Universal single-pot CRISPR / Cas12a sensing strategy and pathogenic bacterium detection application thereof

Abstract

The invention relates to the technical field of food safety detection, in particular to a universal single-pot CRISPR / Cas12a sensing strategy and pathogenic bacterium detection application thereof.The universal single-pot CRISPR / Cas12a sensing strategy comprises the following steps that S1, the 5'end of an upstream primer is marked with a crRNA template region; s2, aiming at the staphylococcus aureus primer group, adding T7 polymerase and NTP mixture, and carrying out RPA (recombinase polymerase amplification) at a constant temperature, so as to obtain an RPA double-chain product and crRNA (complementary ribosomal ribonucleic acid). According to the invention, the upstream primers are specifically functionalized aiming at pathogenic bacteria, and different upstream primers are designed aiming at different pathogenic bacteria, so that different crRNAs are transcribed. According to the detection method, the RPA technology is adopted to amplify the strain to be detected, crRNA can be transcribed by an amplified product in a transcription mode, the transcribed crRNA is complementary with a crRNA template region of an upstream primer, and the trans-cleavage activity of Cas12a can be further activated, so that sensitive detection on various food-borne pathogenic bacteria is realized. The sensitivity can reach 1CFU / mL, the repeatability is good, the operation is simple, the detection time is short, the applicability is strong, and the stability is high.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and specifically to a universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection. Background Art

[0002] Pathogenic bacteria have always been one of the main causes of human diseases, posing a huge threat to global public health. A large part of these pathogens cause diseases by using food as a carrier, also known as foodborne pathogens. Foodborne diseases caused by foodborne pathogens not only cause serious harm to human health, but also have a huge impact on the global economy. According to the World Health Organization, about 600 million people become ill and 420,000 die each year from eating contaminated food. Among them, 25% of food poisoning incidents are caused by Staphylococcus aureus. Staphylococcus aureus is a Gram-positive bacterium that can cause various infections in humans, usually with symptoms such as vomiting, diarrhea, and fever. In severe cases, endocarditis and meningitis may also occur, which may endanger human life and health. Based on this, the development of a method that can sensitively, quickly and accurately detect pathogens in food is of great significance for preventing the spread of foodborne pathogens and maintaining people's health.

[0003] There are many methods for detecting pathogenic bacteria. The traditional culture-based bacteriological examination technology has disadvantages such as long culture time and high labor demand. In addition, with the continuous development of technology, other technologies based on molecular biology such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, surface enhanced Raman scattering, etc. have also been proven to be effective pathogen detection technologies. They can provide rapid detection results, but they require high professionalism of technicians and require expensive PCR instruments, which are difficult to apply in some resource-scarce areas. Since these methods are all based on laboratory analysis, there is an urgent need to develop methods for rapid on-site detection of pathogenic bacteria.

[0004] Recently, the emergence of CRISPR technology has provided an innovative solution for rapid diagnosis. CRISPR / Cas is an adaptive immune system derived from archaea. Researchers have discovered its great application potential in the field of molecular diagnosis and have developed a large number of detection methods based on this. However, it is worth noting that in addition to crRNA, Cas12a also requires the protospacer adjacent motif (PAM) of "TTTN" to recognize dsDNA. It limits the application of CRISPR / Cas12a in dsDNA detection because PAM sequences may be lacking in many targets. At present, researchers have explored many methods that do not rely on PAM. Some add PAM sequences to primers, generate amplification products containing PAM sites through various amplification methods, or add PAM to the end of the target with polyA polymerase, amplify with primers with specific markers, cut one chain with the marker, and the remaining ssDNA activates the cutting activity of Cas12a. However, these methods get rid of the limitation of PAM by increasing or degrading single-stranded DNA. However, these methods are time-consuming and complex in design, which is not conducive to widespread application. Therefore, a fast, sensitive, simple and widely used method is needed to get rid of the dependence of Cas12a on the PAM sequence.

[0005] Although researchers have developed a large number of biosensors for rapid on-site detection of pathogens, most of these methods rely on the recognition of PAM sequences and are easily affected by the target. The crRNA template region is designed at the upstream primer end, and the crRNA can be transcribed after transcription. The transcribed crRNA can bind to the upstream primer crRNA template region, thereby further activating the nonspecific cleavage activity of Cas12a and emitting a fluorescent signal. This method gets rid of the limitations of the PAM sequence in the target and expands the scope of detecting pathogens. Therefore, this detection method can better meet the purpose of rapid and accurate on-site detection, and further prevent the spread of pathogens to maintain people's health. Summary of the invention

[0006] The object of the present invention is to provide a universal single-pot CRISPR / Cas12a sensing strategy and its pathogenic bacteria detection application to solve the problems raised in the above-mentioned background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection, comprising the following steps:

[0008] S1: label the crRNA template region at the 5' end of the upstream primer;

[0009] S2: For the Staphylococcus aureus primer set, add T7 polymerase and NTP mixture to perform RPA at a constant temperature to obtain RPA double-stranded products and crRNA; mix an appropriate amount of Cas12a protein, fluorescent probe, RPA double-stranded product and crRNA, and incubate for a period of time. The transcribed crRNA is complementary to the crRNA template region of the upstream primer, thereby activating the trans-cleavage activity of Cas12a and cleaving the FAM-labeled fluorescent probe. After a few minutes of reaction, the fluorescent signal is observed by the naked eye and then measured by a fluorescence spectrophotometer.

[0010] Preferably, the concentration of the RPA primer set is 0.4-1.4 μmol / L.

[0011] Preferably, the constant temperature of the RPA is 37°C.

[0012] Preferably, the RPA time is 30-35 minutes.

[0013] Preferably, the concentration of Cas12a is 0.014-0.028 μmol / L.

[0014] Preferably, the concentration of the probe is 0.5-2 μmol / L, the reaction time is 3-5 min, and the spectrum measurement conditions of the fluorescence spectrophotometer are an excitation wave of 492 nm and an emission wavelength of 500-650 nm.

[0015] Preferably, the crRNA concentration is 40-400 μmol / L.

[0016] Preferably, the total reaction volume is 20-25 μL, and the reaction time is 30-35 min.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention utilizes functionalized upstream primers specific for pathogenic bacteria, designs different upstream primers for different pathogenic bacteria, and thus transcribes different crRNAs.

[0019] 2. The detection method of the present invention uses RPA technology to amplify the strain to be tested. Since the amplified product can transcribe crRNA by transcription, the transcribed crRNA is complementary to the crRNA template region of the upstream primer, and further activates the trans-cleavage activity of Cas12a, thereby realizing sensitive detection of various foodborne pathogens. The sensitivity can reach 1CFU / mL, with good repeatability, simple operation, short detection time, strong applicability and high stability.

[0020] 3. Compared with traditional nucleic acid amplification technology, the recombinase polymerase isothermal amplification technology used in the present invention has more obvious advantages, such as mild conditions and short time, and the target in the food sample can be amplified in a short time and at a high multiple; the transcribed crRNA is complementary to the designed upstream primer crRNA template region, and further combines with the Cas12a protein to exert its nonspecific cutting function, avoiding the step of repeatedly searching for the PAM sequence.

[0021] 4. The detection method of the present invention does not require complicated steps, and only requires RPA to amplify the target to be detected, so as to achieve rapid and accurate detection of various pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the present invention for detecting food samples contaminated by pathogenic bacteria. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] See also Figure 1 The present invention provides a technical solution: a universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection, comprising the following steps:

[0025] S1: Label the crRNA template region (fixed sequence + T7 promoter) at the 5' end of the upstream primer;

[0026] S2: For the Staphylococcus aureus primer set, add T7 polymerase and NTP mixture to perform RPA at a constant temperature to obtain RPA double-stranded products and crRNA; mix an appropriate amount of Cas12a protein, fluorescent probe, RPA double-stranded product and crRNA, and incubate for a period of time. The transcribed crRNA is complementary to the crRNA template region of the upstream primer, thereby activating the trans-cleavage activity of Cas12a and cleaving the FAM-labeled fluorescent probe. After a few minutes of reaction, the fluorescent signal is observed by the naked eye and then measured by a fluorescence spectrophotometer.

[0027] There is no need to identify the PAM sequence. It is only necessary to mark the crRNA template region (fixed sequence and T7 promoter sequence) at the 5' end of the upstream primer, and transcribe the crRNA through RPA. The transcribed crRNA is complementary to the crRNA template region of the upstream primer, thereby activating the trans-cleavage activity of Cas12a and cutting the FAM-labeled fluorescent probe.

[0028] The primer set sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0029] The primer set concentration of RPA was 0.4-1.4 μmol / L.

[0030] The constant temperature of RPA is 37°C.

[0031] The RPA time is 30-35 minutes.

[0032] The concentration of Cas12a is 0.014-0.028 μmol / L.

[0033] The concentration of the probe is 0.5-2 μmol / L, the reaction time is 3-5 min, and the spectrum measurement conditions of the fluorescence spectrophotometer are an excitation wave of 492 nm and an emission wavelength of 500-650 nm.

[0034] The sequence of crRNA is shown in SEQ ID NO.3, and the crRNA concentration is 40-400 μmol / L.

[0035] The total reaction volume was 20-25 μL and the reaction time was 30-35 min.

[0036] In summary, the implementation steps of the detection method of the present invention are: extraction of genomic DNA; design of upstream primers; crRNA and RPA; changes in fluorescence intensity. The present invention uses functionalized primers specific for pathogenic bacteria, complements the crRNA template region of the crRNA upstream primer by transcribing it, and utilizes the non-specific cutting function of the Cas12a system to cut the fluorescent probe labeled with FAM, which can then be measured by a fluorescence spectrophotometer, thereby achieving the purpose of sensitive detection of pathogenic bacteria in food samples.

[0037] Embodiment 1: The detection platform for detecting Escherichia coli provided in this embodiment comprises the following steps:

[0038] S1. Extraction and RPA amplification of Escherichia coli genes in the pathogenic bacteria to be tested: Escherichia coli genes were extracted using the kit method, and the specific process of RPA reaction was as follows: 10 μL of solvent, 1 μL of Tag-FP, 1 μL of RP, 0.4 μL of T7 RNA polymerase, 0.5 μL of NTP Mixture, 0.7 μL of Cas12a, 1.5 μL of Reporter, 0.5 μL of RNase inhibitor, 1 μL of activator, 1 μL of DNA template, add water to the total system of 25 μL, and react at 37°C for 30 min.

[0039] S2, measuring the change of fluorescence intensity: react at 37° C. for 30 min in the previous step, and then measure it by fluorescence spectrophotometer.

[0040] Embodiment 2: The detection platform for detecting Pseudomonas aeruginosa provided in this embodiment comprises the following steps:

[0041] S1. Extraction and RPA amplification of Pseudomonas aeruginosa genes in the pathogenic bacteria to be tested: Pseudomonas aeruginosa genes were extracted using the kit method, and the specific process of RPA reaction was as follows: 10 μL of solvent, 1 μL of Tag-FP, 1 μL of RP, 0.4 μL of T7 RNA polymerase, 0.5 μL of NTP Mixture, 0.7 μL of Cas12a, 1.5 μL of Reporter, 0.5 μL of RNase inhibitor, 1 μL of activator, 1 μL of DNA template, add water to the total system of 25 μL, and react at 37°C for 30 min.

[0042] S2, measuring the change of fluorescence intensity: react at 37° C. for 30 min in the previous step, and then measure it by fluorescence spectrophotometer.

[0043] By the above technical scheme, the present invention marks the crRNA template region at the upstream primer end, mixes T7 RNA polymerase and NTP Mixture, obtains the corresponding RPA double-stranded product and crRNA by RPA, and the transcribed crRNA is complementary to the crRNA template region of the upstream primer, and Cas12a will be guided by crRNA, and exert its trans-cutting activity, so as to realize the sensitive detection of various foodborne pathogens. The change of fluorescence intensity and the change of color can be observed by the naked eye, and the purpose of pathogen detection is realized. This method has good specificity, high sensitivity, good repeatability, simple operation, strong adaptability, high accuracy, and can detect pathogens with a concentration of 1CFU / mL. This method does not require complicated steps, and only requires ordinary PCR instruments and fluorescence spectrophotometers to achieve rapid and accurate detection of pathogens.

[0044] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection, characterized by: The following steps are included: S1: label the crRNA template region at the 5' end of the upstream primer; S2: For the Staphylococcus aureus primer set, add T7 polymerase and NTP mixture to perform RPA at a constant temperature to obtain RPA double-stranded products and crRNA; mix an appropriate amount of Cas12a protein, fluorescent probe, RPA double-stranded product and crRNA, and incubate for a period of time. The transcribed crRNA is complementary to the crRNA template region of the upstream primer, thereby activating the trans-cleavage activity of Cas12a and cleaving the FAM-labeled fluorescent probe. After a few minutes of reaction, the fluorescent signal is observed by the naked eye and then measured by a fluorescence spectrophotometer.

2. A universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection according to claim 1, characterized in that: The concentration of the primer set of the RPA is 0.4-1.4 μmol / L.

3. A universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection according to claim 1, characterized in that: The constant temperature of the RPA was 37°C.

4. A universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection according to claim 1, characterized in that: The RPA time is 30-35 minutes.

5. A universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection according to claim 1, characterized in that: The concentration of the Cas12a is 0.014-0.028 μmol / L.

6. A universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogen detection according to claim 1, characterized in that: The concentration of the probe is 0.5-2 μmol / L, the reaction time is 3-5 min, and the spectrum measurement conditions of the fluorescence spectrophotometer are an excitation wave of 492 nm and an emission wavelength of 500-650 nm.

7. A universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection according to claim 1, characterized in that: The crRNA concentration is 40-400 μmol / L.

8. A universal single-pot CRISPR / Cas12a sensing strategy and its application in pathogenic bacteria detection according to claim 1, characterized in that: The total reaction volume was 20-25 μL and the reaction time was 30-35 min.

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