Method for detecting campylobacter jejuni based on RAA-CRISPR / Cas13a

Abstract

The invention discloses a method for detecting campylobacter jejuni based on RAA-CRISPR / Cas13a. RAA completes target enrichment within 30 minutes under the constant temperature condition of 42 DEG C, and the instrument limitation of traditional PCR is broken through; according to the CRISPR-Cas13a system, the RNA targeting characteristic of the CRISPR-Cas13a system is utilized, the'incidental cleavage 'effect of an RAA amplification product activated Cas13a protein is accurately recognized through crRNA, hypersensitive detection of 9.3 copies / mu L is achieved, and the template consumption risk is reduced compared with a Cas12a system. The technology breaks through the multi-dimensional technical barriers of sensitivity, timeliness, equipment dependence, operation safety and the like of a traditional method, provides an on-site rapid detection solution which does not need complex instruments and avoids biological safety risks for basic medical institutions and farms, and remarkably improves the early warning capability of campylobacter jejuni infection.

Description

Technical Field

[0001] The present invention belongs to the field of detection, and particularly relates to a method for detecting Campylobacter jejuni based on RAA-CRISPR / Cas13a. Background Art

[0002] Campylobacter jejuni is a Gram-negative bacterium belonging to the genus Campylobacter, presenting an arc shape, a seagull-wing shape, a spiral shape or an S shape. Campylobacter jejuni colonizes the gastrointestinal tracts of various food animals. Clinical symptoms include watery or bloody diarrhea, accompanied by abdominal colic and fever, and a small number of cases can cause miscarriage. Extra-intestinal infections can lead to pericarditis. If people accidentally eat contaminated or undercooked animal products, they are prone to symptoms such as diarrhea, abdominal pain, and arthritis. In addition, long-term sequelae, namely irritable bowel syndrome (IBS), can occur after Campylobacter jejuni infection. Currently, Campylobacter jejuni infection causes approximately 30% of Guillain-Barré syndrome (GBS) cases. From 2020 to 2021, feces were collected and tested at four bird migration routes in China, and the results showed that 73.8% (329 / 446) of the samples were positive for Campylobacter, highlighting the potential public health threat of the avian-human transmission chain.

[0003] The isolation, culture and detection of Campylobacter jejuni are relatively difficult and the operation is cumbersome. Currently, the commonly used detection methods are enzyme-linked immunosorbent assay (ELISA), immunochromatography technology and PCR technology. However, the ELISA detection technology has a long processing time (>24h) and cumbersome operation. The immunochromatography technology is convenient and fast, but has low sensitivity and specificity. The PCR technology has simple primer design and relatively convenient operation, but has a high instrument cost and requires high-temperature reaction conditions. In addition, there are some emerging technologies such as Surface Plasmon Resonance (SPR), which uses the surface plasmon resonance phenomenon to study the interaction between molecules. This method has high selectivity, but low sensitivity and high requirements for equipment. Therefore, establishing a method for detecting Campylobacter jejuni with short time consumption, high sensitivity and accuracy is of great significance for ensuring the sustainable and stable development of animal husbandry and public health safety. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: how to establish a method for detecting Campylobacter jejuni without complex instruments, with short time consumption, high sensitivity and good accuracy.

[0005] The technical solution of the present invention is: a nucleic acid molecule composition, which consists of a primer pair and crRNA. The nucleotide sequences of the primer pair are shown in SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of the crRNA is shown in SEQ ID No.3.

[0006] A kit containing the nucleic acid molecule composition described above.

[0007] Furthermore, the kit further includes one or more of reagents for RAA amplification, T7 RNA polymerase, Cas 13a protein, and reporter RNA.

[0008] Furthermore, the reagents for RAA amplification include one or more of recombinase, DNA polymerase, reverse transcriptase, or dNTP.

[0009] Furthermore, the nucleotide sequence of the reporter RNA is as shown in SEQ ID No. 4, and it has a 6-FAM fluorescent group at the 5' end and a BHQ1 quenching group at the 3' end.

[0010] A method for detecting Campylobacter jejuni based on RAA-CRISPR / Cas13a for non-disease diagnosis purposes, comprising the following steps:

[0011] (1) Extract the total DNA of the sample to be detected;

[0012] (2) Perform RAA amplification on the total DNA extracted in step (1) using the primer pairs shown in SEQ ID No. 1 and SEQ ID No. 2;

[0013] (3) Using the RAA amplification product in step (2) as a template, add the crRNA shown in SEQ ID No. 3, T7 RNA polymerase, Cas 13a protein, and reporter RNA to the CRISPR-Cas13a detection system for reaction;

[0014] (4) Detect the fluorescence intensity of the reaction system obtained in step (3).

[0015] Furthermore, in step (2), the RAA amplification system is: configure the required reaction system: 1 tube of reaction dry powder, 25 μL of A buffer, 2 μL each of the upstream and downstream primers shown in SEQ ID No. 1 and SEQ ID No. 2 with a concentration of 10 μM, add 5 μL of the sample to be detected to the detection unit tube, 2.5 μL of B Buffer, and make up the system to 50 μL with RNase free H2O; the RAA amplification method is: invert the system up and down to mix well, then centrifuge at low speed for 10 s, and incubate it in a 42°C constant temperature metal bath for 30 min.

[0016] Further, in step (3), the composition of the CRISPR-Cas13a detection system is as follows: 2 μL of 10×RNA Polymerase reaction buffer, 2 μL of 10×LwaCas 13a reaction buffer, 2 μL of 50000 U / mL T7 RNA Polymerase, 1.5 μL of 2 μmol / L Cas13a protein, 0.4 μL of Ribonucleotide Solution Mix with NTP Buffer Mix at 25 mmol / L, 0.5 μL of 40000 U / mL RNase inhibitor, 2.4 μL of 80 nmol / L reporter RNA, 5 μL of 10 nmol / L crRNA, and 3 μL of RAA amplification product. Make up to 25 μL with Dnase / Rnase-free water.

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

[0018] (1) The detection method provided by the present invention has high sensitivity. The RT-RAA-CRISPR / Cas13a system maintains high sensitivity within a short reaction time, with a detection limit of 9.3 copies / μL, which is 3 orders of magnitude higher than that of traditional ELISA and 2 orders of magnitude higher than that of conventional PCR.

[0019] (2) The RAA primers are designed for the conserved region of the Map A gene, and the CRISPR system uses specific crRNA, which has no cross-reaction with 4 common pathogenic bacteria, namely Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Clostridium perfringens, and has high specificity.

[0020] (3) Both the RAA amplification and CRISPR detection stages adopt constant temperature conditions (42°C / 37°C), without the need for large instruments such as PCR machines and electrophoresis equipment, and without high temperature and cumbersome equipment. Description of the Drawings

[0021] Figure 1 Optimization results of the RAA-CRISPR / Cas13a system;

[0022] Figure 2 Specificity of the RAA-CRISPR / Cas13a detection method;

[0023] Figure 3 Sensitivity of the RAA-CRISPR / Cas13a system. Detailed Embodiments

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0025] Example 1 Primer Design for Campylobacter jejuni

[0026] According to the Map A gene of Campylobacter jejuni published in GenBank, RAA specific primers containing T7 promoter were designed using the RAA / RPA Primer online design software, and crRNA was designed using the CRISPRTarget software, which was synthesized by Guangzhou Boles Biotechnology Co., Ltd.

[0027] Table 1 Primers and Probes

[0028]

[0029] Example 2 RAA Amplification of Campylobacter jejuni Template

[0030] (1) Prepare the required reaction system (50 μL): 1 tube of reaction dry powder, 25 μL of A buffer, 2 μL each of upstream and downstream primers (10 μM). Add 5 μL of the sample to be tested to the detection unit tube, 2.5 μL of B Buffer, and make up the system to 50 μL with RNase free H2O.

[0031] (2) Invert it up and down to mix well, and then centrifuge at low speed for 10 s.

[0032] (3) Incubate it in a 42°C constant temperature metal bath for 30 min.

[0033] Example 3 Establishment and Optimization of the RAA-CRISPR / Cas13a Detection System

[0034] (1) Rapidly prepare a 25 μL CRISPR-Cas13a detection system on ice: 2 μL of 10×RNA Polymerase reaction buffer, 2 μL of 10×LwaCas 13a reaction buffer, 2 μL of T7 RNA Polymerase (50000 U / mL), 1 μL of LwaCas 13a protein (2 μmol / L), 0.4 μL of Ribonucleotide Solution Mix (NTP Buffer Mix 25 mmol / L), 0.5 μL of RNase inhibitor (40000 U / mL), 0.64 μL of TaqMan probe (80 nmol / L), 1 μL of crRNA (10 nmol / L), 3 μL of RAA amplification product, and make up to 25 μL with Dnase / Rnase-free water.

[0035] (2) After transient centrifugation, place it in a real-time fluorescence PCR instrument (during the process of method establishment and optimization, a real-time fluorescence PCR instrument is needed to read the fluorescence to help us select the best reaction conditions. But after the method is established, a real-time fluorescence PCR instrument can be not used, as long as it can be observed with the naked eye through a small fluorescence observation instrument after reacting at 37 °C for 30 min), react at 37 °C for 30 min, and collect the fluorescence signal of the reaction every 30 s.

[0036] (3) Use the control variable method to gradually optimize the dosages of the main reagents crRNA, TaqMan probe, and LwaCas 13a protein in the CRISPR-Cas13a detection system. The dosage settings are as follows: crRNA (10 nmol / L) 3 μL, 5 μL, 7 μL, 9 μL; TaqMan probe (80 nmol / L) 0.8 μL, 1.2 μL, 1.6 μL, 2.0 μL, 2.4 μL; LwaCas 13a protein (2 μmol / L) 0.5 μL, 1.0 μL, 1.5 μL, 2.0 μL. Taking the shortest time for the strongest fluorescence value to appear in the CRISPR-Cas13a detection system as the judgment criterion, determine that the optimal actual dosages of the CRISPR-Cas13a detection are 5 μL of crRNA (10 nmol / L), 2.4 μL of TaqMan probe (80 nmol / L), and 1.5 μL of Cas13a protein (2 μmol / L). The results are as Figure 1 shown.

[0037] Example 4 Specificity evaluation of the RAA-CRISPR / Cas13a detection system

[0038] Using the pUC57 Campylobacter jejuni plasmid standard containing the target gene and the nucleic acids of 4 other foodborne pathogenic bacteria, namely Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Clostridium perfringens, as the templates for detection, the established RAA-CRISPR-Cas13a detection system was used to detect the above templates, and Dnase / Rnase-free water was used as the negative control for detection. The fluorescence signals of the reaction were collected to analyze the specificity of the detection system. The results are as Figure 2 shown. Only Campylobacter jejuni had fluorescence signals, while Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Clostridium perfringens, and the negative control had no fluorescence signals.

[0039] Example 5 Evaluation of the sensitivity of the RAA-CRISPR / Cas13a detection system

[0040] (1) The pUC57 Campylobacter jejuni plasmid standard containing the target gene was serially diluted 10-fold with Dnase / Rnase-free water, and 9 concentration gradients (108 - 10 -1 copies / μL) were set.

[0041] (2) The optimized CRISPR-Cas13a detection system was used to detect plasmid standards at different concentrations. At the same time, Dnase / Rnase-free water was set as the negative control for detection. The fluorescence signals of the reaction were collected, and the detection sensitivity of the system was determined through the amplification curve. The results are as Figure 3 shown. The lowest detection concentration of this method is 9.3 copies / μL.

[0042] Example 6 Evaluation of the repeatability of the RAA-CRISPR / Cas13a detection system

[0043] Campylobacter jejuni plasmid DNA was repeatedly detected within and between batches at three concentrations: 9.3×10 8 copies / μL (high concentration), 9.3×10 5 copies / μL (medium concentration), and 9.3×10 2 copies / μL (low concentration). The coefficient of variation (CV) of the within- and between-batch repeat tests was determined by calculating the fluorescence values to analyze the repeatability within and between groups of the experiment.

[0044] Table 2: Repeatability of the RAA-CRISPR / Cas13a detection method

[0045]

[0046] The results are shown in Table 2. The within-group and between-group coefficients of variation are lower than 4.15% and 3.51% respectively, indicating good reproducibility.

Claims

1. A nucleic acid molecule composition, consisting of a primer pair and crRNA, characterized in that, The nucleotide sequences of the primer pair are shown in SEQ ID No.1 and SEQ ID No.2, and the nucleotide sequence of the crRNA is shown in SEQ ID No.

3.

2. A kit containing the nucleic acid molecule composition according to claim 1.

3. The kit according to claim 2, wherein The kit further comprises one or more of a reagent for RAA amplification, T7 RNA polymerase, Cas 13a protein, and reporter RNA.

4. The kit according to claim 3, wherein The reagent for RAA amplification comprises one or more of a recombinase, a DNA polymerase, a reverse transcriptase, or dNTPs.

5. The kit according to claim 3, characterized in that, The nucleotide sequence of the reporter RNA is shown in SEQ ID No.4, with a 6-FAM fluorophore at the 5' end and a BHQ1 quencher at the 3' end.

6. A method for detecting Campylobacter jejuni based on RAA-CRISPR / Cas13a for non-disease diagnosis purposes, characterized in that, Comprising the following steps: (1) Extract the total DNA of the sample to be detected; (2) Perform RAA amplification on the total DNA extracted in step (1) using the primer pair shown in SEQ ID No.1 and SEQ ID No.2; (3) Using the RAA amplification product in step (2) as a template, add the crRNA shown in SEQ ID No.3, T7 RNA polymerase, Cas 13a protein, and reporter RNA to the CRISPR-Cas13a detection system for reaction; (4) Detect the fluorescence intensity of the reaction system obtained in step (3).

7. The method according to claim 6, wherein In step (2), the RAA amplification system is as follows: Prepare the required reaction system: 1 tube of reaction dry powder, 25 μL of A buffer, 2 μL each of the upstream and downstream primers shown in SEQ ID No.1 and SEQ ID No.2 with a concentration of 10 μM. Add 5 μL of the sample to be detected to the detection unit tube, 2.5 μL of B buffer, and use RNase-free H2O to make up the system to 50 μL; The method of RAA amplification is: Invert the system up and down to mix well, then centrifuge at low speed for 10 s, and incubate it in a 42 °C constant temperature metal bath for 30 min.

8. The method according to claim 6, characterized in that, In step (3), the composition of the CRISPR-Cas13a detection system is: 2 μL of 10×RNA Polymerase reaction buffer, 2 μL of 10×LwaCas 13a reaction buffer, 2 μL of 50000 U / mL T7 RNA Polymerase, 1.5 μL of 2 μmol / L Cas13a protein, 0.4 μL of NTP BufferMix 25 mmol / L Ribonucleotide Solution Mix, 0.5 μL of 40000 U / mL RNase inhibitor, 2.4 μL of 80 nmol / L reporter RNA, 5 μL of 10 nmol / L crRNA, 3 μL of RAA amplification product, and make up to 25 μL with Dnase / Rnase-free water.

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