Detection method and kit for detecting double-target pathogens by fusing RAA-CRISPR / Cas13a

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a method and a kit for detecting double-target pathogens by fusing RAA-CRISPR / Cas13a. According to the present invention, the fusion RAA, the in vitro transcription and the CRISPR / Cas13a system are combined, such that the rapid and accurate double-target pathogen detection technology, namely the fusion RAA-CRISPR / Cas13a, is developed; for the technology, two important consideration factors exist: firstly, a T7 promoter must be added to the 5'end of FP1 of a fragment A; secondly, a recognition site of the crRNA needs to be in a fragment B; and under the condition that only the A gene exists, a crRNA targeting sequence is not generated. However, if only the B gene exists, transcription does not occur. Therefore, only when the A gene and the B gene exist at the same time, T7 transcription occurs, a transcript containing a crRNA targeting sequence is generated, and a fluorescence signal is generated.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and specifically relates to a dual-target pathogen detection method and a kit integrating RAA-CRISPR / Cas13a. Background Art

[0002] Infectious diseases are one of the most important factors threatening human life and health, and rapid and accurate detection of pathogens is crucial for the prevention and control of infectious diseases. At present, in addition to the single-target characteristics, more and more pathogens also show multi-target characteristics due to factors such as pathogen mutation and changes in pathogenicity. For example, many common pathogens, such as Staphylococcus aureus and Klebsiella pneumoniae, carry drug-resistant genes and develop resistance to antibiotics due to antibiotic abuse and genetic exchange mechanisms, leading to the emergence of drug-resistant bacteria; the presence of different virulence genes in certain microorganisms also leads to the emergence of different pathogenic pathogens. This poses a challenge to "rapid and accurate detection of pathogens". Nucleic acid amplification technology is the primary technology for rapid and accurate detection of pathogens. Depending on the number of targeted genes, nucleic acid amplification technology can achieve single-target pathogen detection and multi-target pathogen detection. At present, nucleic acid amplification technology has basically or completely been competent for rapid and accurate detection of single-target pathogens. However, for multi-target pathogen detection, due to the need to obtain multiple detection signals, nucleic acid amplification technology has problems such as complex operation, high hardware and software requirements, time-consuming, and missed / wrong detection. Therefore, developing a fast, accurate, and simple multi-target (mainly dual-target) pathogen detection technology is of great significance for the prevention and control of related diseases.

[0003] Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen. Due to its severe drug resistance and high mortality rate, it has become a difficult point in clinical anti-infection treatment. Rapid identification of MRSA is of great significance for controlling its spread and reducing its harm. The main MRSA detection methods include isolation culture identification method, immunoassay method and nucleic acid detection method. Isolation culture method and immunoassay method are complicated and time-consuming, and require high professional ability of experimental personnel. Nucleic acid amplification technology is currently the main method for identifying MRSA. However, in order to quickly and accurately identify MRSA, nucleic acid amplification technology needs to simultaneously amplify strain-specific genes (such as nuc gene) and drug-resistant genes (such as mecA gene), and the amplified products need to be indicated by different signals respectively, which reduces the operability of the method.

[0004] At present, the detection methods for multi-target pathogens including MRSA mainly include isolation and culture method, immunological detection method and multiple nucleic acid detection method. Isolation and culture method and immunological detection method are complicated to operate, time-consuming, have poor specificity and low sensitivity, and require high professional ability of experimental personnel, and cannot cope with the infection threat of a large number of pathogenic microorganisms facing humans. Multiple nucleic acid detection method relies on multiple nucleic acid amplification technology, such as multiple qPCR, to identify pathogens by reading the signals of multiple amplified targets. This method requires high skills of instruments and experimental personnel, is complex to operate, and takes a long time, which limits the application and popularization of this method. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a dual-target pathogen detection method and kit integrating RAA-CRISPR / Cas13a.

[0006] In a first aspect of the present invention, a dual-target pathogen detection method integrating RAA-CRISPR / Cas13a is provided, wherein the target pathogen has dual targets of an A gene fragment and a B gene fragment;

[0007] The method comprises the following steps:

[0008] (1) Lysing the sample to be tested to extract the genome and obtain a template;

[0009] (2) Amplifying the template by fusion RAA to obtain a fusion DNA product; the fusion RAA reaction system includes outer primers FP1 and RP2, and inner primers RP1 and FP2, FP1 and RP1 are designed for the A gene segment, and FP2 and RP2 are designed for the B gene segment, and the 5' end of FP1 carries a T7 promoter, and FP2 contains a 15nt sequence complementary to RP1;

[0010] (3) The fusion DNA product is transcribed in vitro by T7 polymerase and recognized and detected by CRISPR / Cas13a; the CRISPR / Cas13a reaction system includes crRNA, Cas13a, and a fluorescent reporter probe, and the B gene fragment contains a recognition site for crRNA;

[0011] (4) Detect fluorescence intensity.

[0012] The pathogen detection method of the present invention is for purposes other than disease diagnosis and treatment. In actual application, it can be used for the detection of food, clothing, environmental pathogens, and the like.

[0013] The target pathogen for detection may be methicillin-resistant Staphylococcus aureus. Specific genes (such as nuc gene) and drug-resistant genes (such as mecA gene) may be used as target genes.

[0014] The present invention designs two sets of primers for methicillin-resistant Staphylococcus aureus, and screens them to obtain the following primer sets with better detection effect: the sequence of FP1 is shown in SEQ ID NO.6, the sequence of FP2 is shown in SEQ ID NO.7, the sequence of RP1 is shown in SEQ ID NO.8, and the sequence of RP2 is shown in SEQ ID NO.9; in step (3), the sequence of crRNA is shown in SEQ ID NO.10.

[0015] Preferably, in step (2), the RAA fusion reaction time is 35 minutes.

[0016] Preferably, in step (3), the Cas13a cutting time is 15 minutes.

[0017] In a second aspect of the present invention, based on the principle of the above method, the present invention provides a dual-target pathogen detection kit fused with RAA-CRISPR / Cas13a, the kit comprising:

[0018] The fusion RAA reagent set includes outer primers FP1 and RP2, and inner primers RP1 and FP2. FP1 and RP1 are designed for the A gene segment, and FP2 and RP2 are designed for the B gene segment. The 5' end of FP1 carries a T7 promoter, and FP2 contains a 15nt sequence complementary to RP1.

[0019] T7 polymerase;

[0020] The CRISPR / Cas13a reagent set includes crRNA, Cas13a, and a fluorescent reporter probe, and the B gene fragment contains a recognition site for crRNA.

[0021] CRISPR / Cas13a is a highly specific RNA detection system, which consists of RNA-targeted ribonuclease Cas13a protein and CRISPR RNA (crRNA). By combining with a DNA amplification technology, a variety of rapid, sensitive and specific nucleic acid detection methods have been developed. The present invention combines fusion RAA, in vitro transcription and CRISPR / Cas13a system to develop a rapid and accurate dual-target pathogen detection technology-fusion RAA-CRISPR / Cas13a. There are two important considerations for this technology: first, the T7 promoter must be added to the 5' end of FP1 of fragment A; second, the recognition site of crRNA needs to be in fragment B. In the presence of only gene A, no crRNA targeting sequence is generated. But if only gene B is present, transcription will not occur. Therefore, only when gene A and gene B are present at the same time, T7 transcription will occur, producing transcripts containing crRNA targeting sequences, thereby generating fluorescent signals.

[0022] The novel dual-target pathogen detection platform developed by the present invention can solve the problems faced by MRSA detection, provide a new idea for rapid and accurate MRSA detection, and has important commercial value. Based on the concept of the present invention, it can also be applied to the detection of other pathogens with dual target genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0024] Figure 1 Schematic diagram of the principle of RAA-CRISPR / Cas13a fusion;

[0025] Figure 2 are two groups of corresponding target sequences;

[0026] Figure 3 Optimize the reaction time for RAA-CRISPR / Cas13a fusion;

[0027] Figure 4 For sensitivity exploration;

[0028] Figure 5 To explore the results for specificity;

[0029] Figure 6 The accuracy evaluation results. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0031] The present invention provides a dual-target pathogen detection method integrating RAA-CRISPR / Cas13a, comprising the following steps:

[0032] (1) Lysing the sample to be tested to extract the genome and obtain a template;

[0033] (2) Amplifying the template by fusion RAA to obtain a fusion DNA product; the fusion RAA reaction system includes outer primers FP1 and RP2, inner primers RP1 and FP2, the 5' end of FP1 carries a T7 promoter, and FP2 contains a 15nt sequence complementary to RP1;

[0034] (3) The fusion DNA product is transcribed in vitro by T7 polymerase and recognized and detected by CRISPR / Cas13a; the CRISPR / Cas13a reaction system includes crRNA, Cas13a, and a fluorescent reporter probe, and the B gene fragment contains a recognition site for crRNA;

[0035] (4) Detecting fluorescence intensity;

[0036] The target pathogen detected has A gene fragment and B gene fragment.

[0037] The principle of the above method to achieve detection is as follows: Figure 1 As shown, the fusion DNA product of gene A and gene B is obtained by fusing RAA, and then transcribed with T7 RNA polymerase. The resulting RNA is recognized by the Cas13a-crRNA complex, and finally the fluorescent signal is released by activating the side cutting ability of Cas13a. There are two important considerations for this technology: first, the T7 promoter must be added to the 5' end of FP1 of fragment A; second, the recognition site of crRNA needs to be in fragment B. In the presence of only gene A, no crRNA targeting sequence is generated. But if only gene B is present, transcription will not occur. Therefore, only when genes A and B are present at the same time, T7 transcription will occur, producing transcripts containing crRNA targeting sequences, thereby generating fluorescent signals.

[0038] The present invention applies the above method to the detection of methicillin-resistant Staphylococcus aureus (MRSA), performs fusion RAA amplification on its strain-specific gene (nuc gene) and drug-resistant gene (mecA gene) to form a fusion DNA product, and then performs transcription and CRISPR / Cas13a recognition detection. The following examples are all feasibility verification contents of the above method for MRSA detection.

[0039] The materials used in the following examples were prepared as follows:

[0040] (1) Primer and crRNA preparation

[0041] All primers used in the present invention were synthesized by Qingke Biotechnology Co., Ltd. Primer dry powder was dissolved with a certain volume of ultrapure water (ThermoFisher Scientific), diluted to 10 μM and stored at -20°C.

[0042] Preparation of crRNA: 2 μL T7 promoter primer (100 μM), 2 μL ssDNA template (100 μM), 2 μL 1×NEBuffer 2.1 and 8 μL nuclease-free water were mixed and heated to 95 ° C, and then slowly cooled to room temperature for annealing. The annealed product was transcribed in vitro using the T7 RNA transcription kit (New England Biolabs) and incubated overnight at 37 ° C. After incubation for 16 hours, 3 μL DNase I and 45 μL nuclease-free water were added, incubated at 37 ° C for 60 minutes, and then incubated at 65 ° C for 15 minutes to inactivate excess enzyme. Subsequently, crRNA was purified using an RNA concentration kit (Sangon Biotech). After purification, the concentration of crRNA was measured, diluted to 1 μM with ultrapure water, and stored in aliquots at -80 ° C until use.

[0043] (2) Bacterial genome preparation

[0044] The bacterial strains used in the present invention include MRSA (ATCC43300), methicillin-sensitive Staphylococcus aureus (MSSA; ATCC29213), methicillin-resistant Staphylococcus epidermidis (MRCoNS; ATCC35984), Salmonella (ATCC14028), methicillin-sensitive Staphylococcus epidermidis (clinical strain), Streptococcus agalactiae (ATCC12386), Klebsiella pneumoniae (ATCC13883), Vibrio vulnificus (ATCC27562), Pseudomonas aeruginosa (ATCC27853) and Escherichia coli (ATCC25922). The bacterial genome was extracted using a bacterial genomic DNA extraction kit (Takara Biomedical Technology) and stored at -20°C until use.

[0045] The 19 clinical isolates of Staphylococcus aureus included 14 MRSA and 5 MSSA. To explore the accuracy of the established method in clinical strain detection, a single colony was first inoculated into LB medium and cultured at 250rpm for 12 hours, and then the bacteria were washed three times with PBS at 3500rpm. The bacterial genome was extracted using the boiling lysis method: the washed bacteria were resuspended in 1mL of sterile water, boiled at 100℃ for 20 minutes, and then centrifuged at 12000g for 5 minutes, and the supernatant was collected. The resulting supernatant lysate was used as a template for the detection of MRSA by the technology of the present invention.

[0046] (3) Fusion RAA reactions were all performed using the RAA kit of Jiangsu Qitian Gene Biotechnology Co., Ltd., which includes enzyme components, specifically recombinase, single-stranded binding protein (SSB) and DNA polymerase. Recombinase is one of the core components, which is responsible for recognizing the specific sequence of double-stranded DNA and initiating the reaction; single-stranded binding protein can stabilize single-stranded DNA to prevent it from re-forming double strands, ensuring the smooth progress of the reaction; DNA polymerase is used to extend the DNA chain and synthesize a new DNA chain based on the template chain.

[0047] Example 1 Primer screening for MRSA detection:

[0048] In order to obtain better detection performance, two sets of primers and two crRNA sequences were designed in this embodiment, as shown in Table 1. In the first group, the mecA gene was selected as fragment A, the nuc gene was selected as fragment B, and the crRNA recognition target was located on the nuc gene. In contrast, in the second group, the nuc gene was selected as fragment A, the mecA gene was selected as fragment B, and the crRNA recognition target was located on the mecA gene. The target sequence information is shown in Table 2.

[0049] Table 1. Primers and crRNA sequences

[0050]

[0051]

[0052] Table 2. Target sequences corresponding to the two groups

[0053]

[0054] (1) In order to reduce the detection cost and at the same time not reduce the detection performance, the lyophilized powder in each test tube was dissolved and divided into two parts. The specific operation was as follows: 25 μL of buffer V and a certain volume of purified water were added to the test tube to dissolve the particles in the tube, and after mixing, the solution was equally divided into two new reaction tubes. Each reaction system included 2 μL of 10 μM external primers FP1 and RP2, 1 μL of 10 μM internal primers RP1 and FP2, and then according to the instructions, 2.5 μL of magnesium acetate I and a certain volume of the sample to be tested were added to form a total reaction system of 25 μL.

[0055] (2) After the RAA reaction, the amplified product was subjected to in vitro transcription and CRISPR / Cas13a, and the reaction time was 20 minutes.

[0056] LwaCas13a protein (Easy to Make) and crRNAs were diluted to 1 μM using 1×LwaCas13a protein dilution buffer and nuclease-free water, respectively. Fluorescent reporter probe (5'-FAM-UUUUUU-quencher-3') was diluted to 1 μM with nuclease-free water and stored at -80 °C until use. The Cas13a cleavage system included: 3 μL of 1 μM Cas13a, 3 μL of 10×Cas13a reaction buffer, 3 μL of 1 μM crRNA, 3 μL of 1 μM fluorescent reporter probe, 1 μL of T7 polymerase (NewEngland Biolabs), 2 μL of 20mM rNTPs (New England Biolabs), 1 μL of 40U / μL RNase inhibitor (Biyuntian Biotechnology), 3 μL of amplification product and 11 μL of nuclease-free water, constituting a total reaction system of 30 μL.

[0057] (3) Detect the fluorescence intensity using an enzyme-labeled instrument.

[0058] These two sets of primers were used to detect MRCoNS, MSSA and MRSA. MRCoNS and MSSA only contain mecA gene and nuc gene respectively, while MRSA contains both genes.

[0059] This example uses two sets of primers in Table 1 for testing. After 30 minutes of RAA fusion reaction, the purified RAA fusion product is subjected to 2% gel electrophoresis analysis, or the RAA fusion product is directly subjected to 20 minutes of in vitro transcription and CRISPR / Cas13a reaction. Finally, the best primer set and crRNA are selected based on the fluorescence intensity and signal-to-noise ratio.

[0060] Among all three samples, only the MRSA sample showed a fusion band of the expected size in both primer pairs ( Figure 2 a), indicating that fusion RAA can be used for MRSA detection. Next, the amplified product was transcribed into RNA and detected by CRISPR / Cas13a, and the fluorescent signal was only observed in the MRSA sample ( Figure 2 b), demonstrating that the fusion RAA-CRISPR / Cas13a can be used to detect MRSA. In addition, the technology showed a satisfactory signal-to-noise ratio in MRSA detection, with the signal-to-noise ratio of primer set 2 being higher ( Figure 2 c), this primer set was selected for subsequent experiments.

[0061] Example 2 Optimizing the fusion RAA reaction time:

[0062] In order to achieve the best detection performance of the fusion RAA-CRISPR / Cas13a system while shortening the detection time, this example tests the effect of the fusion RAA reaction time. The general steps are roughly the same as those in Example 1. The specific test steps that are mainly different in this example are as follows:

[0063] a. Place the fusion RAA reaction tube in a 37°C water bath and time for 40 minutes. At the same time, aspirate 3 μL as the RAA product at 0 minutes.

[0064] b. 3 μL RAA product was aspirated every 5 min. The set time gradient was: 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min and 40 min.

[0065] c. After the gradient RAA reaction, the products were subjected to in vitro transcription and CRISPR / Cas13a, and the reaction time was 30 minutes.

[0066] d. Detect the fluorescence intensity with an enzyme-labeled instrument.

[0067] This example tested 8 time points from 5 to 40 minutes and observed high concentrations of MRSA genomic DNA (1.5×10 7 The fluorescence signal of the high concentration (1.5×10 3 The fluorescence signal of 100 copies / μL reached a steady state at 35 minutes ( Figure 3 a). Therefore, we determined 35 minutes as the optimal reaction time for RAA fusion and used this time as the reaction time for RAA in the subsequent RAA-CRISPR / Cas13a fusion detection method.

[0068] Example 3 Optimization of Cas13a cutting time

[0069] In order to achieve the best detection performance of the fusion RAA-CRISPR / Cas13a system while shortening the detection time, this embodiment tests the effect of Cas13a cutting time. The general steps are roughly the same as those in Example 1. The specific test steps that are mainly different in this embodiment are as follows:

[0070] a. Carry out RAA reaction at the optimal RAA reaction time. After the reaction is completed, place it at -20℃ for later use.

[0071] b. The products were added to the in vitro transcription and CRISPR reaction systems respectively, and the reactions were carried out in a 37°C water bath for 0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min.

[0072] c. Collect the corresponding samples at the corresponding time, and finally use the ELISA reader to detect the fluorescence intensity.

[0073] The best performance was found at the 15-minute time point ( Figure 3 b).

[0074] Example 4 Sensitivity test:

[0075] In order to test the sensitivity of the fusion RAA-CRISPR / Cas13a system, this method detects different genomic DNA concentrations (1.5×10 0 Copies / μL to 1.5×10 5 The MRSA sample with a concentration of 100 copies / μL was tested using the above-mentioned optimal primer set and fusion RAA-CRISPR / Cas13a reaction time, and the general steps were roughly the same as those in Example 1.

[0076] The results showed that the platform can sensitively detect genomic DNA concentrations as low as 1.5×10 2 Copies / μL of MRSA sample ( Figure 4 ).

[0077] Example 5 Specificity Evaluation:

[0078] In order to achieve the specificity test of the fusion RAA-CRISPR / Cas13a system, the method in this embodiment detects MRSA, two strains with microbial characteristics similar to MRSA, and 7 common clinical bacteria. The above-mentioned optimal primer set and fusion RAA-CRISPR / Cas13a reaction time are used for testing, and the general steps are roughly the same as those in Example 1.

[0079] The data showed that only MRSA triggered a strong fluorescent signal ( Figure 5 ), indicating that the platform has high specificity in detecting MRSA.

[0080] Example 6 Accuracy Evaluation:

[0081] In order to test the accuracy of the fusion RAA-CRISPR / Cas13a system, the method in this embodiment detected 19 clinical isolates of Staphylococcus aureus, including 14 MRSA strains and 5 MSSA strains. The above-mentioned optimal primer set and fusion RAA-CRISPR / Cas13a reaction time were used for testing, and the general steps were roughly the same as those in Example 1.

[0082] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A dual-target pathogen detection method integrating RAA-CRISPR / Cas13a, which is for non-disease diagnosis and treatment purposes, characterized in that: The detection target pathogen has dual targets of A gene fragment and B gene fragment; The method comprises the following steps: (1) Lysing the sample to be tested to extract the genome and obtain a template; (2) Amplifying the template by fusion RAA to obtain a fusion DNA product; the fusion RAA reaction system includes outer primers FP1 and RP2, and inner primers RP1 and FP2, FP1 and RP1 are designed for the A gene segment, and FP2 and RP2 are designed for the B gene segment, and the 5' end of FP1 carries a T7 promoter, and FP2 contains a 15nt sequence complementary to RP1; (3) The fusion DNA product is transcribed in vitro by T7 polymerase and recognized and detected by CRISPR / Cas13a; the CRISPR / Cas13a reaction system includes crRNA, Cas13a, and a fluorescent reporter probe, and the B gene fragment contains a recognition site for crRNA; (4) Detect fluorescence intensity.

2. The dual-target pathogen detection method of fusion RAA-CRISPR / Cas13a according to claim 1, characterized in that: The detection target pathogen is methicillin-resistant Staphylococcus aureus.

3. The dual-target pathogen detection method of fusion RAA-CRISPR / Cas13a according to claim 2, characterized in that: In step (2), the sequence of FP1 is shown as SEQ ID NO.6, the sequence of FP2 is shown as SEQ ID NO.7, the sequence of RP1 is shown as SEQ ID NO.8, and the sequence of RP2 is shown as SEQ ID NO.9; in step (3), the sequence of crRNA is shown as SEQ ID NO.

10.

4. The dual-target pathogen detection method of fusion RAA-CRISPR / Cas13a according to claim 3, characterized in that: In step (2), the RAA fusion reaction time is 35 minutes.

5. The dual-target pathogen detection method of fusion RAA-CRISPR / Cas13a according to claim 1, characterized in that: In step (3), the Cas13a cutting time is 15 minutes.

6. A dual-target pathogen detection kit integrating RAA-CRISPR / Cas13a, characterized in that: The detection target pathogen has dual targets of A gene fragment and B gene fragment; The kit comprises, The fusion RAA reagent set includes outer primers FP1 and RP2, and inner primers RP1 and FP2. FP1 and RP1 are designed for the A gene segment, and FP2 and RP2 are designed for the B gene segment. The 5' end of FP1 carries a T7 promoter, and FP2 contains a 15nt sequence complementary to RP1. T7 polymerase; The CRISPR / Cas13a reagent set includes crRNA, Cas13a, and a fluorescent reporter probe, and the B gene fragment contains a recognition site for crRNA.

7. The fusion RAA-CRISPR / Cas13a dual-target pathogen detection kit according to claim 6, characterized in that: The detection target pathogen is methicillin-resistant Staphylococcus aureus.

8. The fusion RAA-CRISPR / Cas13a dual-target pathogen detection kit according to claim 7, characterized in that: In step (2), the sequence of FP1 is shown as SEQ ID NO.6, the sequence of FP2 is shown as SEQ ID NO.7, the sequence of RP1 is shown as SEQ ID NO.8, and the sequence of RP2 is shown as SEQ ID NO.9; in step (3), the sequence of crRNA is shown as SEQ ID NO.10.

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