CN110241182B - Synthesis method of quenched fluorescent RNA marker and method applied to detection of food-borne pathogenic bacteria - Google Patents
Synthesis method of quenched fluorescent RNA marker and method applied to detection of food-borne pathogenic bacteria Download PDFInfo
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- CN110241182B CN110241182B CN201910375397.5A CN201910375397A CN110241182B CN 110241182 B CN110241182 B CN 110241182B CN 201910375397 A CN201910375397 A CN 201910375397A CN 110241182 B CN110241182 B CN 110241182B
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses a method for synthesizing a quenched fluorescent RNA marker and a method for detecting food-borne pathogenic bacteria, wherein an up-conversion nano particle with amino modification is synthesized by adopting a one-step solvothermal method, a specific RNA and a quencher are combined to synthesize the RNA fluorescent marker, and the RNA fluorescent marker and the up-conversion nano particle with amino modification are connected together by adopting a glutaraldehyde crosslinking method to obtain the quenched fluorescent RNA marker; the quenched fluorescent RNA marker is applied to the detection of food-borne pathogenic bacteria, nuclease Cas13a precisely cuts a specific nucleic acid target in trace pathogenic bacteria cells, and the auxiliary cutting effect of the nuclease is utilized to cut the quenched fluorescent RNA marker and release the fluorescence which can be detected; and collecting fluorescence spectrum data, constructing a quantitative detection model of a fluorescence intensity change value and different amounts of food-borne pathogenic bacteria nucleic acid targets, and realizing nano fluorescence trace detection of the food-borne pathogenic bacteria nucleic acid.
Description
Technical Field
The invention belongs to the technical field of food-borne pathogenic bacteria detection, and particularly relates to a method for synthesizing a quenched fluorescent RNA marker and a method applied to food-borne pathogenic bacteria detection.
Background
Food-borne pathogenic bacteria are one of the main causes of food-borne diseases, are important for world food safety, and have seriously threatened human health. Detection of food-borne pathogenic bacteria is an important means of food safety assurance. Along with the rapid development of nucleic acid detection technology, rapid detection methods for various food-borne pathogens are developed. The polymerase chain reaction and its derivative technology, nucleic acid isothermal amplification technology, oligonucleotide microarray technology, immunomagnetic cell separation technology, etc. are commonly used. Although the existing methods have advantages, the defects of long detection period, too strong fluorescence background during detection and the like exist. In view of the good experience accumulated in the food nondestructive testing field of the applicant and the molecular biology foundation of team member proficiency, particularly the up-conversion fluorescence detection technology and the deep research in the CRISPR/Cas technical field, the project is to construct an up-conversion nano fluorescence ultrasensitive detection method for food-borne pathogenic bacteria, and the rapid and sensitive food-borne pathogenic bacteria nucleic acid quantitative detection method is deeply explored, and is suitable for the technical fields of food safety, environmental monitoring and the like.
At present, a method for realizing rapid detection of food-borne pathogenic bacteria nucleic acid by using an upconversion fluorescence nanotechnology is not reported yet. The invention is used as a novel food-borne pathogenic bacteria nucleic acid quantification method to realize the rapid detection of the food-borne pathogenic bacteria nucleic acid
Disclosure of Invention
According to the problems in the prior art, the invention provides a method for synthesizing a quenching fluorescent RNA marker and a method for detecting food-borne pathogenic bacteria.
The technical scheme adopted by the invention is as follows:
a method for synthesizing a quenching fluorescent RNA marker;
synthesizing up-conversion nano particles with amino modification by adopting a one-step solvothermal method, combining specific RNA with a quencher to synthesize an RNA fluorescent marker, and adopting a glutaraldehyde crosslinking method to crosslink the RNA fluorescent marker (NH) 2 RNA-BHQ) with up-conversion Nanoparticles (NH) with amino modification 2 -UCNP S ) Connecting together to obtain a quenched fluorescent RNA marker;
further, the up-conversion nanoparticle particle size<100nm by adding NH 4 The amount of F is used for adjusting the particle size;
further, the fluorescence color of the up-conversion nano-particles is controlled by adjusting the doping proportion of rare earth elements;
further, up-converting fluorescent nanoparticles to NaGdF 4 :Yb/Er、NaGdF 4 Yb/Tm or NaGdF 4 :Yb/Ho；
Further, the quencher adopts BHQ series fluorescence quencher, so as to improve detection sensitivity.
An up-conversion nano-fluorescence ultrasensitive detection method for food-borne pathogenic bacteria uses novel nuclease Cas13a to precisely cut specific nucleic acid targets in trace pathogenic bacteria cells, and uses the incidental cutting effect of the enzyme to cut and quench fluorescent RNA markers to release detectable fluorescence; and acquiring fluorescence spectrum data by means of an up-conversion fluorescence spectrum system, acquiring a fluorescence intensity value of a maximum absorption peak of the up-conversion fluorescent nanomaterial, and constructing quantitative detection models of the fluorescence intensity change value and different amounts of food-borne pathogenic bacteria nucleic acid targets to realize nano-fluorescence trace detection of the food-borne pathogenic bacteria nucleic acid.
The invention has the beneficial effects that:
because the quenched fluorescent RNA marker synthesized by the invention can be specifically combined with RNA of the food-borne pathogenic bacteria to be detected. In the detection process, nuclease Cas13a participates in the maturation process of crRNA, and simultaneously specifically cleaves specific nucleic acid target RNA, and activated Cas13a has an attached cleavage activity, so that other non-target RNA can be sheared, namely, a quenched fluorescent RNA marker is cleaved, and fluorescence which can be detected is released. Finally, nano fluorescence trace detection of the food-borne pathogenic bacteria nucleic acid is realized.
Drawings
FIG. 1 is UCNP S Is a transmission electron microscope image;
FIG. 2 is a graph showing the detection results of E.coli: a fluorescence spectrum curve and B standard curve.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention is suitable for detecting food-borne pathogenic bacteria, in the embodiment, only E.coli (E.coli) is taken as an example, and the specific operation steps are as follows:
up-conversion nano-preparation with amino modification by solvothermal methodRice grains. The specific process is as follows: NH is added to 4 F (6.24 mmol) was dissolved in 12 mLEG. Taking NaCl (1 mmol), PEI (0.5 g), gd (NO) 3 ) 3 (0.8mmol)、Yb(NO 3 ) 3 (0.17 mmol) and Er (NO 3 ) 3 (0.03 mmol) was dissolved in EG (38 mL) and magnetically stirred for 30min. When the solution is transparent, adding NH 4 F, stirring the solution for 10min, and transferring the solution into a stainless steel high-pressure reaction kettle. The reaction vessel was heated at 200℃for 1.5h and cooled to room temperature. The nanoparticles were separated by centrifugation, washed three times with deionized water, and dried in a vacuum oven for 6 hours to obtain the up-conversion Nanoparticles (NH) with amino modification in powder form as shown in fig. 1 2 -UCNP S )。
In the preparation of up-conversion nanoparticles with amino modification, by adding NH 4 F, adjusting the particle size and ensuring the up-conversion of the particle size of the nano-particles<100nm. The fluorescent color is controlled by adjusting the doping proportion of the rare earth element.
Combining specific RNAs with a quencher to synthesize an RNA fluorescent marker, in this example, a quencher using BHQ series fluorescence quencher which does not itself emit fluorescence, in this example, an RNA fluorescent marker using NH synthesized by TaKaRa Co 2- RNA-BHQ1。
NH is crosslinked by glutaraldehyde 2 RNA-BHQ1 and NH 2 -UCNP S Are connected together to finally obtain the quenching fluorescent RNA marker UCNP S --RNA-BHQ1。
The quenched fluorescent RNA reporter marker has a signal reporting function, releasing a green fluorescent signal that can be detected when Cas13a cleaves the RNA sequence therein.
Based on the designed quenched fluorescent RNA report marker, the invention also designs an up-conversion nano fluorescent ultrasensitive detection method of the pathogenic bacteria, which comprises the following specific processes:
(1) Preparation of E.coli nucleic acid target fragment: the E.coli was first inoculated on Luria-Bertani medium, cultured at 37℃and 200rpm/min for 24 hours, then 1mL of the fungus night was taken, centrifuged at 12000/min for 1min, and the supernatant was discarded. Pathogenic nucleic acids are extracted using a bacterial genome extraction kit. The target sequence is precisely positioned by combining the recombinase and the oligonucleotide primer, the template double strand is unwound under the assistance of the single-strand DNA binding protease, then the exponential amplification of the target sequence is started under the action of the DNA polymerase, the whole reaction can be carried out at normal temperature without denaturation, and the detection level of an amplified product can be obtained within 20 min.
(2) Preparation of E.coli crRNA: crRNA was synthesized using chemical synthesis. The crRNA structure is 5 '-anchor sequence-guide sequence-3'. The anchoring sequence depends on the source of Cas13a, and when Cas13a is LshCas13a, the anchoring sequence is 5'-CCACCCCAAUAUCGAAGGGGACUAAAAC-3'; when Cas13a is LwCas13a, the anchoring sequence is 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3'. The guide sequence is designed to be 21-28 nucleotides in length, which is complementary to the in vitro transcribed RNA fragment. And then reverse transcribing the designed 5' -anchor sequence-guide sequence-3 ', adding a T7 promoter sequence to the 5' of the target sequence, and rapidly synthesizing a large amount of crRNA from the 5' -T7 promoter sequence-anchor sequence-guide sequence-3 ' DNA under the action of T7RNA polymerase. The synthesized E.coli nuclear crRNA was purified using RNAXP cleaning beads.
(3) Purification of Cas13a protein: cas13a bacterial expression vectors were transformed into competent cells and 16mL cultures were grown overnight in Terrific Broth 4 growth medium. IPTG was then supplemented and the cells were cooled to 18 ℃ for 16 hours for protein expression. Cell pellets were collected and crushed for protein purification by centrifugation at 5200g at 4℃for 15 min.
Purified Cas13a protein, crRNA, quenched fluorescent RNA reporter marker, rnase inhibitor, background RNA and varying amounts of e.coli in vitro transcript RNA were mixed in nuclease assay buffer (40 mM Tris-HCl,60mM NaCl,6mM MgCl 2 pH 7.3), and recording the fluorescence intensities corresponding to the different contents of S.aureus nucleic acid targets by an up-conversion fluorescence spectrometer. Fluorescence spectrum data are acquired by means of an up-conversion fluorescence spectrum system, and the fluorescence intensity value of the maximum absorption peak of the up-conversion fluorescent nanomaterial is acquired, as shown in figure 2,and constructing a quantitative analysis model of the fluorescence intensity variation value and the escherichia coli nucleic acid targets with different contents, thereby realizing nano fluorescence trace detection of the escherichia coli target nucleic acid.
The above embodiments are merely for illustrating the design concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, the scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications according to the principles and design ideas of the present invention are within the scope of the present invention.
Claims (1)
1. An up-conversion nano-fluorescent ultrasensitive detection method of food-borne pathogenic bacteria not used for disease diagnosis and treatment is characterized in that nuclease Cas13a is used for precisely cutting a specific nucleic acid target in trace pathogenic bacteria cells, and by utilizing the incidental cutting effect of the nuclease Cas13a, fluorescent RNA markers are sheared and quenched to release detectable fluorescence; collecting fluorescence spectrum data by means of an up-conversion fluorescence spectrum system, obtaining a fluorescence intensity value of a maximum absorption peak of the up-conversion fluorescent nanomaterial, constructing quantitative detection models of fluorescence intensity change values and different amounts of food-borne pathogenic bacteria nucleic acid targets, and realizing nano-fluorescence trace detection of the food-borne pathogenic bacteria nucleic acid;
the method for synthesizing the quenched fluorescent RNA marker comprises the following steps: synthesizing up-conversion nano particles with amino modification by adopting a one-step solvothermal method, combining specific RNA with a quencher to synthesize an RNA fluorescent marker, and connecting the RNA fluorescent marker with the up-conversion nano particles with amino modification by adopting a glutaraldehyde crosslinking method to obtain the quenched fluorescent RNA marker; in the detection process, the quenched fluorescent RNA marker is specifically combined with RNA of food-borne pathogenic bacteria to be detected, nuclease Cas13a specifically cuts specific nucleic acid target RNA, activated Cas13a has auxiliary cutting activity, cuts the quenched fluorescent RNA marker, and releases fluorescence which can be detected;
the up-conversion nanoparticle particle size<50-100nm by adding NH 4 The amount of F is used for adjusting the particle size;
the fluorescence color of the up-conversion nano-particles is controlled by adjusting the doping proportion of rare earth elements;
the up-conversion fluorescent nanoparticle is NaGdF 4 :Yb/Er、NaGdF 4 Yb/Tm or NaGdF 4 :Yb/Ho；
The quencher adopts BHQ series fluorescence quencher.
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