WO2020224164A1

WO2020224164A1 - Method for detecting nano fluorescent traces in nucleic acids of food-borne pathogenic bacteria on basis of crispr/cas13a

Info

Publication number: WO2020224164A1
Application number: PCT/CN2019/107184
Authority: WO
Inventors: 陈全胜; 李欢欢; 吕鹏; 刘蕊; 欧阳琴
Original assignee: 江苏大学
Priority date: 2019-05-07
Filing date: 2019-09-23
Publication date: 2020-11-12
Also published as: CN110205360A

Abstract

Disclosed by the present invention is a method for detecting nano fluorescent traces in nucleic acids of food-borne pathogenic bacteria on the basis of CRISPR/Cas13a. In the present invention: nucleic acids of food-borne pathogenic bacteria are used as a study object, and food-borne pathogenic bacteria nucleic acid targets, food-borne pathogenic bacteria crRNA, and nuclease Cas13a protein purification and fluorescence quenching RNA reporting markers are prepared; nuclease Cas13a is used to cut a specific nucleic acid target in trace pathogenic bacteria cells, and the fluorescence quenching RNA reporting markers are sheared by using the incidental cutting effect of the nuclease to release fluorescence that can detected; and fluorescence spectrum data is collected by virtue of a fluorescence spectrum system, a fluorescence intensity value of the maximum absorption peak of an up-conversion fluorescent nanomaterial is acquired, and a quantitative determination model of up-conversion nano fluorescence intensity and food-borne pathogenic bacteria nucleic acid target content is constructed so that the nucleic acids in the food-borne pathogenic bacteria are detected.

Description

A CRISPR/Cas13a-based method for detecting food-borne pathogenic bacteria nucleic acid nano-fluorescence trace

Technical field

The invention belongs to the technical field of food-borne pathogenic bacteria trace detection, and in particular relates to a CRISPR/Cas13a-based food-borne pathogenic bacteria nucleic acid nano-fluorescence trace detection method.

Background technique

Food-borne pathogens are one of the main causes of food-borne diseases. They are important for world food safety and have seriously threatened human health. The detection of food-borne pathogens is an important means of food safety. With the rapid development of nucleic acid detection technology, rapid detection methods for various food-borne pathogens have emerged one after another. Commonly used are polymerase chain reaction and its derivative technology, nucleic acid constant temperature amplification technology, oligonucleotide microarray technology and immunomagnetic cell separation technology. Although these existing methods have their own advantages, they have disadvantages such as long detection cycle or too strong fluorescent background during detection. In view of the applicant's accumulated good experience in the field of food non-destructive testing and the skilled molecular biology basis of team members, especially in-depth research in the field of up-conversion fluorescence detection technology and CRISPR/Cas technology, this project plans to build a CRISPR/Cas13a Food-borne pathogenic bacteria nucleic acid nano-fluorescence trace detection method, in-depth exploration of rapid and sensitive food-borne pathogen nucleic acid quantitative detection method, this method is suitable for food safety, environmental monitoring and other technical fields.

At present, the use of up-conversion fluorescent nanotechnology to achieve rapid detection of nucleic acids of food-borne pathogens has not yet been reported. As a novel quantitative method for nucleic acid of food-borne pathogenic bacteria, the invention realizes rapid detection of nucleic acid of food-borne pathogenic bacteria.

Summary of the invention

According to the existing problems in the prior art, the present invention proposes a CRISPR/Cas13a-based food-borne pathogenic bacteria nucleic acid nano-fluorescence trace detection method. The present invention has the advantages of short detection cycle, strong specificity and high sensitivity, and can be Realize trace detection and analysis of food-borne pathogenic bacteria nucleic acid, which is suitable for food safety, environmental monitoring and other technical fields.

The technical scheme adopted by the present invention is as follows:

A CRISPR/Cas13a-based nano-fluorescence trace detection method for nucleic acid of food-borne pathogenic bacteria. The nucleic acid of food-borne pathogenic bacteria is used as the research object. The nucleic acid target is prepared by using CRISPR/Cas13a technology and recombinase polymerase amplification technology. , And construct a set of powerful nucleic acid detection tools by synthesizing quenched fluorescent RNA reporter markers, using the new nuclease Cas13a to accurately cut specific nucleic acid targets in trace pathogenic bacteria cells, and use the enzyme's incidental cutting effect , Shear quenching the fluorescent RNA reporter marker, release the detectable fluorescence; use the fluorescence spectroscopy system to collect fluorescence spectrum data, obtain the fluorescence intensity value of the maximum absorption peak of the up-conversion fluorescent nanomaterial, and construct the up-conversion nano-fluorescence intensity and food source A quantitative detection model for the content of nucleic acid targets of pathogenic bacteria to achieve rapid detection of nucleic acid nano-fluorescence traces of food-borne pathogens.

Further, the method for collecting fluorescence spectrum data is: mixing purified Cas13a protein, crRNA, quenched fluorescent RNA reporter marker, RNase inhibitor, background RNA and different content of pathogenic bacteria in vitro transcription product RNA into nucleic acid Incubate in enzyme assay buffer, and record the fluorescence intensity corresponding to the nucleic acid target of different pathogenic bacteria through an up-conversion fluorescence spectrometer.

Further, the nuclease Cas13a specifically cuts the target RNA, and the activated Cas13a has incidental cutting activity and can cut other non-target RNAs.

Further, the nucleic acid target preparation method: extract the nucleic acid of food-borne pathogenic bacteria, use RPA/RT-PCR recombinase polymerase amplification technology to amplify trace nucleic acid fragments of pathogenic bacteria, and then perform in vitro transcription to obtain RNA The fragment serves as a nucleic acid target piece of food-borne pathogenic bacteria.

Further, the preparation method of the food-borne pathogenic bacteria crRNA is: synthesizing a crRNA with a structure of 5'-anchor sequence-guide sequence-3', the anchor sequence depends on the source of Cas13a, the guide sequence and in vitro transcribed RNA fragments Complementary, reverse transcription of 5'-anchor sequence-guide sequence-3', add T7 promoter sequence to its 5', and place 5'-T7 promoter sequence-anchor sequence-guide sequence-3' DNA in Under the action of T7RNA polymerase, a large amount of crRNA is synthesized rapidly.

Further, the preparation method of the quenched fluorescent RNA reporter marker:

The upconversion nanoparticles with amino modification were prepared by solvothermal method, and the glutaraldehyde cross-linking method was used to combine the RNA fluorescent marker (NH ₂ -RNA-BHQ) with the upconversion nanoparticles with amino modification (NH ₂ -UCNP). _S ) is connected together as a quenching fluorescent RNA marker. The quenching fluorescent RNA reporter marker has a signal reporting function. When Cas13a cuts the RNA sequence therein, it will release a detectable fluorescent signal.

Further, the up-conversion fluorescent nanoparticles are NaGdF ₄ :Yb/Er, NaGdF ₄ :Yb/Tm or NaGdF ₄ :Yb/Ho.

The beneficial effects of the present invention:

The invention relates to a CRISPR/Cas13a-based food-borne pathogenic bacteria nucleic acid nano-fluorescence trace detection method, which is simple and easy to operate, has a short detection period and high sensitivity, and can be widely used in technical fields such as food safety and environmental monitoring.

The novel nuclease Cas13a in the CRISPR-Cas13a technology involved in the present invention has a dual function. It not only participates in the maturation process of crRNA, but also specifically cuts the target RNA, and the activated Cas13a has incidental cutting activity and can cut other non-target RNAs. . CRISPR/Cas13a technology extends the original DNA-targeting gene editing tool to a new detection system targeting RNA, which has the advantages of fast detection speed, strong specificity and high sensitivity.

The RPA technology of the combined recombinase polymerase amplification technology involved in the present invention does not rely on thermal cycle amplification of template sequences, uses the combination of recombinase and oligonucleotide primers to accurately locate the target sequence, and unwinds the template with the assistance of single-stranded DNA combined with protease Double-stranded, and then start the exponential amplification of the target sequence under the action of DNA polymerase. The entire reaction can be carried out at room temperature without denaturation. The detection level of the amplified product can be obtained within 20 minutes, with strong specificity and high sensitivity. Rapid response, low equipment dependence and precise amplification are advantages that traditional in vitro nucleic acid amplification technology does not have.

Description of the drawings

Figure 1 is a technical roadmap of CRISPR/Cas13a-based nucleic acid nanofluorescence trace detection of food-borne pathogens;

FIG 2 is a characterization of the converter of FIG fluorescent nanoparticles of UCNP _S, A is a TEM FIG upconversion fluorescent nanoparticles UCNP _S, B is a 0.1mg / mL UCNP _S conversion fluorescent nanoparticle fluorescence intensity of the particles.

Detailed ways

In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not to limit the present invention.

As shown in Figure 1, the CRISPR/Cas13a-based food-borne pathogenic bacteria nucleic acid nano-fluorescence trace detection method proposed by the present invention further verifies the detection method proposed by the present invention. The solution designed by the present invention is suitable for food-borne disease For the detection of bacteria, only S. aureus is used as an example in this embodiment. The specific operation process is as follows:

Preparation of Staphylococcus aureus nucleic acid target fragment: first inoculate Staphylococcus aureus on Luria-Bertani medium, culture at 37°C, 200rpm/min for 24h, then take 1mL of bacterial night, centrifuge at 12000/min for 1min, discard the supernatant liquid. Use bacterial genome extraction kit to extract nucleic acid from Staphylococcus aureus. RPA/RT-PCR technology was used to amplify the trace nucleic acid target fragments of Staphylococcus aureus, followed by in vitro transcription to obtain RNA fragments. RPA (recombinase polymerase amplification) technology is known as the only new constant temperature nucleic acid amplification method that can replace PCR technology. It does not rely on thermal cycling to amplify template sequences, but uses recombinase and oligonucleosides. The acid primer binds to accurately locate the target sequence, unwinds the template duplex with the help of single-stranded DNA binding protease, and then initiates the exponential amplification of the target sequence under the action of DNA polymerase. The entire reaction can be carried out at room temperature without denaturation , The detection level of the amplified product can be obtained within 20-30min, which has the advantages of strong specificity, high sensitivity, rapid reaction, low equipment dependence and accurate amplification, etc., which traditional in vitro nucleic acid amplification technology does not have.

Preparation of Staphylococcus aureus crRNA: synthetic crRNA by chemical synthesis. The crRNA structure is 5'-anchor sequence-guide sequence-3'. The anchor sequence depends on the source of Cas13a. When Cas13a is LshCas13a, the anchor sequence is 5’-CCACCCCAAUAUCGAAGGGGACUAAAAC-3’; when Cas13a is LwCas13a, the anchor sequence is 5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3’. The designed length of the guide sequence is 21-28 nucleotides, which is complementary to the in vitro transcribed RNA fragment. Then reverse transcribed the designed 5'-anchor sequence-guide sequence-3', and add the T7 promoter sequence to its 5', and change the 5'-T7 promoter sequence-anchor sequence-guide sequence-3' DNA Under the action of T7RNA polymerase, a large amount of crRNA is synthesized rapidly. Purify the synthesized crRNA using RNAXP cleaning beads.

Purification of Cas13a protein: Transform the Cas13a bacterial expression vector into competent cells, and take 16 mL of the culture and culture it overnight in Terrific Broth 4 growth medium. Then supplement with IPTG, and cool the cells to 18°C for 16 hours for protein expression. Centrifuge at 5200g for 15min at 4°C, collect the cell pellet and break it for protein purification.

Preparation of quenched fluorescent RNA reporter markers: Upconversion nanoparticles UCNP _S were prepared by solvothermal method, and the prepared nanoparticles were amino-modified nanoparticles. The specific process is: dissolving NH ₄ F (6.24 mmol) in 12 mL EG. Take NaCl (1mmol), PEI (0.5g), Gd(NO ₃ ) ₃ (0.8mmol), Yb(NO ₃ ) ₃ (0.17mmol) and Er(NO ₃ ) ₃ (0.03mmol) and dissolve in EG (38mL) In, magnetic stirring for 30min. When the solution is transparent, add NH ₄ F solution, stir for 10 min, and transfer the solution to a stainless steel autoclave. The reaction kettle was heated at 200°C for 1.5 hours and cooled to room temperature. The nanoparticles were separated by centrifugation, washed three times with deionized water, and dried in a vacuum drying oven for 6 hours to obtain powdered upconversion nanoparticles. In this embodiment, the particle size (<100 nm) is adjusted by adding the amount of NH ₄ F, and the fluorescent color is controlled by adjusting the doping ratio of rare earth elements. The glutaraldehyde cross-linking method was used to connect the RNA fluorescent marker NH ₂ -RNA-BHQ1 and the amino-modified nanoparticles NH ₂ -UCNP _S to obtain the quenched fluorescent RNA reporter marker UCNP _S --RNA-BHQ1. In this embodiment, the RNA fluorescent marker uses NH _2- RNA-BHQ1 synthesized by TaKaRa Company. The quenched fluorescent RNA reporter marker has a signal reporting function, and when Cas13a cuts the RNA sequence therein, it releases a detectable green fluorescent signal.

Fluorescence spectroscopy data collection and analysis: The purified Cas13a protein, crRNA, quenched fluorescent RNA reporter marker, RNase inhibitor, background RNA and different content of Staphylococcus aureus in vitro transcription product RNA are mixed in nuclease assay buffer (40mM Incubate in Tris-HCl, 60mM NaCl, 6mM MgCl ₂ , pH 7.3), and record the fluorescence intensity corresponding to different contents of Staphylococcus aureus nucleic acid targets by an up-conversion fluorescence spectrometer.

Nano-fluorescence trace detection of Staphylococcus aureus nucleic acid target: Construct a quantitative analysis model of the fluorescence intensity change value and different content of Staphylococcus aureus nucleic acid target, so as to realize the nano-fluorescence trace detection of Staphylococcus aureus target nucleic acid, as shown in Figure 2 Shown.

The CRISPR/Cas13a-based food-borne pathogenic bacteria nucleic acid nano-fluorescence trace detection method proposed in the present invention is suitable for the technical fields of food safety and environmental monitoring. The nuclease Cas13a in the present invention not only participates in the crRNA maturation process, but also For specific cutting of target RNA, the activated Cas13a has incidental cutting activity, which can cut other non-target RNAs. CRISPR/Cas13a technology extends the original DNA-targeting gene editing tool to a new detection system targeting RNA. It has the advantages of fast detection speed, specific detection of single base mismatches and sensitivity detection of single target nucleic acid molecules.

The detection method designed by the present invention has strong specificity. When multiple food-borne pathogenic bacteria exist, only the in vitro transcript of the target pathogenic bacteria nucleic acid after amplification matches the designed crRNA, and the incidental cutting activity of Cas13a Is activated to release detectable fluorescence.

The above embodiments are only used to illustrate the design ideas and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement them accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention fall within the protection scope of the present invention.

Claims

A CRISPR/Cas13a-based method for detecting food-borne pathogenic bacteria nucleic acid nano-fluorescence trace, which is characterized in that the nucleic acid of food-borne pathogenic bacteria is used as the research object, using CRISPR/Cas13a technology, combined with recombinase polymerase amplification Technology to prepare nucleic acid targets and synthesize quenching fluorescent RNA reporter markers to construct a powerful nucleic acid detection tool. Use nuclease Cas13a to accurately cut specific nucleic acid targets in trace pathogenic bacteria cells, and use nuclease Cas13a to attach Cleavage effect, shear quenching the fluorescent RNA reporter marker, release the detectable fluorescence; use the fluorescence spectroscopy system to collect fluorescence spectrum data, obtain the fluorescence intensity value of the maximum absorption peak of the up-conversion fluorescent nanomaterial, and construct the up-conversion nano-fluorescence intensity and The quantitative detection model of food-borne pathogenic bacteria nucleic acid target content realizes the rapid detection of food-borne pathogenic bacteria nucleic acid nano-fluorescence trace.
The CRISPR/Cas13a-based method for detecting nucleic acid nano-fluorescence traces of food-borne pathogenic bacteria according to claim 1, wherein the method for collecting fluorescence spectrum data is: combining purified Cas13a protein, crRNA, Quenching fluorescent RNA reporter markers, RNase inhibitors, background RNA and different content of pathogenic bacteria in vitro transcribed RNA nucleic acid targets are mixed and incubated in nuclease assay buffer, and recorded with different content of pathogenic bacteria through an up-conversion fluorescence spectrometer The fluorescence intensity corresponding to the nucleic acid target.
The CRISPR/Cas13a-based method for detecting nucleic acid nano-fluorescence traces of food-borne pathogenic bacteria according to claim 1 or 2, wherein the nuclease Cas13a specifically cleaves the nucleic acid target RNA to activate the Cas13a has incidental cutting activity and can cut other non-target RNAs.
The CRISPR/Cas13a-based method for detecting food-borne pathogenic bacteria nucleic acid nano-fluorescence trace according to claim 2, wherein the nucleic acid target preparation method: extracting nucleic acid of food-borne pathogenic bacteria, using recombinant Enzyme polymerase amplification technology performs amplification reaction on trace nucleic acid fragments of pathogenic bacteria, followed by in vitro transcription to obtain RNA fragments as nucleic acid targets of food-borne pathogenic bacteria.
The CRISPR/Cas13a-based method for detecting nucleic acid nano-fluorescence traces of food-borne pathogenic bacteria according to claim 2, wherein the preparation method of the crRNA is: the synthetic structure is a 5'-anchor sequence- Guide sequence-3' of crRNA, anchor sequence depends on the source of Cas13a, guide sequence is complementary to in vitro transcribed RNA fragment, reverse transcription of 5'-anchor sequence-guide sequence-3', and add T7 to its 5' Promoter sequence, the 5'-T7 promoter sequence-anchor sequence-guide sequence-3'DNA is rapidly synthesized under the action of T7RNA polymerase.