CN110241182A - Quench fluorescent rna marker synthetic method and the method applied to food-borne pathogens detection - Google Patents
Quench fluorescent rna marker synthetic method and the method applied to food-borne pathogens detection Download PDFInfo
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Abstract
The invention discloses a kind of quenching fluorescent rna marker synthetic method and applied to the method for food-borne pathogens detection, amido modified upper conversion nano particle is had using a step solvent structure, specific RNA is synthesized into RNA fluorescent marker in conjunction with quencher, links together to obtain quenching fluorescent rna marker with amido modified upper conversion nano particle is had by RNA fluorescent marker using Euplotes woodruffi；And detection of the fluorescent rna marker applied to food-borne pathogens will be quenched, nuclease Cas13a accurately cuts the specific nucleic acid target in trace pathogen cell, and using the subsidiary cutting effect of the enzyme, shearing quenching fluorescent rna marker discharges the fluorescence that can be detected；Fluorescence data is acquired, the quantitative detection model of building fluorescence intensity change value and not same amount food-borne pathogen nucleic acid target realizes the nano fluorescent trace detection of food-borne pathogen nucleic acid.
Description
Technical field
The invention belongs to food-borne pathogens detection technique fields more particularly to a kind of quenching fluorescent rna marker to synthesize
Method and the method detected applied to food-borne pathogens.
Background technique
One of the main reason for food-borne pathogens are initiation food origin diseases is the important of World of Food safety, sternly
Human health is threatened again.The detection of food-borne pathogens is the important means of food safety guarantee.Along with detection of nucleic acids skill
The rapid detection method of the fast development of art, various food-borne pathogens is mutually then given birth to.There are commonly polymerase chain reaction and
Its deriving technology, nucleic acid constant-temperature amplification technology, oligonucleotide microarray technique and immunomagnetic cell isolation technics etc..It is existing
The defects of though these methods are each advantageous, too strong there are fluorescence background when detection cycle length or detection.Exist in view of applicant
The skilled molecular biology mechanism of the good experience and Team Member that food field of non destructive testing accumulates, especially in upper conversion
The further investigation of detection technique of fluorescence and CRISPR/Cas technical field, this project is quasi- to construct a kind of the upper of food-borne pathogens
Conversion nano fluorescence super sensitivity detection method, deeply probes into quick, sensitive food-borne pathogen nucleic acid quantitative detecting method, should
Method is suitable for the technical fields such as food safety, environmental monitoring.
Currently, realizing that the rapid detection method of food-borne pathogen nucleic acid is not reported yet with up-conversion fluorescence nanotechnology
Road.The present invention realizes the quick inspection of food-borne pathogen nucleic acid as a kind of novel food-borne pathogen nucleic acid quantitative approach
It surveys
Summary of the invention
The present invention according to problems of the prior art, propose a kind of quenching fluorescent rna marker synthetic method and
Method applied to food-borne pathogens detection.
The technical solution adopted in the present invention is as follows:
A kind of quenching fluorescent rna marker synthetic method；
Amido modified upper conversion nano particle is had using a step solvent structure, by specific RNA and quencher
In conjunction with synthesis RNA fluorescent marker, using Euplotes woodruffi by RNA fluorescent marker (NH2- RNA-BHQ) and with amino
The upper conversion nano particle (NH of modification2-UCNPS) link together to obtain quenching fluorescent rna marker；
Further, the upper conversion nano grain diameter size < 100nm, by adding NH4The amount of F is big to adjust partial size
It is small；
Further, the upper conversion nano particle fluorescence color is controlled by adjusting rare earth doped ratio；
Further, upper conversion fluorescent nano particle NaGdF4:Yb/Er、NaGdF4: Yb/Tm or NaGdF4:Yb/Ho；
Further, the quencher uses BHQ line fluorescent quencher, improves detection sensitivity.
A kind of upper conversion nano fluorescence super sensitivity detection method of food-borne pathogens, with Cas13a pairs of novel nucleic acids enzyme
Specific nucleic acid target in trace pathogen cell is accurately cut, and utilizes the subsidiary cutting effect of the enzyme, shearing quenching
Fluorescent rna marker discharges the fluorescence that can be detected；Fluorescence data is acquired by up-conversion fluorescence spectroscopic system, is obtained
The fluorescence intensity level of upconversion fluorescence nano material maximum absorption band, building fluorescence intensity change value and not same amount food-borne pathogenic
The quantitative detection model of bacterium nucleic acid target realizes the nano fluorescent trace detection of food-borne pathogen nucleic acid.
Beneficial effects of the present invention:
The quenching fluorescent rna marker as synthesized by the present invention can be with the RNA specificity of food-borne pathogens to be measured
In conjunction with.In the detection process, nuclease Cas13a participates in the maturation of crRNA, while carrying out to specific nucleic acid target RNA special
Opposite sex cutting, and the Cas13a activated has subsidiary cleavage activity, can shear other non-target RNA, i.e., to quenching fluorescence
RNA marker is cut, and the fluorescence that can be detected is discharged.The final nano fluorescent trace inspection for realizing food-borne pathogen nucleic acid
It surveys.
Detailed description of the invention
Fig. 1 is UCNPSTransmission electron microscope picture；
Fig. 2 is the testing result figure of Escherichia coli: A fluorescent spectrum curve, B standard curve.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that described herein, the specific embodiments are only for explaining the present invention, not
For limiting the present invention.
The present invention is suitable for the detection of food-borne pathogens, in the present embodiment, only by taking Escherichia coli (E.coli) as an example,
Specific steps are as follows:
Amido modified upper conversion nano particle is had using solvent-thermal method preparation.Detailed process is as follows: by NH4F
(6.24mmol) is dissolved in 12mLEG.Take NaCl (1mmol), PEI (0.5g), Gd (NO3)3(0.8mmol)、Yb(NO3)3
(0.17mmol) and Er (NO3)3(0.03mmol) is dissolved in EG (38mL), magnetic agitation 30min.When solution is transparent, it is added
NH4F solution stirs 10min, transfers the solution into stainless steel autoclave.Reaction kettle heats under the conditions of 200 DEG C
1.5h is cooled to room temperature.By being centrifugated nano particle, it is washed with deionized three times, and dry in a vacuum drying oven
6h is obtained as shown in Figure 1 powdered with amido modified upper conversion nano particle (NH2-UCNPS)。
When preparation is with amido modified upper conversion nano particle, by adding NH4The amount of F adjusts particle size,
And guarantee upper conversion nano grain diameter size < 100nm.It is controlled by fluorescence color by adjusting rare earth doped ratio
System.
Specific RNA and quencher are combined into synthesis RNA fluorescent marker, in the present embodiment, quencher uses itself
The BHQ line fluorescent quencher of fluorescence is not emitted, in the present embodiment, RNA fluorescent marker is used and synthesized by TaKaRa company
NH2-RNA-BHQ1。
Using Euplotes woodruffi by NH2- RNA-BHQ1 and NH2-UCNPSIt links together, finally obtains quenching fluorescence
RNA marker UCNPS--RNA-BHQ1。
The quenching fluorescent rna reporting flag object has the function of signal reports, when Cas13a cuts RNA sequence therein,
Discharge the green florescent signal being able to detect that.
Based on above-mentioned designed quenching fluorescent rna reporting flag object, a kind of borne pathogen is had also been devised in the present invention
Upper conversion nano fluorescence super sensitivity detection method, detailed process is as follows:
(1) Escherichia coli the preparation of Escherichia coli nucleic acid target fragments: are seeded in Luria-Bertani culture medium first
On, 37 DEG C, 200rpm/min is cultivated for 24 hours, and bacterium night 1mL, 12000/min is then taken to be centrifuged 1min, discard supernatant liquid.Use bacterium
Genome extraction kit extracts pathogenic bacteria nucleic acid.Target sequence is pin-pointed in conjunction with Oligonucleolide primers using recombinase,
It untwists template double-strand under single-stranded DNA binding protein enzyme auxiliary, then starts the exponential of target sequence under archaeal dna polymerase effect
Amplification, entire react can carry out at normal temperature, without denaturation, can get amplified production detection level in 20min, have special
Property strong, high sensitivity, be swift in response, device dependence is low and the accurate equal traditional isothermal DNA amplification of amplification not available for
Advantage.
(2) crRNA the preparation of Escherichia coli crRNA: is synthesized using chemical synthesis.CrRNA structure is 5 '-anchoring sequences
Column-go-ahead sequence -3 '.Anchor series are depending on the source Cas13a, when Cas13a is LshCas13a, anchor series 5 '-
CCACCCCAAUAUCGAAGGGGACUAAAAC-3'；When Cas13a is LwCas13a, 5 '-GAUUUAGACUAC of anchor series
CCCAAAAACGAAGGGGACUAAAAC-3'.The length of go-ahead sequence design is 21-28 nucleotide, with in-vitro transcription RNA piece
Section is complementary.Then by the 5 ' of design-anchor series-go-ahead sequence -3 ' carry out reverse transcription, its 5 ' addition T7 promoter sequence,
By the 5 '-T7 promoter sequence-anchor series-DNA of go-ahead sequence -3 ', rapid synthesis is a large amount of under t7 rna polymerase effect
crRNA.Use the Escherichia coli core crRNA of RNAXP cleaning pearl purifying synthesis.
(3) purifying of Cas13a albumen: Cas13a bacterial expression vector is transformed into competent cell, 16mL is taken to cultivate
Object overnight incubation in 4 growth medium of Terrific Broth.Then IPTG is supplemented, and cell is cooled to 18 DEG C and is continued
16 hours progress protein expressions.4 DEG C, 5200g is centrifuged 15min, collects cell precipitation and is crushed progress protein purification.
By the Cas13a albumen of purifying, crRNA, quenching fluorescent rna reporting flag object, RNase inhibitor, background RNA and
The Escherichia coli of different content are transcribed in vitro product RNA and are blended in nucleic acid enzymatic determination buffer (40mM Tris-HCl, 60mM
NaCl, 6mM MgCl2, pH 7.3) in be incubated for, pass through up-conversion fluorescence spectrometer record different content S.aureus nucleic acid target
Corresponding fluorescence intensity.Fluorescence data is acquired by up-conversion fluorescence spectroscopic system, obtains up-conversion fluorescence nanometer material
Expect the fluorescence intensity level of maximum absorption band, such as Fig. 2, building fluorescence intensity change value and different content Escherichia coli nucleic acid target
Quantitative Analysis Model, to realize the nano fluorescent trace detection of Escherichia coli target nucleic acids.
Above embodiments are merely to illustrate design philosophy and feature of the invention, and its object is to make technology in the art
Personnel can understand the content of the present invention and implement it accordingly, and protection scope of the present invention is not limited to the above embodiments.So it is all according to
It is within the scope of the present invention according to equivalent variations made by disclosed principle, mentality of designing or modification.
Claims (7)
1. a kind of quenching fluorescent rna marker synthetic method, which is characterized in that synthesis has amido modified upper conversion nano
Grain, synthesizes RNA fluorescent marker for specific RNA with quencher in conjunction with, using Euplotes woodruffi by RNA fluorescent marker and
It links together to obtain quenching fluorescent rna marker with amido modified upper conversion nano particle.
2. a kind of quenching fluorescent rna marker synthetic method according to claim 1, which is characterized in that synthesize the band
The method for having amido modified upper conversion nano particle is a step solvent-thermal method.
3. a kind of quenching fluorescent rna marker synthetic method according to claim 1 or 2, which is characterized in that described upper turn
Nano particle diameter size < 50-100nm is changed, by adding NH4The amount of F adjusts particle size.
4. a kind of quenching fluorescent rna marker synthetic method according to claim 3, which is characterized in that the upper conversion
Nano-particle fluorescence color is controlled by adjusting rare earth doped ratio.
5. a kind of quenching fluorescent rna marker synthetic method according to claim 4, which is characterized in that up-conversion fluorescence
Nano particle is NaGdF4:Yb/Er、NaGdF4: Yb/Tm or NaGdF4:Yb/Ho。
6. a kind of quenching fluorescent rna marker synthetic method according to claim 1, which is characterized in that the quencher
Using BHQ line fluorescent quencher.
7. a kind of upper conversion nano using the food-borne pathogens for quenching fluorescent rna marker described in the claims 1 is glimmering
Light super sensitivity detection method, which is characterized in that with nuclease Cas13a to the specific nucleic acid target in trace pathogen cell
It is accurately cut, and using the subsidiary cutting effect of nuclease Cas13a, shearing quenching fluorescent rna marker, release can quilt
The fluorescence of detection；Fluorescence data is acquired by up-conversion fluorescence spectroscopic system, it is maximum to obtain upconversion fluorescence nano material
The fluorescence intensity level of absorption peak, the quantitative detection mould of building fluorescence intensity change value and not same amount food-borne pathogen nucleic acid target
Type realizes the nano fluorescent trace detection of food-borne pathogen nucleic acid.
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| WO2020224164A1 (en) * | 2019-05-07 | 2020-11-12 | 江苏大学 | Method for detecting nano fluorescent traces in nucleic acids of food-borne pathogenic bacteria on basis of crispr/cas13a |
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