A composition for one-pot detection of melioidosis based on rpa-cas12a and its use

Abstract

The application provides a composition and a method for RPA-Cas12a-based leptospirosis nucleic acid one-pot detection. The detection method can simultaneously perform double RPA reaction and CRISPR reaction in a single reaction system, only needs a starting step of sample adding, performs reaction in a closed reaction tube, does not need any manual operation in the middle of the reaction, and does not need an additional reaction heat source and a detection light source.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of gene diagnosis, and particularly relates to a composition for one-pot detection of melioidosis nucleic acid based on RPA-Cas12a and application thereof. BACKGROUND

[0002] Melioidosis is a disease caused by Burkholderia pseudomallei (Bp) infection. At present, there is no effective treatment for BP infection, and there is no vaccine protection. Bp is easy to obtain, easy to spread, resistant to a wide range of drugs, and easy to relapse. The clinical manifestations of infection are "hidden" or "similar to a hundred diseases". If not treated in time after infection, once developed into organ abscess or even sepsis, the clinical mortality rate is as high as 10% to 60%. Therefore, early screening and rapid detection of melioidosis bacteria are extremely important.

[0003] Existing laboratory detection methods such as bacterial isolation and culture have long detection time and high biological safety requirements. Serological detection has poor specificity and low sensitivity due to high background of serum of epidemic population. Proteomics analysis has high requirements for sample concentration and purity. Molecular biology methods have high requirements for instrument conditions and personnel operation. The existing detection methods are not suitable for bedside detection or rapid detection in the field. Therefore, it is urgent to provide a rapid, sensitive, specific and simple detection method for melioidosis.

[0004] Recombinase Polymerase Amplification (RPA) can complete nucleic acid amplification at a reaction temperature of 37-42℃, and is the most likely nucleic acid amplification detection technology to replace traditional PCR. However, due to the reaction temperature and the fidelity of the polymerase, the specificity of the RPA amplification reaction is slightly lower than that of the qPCR reaction. Although the CRISPR / Cas12 nucleic acid detection technology has high specificity, its sensitivity is not sufficient to directly detect pathogenic nucleic acids, and it relies on the amplification of target sequence nucleic acids. The combination of the two technologies not only solves the problem of CRISPR / Cas12 relying on nucleic acid amplification, but also improves the specificity of detection by double checking the target sequence through two reactions. However, the conventional RPA-CRISPR combined nucleic acid detection technology needs to perform an RPA amplification reaction first, then open the cover to transfer the amplification product for CRISPR detection, and the transfer of the amplification product from the open cover is prone to aerosol pollution and potential bio-safety risks. In recent years, some one-step RPA-CRISPR nucleic acid detection methods based on RPA-CRISPR have physically isolated the RPA reaction system and the CRISPR reaction system through special custom reaction containers or non-water-soluble solvents, but in fact, the detection is still completed by two independent reactions. Although this detection method solves the problem of aerosol pollution, the operation is still cumbersome (centrifugation and mixing in the middle), special containers are needed, and the detection reaction time is still relatively long. SUMMARY

[0005] The present application provides a composition and method for one-pot Burkholderia pseudomallei nucleic acid detection based on RPA-Cas12a. The detection method can simultaneously perform double RPA reaction and CRISPR reaction in a single reaction system, only needs one-step sample addition, and performs reaction in a closed reaction tube without any manual operation in the middle of the reaction and without additional reaction heat source and detection light source.

[0006] The present application provides a composition for detecting Burkholderia pseudomallei, which comprises: a Burkholderia pseudomallei nucleic acid target sequence RPA primer, a target sequence crRNA, an LbCas12a protease, and an ssDNA fluorescent probe.

[0007] In an embodiment according to the present application, the nucleotide sequence of the target sequence RPA primer is SEQ ID NO: 1 and SEQ ID NO: 4; and the nucleotide sequence of the transcription template of the crRNA is SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0008] In an embodiment according to the present application, the nucleotide sequence of the ssDNA fluorescent probe is TTATT; preferably, the ssDNA fluorescent probe is labeled with a FAM fluorescent reporter group at the 5' end and a BHQ1 quenching group at the 3' end.

[0009] In an embodiment according to the application, the composition further comprises: RPA primers of the internal reference gene ACTB gene and / or GAPDH gene;

[0010] Preferably, when the internal reference gene is the ACTB gene, the primer pair is selected from the primer combinations shown in the following table:

[0011]

[0012] Preferably, when the internal reference gene is the ACTB gene, the nucleotide sequence of the upstream primer is SEQ ID NO: 17 and the nucleotide sequence of the downstream primer is SEQ ID NO: 18; preferably, when the internal reference gene is the GAPDH gene, the nucleotide sequence of the upstream primer is SEQ ID NO: 20 and the nucleotide sequence of the downstream primer is SEQ ID NO: 21 or SEQ ID NO: 22; preferably, the molar concentration ratio of the RPA primers of the nucleic acid target sequence of B. pseudomallei to the RPA primers of the internal reference gene (ACTB) is 1:1-4:1.

[0013] The application also provides the use of the above-mentioned composition in the preparation of a reagent or kit for detecting B. pseudomallei.

[0014] The application further provides a reagent or kit for detecting B. pseudomallei, characterized in that it comprises the above-mentioned composition.

[0015] In another aspect, the application provides a method for detecting B. pseudomallei, comprising:

[0016] 1) extracting genomic DNA from the sample to be tested; preferably, the sample is selected from a whole blood sample.

[0017] 2) sample addition reaction stage:

[0018] After mixing the genomic DNA with the composition as described above, RPA reaction and CRISPR / Cas12a reaction are carried out, and in the reaction with the nucleic acid sample to be tested, RPA-Cas12a reaction products are obtained, and fluorescence signals (ΔRn) are collected; preferably, the RPA reaction and the CRISPR / Cas12a reaction are carried out simultaneously.

[0019] In an embodiment according to the application, it further comprises 3) detecting the fluorescence signal (ΔRn) of the RPA-Cas12a reaction product obtained in step 2).

[0020] In an embodiment according to the present application, the detection result is judged by naked eye observation of the fluorescence of the RPA-Cas12a reaction product; preferably, if the reaction product can detect fluorescence, and the color of the HNB dye changes from dark blue to light blue or green by naked eye observation, it is judged that the Burkholderia pseudomallei in the sample contains Burkholderia pseudomallei; if the reaction product has no fluorescence detection, and the color of the HNB dye changes from dark blue to light blue or green by naked eye observation, it is judged that the sample does not contain Burkholderia pseudomallei or the concentration of the target sequence nucleic acid in the sample is lower than the minimum detection limit of the method; if the reaction product has no fluorescence production, and the color of the HNB dye has no change by naked eye observation, it is judged that the detection is invalid, and no effective sample is collected.

[0021] In an embodiment according to the present application, the method for detecting the fluorescence signal (ΔRn) is as follows a), b) or c)

[0022] a) placing the reaction product under LED blue light, and naked eye observing whether the reaction product has fluorescence production;

[0023] b) placing the reaction product under natural light, and naked eye observing whether the HNB in the reaction product has color change.

[0024] c) placing the reaction product in a fluorescence quantifier (ABI) to detect the fluorescence signal (ΔRn); if the fluorescence signal (ΔRn) is higher than 599724, it is judged that the sample contains Burkholderia pseudomallei; if the fluorescence signal (ΔRn) is lower than 599724, it is judged that the sample does not contain Burkholderia pseudomallei, or the concentration of Burkholderia pseudomallei contained in the sample is lower than the detection lower limit.

[0025] The beneficial effects of the above technical solutions of the present application are as follows:

[0026] The present application overcomes the possibility of contamination of RPA product transfer operation and improves the reaction efficiency, shortens the reaction time, and the integrated RPA-Cas12a reaction realizes double specific recognition of target sequence RPA primer and crRNA and double signal amplification, confirmation of negative samples by internal reference quality control sequence, stronger specificity and higher sensitivity, and further improvement of specificity and sensitivity of nucleic acid detection. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 One-pot nucleic acid detection visual detection flowchart of Burkholderia pseudomallei.

[0028] Figure 2 SDS-PAGE electrophoresis result map of purified LbCas12a protein; the left lane is the protein molecular weight marker, and the right lane is the purified LbCas12a protein.

[0029] Figure 3 LED blue light visual detection results of RPA-Cas12a one-pot reaction of different RPA primers combined with crRNA.

[0030] Figure 4 Agarose electrophoresis map of RPA primer screening for internal reference gene.

[0031] Figure 5 Fluorescent signal (ΔRn) and reaction time relationship diagram of RPA-Cas12a one-pot reaction of different concentrations of RPA primers combined with internal reference gene RPA primers for nucleic acid target sequence of Burkholderia pseudomallei.

[0032] Figure 6 Result diagram of sensitivity evaluation experiment of a Burkholderia pseudomallei nucleic acid one-pot detection system based on RPA-Cas12a.

[0033] Figure 7 Result diagram of specificity evaluation experiment of a Burkholderia pseudomallei nucleic acid one-pot detection system based on RPA-Cas12a.

[0034] Figure 8 ROC curve diagram of a Burkholderia pseudomallei nucleic acid one-pot detection system based on RPA-Cas12a.

[0035] Figure 9 Characteristic change diagram of color and fluorescence of Burkholderia pseudomallei RPA-Cas12a one-pot reaction product under various light sources. DETAILED DESCRIPTION

[0036] To make the technical problems, technical solutions and advantages to be solved by the present application clearer, the following will be described in detail in combination with the drawings and specific embodiments.

[0037] Unless otherwise specified, the reagents used in the following examples are conventional reagents in the art, which can be commercially available or prepared according to conventional methods in the art; the experimental methods and conditions used are conventional experimental methods and conditions in the art, which can be referred to relevant experimental manuals, known documents or manufacturer's instructions. In the quantitative test in the following examples, three repeated experiments were set, and the average value was taken. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0038] The Burkholderia pseudomallei BPC006 strain used in the following examples is the BPC006 strain described in the literature (Yao Fang, et al. 2012. First Genome Sequence of a Burkholderia pseudomallei Isolate in China, Strain BPC006, Obtained from a Melioidosis Patient in Hainan. Journal of Bacteriology. 194(23): 6604-6605.) and provided by the Laboratory of Clinical Microbiology and Immunology, Department of Pharmacal and Medical Laboratory Science, Army Medical University.

[0039] The Burkholderia pseudomallei clinical isolate BP-hs used in the following examples was provided by the Laboratory of Clinical Microbiology and Immunology, Department of Pharmacal and Medical Laboratory Science, Army Medical University.

[0040] The Burkholderia thailandensis (BT) used in the following examples is the ATCC700388 strain, which was provided by the Laboratory of Clinical Microbiology and Immunology, Department of Pharmacal and Medical Laboratory Science, Army Medical University.

[0041] The Burkholderia cepacia (BC) used in the following examples is the ATCC25416 strain, which was provided by the Laboratory of Clinical Microbiology and Immunology, Department of Pharmacal and Medical Laboratory Science, Army Medical University.

[0042] The Staphylococcus aureus used in the following examples is the ATCC25923 strain, which was provided by the Laboratory of Clinical Microbiology and Immunology, Department of Pharmacal and Medical Laboratory Science, Army Medical University.

[0043] The Acinetobacter baumannii used in the following examples is the ATCC19606 strain, which was provided by the Laboratory of Clinical Microbiology and Immunology, Department of Pharmacal and Medical Laboratory Science, Army Medical University.

[0044] The E. coli (BL21DE3) of LbCas12a expression plasmid (PUC57-ZB011) used in the following examples was purchased from Nanjing Mai Xike Biological Technology Co., Ltd.

[0045] Main reagents and consumables:

[0046] Quick DNA / RNA Pathogen miniprep Kit: ZYMO RESEARCH, Cat. No. R1042. Human DNA Quantitation Standard: NIST, Cat. No. SRM2372a. liquid basic kit: TwistDx, Cat. No. TALQBAS01. T7 RiboMAX TM Express Large Scale RNA Production System: Promega, Cat. No. P1320. RNA Clean & Concentrator Kits: ZYMO RESEARCH, Cat. No. R1015. DNase I Reaction Buffer: NEB, Cat. No. B0303S. NEB buffer: NEB, Cat. No. B7203S

[0047] Example 1, Establishment of a RPA-Cas12a-based Melioidosis nucleic acid one-pot detection system

[0048] 1. Selection of target sequence

[0049] Through analysis, the nucleotide sequence of 135926-136130 bp on Burkholderia pseudomallei BPC006 strain chromosome 2 (GenBank: CP003782.1) was selected as the target sequence for Burkholderia pseudomallei gene detection, as shown in SEQ ID NO: 10.

[0050] Burkholderia pseudomallei gene detection target sequence (205 bp):

[0051]

[0052] 2. Design of Burkholderia pseudomallei gene target sequence RPA primers and crRNA

[0053] For the selected target sequence (SEQ ID NO: 10), the present application designed a plurality of theoretically feasible candidate RPA primers and crRNA, and most of them were not effective after experimental verification. Some usable sequences are shown in Table 1 and Table 2.

[0054] Table 1 Candidate RPA primers

[0055]

[0056] Table 2 Transcription templates of candidate crRNA

[0057]

[0058] Note: In the transcription template sequence of crRNA, the underlined part is the anchor sequence combined with LbCas12a protein and the transcription promoter sequence, and the remaining part is the guide sequence, which is complementary to the target sequence RPA amplification product.

[0059] Universal reporter probe (ssDNA): 5'-FAM-TTATT-BHQ1-3'. FAM at the 5' end is a fluorescent reporter group, and BHQ1 at the 3' end is a quencher group.

[0060] The RPA primers in Table 1, the transcription template of crRNA in Table 2, and the universal reporter probe (ssDNA) described above were synthesized by Shanghai Generay Biotech Co., Ltd.

[0061] 3. Preparation of bacterial genomic DNA

[0062] Test bacteria: Burkholderia pseudomallei BPC006 strain, Burkholderia pseudomallei clinical isolate BP-hs, Burkholderia thailandensis, Burkholderia cepacia, Staphylococcus aureus, Acinetobacter baumannii.

[0063] Bp bacterial template: BPC006 strain was extracted with Quick DNA / RNA Pathogen miniprep Kit (Zymo, R1042) kit to extract genomic DNA.

[0064] Bp mock sample template: BPC006 strain was added to normal human blood samples, and genomic DNA was extracted with Quick DNA / RNA Pathogen miniprep Kit (Zymo, R1042) kit

[0065] Negative control sample: Any one of Burkholderia thailandensis, Burkholderia cepacia, Staphylococcus aureus, and Acinetobacter baumannii was added to normal human blood samples, and genomic DNA was extracted with Quick DNA / RNA Pathogen miniprep Kit (Zymo, R1042) kit.

[0066] 4. Expression and purification of LbCas12a protein

[0067] E. coli BL21 (DE3) containing LbCas12a expression plasmid (PUC57-ZB011) was inoculated in LB medium (final concentration of kanamycin was 50 μg / μL) for overnight culture, and then the bacterial liquid obtained by overnight culture was inoculated in 1 L of new LB medium at a volume ratio of 1:100, and cultured to OD600=0.5-0.6, then 400 μL of 0.5 M IPTG was added, and the culture was induced for 20 h. The bacterial body after induction culture was ultrasonically broken and centrifuged to obtain the supernatant. The LbCas12a protein in the supernatant was purified by Ni column (GE Healthcare), and after concentration using ultrafiltration tube, the protein was divided and stored in -80℃ refrigerator. The purified LbCas12a protein was detected by SDS-PAGE. The results are shown in Figure 2 Figure 2, and the LbCas12a protein band can be seen at 170 kD.

[0068] 5. Screening of RPA primer pair of X. oryzae gene target sequence and crRNA

[0069] 5.1 Preparation of crRNA

[0070] 1 μL of Bp-T6-crRNA1, Bp-T6-crRNA2, Bp-T6-crRNA3 transcription template (10 μM) and 1.5 μL of Bp-crRNA upstream transcription template (10 μM) were mixed to prepare the annealing reaction system. The annealing reaction system was placed in a PCR instrument, preheated at 95℃ for 2 min; then slowly cooled to 25℃ at a cooling rate of 0.1℃ / s. Linear DNA template was obtained.

[0071] T7 RiboMAX TMThe in vitro transcription reaction system was prepared according to the product instructions of Express Large Scale RNA Production System (Promega, P1320): Ribomax express T7 2x buffer 10 μL, linear DNA template 2.5 μL, Enzyme mix T7 express 2 μL, Nuclese-free water to 20 μL. The in vitro transcription reaction system was placed at 37°C for 30 min to obtain the in vitro transcription reaction solution. Then prepare the following mixture: NEB buffer (B7203S) 5 μL, DNase I 4 μL, in vitro transcription reaction solution 20 μL, DEPC-treated water to 100 μL. The mixture was placed at 37°C for 20-30 min, then 1 μL of 0.5M EDTA was added to the mixture, mixed and centrifuged, and then reacted at 75°C for 10 min. The crRNA in the mixture was purified using RNA Clean & Concentrator Kits (ZYMO RESEARCH, R1015) according to the product instructions to obtain purified Bp-T6-crRNA1, Bp-T6-crRNA2, and Bp-T6-crRNA3, which were quantified and stored at -80°C for later use.

[0072] 5.2 Screening of RPA primers and crRNA

[0073] Reaction solution A (17.15 μl): Bp-p1-F (40 μM) 0.345 μl, Bp-p2-R (40 μM) 0.345 μl, 2*Reaction buffer 12.5 μl, 10*Basic E-mix 2.5 μl, dNTPs (10 mM) 1.25 μl, HNB (6 mM) 0.5 μl, B solution (3.25 μl): ssDNA (100 μM) 2 μl, 20*Core Buffer 1.25 μl, C solution (1.9 μl): Bp-T6-crRNA1-3 (32.31 μM) 0.425, cas12a (9.32 μM) 1.475 μl. After preparing the ABC three Mix, take the corresponding volume, then take Mgo Ac280 mM 1.25 ul, ddH20 0.26 μl, Bp bacterial template 1 ul, add to the top cover of the two ends, do not mix before starting the reaction, cover the tube cap, shake the liquid in the tube on the cap and then centrifuge, avoid the generation of bubbles, 37°C reaction for 30 min, observe the fluorescence result under LED blue light.

[0074] The results are as follows Figure 3As shown, Bp-p1-crRNA1 represents the combination of Bp-p1-F / Bp-p1-R primer pair and Bp-T6-crRNA1; Bp-p2-crRNA2 represents the combination of Bp-p2-F / Bp-p2-R primer pair and Bp-T6-crRNA2; Bp-p2-crRNA3 represents the combination of Bp-p2-F / Bp-p2-R primer pair and Bp-T6-crRNA3; Bp-p1F2R-crRNA2 represents the combination of Bp-p1-F / Bp-p2-R primer pair and Bp-T6-crRNA2; Bp-p1F2R-crRNA3 represents the combination of Bp-p1-F / Bp-p2-R primer pair and Bp-T6-crRNA3. NC represents a negative control group without Bp nuclease template, and blank represents an experimental group using enzyme-free water instead of template. As can be seen from the results, only Bp-p1F2R-crRNA3 detects fluorescence, and NC and blank are both true, therefore, Bp-p1-F / Bp-p2-R is preferably used as the target gene primer for RPA-CRISPR one-pot method detection of nocardiosis, and Bp-T6-crRNA3 is used as the crRNA for RPA-CRISPR one-pot reaction.

[0075] 6. Optimization of RPA-Cas12a one-pot reaction system

[0076] 6.1 Primer and ssDNA concentration ratio

[0077] The RPA-Cas12a one-pot reaction system is configured as follows: Reaction solution A: Bp-p1-F (40 μM) 0.1-0.588 μl, Bp-p2-R (40 μM) 0.1-0.588 μl, 2*Reaction buffer 12.5 μl, 10*Basic E-mix 2.5 μl, dNTPs (10 mM) 1.25 μl; B liquid: ssDNA (100 μM) 0.5-2.5 μl, 20*Core Buffer 1.25 μl, C liquid (1.9 μl): crRNA (32.31 μM) 0.425, cas12a (9.32 μM) 1.475 μl. After the three Mixes A, B and C are configured, the corresponding volume is taken, and then MgoAc 280 mM 1.25 ul, Bp bacterial template 1 ul, and H20 are added to 25 μl. Do not mix before starting the reaction. After covering the tube cap, shake the liquid in the tube on the cap and then centrifuge momentarily to avoid the generation of air bubbles. React at 37°C for 30 min. Collect the fluorescence signal (ΔRn) by ABI fluorescence quantitative pcr instrument.

[0078] The results are shown in Table 3. When the Bp primer concentration is 0.55 μM and the ssDNA primer concentration is 8 μM, the reaction system has a fluorescence signal (ΔRn) which is significantly higher than that of other groups.

[0079] 6.2dNTP and ssDNA concentration ratio

[0080] The following RPA-Cas12a one-pot reaction system was configured: reaction solution A: Bp-p1-F (40 mM) 0.345 ml, Bp-p2-R (40 mM) 0.345 ml, 2*Reaction buffer 12.5 ml, 10*Basic E-mix 2.5 ml, dNTPs (10 mM) 1.25-3 ml; B solution: ssDNA (100 mM) 1-2 ml, 20*Core Buffer 1.25 ml, C solution (1.9 ml): crRNA (32.31 mM) 0.425, cas12a (9.32 mM) 1.475 ml. After the three Mixes A, B and C were configured, the corresponding volumes were taken, and then MgoAc280mM 1.25ul, Bp bacterial template 1ul, H20 were added to 25ul, which were added to the two ends of the top cover. The mixture was not mixed before the reaction started. After the tube cover was covered, the liquid in the tube was shaken on the cover and then centrifuged instantly to avoid the generation of bubbles. The reaction was carried out at 37°C for 20 min, and the fluorescence signal (ARn) was collected by ABI fluorescence quantitative pcr instrument.

[0081] The results are shown in Table 4. When the dNTP working solution concentration is 0.5 mM and the ssDNA working solution concentration is 8 mM, the reaction system has a fluorescence signal (ARn) which is significantly higher than that of other groups.

[0082] 6.3 crRNA and LbCas12a concentration ratio

[0083] The following RPA-Cas12a one-pot reaction system was configured: reaction solution A: Bp-p1-F (40 mM) 0.345 ml, Bp-p2-R (40 mM) 0.345 ml, 2*Reaction buffer 12.5 ml, 10*Basic E-mix 2.5 ml, dNTPs (10 mM) 1.25 ml; B solution: ssDNA (100 mM) 2 ml, 20*Core Buffer 1.25 ml, C solution (1.9 ml): crRNA (32.31 mM) 0.425, cas12a (18.64 mM) 0.74-1.475 ml. After the three Mixes A, B and C were configured, the corresponding volumes were taken, and then MgoAc280mM 1.25ul, Bp bacterial template 1ul, H20 were added to 25ul, which were added to the two ends of the top cover. The mixture was not mixed before the reaction started. After the tube cover was covered, the liquid in the tube was shaken on the cover and then centrifuged instantly to avoid the generation of bubbles. The reaction was carried out at 37°C for 20 min, and the fluorescence signal (ARn) was collected by ABI fluorescence quantitative pcr instrument.

[0084] The results are shown in Table 5. When the concentration ratio of crRNA and LbCas12a is 1:1, the reaction system has a fluorescence signal (ΔRn) and is significantly higher than that of the 1:2 group; based on the above analysis, the optimal reaction system has an LbCas12a protein concentration of 0.55 μM, a Bp-crRNA1 concentration of 0.55 μM, a primer concentration of 0.55 μM, and an ssDNA concentration of 8 μM (all of which are the concentrations in the reaction system).

[0085] Table 3Bp primer and ssDNA working solution concentration ratio optimization experiment results in the reaction system

[0086]

[0087] Note: The primer concentration and ssDNA concentration in the table are the final concentrations in the reaction system, and the concentration in the brackets is the mother liquor concentration.

[0088] Table 4 dNTP and ssDNA concentration optimization experiment results in the reaction system

[0089]

[0090] Table 5 crRNA and LbCas12a concentration ratio optimization experiment results in the reaction system

[0091] crRNA to LbCas12a ratio Fluorescent signal (ARn) 1：1 1755322 1：2 1127543

[0092] Note: The working solution concentration of LbCas12a and crRNA is 0.64 μM

[0093] 7. Selection of internal reference gene RPA primers

[0094] 7.1 Selection of target sequences

[0095] Through analysis, the present application selects human ACTB gene and GAPDH gene as the target sequences of the alternative internal reference genes of the "one-pot nucleic acid detection composition and method based on RPA-Cas12a", as shown in the ACTB target sequence SEQ ID NO: 11 and the GAPDH target sequence SEQ ID NO: 23.

[0096]

[0097]

[0098] 7.2 Internal reference gene RPA primer design and screening

[0099] For the selected target sequences (SEQ ID NO: 10) and (SEQ ID NO: 23), the present application designed a pair of theoretically feasible candidate RPA primers and crRNA, and most of them were not effective after experimental verification. Some usable sequences are shown in Table 6.

[0100] Table 6 ACTB primer candidate internal reference gene

[0101]

[0102] The internal reference gene primer (ACTB-RPA-1F, ACTB-RPA-1R, ACTB-RPA-2F, ACTB-RPA-2F-1, ACTB-RPA-2R, ACTB-RPA-3F, ACTB-RPA-3R, ACTB-RPA-3R-1, GAPDH-RPA-1F, GAPDH-RPA-1R, GAPDH-RPA-1R-1) solution with a concentration of 10 μM was prepared. After diluting the human blood genomic DNA solution 10 3 times as template DNA, ddH2O instead of template DNA as negative control, using liquid basic kit (TwistDx, TALQBAS01) to perform RPA amplification reaction according to the kit instructions. First, prepare the following 25 μL premix, A liquid (21.5 μL): 2x Reaction Buffer 12.5 μL, 10x Basic E-mix 2.5 μL, forward and reverse primers (10 μM) 1.38 μL each, dNTPs (10 mM) 1.25 μL, RNase free ddH2O 2.49 μL, B (1.25 μL) liquid: 20*Core Buffer 1.25 μL, then take MgoAc 280 mM 1.25 ul, Bp analog sample template 1 ul, add to the top cover of both ends, do not mix before reaction starts, cover the tube cap, shake the liquid in the tube on the cap and then centrifuge, avoid the generation of bubbles, 37°C reaction for 30 min, after the reaction is completed, the product is subjected to agarose gel electrophoresis to judge the amplification.

[0103] The results are shown in Figure 4 ACTB-RPA-2F-1 / ACTB-RPA-2R, ACTB-RPA-3F / ACTB-RPA-3R, ACTB-RPA-3F / ACTB-RPA-3R-1, GAPDH-RPA-1F / GAPDH-RPA-1R-1, respectively, amplified bands of 278 bp, 266 bp, 267 bp, 411 bp, respectively. Next, three pairs of RPA primers were used for RPA-Cas12a one-pot reaction.

[0104] The RPA-CRISPR one-pot reaction system was configured as follows: reaction solution A (17.67 μl): Bp-p1-F (60 μM) 0.23 μl, Bp-p2-R (60 μM) 0.23 μl, internal reference gene upstream primer (15 μM-60 μM) 0.23 μl, internal reference gene downstream primer (15 μM-60 μM) 0.23 μl, 2*Reaction buffer 12.5 μl, 10*Basic E-mix 2.5 μl, dNTPs (10 mM) 1.25 μl; HNB (6 mM) 0.5 μl, B solution (3.25 μl): ssDNA (100 μM) 2 μl, 20*CoreBuffer 1.25 μl, C solution (1.82 μl): crRNA (32.31 μM) 0.425, cas12a (9.32 μM) 1.475 μl. After the three Mixes A, B and C were configured, the corresponding volume was taken, and then MgoAc 280 mM 1.25 ul and Bp simulation sample template 1 ul were added to the two ends of the top cover. The mixture was not mixed before the reaction was started. After the tube cover was covered, the liquid in the tube was shaken on the cover and then centrifuged for a moment to avoid the generation of bubbles. The reaction was carried out at 37°C for 30 min.

[0105] The results are shown in Table 1. Figure 5 As shown in Table 1, the fluorescence signal (ΔRn) of the Bp-p1-F / Bp-p2-R primer pair with a concentration ratio of 4:1 was significantly higher than that of the 1:1 group, and the fluorescence signal (ΔRn) of the ACTB-RPA-3F / ACTB-RPA-3R group was significantly higher than that of the other groups. Therefore, the ACTB-RPA-3F / ACTB-RPA-3R group was selected as the preferred internal reference primer, and a double RPA reaction with the optimal primer concentration ratio (Bp-p1-F / Bp-p2-R: ACTB-RPA-3F / ACTB-RPA-3R = 4:1) was established.

[0106] Example 2 Sensitivity and specificity evaluation of a RPA-Cas12a-based melioidosis nucleic acid one-pot detection system

[0107] Plasmid copy number calculation: The sequence of SEQ ID NO: 10 was sent to Shanghai Biosciences to synthesize a PUC57 plasmid, and the plasmid copy number was calculated using the formula copies / μl = 6.02*10 23 *(ng / μl*10 -9 ) / (DNA length*660) to be 1.21*10 10 copies / μl

[0108] The negative control sample DNA prepared in Column 1 was mixed with plasmid 1:1, so that the copy number of plasmid in the detection sample was 1300 copies / μl, 130 copies / μl, 13 copies / μl, 1.3 copies / μl, while the normal blood genomic DNA was used as a negative control template, and the RPA-Cas12a one-pot method for detecting melioidosis nucleic acid detection system established in Example 1 was used for detection.

[0109] Table 7

[0110]

[0111] Figure 6 As shown in Table 7, the lowest detection line is 1.2 copies / μl, the average fluorescence signal (ΔRn) of the lowest detection limit is greater than 599724, and the detection rate of 20 repeated experiments is 100%, so the composition and method for detecting melioidosis nucleic acid one-pot method based on RPA-CRISPR provided by the present application have very high sensitivity for detecting Burkholderia pseudomallei.

[0112] Note: Figure 6 The abscissa is the reaction time (min) of the RPA-CRISPR one-pot method, and the ordinate is the fluorescence signal (ΔRn).

[0113] 1300 copies / μL represents the concentration of Burkholderia pseudomallei plasmid (Bp-T6SS plasmid) in the nucleic acid sample.

[0114] 2. Specificity evaluation

[0115] The normal blood was added with non-melioidosis bacterial genomic DNA as a negative control (NC), and the genomic DNA of Burkholderia pseudomallei BPC006 strain was used as a positive control. A one-pot method for detecting melioidosis nucleic acid based on RPA-CRISPR was established in Example 1, and the blood samples added with Staphylococcus aureus (Sau), Escherichia coli (E. coli), Thai Burkholderia (Bt), onion Burkholderia (Bc), Pseudomonas aeruginosa (Pae), Acinetobacter baumannii (Ab), and Fusobacterium nucleatum (Fn) genomic DNA were detected according to the RPA-CRISPR one-pot method for detecting melioidosis nucleic acid detection system established in Example 1.

[0116] The specificity of the RPA-CRISPR one-pot method detection system for Burkholderia pseudomallei was evaluated.

[0117] The results are as follows: Figure 7As shown, the abscissa is the RPA-CRISPR one-pot reaction time (min), and the ordinate is the fluorescence signal (ΔRn). The detection results of the negative control and the genomic DNA samples of Staphylococcus aureus (Sau), Escherichia coli (E. coli), Burkholderia thailandensis (Bt), Burkholderia cenocepacia (Bc), Pseudomonas aeruginosa (Pae), Acinetobacter baumannii (Ab), and Fusobacterium nucleatum (Fn) were all negative, and the detection result of the positive control was correct. It can be seen that the established RPA-CRISPR one-pot detection system has good specificity for Burkholderia pseudomallei (Bp), and other strains of Burkholderia and other bacteria such as Staphylococcus aureus and Acinetobacter baumannii do not interfere with the detection and analysis of Burkholderia pseudomallei (Bp).

[0118] Example 3, ROC curve verification of a RPA-CRISPR-based one-pot nucleic acid detection system for glanders

[0119] The human normal blood used in this experiment was a human normal blood sample provided by the Laboratory of the Department of Clinical Microbiology and Immunology, School of Pharmacy and Laboratory Medicine, Army Medical University. The Burkholderia pseudomallei liquid used in this experiment was the Burkholderia pseudomallei BPC006 strain.

[0120] First, the following samples were prepared:

[0121] Sample 1: 20 normal blood samples were taken, and genomic DNA was extracted respectively;

[0122] Sample 2: 20 normal blood samples were taken, and 1-2*10 2 CFU of Burkholderia pseudomallei liquid was added respectively, and the template DNA was extracted by the kit;

[0123] Sample 3: 20 normal blood samples were taken, and 1-2*10 3 CFU of Burkholderia pseudomallei liquid was added respectively, and the template DNA was extracted by the kit;

[0124] Sample 4: 20 normal blood samples were taken, and 1-2*10 4 CFU of Burkholderia pseudomallei liquid was added respectively, and the template DNA was extracted by the kit;

[0125] Sample 5: 5 samples of Helicobacter pylori, Pseudomonas aeruginosa, Acinetobacter baumannii, and Fusobacterium nucleatum, 10 samples of Burkholderia thailandensis, and 10 samples of Burkholderia cenocepacia were added, and the template DNA was extracted respectively.

[0126] Then, each sample was detected by the RPA-CRISPR-based one-pot nucleic acid detection system for glanders established in Example 1, and the obtained fluorescence signal (ΔRn) was input into the SPSS software to make a ROC curveFigure 8 ). The results of the fluorescence signal (ΔRn) of each sample detected by the ABI real-time fluorescence quantitative PCR instrument were analyzed by SPSS software as shown in Table 8. The AUC (area under ROC) was 0.923, and the maximum Youden index (correct index) was 0.8. The fluorescence signal (ΔRn) 599724 corresponding to the maximum Youden index was the positive judgment value (cutoff value).

[0127] Table 8

[0128]

[0129] Note: a. Assuming in the case of non-parametric; b. Null hypothesis: true area = 0.5.

[0130] Example 4, a composition and method for RPA-CRISPR-based melioidosis nucleic acid one-pot detection

[0131] 1. A composition and method for RPA-CRISPR-based melioidosis nucleic acid one-pot detection

[0132] The composition comprises melioidosis gene nucleic acid RPA primers, internal reference gene (ACTB) RPA primers, crRNA, ssDNA fluorescent probes, and reagents required for RPA-CRISPR reaction system;

[0133] The RPA primers are as follows:

[0134] Bp-p1-F: 5'-CTCACAGTTCCTTTCCCATATCCTTCTCTTC-3' (SEQ ID NO: 1), Bp-p2-R: 5'-GTTTCATAGATCGAATTTCCGAGTTCCGAC-3' (SEQ ID NO: 4);

[0135] The transcription template sequence of the crRNA is as follows:

[0136] Upstream template: 5'-TAATACGACTCACTATAGGT-3' (SEQ ID NO: 8);

[0137] Downstream template:

[0138] 5'-CAGCGGGGACGAATTGAGCGATCTACACTTAGTAGAAATTACCTATAGTGAGTCGTATTA-3' (SEQ ID NO: 7);

[0139] The ssDNA fluorescent probe is as follows:

[0140] 5'-FAM-TTATT-BHQ1-3';

[0141] Wherein the FAM and BHQ1 modified ssDNA fluorescent probe is used for naked eye judgment of whether Burkholderia pseudomallei exists in the target system under LED blue light. HNB in the reaction system is used for naked eye judgment of whether the internal reference gene in the negative sample is amplified under natural light.

[0142] 2. Composition and method for RPA-CRISPR-based one-pot detection of Burkholderia pseudomallei nucleic acid

[0143] The method for visualizing detection of Burkholderia pseudomallei using the composition comprises the following steps:

[0144] (1) Extracting template DNA of the sample to be tested (for example: whole blood sample);

[0145] (2) Performing RPA-CRISPR one-pot detection of Burkholderia pseudomallei nucleic acid

[0146] Preparation of reaction liquid A (17.67 μl): Bp-p1-F (60 μM) 0.23 μl, Bp-p2-R (60 μM) 0.23 μl, ACTB-RPA-3F (15 μM) 0.23 μl, ACTB-RPA-3F (15 μM) 0.23 μl, 2*Reaction buffer 12.5 μl, 10*BasicE-mix 2.5 μl, dNTPs (10 mM) 1.25 μl; HNB (6 mM) 0.5 μl, B liquid (3.25 μl): ssDNA (100 μM) 2 μl, 20*Core Buffer 1.25 μl, C liquid (1.9 μl): crRNA (32.31 μM) 0.425, cas12a (9.32 μM) 1.475 μl. After preparation of the three Mixes A, B and C, take the corresponding volume, then take MgoAc 280 mM 1.25 ul, add 1 ul of Bp simulated sample template to the two ends of the top cover, do not mix before starting the reaction, cover the tube cover, shake the liquid in the tube on the cover, and then centrifuge momentarily to avoid the generation of bubbles, and react at 37°C for 30 min

[0147] (3) placing the RPA-CRISPR product above under LED blue light to make naked eye judgment, or placing the RPA-CRISPR product in a real-time fluorescent quantitative PCR instrument to make detection; the RPA-CRISPR product produces fluorescence, and the color of the HNB dye is changed from dark blue to light blue (or green) by naked eye observation, indicating that the Burkholderia pseudomallei in the sample is detected; if no fluorescence is detected in the reaction product, and the color of the HNB dye is changed from dark blue to light blue (or green) by naked eye observation, it is determined that the Burkholderia pseudomallei is not contained in the sample (or the concentration of the target sequence nucleic acid in the sample is lower than the minimum detection limit of the method); if no fluorescence is produced in the enzyme digestion product, and the color of the HNB dye has no change by naked eye observation, it is determined that the detection is invalid, and no effective sample to be detected is collected; if the RPA-CRISPR product has fluorescence or fluorescence signal (ΔRn) greater than the positive judgment value (599724) by naked eye observation, it indicates that the Burkholderia pseudomallei is contained in the sample.

[0148] Figure 9 The typical results of placing the RPA-CRISPR reaction product under LED blue light, natural light and gel imaging system for naked eye judgment are shown, wherein the bp+ / IAC+(HNB) group contains Bp, blood genome and HNB dye, the Bp- / IAC+(HNB) group does not contain Bp, contains blood genome and HNB dye, the Bp- / IAC-(HNB) group does not contain Bp and blood genome, contains HNB dye, the bp+ / IAC+ group contains Bp, blood genome and does not contain HNB dye, and the bp- / IAC+ group does not contain Bp and contains blood genome. From the results, it can be seen that only the BP+ group can produce obvious fluorescence under LED blue light after reaction, and the gray scale of the gel imaging system is higher than that of other groups. The groups containing HBN dye are blue before reaction, and only the BP+ or IAC+ group is changed to green after reaction.

[0149] The above is the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A composition for detecting Burkholderia melioides, characterized in that, Includes: RPA primers for the target nucleic acid sequence of melioidosis, target sequence crRNA, LbCas12a protease, ssDNA fluorescent probe, and RPA primers for the internal reference gene; The nucleotide sequence of the upstream primer of the target sequence RPA primer is SEQ ID NO:1; the nucleotide sequence of the downstream primer of the target sequence RPA primer is SEQ ID NO:4; and the nucleotide sequence of the transcription template of the crRNA is SEQ ID NO:

7. The internal reference gene is the ACTB gene, the nucleotide sequence of the upstream primer of the internal reference gene RPA primer is SEQ ID NO:17, and the nucleotide sequence of the downstream primer of the internal reference gene RPA primer is SEQ ID NO:18; The molar ratio of the target sequence RPA primer to the internal reference gene RPA primer is 4:

1.

2. The composition according to claim 1, characterized in that, The nucleotide sequence of the ssDNA fluorescent probe is TTATT.

3. The composition according to claim 2, characterized in that, The ssDNA fluorescent probe is labeled with a FAM fluorescent reporter group at its 5' end and a BHQ1 quencher group at its 3' end.

4. Use of the composition according to any one of claims 1-3 in the preparation of a reagent or kit for detecting Burkholderia melioides.

5. A reagent or kit for detecting Burkholderia melioides, characterized in that, It comprises the composition as described in any one of claims 1-3.

6. A method for detecting Burkholderia melioides for non-diagnostic purposes, characterized in that, include: 1) Extract genomic DNA from the sample to be tested; 2) Sample addition reaction stage: The genomic DNA is mixed with the composition as described in any one of claims 1-3, and then subjected to an RPA reaction and a CRISPR / Cas12a reaction to obtain the RPA-Cas12a reaction product, and the fluorescence signal ΔRn is collected.

7. The method according to claim 6, characterized in that, The sample to be tested was selected from whole blood samples.

8. The method according to claim 6, characterized in that, The RPA reaction and the CRISPR / Cas12a reaction are carried out simultaneously.

9. The method as described in claim 6, characterized in that, The method also includes placing the reaction product in an ABI fluorescence quantitative analyzer to detect the fluorescence signal ΔRn; if ΔRn is higher than 599724, it is determined that the sample to be tested contains Burkholderia melioides; if ΔRn is lower than 599724, it is determined that the sample to be tested does not contain Burkholderia melioides or that the concentration of Burkholderia melioides in the sample to be tested is lower than the detection limit.

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