloids gold is added, sample 3 is that template golden yellow Portugal is added
Grape coccus DNA, sample 4 are the negative control for being added without template L-form staphylococcus aureus；M: (TakaRa is public for molecular weight marker
Take charge of DL2000, respectively 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp)；It can be seen from the figure that not being added excellent
System (sample 2) colour developing for changing material colloids gold is bright green, and the trapezoid-shaped strips of electrophoresis expression characteristics show non-specificity
Amplification；And be added optimization material colloids gold system (sample 4) colour developing be it is orange, electrophoresis does not occur characteristic trapezoid-shaped strips,
Display eliminates non-specific amplification.The system (sample 1 and sample 3) that template L-form staphylococcus aureus is added, which develops the color, is
Bright green, the trapezoid-shaped strips of electrophoresis expression characteristics.The LAMP amplification shows that the present invention can eliminate non-specific expansion
Increase, avoids false positive, effect of optimization is significant.
Embodiment 2
A method of food-borne pathogenic microorganism LAMP amplified reaction efficiency is improved, colloidal gold is added in salmonella LAMP
In amplification reaction system, the optimization expanded to LAMP is realized, as follows:
(1) LAMP reaction system, composition such as embodiment 1, the difference is that primer LF, FIP, BIP, F3 and B3 are sramana are prepared
Salmonella specificity LAMP amplimer, template are that salmonella DNA(derives from Chinese industrial Microbiological Culture Collection administrative center
CICC21560), template quantity is serially diluted, and colloidal gold sample-adding amount is 0.3 μ L, final concentration of 1.0nM；
(2) processed optimization material colloids gold is added into the above system, the colloid of 0.3 μ L is added in the system of each 25 μ L
Gold solution, at the same be not added optimization material corresponding control experiment, then under the conditions of 63 DEG C carry out LAMP react 60min, 80
DEG C terminate reaction 10min；
(3) SYBR Green I is added in amplified production and carries out color developing detection:
Amplification is as shown in figure 3, template quantity is respectively 1000fg, 100fg, 10fg, 5fg in each reaction system from left to right
With 0(, that is, negative control), it can be seen that in the processing of no addition colloidal gold, template quantity is the reaction of 1000fg and 100fg
System is in bright green, is judged as positive；Template quantity be 10fg, 5fg and 0 reaction system in orange, be judged as negative, show not
The template (being equivalent to 20 bacteriums) of the minimum detectable 100fg of reaction system of addition optimization material colloids gold, in addition glue
In the processing of body gold, the reaction system of template quantity 1000fg, 100fg, 10fg, 5fg are in bright green, are judged as positive, template
Amount for 0 reaction system in orange, be judged as negative, show that the reaction system of addition optimization material colloids gold is minimum detectable
The template (being equivalent to 1 bacterium) of 5fg, which shows that reaction sensitivity, effect of optimization can be improved in the present invention
Significantly.
Embodiment 3
A method of food-borne pathogenic microorganism LAMP amplified reaction efficiency is improved, colloidal gold is added in monocyte hyperplasia
In Listeria LAMP amplification reaction system, the optimization expanded to LAMP is realized, as follows:
(1) LAMP reaction system, composition such as embodiment 2, the difference is that primer LF, FIP, BIP, F3 and B3 are monokaryon are prepared
Hyperplasia Listeria specificity LAMP amplimer, template are that Listeria monocytogenes DNA(derives from Chinese work
Industry Microbiological Culture Collection administrative center CICC21635)；
(2) processed optimization material colloids gold is added into the above system, the colloid of 0.3 μ L is added in the system of each 25 μ L
Gold solution, at the same be not added optimization material corresponding control experiment, then under the conditions of 63 DEG C carry out LAMP react 60min, 80
DEG C terminate reaction 5min；
(3) SYBR Green I is added in amplified production and carries out color developing detection: amplification is as shown in figure 4, every from left to right
Template quantity is respectively 0(negative control in a reaction system), 1000fg, 100fg, 10fg and 5fg.It can be seen that not adding
In the processing of colloidal gold, the reaction system that template quantity is 1000fg and 100fg is in bright green, is judged as positive；Template quantity is
10fg, 5fg and 0 reaction system in orange, be judged as negative, show that the reaction system for not adding optimization material colloids gold is minimum
The template (being equivalent to 20 bacteriums) of 100fg can be detected.In the processing of addition colloidal gold, template quantity 1000fg, 100fg
It is in bright green with the reaction system of 10fg, is judged as positive, the reaction system that template quantity is 0 is judged as negative, shows in orange
The template (being equivalent to 2 bacteriums) of the minimum detectable 10fg of reaction system of addition optimization material colloids gold.LAMP amplification
The results show that reaction sensitivity can be improved in the present invention, effect of optimization is significant.
Claims (8)
1. a kind of method for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency, with ring mediated isothermal amplification LAMP system
Based on, comprising: outer primer F3 and B3, inner primer FIP and BIP, Bst archaeal dna polymerase, 10 × polymerase buffer, feature
It is, by colloidal gold addition in food-borne pathogenic microorganism LAMP amplification reaction system, realizes the optimization expanded to LAMP, packet
It includes as follows:
(1) LAMP amplification reaction system optimizes: colloidal gold is added in food-borne pathogenic microorganism LAMP amplification reaction system,
Then LAMP amplification, isothermal amplification reactions, wherein in the food-borne pathogenic microorganism LAMP amplification reaction system of 25 μ L are carried out
The final concentration of 0.25-15nmol/L of colloidal gold is added；
(2) LAMP amplified production detects: passing through electrophoresis detection, Turbidity measurement or color developing detection.
2. a kind of method for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency according to claim 1, special
Sign is, the LAMP amplification reaction system includes outer primer F3 and B3, inner primer FIP and BIP, Bst archaeal dna polymerase,
DNTP, Mg2+, glycine betaine, colloidal gold, one of distilled water or a variety of, wherein 1.0-1.6mmol/L dNTP, 2-9mmol/
L Mg2+, 0-1.5mol/L glycine betaine, 0.25-15nmol/L colloidal gold, the ratio between the outer primer and the concentration of inner primer are 1:
(2-8).
3. a kind of method for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency according to claim 2, special
Sign is, further includes ring primer LF and LB, and the ratio between the outer primer and the concentration of ring primer are 1:(2-8).
4. a kind of method for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency according to claim 2, special
Sign is that 1 × Bst DNA polymerase reaction buffer selects 1 × Thermopol reaction buffer, includes 20mmol/L
Tris-HCl (pH 8.8), 10mmol/L KCl, 10 mmol/L (NH4)2SO4, 0.1% Triton X-100,2mM
MgSO4；MgSO in 1 × Bst DNA polymerase reaction buffer4With the magnesium ion Mg in enzyme reaction system2+It does at merging
Reason.
5. a kind of method for improving food-borne pathogenic microorganism LAMP amplified reaction efficiency according to claim 2, special
Sign is that the response procedures of the isothermal amplification reactions are 1. 60~65 DEG C of 10~90min of incubation；2. 80 DEG C of termination reactions 2 ~
20min。
6. purposes of -2 any the methods in detection food-borne pathogenic microorganism according to claim 1.
7. purposes according to claim 6, it is characterised in that: the food-borne pathogenic microorganism is at least golden yellow grape
It is coccus, salmonella, one or more in Listeria monocytogenes.
8. the application of -3 any the methods in the detection according to claim 1.
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