Establishment and optimization method of mycoplasma pneumoniae detection system based on ERA-CRISPR / Cas12a

Abstract

The invention provides a method for establishing and optimizing a mycoplasma pneumoniae detection system based on ERA-CRISPR / Cas12a (Enhanced Reduced Receptor-CRISPR / CRISPR / Cas12a). The invention provides a method for detecting whether mycoplasma pneumoniae exists in a sample and a corresponding kit thereof. The method and the kit disclosed by the invention can be used for rapidly and economically detecting the mycoplasma pneumoniae with high sensitivity and high specificity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a method for the establishment and optimization of a Mycoplasma pneumoniae detection system based on ERA-CRISPR / Cas12a. Background Art

[0002] Mycoplasma pneumoniae (MP) was first identified by Eaton et al. in 1944 when the pathogen was isolated from sputum samples of patients with atypical pneumonia. MP presents as short and thin filamentous structures 2-5 μm in length, which are difficult to stain in Gram staining but appear light purple in Giemsa staining. MP infections originate from patients or carriers, and the pathogen is transmitted through their nasal, pharyngeal, laryngeal, and tracheal secretions, mainly through droplets or aerosols. This infection is contagious during both the incubation period and the treatment period. MP can cause various respiratory diseases and lead to a variety of extrapulmonary complications. Preschool children and adolescents with immature immune systems are particularly prone to clinical symptoms and are the main affected population by MP. Although MP infections are generally considered mild and self-limiting, their severity is often overlooked, and more than 10% of children hospitalized due to MP infections need to be admitted to the intensive care unit. The clinical symptoms of MP infections are diverse and often similar to those of other respiratory pathogens, making clinical diagnosis complex. Therefore, laboratory diagnosis is crucial for effective clinical treatment. Laboratory diagnostic methods for MP include isolation and culture, serological testing, antibody testing, and molecular biology techniques, etc. Although isolation and culture are considered the "gold standard" for diagnosing MP infections, the cultivation and morphological observation of MP are challenging, and the cultivation process is time-consuming. To achieve optimal serological testing for MP infections, it is usually necessary to collect at least two serum samples every two weeks, which is a time-consuming procedure. In addition, the limited immune responses observed in infants and the elderly population, as well as potential cross-reactions with infections such as cytomegalovirus or Epstein-Barr virus, pose significant challenges to the sensitivity and specificity of MP detection. Therefore, this method often shows a low detection rate and cannot meet the needs of rapid clinical diagnosis. Antibody detection methods, including enzyme-linked immunosorbent assay (ELISA), colloidal gold lateral flow chromatography, and fluorescent antibody testing, are usually simple and rapid but have low sensitivity. These methods may also produce high background levels of interfering antibodies in healthy individuals and result in non-specific reactions, thus producing false-positive results. Therefore, they are not suitable for early screening of MP but are more suitable for retrospective investigations of MP infections. Molecular biology techniques are the main methods for detecting MP nucleic acids, mainly including PCR technology and isothermal amplification techniques. However, due to the complex operation process, long detection cycle, and dependence on professional instruments and electricity, various PCR-based detection methods are not suitable for rapid detection and wide application in resource-limited areas. Isothermal amplification techniques cover a class of emerging molecular biotechnology, including rapid detection methods such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification technology (RPA). Compared with traditional nucleic acid detection techniques, these methods significantly shorten the reaction time and eliminate the need for large equipment, providing a promising solution for the rapid diagnosis of MP.

[0003] Enzymatic Recombinase Amplification (ERA) is a patented technology independently developed by GenDx Biotechnology Co., Ltd. in 2019. This technology is an enhanced version of RPA that utilizes engineered enzymes from different species to accelerate the reaction. The engineered DNA recombinase interacts with the primers to form a protein-DNA complex, which then localizes to the homologous sequence in double-stranded DNA and initiates DNA synthesis, thus initiating the exponential amplification of the target gene template. Due to the high specificity of the enzyme, the occurrence of mismatches is minimized, thereby reducing the possibility of detection errors. ERA exhibits high detection sensitivity and can amplify trace amounts of nucleic acid templates to detectable levels. At a constant temperature of 35 to 42 °C, specific genes can be amplified and qualitatively detected, and the results can be observed by methods such as agarose gel electrophoresis. In most cases, trace nucleic acid samples can be amplified to detectable levels within 15 minutes. The characteristics of this technology are high stability and high sensitivity, with a detection limit of 10 1 -10 2 copies per reaction. The entire process is simple, requiring no professional equipment, and the operation steps are convenient, without the need for professional skill training.

[0004] Clustered regularly interspaced short palindromic repeats (CRISPRs) are specific DNA sequences identified in prokaryotes. CRISPRs and their associated proteins (Cas proteins) play a key role in the adaptive immune systems of archaea and bacteria, providing defense against exogenous plasmids and phage invasive nucleic acids. In 2015, Feng Zhang and his team identified the type V Cas12a (also known as Cpf1) CRISPR / Cas system in Prevotella and Francisella. This protein is a single-RNA-guided CRISPR effector with DNA endonuclease activity, which may pave the way for new gene editing tools. In April 2018, Doudna and her team discovered that the Cas12 protein has a "trans cleavage" effect, thus developing the DETECTR system. Cas12a can recognize and bind to target DNA under the guidance of crRNA. When the Cas12 protein, crRNA, and target sequence bind to form a ternary complex, the complex exhibits significant "trans cleavage" activity. Using this property of CRISPR / Cas12a, a pathogen detection method has been developed.

[0005] Therefore, there is an urgent need in the art to develop a rapid, economical, highly sensitive, and highly specific MP detection method. Summary of the Invention

[0006] The object of the present invention is to provide a rapid, economical, highly sensitive, and highly specific MP detection method.

[0007] In the first aspect of the present invention, there is provided a method for detecting whether Mycoplasma pneumoniae exists in a sample, and the detection method includes:

[0008] (a) Providing a reaction system, the reaction system includes: a detection system, an amplification system, and a Cas12 activator, wherein the detection system, the amplification system, and the Cas12 activator are located in the same tube, the Cas12 activator is located in the tube cap, the detection system and the amplification system are located at the bottom of the tube, and the Cas12 activator contains a guide RNA and a nucleic acid probe, the detection system contains Cas12 protein, the amplification system contains a nucleic acid molecule of Mycoplasma pneumoniae to be detected from the sample and an amplification primer for isothermal amplification of the nucleic acid molecule of Mycoplasma pneumoniae to be detected, wherein the guide RNA guides the Cas12 protein to specifically bind to the nucleic acid molecule of Mycoplasma pneumoniae to be detected;

[0009] (b) At the bottom of the same tube, nucleic acid amplification of the nucleic acid molecule of Mycoplasma pneumoniae in the amplification system is carried out to obtain an amplification product of the nucleic acid molecule of Mycoplasma pneumoniae;

[0010] (c) In the same tube, the Cas12 activator in the tube cap is added to the detection system at the bottom of the tube and the amplification system containing the amplification product, and a detectable signal emitted by the nucleic acid probe is detected;

[0011] Wherein, if the nucleic acid probe is cleaved by the Cas12 protein, it indicates that Mycoplasma pneumoniae exists in the sample; if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that Mycoplasma pneumoniae does not exist in the sample.

[0012] In another preferred example, the detection includes: qualitative detection or quantitative detection.

[0013] In another preferred example, the detection includes a fluorescence detection method and a colloidal gold detection method.

[0014] In another preferred example, the fluorescence detection method is carried out using an enzyme-labeled instrument or a fluorescence spectrophotometer.

[0015] In another preferred example, the nucleic acid molecule of Mycoplasma pneumoniae to be detected is selected from the group consisting of: single-stranded DNA, double-stranded DNA, or a combination thereof.

[0016] In another preferred example, the nucleic acid molecule of Mycoplasma pneumoniae to be detected is naturally occurring DNA or artificially synthesized DNA.

[0017] In another preferred example, the nucleic acid molecule of the Mycoplasma pneumoniae to be detected is derived from a non-cultured sample or a sample obtained by a culturing method selected from the group consisting of: cell culture, bacterial culture, viral culture, fungal culture, microbial culture, organoid culture, in vivo enrichment culture in animals, and plant culture.

[0018] In another preferred example, the nucleic acid molecule of the Mycoplasma pneumoniae to be detected is derived from DNA extracted from a sample.

[0019] In another preferred example, the sample is an in vitro or ex vivo sample.

[0020] In another preferred example, the sample is a nucleic acid sample prepared from a sample selected from the group consisting of: throat swab, bronchoalveolar lavage fluid, and nasal swab.

[0021] In another preferred example, the length of the guide RNA is 55 - 60 nt, such as 59 nt.

[0022] In another preferred example, the guide RNA includes the sequences shown in SEQ ID NO.1 - 5.

[0023] In another preferred example, the final concentration of the guide RNA is 250 - 400 nM, preferably 300 - 350 nM, such as 312.5 nM.

[0024] In another preferred example, the nucleic acid probe is labeled with a detectable label.

[0025] In another preferred example, the detectable label includes a fluorophore and a quencher group.

[0026] In another preferred example, the fluorophore is selected from the group consisting of: FAM, biotin, HEX, Cy3, Cy5, Cy5.5, Cy7, ROX, VIC, JOE, TET, Texas Red, FITC, LC RED640, RB200, NED, Atto 425, Quasar 670, or a combination thereof.

[0027] In another preferred example, the quencher group is selected from the group consisting of: TAMARA, BHQ1, BHQ2, BHQ3, DABSYL, Dabcyl, eclipse, MGB, or a combination thereof.

[0028] In another preferred example, the fluorophore and the quencher group are each independently located at the 5'-end, 3'-end, and middle of the nucleic acid of the nucleic acid probe.

[0029] In another preferred example, the length of the nucleic acid probe is 5 - 50 nt, preferably 5 - 30 nt, more preferably 5 - 20 nt, even more preferably 5 - 15 nt.

[0030] In another preferred embodiment, the nucleic acid probe comprises single-stranded DNA or single-stranded RNA.

[0031] In another preferred embodiment, the nucleic acid probe comprises single-stranded DNA or single-stranded RNA with a detectable label.

[0032] In another preferred embodiment, the nucleic acid probe is single-stranded DNA or single-stranded RNA labeled with a fluorescent group and biotin.

[0033] In another preferred embodiment, the nucleic acid probe is single-stranded DNA or single-stranded RNA labeled with a fluorescent group and a quenching group.

[0034] In another preferred embodiment, when detected by fluorescence detection, the final concentration of the nucleic acid probe is 200-400 nM, preferably 250-350 nM, for example, 300 nM.

[0035] In another preferred embodiment, when detected by colloidal gold detection, the concentration of the nucleic acid probe is 20-60 nM, preferably 30-50 nM, more preferably 35-45 nM.

[0036] In another preferred embodiment, in step (b), an incubation step is further included before amplification.

[0037] In another preferred embodiment, the incubation time is 20-60 minutes, preferably 25-35 minutes, more preferably 25-30 minutes.

[0038] In another preferred embodiment, the detection system contains Cas12 protein and buffer.

[0039] In another preferred embodiment, the buffer includes HOLMES Buffer.

[0040] In another preferred embodiment, in the detection system, the final concentration of Cas12 protein is 100-250 nM, preferably 110-200 nM, preferably 120-150 nM, more preferably 125-150 nM.

[0041] In another preferred embodiment, the ratio of Cas12 protein to guide RNA is 1:1-3, preferably 1:2-2.5.

[0042] In another preferred embodiment, the Cas12 protein is selected from the group consisting of: Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas12l or a combination thereof.

[0043] In another preferred example, the Cas12a is selected from the following group: FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, CeCas12a, PrCas12a, CsbCas12a, BhCas12a, SsCas12a, Lb3Cas12a, BpCas12a, PdCas12a, BfCas12a, PcCas12a, cMtCas12a, PeCas12a, LiCas12a, Lb2Cas12a, PmCas12a, MbCas12a, EeCas12a, CsbCas12a, ErCas12a, ArCas12a, BsCas12a, AbCas12a or a combination thereof.

[0044] In another preferred example, the source of the Cas12a is selected from the following group: Leptotrichia, Listeria, Corynebacterium, Sutterella, Legionella, Treponema, Lineola, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospirillum, Sphaerochaeta, Gluconacetobacter, Neisseria, Rothia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter, Lachnospira, or a combination thereof.

[0045] In another preferred example, the source of the Cas12a is selected from the group consisting of: Francisella tularensis (FnCas12a), Acidaminococcus sp. BV3L6 (AsCas12a), Lachnospiraceae bacterium ND2006 (LbCas12a), Lachnospiraceae bacterium NC2008 (Lb5Cas12a), Helcococcus sp kunzii (HkCas12a), Oribacterium sp. NK2B42 (OsCas12a), Thiomicrospira sp. XS5 (TsCas12a), Bacteroidales bacterium KA00251 (BbCas12a), Bacteroidetes oral taxon 274 (BoCas12a), Lachnospiraceae bacterium MC2017 (Lb4Cas12a), Coprococcus eutactus (CeCas12a), Prevotella ruminicola strain BPI-34 (PrCas12a), Candidatus Saccharibacteria bacterium (CsbCas12a), Butyrivibrio hungatei strain MB2003 (BhCas12a), Smithella sp. SC_K08D17SC_K08D17) (SsCas12a), Lachnospiraceae bacterium MC2017 (Lb3Cas12a), Butyrivibrio proteoclasticus (BpCas12a), Prevotella disens (PdCas12a), Butyrivibrio fibrisolvens MD2001 (BfCas12a), Porphyromonas crevioricanis PcCas12a, Candidatus Methanoplasma termitum (CMtCas12a), Peregrinibacteria bacterium (PeCas12a), Leptospira inadai serovar Lyme (LiCas12a), Lachnospiraceae bacterium MA2020 (Lb2Cas12a), Porphyromonas macaca (PmCas12a), Moraxella bovoculi 237 (MbCas12a), Eubacterium eligens (EeCas12a), Candidatus Saccharibacteria bacterium (CsbCas12a), Eubacterium rectale (ErCas12a), Agathobacter rectalis strain (ArCas12a), Butyrivibrio sp. NC3005 (BsCas12a), Arcobacter butzleri (AbCas12a), or a combination thereof.

[0046] In another preferred example, the source of the Cas12b is selected from the group consisting of: Alicyclobacillus kakegawensis, Bacillus sp. V3-13, Bacillus hisashii, Lentisphaeria bacterium, Laceyella sediminis, or a combination thereof.

[0047] In another preferred example, the amplification primers include:

[0048] (P1) F1R1 primers for Mycoplasma pneumoniae P1 gene: The sequences are shown in SEQ ID NO: 6 and 7;

[0049] (P2) F2R2 primers for Mycoplasma pneumoniae P1 gene: The sequences are shown in SEQ ID NO: 8 and 9;

[0050] (P3) F3R3 primers for Mycoplasma pneumoniae P1 gene: The sequences are shown in SEQ ID NO: 10 and 11.

[0051] In another preferred example, the combination of the amplification primers and the guide RNA is selected from the following group:

[0052] (Z1) F1R1 primers for Mycoplasma pneumoniae P1 gene: The sequences are shown in SEQ ID NO. 6 and 7, and the guide RNA crRNA1, the sequence is shown in SEQ ID NO: 1;

[0053] (Z2) F2R2 primers for Mycoplasma pneumoniae P1 gene: The sequences are shown in SEQ ID NO: 8 and 9, and the guide RNA crRNA2-1, the sequence is shown in SEQ ID NO: 2;

[0054] (Z3) F2R2 primers for Mycoplasma pneumoniae P1 gene: The sequences are shown in SEQ ID NO: 8 and 9, and the guide RNA crRNA2-2, the sequence is shown in SEQ ID NO: 3;

[0055] (Z4) F3R3 primers for Mycoplasma pneumoniae P1 gene: The sequences are shown in SEQ ID NO: 10 and 11, and the guide RNA crRNA3-1, the sequence is shown in SEQ ID NO: 4.

[0056] In another preferred example, the concentration of the amplification primers is 200 - 300 nM, more preferably, 220 - 280 nM, even more preferably, 240 - 260 nM.

[0057] In another preferred example, the amplification system further includes an activator.

[0058] In another preferred example, the activator is from the activator in the ERA isothermal amplification kit of GenDx Biotech Co., Ltd.

[0059] In another preferred example, the isothermal amplification is carried out at 37 - 42 °C, preferably, 37 - 40 °C.

[0060] In another preferred example, the isothermal amplification is selected from the group consisting of: ERA (Enzyme Recombinase Amplification), RPA (Recombinase Polymerase Amplification), and RAA (Recombinase Mediated Isothermal Amplification of Nucleic Acids).

[0061] In another preferred example, the amplification system further comprises reagents for isothermal amplification.

[0062] In another preferred example, the reagents for isothermal amplification include:

[0063] (a1) DNA polymerase from the ERA Isothermal Amplification Kit of GenDx Biotech B.V. for amplifying the nucleic acid molecule of Mycoplasma pneumoniae to be detected;

[0064] (a2) Lyophilized microspheres for the amplification reaction (from the ERA Isothermal Amplification Kit of GenDx Biotech B.V.);

[0065] (a3) Activator MC for activating the amplification reaction (from the ERA Isothermal Amplification Kit of GenDx Biotech B.V.);

[0066] (a4) 6*Loading Buffer for the amplification reaction (from the ERA Isothermal Amplification Kit of GenDx Biotech B.V.).

[0067] In another preferred example, in step (b), the nucleic acid molecule of Mycoplasma pneumoniae in the amplification system is subjected to nucleic acid amplification at 37°C - 42°C, preferably 37 - 40°C.

[0068] In another preferred example, the concentration of the nucleic acid molecule of Mycoplasma pneumoniae to be detected in step (a) is 10 0 -10 7 , preferably 10 3 -10 7 copies / μL, more preferably 10 6 -10 7 copies / μL.

[0069] In another preferred example, in step (c), a centrifugation step is further included to make the Cas12a activator sink to the bottom of the tube.

[0070] In another preferred example, the method is an in vitro method.

[0071] In another preferred example, the method is a one-tube method.

[0072] In another preferred example, the method is non-diagnostic and non-therapeutic.

[0073] The second aspect of the present invention provides a kit for detecting Mycoplasma pneumoniae, the kit comprising:

[0074] (i) A first container and a detection system located within the first container, the detection system containing Cas12 protein;

[0075] (ii) A second container and an amplification system located within the second container, the amplification system containing amplification primers for amplifying nucleic acid molecules of Mycoplasma pneumoniae to be detected;

[0076] (iii) A third container and a Cas12 activator located within the third container, the Cas12 activator containing guide RNA and a nucleic acid probe;

[0077] And a label or an instruction manual.

[0078] In another preferred embodiment, in the detection system, the final concentration of Cas12 protein is 100 - 250 nM, preferably 110 - 200 nM, more preferably 120 - 150 nM, and even more preferably 125 - 150 nM.

[0079] In another preferred embodiment, the amplification primers include:

[0080] (P1) F1R1 primers of the P1 gene of Mycoplasma pneumoniae: the sequences are as shown in SEQ ID NO:6 and 7;

[0081] (P2) F2R2 primers of the P1 gene of Mycoplasma pneumoniae: the sequences are as shown in SEQ ID NO:8 and 9;

[0082] (P3) F3R3 primers of the P1 gene of Mycoplasma pneumoniae: the sequences are as shown in SEQ ID NO:10 and 11.

[0083] In another preferred embodiment, the combination of the amplification primers and the guide RNA is selected from the following groups:

[0084] (Z1) F1R1 primers of the P1 gene of Mycoplasma pneumoniae: the sequences are as shown in SEQ ID NO.6 and 7, and the guide RNA crRNA1, the sequence is as shown in SEQ ID NO:1;

[0085] (Z2) F2R2 primers of the P1 gene of Mycoplasma pneumoniae: the sequences are as shown in SEQ ID NO:8 and 9, and the guide RNA crRNA2-1, the sequence is as shown in SEQ ID NO:2;

[0086] (Z3) F2R2 primers of the P1 gene of Mycoplasma pneumoniae: the sequences are as shown in SEQ ID NO:8 and 9, and the guide RNA crRNA2-2, the sequence is as shown in SEQ ID NO:3;

[0087] (Z4) F3R3 primers for Mycoplasma pneumoniae P1 gene: The sequences are shown in SEQ ID NO: 10 and 11, and the guide RNA crRNA3-1 has a sequence shown in SEQ ID NO: 4.

[0088] In another preferred embodiment, the concentration of the amplification primers is 500 - 550 nM, more preferably 500 - 530 nM, even more preferably 500 - 510 nM.

[0089] In another preferred embodiment, the kit further comprises:

[0090] (iv) A fourth container and a nucleic acid molecule of Mycoplasma pneumoniae to be detected located within the fourth container.

[0091] In another preferred embodiment, the kit further comprises:

[0092] (v) A fifth container and reagents for isothermal amplification of the nucleic acid molecule of Mycoplasma pneumoniae to be detected located within the fifth container.

[0093] In another preferred embodiment, the kit further comprises:

[0094] (vi) A PCR tube.

[0095] In another preferred embodiment, any two, three (or all) of the first container, the second container, and the third container can be the same (identical) or different containers.

[0096] In another preferred embodiment, the components in the first container, the second container, and the third container are all located in the PCR tube, the Cas12 activator is located on the tube cap, and the detection system and the amplification system are located at the bottom of the tube.

[0097] In another preferred embodiment, any two (or all) of the fourth container and the fifth container can be the same (identical) or different containers.

[0098] In another preferred embodiment, two, more, or all of the first container to the fifth container can be the same (identical) container or different containers.

[0099] In another preferred embodiment, the components in the first container to the fifth container are all located in the PCR tube, the Cas12 activator is located on the tube cap, and the detection system and the amplification system are located at the bottom of the tube.

[0100] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0101] Figure 1 It shows that three target fragments are selected from the P1 gene and integrated into three different recombinant plasmids. This image shows the final positive recombinant plasmids used.

[0102] Figure 2 It shows the combination of 3 designed targets, 3 pairs of primers and 5 crRNAs. crRNA1 is used to recognize Target1, which is amplified by primer F1R1; crRNA2-1 and crRNA2-2 are used to recognize Target 2 and Target 3, which are amplified by primer F2R2; crRNA3-1 and crRNA3-2 are used to recognize Target 4 and Target 5, which are amplified by primer F3R3.

[0103] Figure 3 It shows the schematic diagram of the MP-ERA-Cas12a system for detecting MP. After extracting DNA from the sample, the target sequence is amplified by ERA for 20 to 30 minutes. Before the start of the amplification process, crRNA and ssDNA are added to the reaction tube cap. After the completion of ERA amplification, the LbCas12a-crRNA complex recognizes the amplification product, thus triggering "trans cleavage" and cleaving the ssDNA reporter probe within about 35 minutes. Subsequent analysis can be carried out by observing the fluorescence signal or using an LFA test strip. T: Test line, C: Control line.

[0104] Figure 4 It shows different sampling methods tried when establishing the MP-ERA-Cas12a one-tube method system. A: The detection system is added to the tube cap and the amplification system is added to the tube bottom. B: The Cas12a activator is added to the tube cap, and the detection system and the amplification system are added to the tube bottom. C: The detection system and the amplification system are added to the tube bottom. D: The amplification system is added to the tube bottom and separated from the detection system by 15% glycerol. E: The detection system is added to the tube bottom and separated from the amplification system by 15% glycerol.

[0105] Figure 5 It shows the fluorescence curves generated by detecting positive samples for each combination. NTC: No-template control.

[0106] Figure 6 It shows the fluorescence curves generated using different concentrations of F-Q and different Cas12a / crRNA ratios. A: Fluorescence curves generated using different concentrations of F-Q. B: Fluorescence values generated by different concentrations of F-Q at 50 minutes. C: Fluorescence curves generated using different Cas12a / crRNA ratios. D: Fluorescence values generated by different ratios of Cas12a / crRNA at 35 minutes. NTC: No-template control.

[0107] Figure 7 The fluorescence curves generated by five sample addition methods are shown. A: The unlabeled curve corresponds to methods C, D, and E. B: Comparison of the effects of the optimized one-tube system with other methods. NTC: No-template control.

[0108] Figure 8 The optimized MP-ERA-Cas12a LFA system is shown. A: The LFA system with different concentrations of F-B added. B: The LFA system after different incubation times.

[0109] Figure 9 The sensitivity analysis of the MP-ERA-Cas12a system is shown. A: The fluorescence curves generated by the two-tube system with different copy numbers of templates. B: The fluorescence curves generated by the two-tube system at 35 min with different copy numbers of templates. C: The fluorescence curves generated by the one-tube system with different copy numbers of templates. D: The fluorescence curves generated by the one-tube system at 35 min with different copy numbers of templates. E: The fluorescence curves generated by the LFA system with different copy numbers of templates. F: Specificity analysis of the MP-ERA-Cas12a fluorescence system, MP: Mycoplasma pneumoniae, UU: Ureaplasma urealyticum, CAL: Candida albicans, CTR: Candida tropicalis, AF: Aspergillus fumigatus, E.coli: Escherichia coli, SE: Salmonella enteritidis, SAU: Staphylococcus aureus, NTC: No-template control. G: Specificity analysis of the MP-ERA-Cas12a LFA system.

[0110] Figure 10 The results of detecting 34 known positive samples by the MP-ERA-Cas12a system are shown. +: Positive, -: Negative. Detailed implementation mode

[0111] Through extensive and in-depth research, the present inventors have developed for the first time a detection method for MP that is rapid, economical, highly sensitive, and highly specific. The present invention integrates the detection system, amplification system, and Cas12 activator in the same tube for the first time. The Cas12 activator is located on the tube cap, and the detection system and the amplification system are located at the bottom of the tube. The amplification and detection steps are integrated in one reaction container, effectively reducing the risk of contamination and false positives caused by handling multiple test tubes. Moreover, the present invention can be detected by fluorescence detection method and colloidal gold detection method. And the experimental results of the present invention show that the one-tube method of the present invention can generate fluorescence signals within 1 h, and the fluorescence signal intensity is about 1.6 times higher than that of the two-tube method, and the detection limit is 1 copy / μL. In addition, the colloidal gold detection method (LFA method) of the present invention can achieve rapid on-site screening. Within 5 min, visible bands will appear on the test strip, and the detection limit is 10 2copies / μL. All methods showed high specificity for MP. The MP-ERA-Cas12a detection system of the present invention has significant advantages, including fast processing speed, no need for complex instruments and easy operation, and is particularly suitable for clinical environments with limited resources. This system is an efficient tool for early diagnosis of MP and has important public health and clinical significance. The present invention is completed on this basis.

[0112] Term

[0113] The term "PCR" refers to the polymerase chain reaction technology, which is a technology suitable for amplifying target nucleic acids.

[0114] As used herein, the term "CRISPR" refers to Clustered regularly interspaced short palindromic repeats, which come from the immune system of microorganisms.

[0115] As used herein, "biotin" is also called vitamin H, which is a small molecule vitamin with a molecular weight of 244 Da. The extremely strong affinity of biotin can be used to amplify or enhance the detection signal in the detection system. For example, biotin can easily bind to proteins (such as antibodies) by covalent bonds, and the avidin molecule conjugated with an enzyme reacts with the biotin molecule conjugated with a specific antibody, which not only plays a multi-stage amplification role, but also colors due to the catalytic action of the enzyme when encountering the corresponding substrate, so as to achieve the purpose of detecting unknown antigen (or antibody) molecules.

[0116] CRISPR-Cas: A unique genomic element derived from bacteria and archaea, which acts as an adaptive immune defense system to resist invading phages or foreign nucleic acids. This system consists of Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (abbreviated as Cas proteins, Cas).

[0117] The term "Cas protein" refers to CRISPR-associated protein, which is a related protein in the CRISPR system.

[0118] The term "Cas12a" (formerly known as "Cpf1") refers to a crRNA-dependent endonuclease, which is an enzyme of type V-A in the CRISPR system classification.

[0119] The terms "Cas12b" and "C2c1" are used interchangeably and refer to an sgRNA-dependent endonuclease, which is an enzyme of type V-B in the CRISPR system classification.

[0120] The term "PAM" refers to the protospacer - adjacent motif, which is a short DNA sequence directly adjacent to the DNA sequence targeted by the CRISPR effector protein and is necessary for Cas12a or Cas12b to cleave double - stranded DNA. For example, the PAM of Cas12a is TTTV.

[0121] The term "target DNA or RNA molecule", when the molecule to be detected is a nucleic acid molecule, refers to the DNA or RNA to be detected or its specific part; when the molecule to be detected is a non - nucleic acid molecule, the target DNA or RNA molecule is a pre - designed nucleic acid sequence.

[0122] Cas protein

[0123] As used herein, the term "Cas protein" refers to CRISPR - associated proteins (some literature translates it as CRISPR - Cas effector protein, CRISPR / Cas effector protein, CRISPR - Cas effector, CRISPR / Cas effector), which can be type V Cas proteins or type VI Cas proteins. For type V Cas proteins, once they bind to the cis - cleavage substrate under the guidance of the guide RNA to form a ternary complex of Cas protein - guide RNA - cis - cleavage substrate, they can induce their trans - cleavage activity, that is, randomly cleave single - stranded nucleic acids and their equivalents (nucleic acid equivalents such as nucleic acid analogs).

[0124] The Cas protein described in this specific embodiment is a protein with trans - cleavage activity. In particular, it still has activity, especially trans - cleavage activity, at a temperature higher than the temperature of the system for the isothermal amplification reaction.

[0125] The Cas protein described in this specific embodiment can be a type V Cas protein; the Cas protein is selected from the following groups: type V - A Cas protein, type V - B Cas protein, type V - C Cas protein, type V - D Cas protein, type V - E Cas protein, type V - F Cas protein, type V - G Cas protein, type V - H Cas protein, type V - I Cas protein, type V - J Cas protein, type V - L, type V - M Cas protein or a combination thereof; the Cas protein described in this specific embodiment includes Cas12, such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12l, Cas12m or a combination thereof.

[0126] In a specific embodiment, the Cas protein referred to herein has trans-cleavage activity, such as Cas12, and also encompasses functional variants of the Cas protein or its homologs or orthologs. As used herein, a "functional variant" of a protein refers to a variant of such a protein that retains at least partially the trans-cleavage activity of the protein. Functional variants can include mutants (which can be insertion, deletion, or substitution mutants), including polymorphs, etc. Also included among functional variants are fusion products of such a protein with another nucleic acid, protein, polypeptide, or peptide that is not normally related. Functional variants can be naturally occurring or can be artificial. Advantageous embodiments can relate to engineered or non-naturally occurring type V DNA-targeting effector proteins.

[0127] In one embodiment, a type V Cas protein or its ortholog or homolog can comprise one or more mutations, and thus the nucleic acid molecule encoding it can have one or more mutations. The mutations can be artificially introduced mutations and can include, but are not limited to, one or more mutations in the catalytic domain.

[0128] In one embodiment, the type V Cas protein can be from: Leptotrichia, Listeria, Corynebacterium, Sutterella, Legionella, Treponema, Lineola, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospirillum, Sphaerochaeta, Gluconacetobacter, Neisseria, Rothia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter, Lachnospira, or a combination thereof.

[0129] Table I Type V family effector properties (from: doi:10.3389 / fcell.2020.622103)

[0130]

[0131]

[0132] a V represents A, C, and G.

[0133] b R represents A and G C B represents C, G, and T.

[0134] Guide RNA

[0135] As used herein, the "guide RNA" is a mature crRNA fused with a tracrRNA as the guide RNA, or a mature crRNA fused with a scoutRNA as the guide RNA, or the crRNA alone as the guide RNA.

[0136] Generally speaking, the guide RNA can contain direct repeat sequences (also known as DR sequences) and a guide sequence, or is substantially composed of or composed of direct repeat sequences and a guide sequence (also called a spacer in the context of the endogenous CRISPR system). In different CRISPR systems, depending on the Cas protein it depends on, the gRNA can include crRNA and tracrRNA, can also include crRNA and scoutRNA, or can only contain crRNA. crRNA and tracrRNA can be artificially modified and fused to form a single guide RNA (sgRNA). In some cases, the guide sequence is a polynucleotide sequence that has sufficient complementarity with the cis-cleavage substrate DNA to hybridize with the cis-cleavage substrate DNA and guide the specific binding of the CRISPR / Cas protein-guide RNA complex to the cis-cleavage substrate DNA, and usually has a sequence length of 15-28 nt. The aforementioned direct repeat sequence can fold into a specific structure (such as a stem-loop structure) for Cas protein recognition to form a complex. The guide sequence does not need to be 100% complementary to the cis-cleavage substrate DNA. The guide sequence is not complementary to the nucleic acid in the trans-cleavage reporter molecule.

[0137] In certain embodiments, when optimally aligned, the degree of complementarity (match degree) between the guide sequence and its corresponding cis-cleavage substrate DNA is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining the optimal alignment is within the ability of those of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs, such as but not limited to ClustalW, the Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0138] The terms "polynucleotide", "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule", and "nucleic acid" can be used interchangeably and include DNA, RNA, or their hybrids, and can be double-stranded or single-stranded.

[0139] The terms "homology" or "identity" are used to refer to the sequence match between two polypeptides or between two nucleic acids. When the same base or amino acid monomer subunit occupies a position in both of the two sequences being compared (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position between the two sequences. Usually, the comparison is made by aligning the two sequences to yield maximum identity. Such alignments can be performed using, for example, the identity of amino acid sequences can be determined by conventional methods, referring to, for example, the teachings of Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:244; Thompson et al., 1994, Nucleic Acids Res 22:467380, etc., by computerized algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group). The BLAST algorithm, available from the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov / ), can also be used with default parameters to determine it.

[0140] Target nucleic acid molecule (nucleic acid molecule of Mycoplasma pneumoniae to be detected)

[0141] As used herein, when the nucleic acid molecule is to be detected, the "target nucleic acid molecule" refers to a polynucleotide molecule extracted from a biological sample (sample to be tested) or its amplification product, transcription product, or reverse transcription product. When the non-nucleic acid molecule is to be detected, the "target nucleic acid molecule" is a nucleic acid sequence designed in advance. The biological sample is any solid or fluid sample obtained, excreted, or secreted from any organism, including but not limited to single-celled organisms such as bacteria, yeast, protozoa, and amoeba, etc., and multicellular organisms (such as plants or animals, including samples from healthy or seemingly healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, such as infections by pathogenic microorganisms such as pathogenic bacteria or viruses). For example, the biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any body secretion, exudate, effusion (e.g., fluid obtained from an abscess or any other infected or inflamed site), or a fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab of the skin or mucosal surface. The sample can also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens such as tumor biopsies) or can contain cells (primary cells or cultured cells) or a culture medium conditioned by any cells, tissues, or organs. Exemplary samples include but are not limited to cells, cell lysates, blood smears, cytocentrifuge preparations, cytology smears, body fluids (such as blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (such as tumor biopsies), fine needle aspirates, and / or tissue sections (such as cryostat tissue sections and / or paraffin-embedded tissue sections).

[0142] In other embodiments, the biological sample can be plant cells, callus, tissues, or organs (such as roots, stems, leaves, flowers, seeds, fruits), etc.

[0143] In the present invention, the target nucleic acid molecule includes a DNA molecule, and also includes an RNA molecule or a DNA molecule formed by reverse transcription of RNA. Further, the target nucleic acid molecule is amplified by a technique well-known in the art, and the amplification technique is an isothermal amplification technique. The isothermal amplification can be ERA, RPA, or RAA.

[0144] In the present invention, the target nucleic acid molecule is amplified using isothermal amplification reagents, and the isothermal amplification reagents include one or more selected from the following groups of the ERA isothermal amplification kit from GenDx Biotech Co., Ltd.: (a1) an enzyme-engineered DNA polymerase for amplifying the nucleic acid molecule of Mycoplasma pneumoniae to be detected; (a2) freeze-dried microspheres for the amplification reaction; (a3) activator MC for activating the amplification reaction; (a4) 6*Loading Buffer for the amplification reaction.

[0145] A method for amplification and detection in a single tube without opening the lid

[0146] This specific embodiment discloses a detection method for target nucleic acid molecules (nucleic acid molecules of Mycoplasma pneumoniae to be detected) that are amplified and detected in a single tube without opening the lid.

[0147] In a preferred embodiment, this specific embodiment provides a method for detecting whether Mycoplasma pneumoniae is present in a sample, and the detection method includes:

[0148] (a) Providing a reaction system, the reaction system includes: a detection system, an amplification system, and a Cas12 activator, wherein the detection system, the amplification system, and the Cas12 activator are located in the same tube, the Cas12 activator is located on the tube lid, the detection system and the amplification system are located at the bottom of the tube, and the Cas12 activator contains a guide RNA and a nucleic acid probe, the detection system contains Cas12 protein, the amplification system contains the nucleic acid molecule of Mycoplasma pneumoniae to be detected from the sample and amplification primers for isothermal amplification of the nucleic acid molecule of Mycoplasma pneumoniae to be detected, wherein the guide RNA guides the Cas12 protein to specifically bind to the nucleic acid molecule of Mycoplasma pneumoniae to be detected;

[0149] (b) At the bottom of the same tube, nucleic acid amplification is performed on the nucleic acid molecule of Mycoplasma pneumoniae in the amplification system to obtain an amplification product of the nucleic acid molecule of Mycoplasma pneumoniae;

[0150] (c) In the same tube, the Cas12 activator on the tube lid is added to the detection system at the bottom of the tube and the amplification system containing the amplification product, and a detectable signal emitted by the nucleic acid probe is detected;

[0151] Wherein, if the nucleic acid probe is cleaved by the Cas12 protein, it indicates that Mycoplasma pneumoniae is present in the sample; if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that Mycoplasma pneumoniae is not present in the sample.

[0152] In a preferred embodiment, the present invention combines the ERA isothermal amplification technology with the CRISPR / Cas12a system, develops and evaluates a novel method for rapid detection of MP, called the MP-ERA-Cas12a system. By utilizing the "trans-cleavage" effect generated after the target gene is recognized and cleaved by the CRISPR / Cas12a system, the present invention introduces two types of reporter probes (fluorescent probe and LFA probe), and accordingly establishes the MP-ERA-Cas12a fluorescence system and the LFA test strip system. In addition, the present invention develops a one-tube detection method, which separates the ERA amplification from the CRISPR detection without opening the reaction tube for liquid transfer. This design not only improves the sensitivity at 37°C - 42°C, but also simplifies the operation process, significantly reducing the possibility of false positive results caused by aerosol contamination, thus ensuring the accuracy of the detection results.

[0153] In the present invention, representative nucleic acid probes are single-stranded DNA or single-stranded RNA with a luminescent group and a quenching group attached to both ends respectively, and single-stranded DNA or single-stranded RNA with a luminescent group and a biotin attached to both ends respectively. Therefore, once the probe is cleaved, the luminescent group can emit light or form a band on the T line.

[0154] In this specific embodiment, it can be known whether the test system contains target nucleic acid molecules, such as nucleic acid molecules of Mycoplasma pneumoniae, by detecting fluorescence.

[0155] In this specific embodiment, the presence or absence of the T line band determines whether Cas12 is activated. By observing the presence or absence of the T line band, it can be known whether the test system contains target nucleic acid molecules, such as nucleic acid molecules of Mycoplasma pneumoniae.

[0156] In this specific embodiment, a suitable Cas protein is a type V Cas protein with trans-cleavage activity, preferably Cas12a or Cas12b. More preferably, the Cas12a is preferably FnCas12a, LbCas12a, ErCas12a, Evcas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, CeCas12a, PrCas12a, CsbCas12a, BhCas12a, SsCas12a, Lb3Cas12a, BpCas12a, PdCas12a, BfCas12a, PcCas12a, cMtCas12a, PeCas12a, LiCas12a, Lb2Cas12a, PmCas12a, MbCas12a, EeCas12a, CsbCas12a, ArCas12a, BsCas12a, AbCas12a, AsCas12a, or a combination thereof.

[0157] The method of this specific embodiment can be used to quickly detect whether a sample contains a target nucleic acid molecule (Mycoplasma pneumoniae). In addition, by combining with isothermal amplification techniques (such as any one of ERA, RPA, and RAA), the sensitivity of this detection method can be greatly improved. Various isothermal amplification techniques in the prior art can theoretically be used in the present invention, and only preferred embodiments are listed in this specific embodiment. Components used in various amplification techniques in this application, such as:

[0158] NTP, buffer, Mg required for RNA amplification 2+ etc., and RNase H required when the reverse transcriptase has no function of digesting single-stranded RNA;

[0159] dNTP, buffer, Mg required for DNA amplification 2+ etc.;

[0160] These are common general knowledge in the art, so they are not specifically described in this application.

[0161] The main advantages of the present invention include:

[0162] (1) The present invention has for the first time developed a rapid, economical, highly sensitive and highly specific detection method for Mycoplasma pneumoniae (MP). For the first time, the present invention places the detection system, the amplification system and the Cas12 activator in the same tube, with the Cas12 activator located on the tube cap and the detection system and the amplification system located at the bottom of the tube. By integrating the amplification and detection steps in one reaction container, it effectively reduces the risk of contamination and false positives caused by handling multiple test tubes. Moreover, the present invention can be detected by fluorescence detection method and colloidal gold detection method. And the experimental results of the present invention show that the one-tube method of the present invention can generate fluorescence signals within 1 h, and the fluorescence signal intensity is about 1.6 times higher than that of the two-tube method, with a detection limit of 1 copy / μL. In addition, the colloidal gold detection method (LFA method) of the present invention can achieve rapid on-site screening. Within 5 min, visible bands will appear on the test strip, with a detection limit of 10 2 copies / μL. All methods show high specificity for MP. The MP-ERA-Cas12a detection system of the present invention has significant advantages, including fast processing speed, no need for complex instruments and simple operation, and is particularly suitable for clinical environments with limited resources. This system is an efficient tool for the early diagnosis of MP and has important public health and clinical significance.

[0163] (2) The present invention combines the ERA isothermal amplification technology with the CRISPR / Cas12a system to develop and evaluate a new rapid detection method for MP, called the MP-ERA-Cas12a system. Utilizing the "trans-cleavage" effect generated after the target gene is recognized and cleaved by the CRISPR / Cas12a system, we introduced two types of reporter probes (fluorescent probes and LFA probes) and accordingly established the MP-ERA-Cas12a fluorescence system and the LFA test strip system. In addition, we developed a one-tube detection method that separates ERA amplification from CRISPR detection without opening the reaction tube for liquid transfer. This design not only improves sensitivity at 37 °C - 42 °C, but also simplifies the operation process and significantly reduces the possibility of false positive results caused by aerosol contamination, thus ensuring the accuracy of the detection results. Our goal is to create a rapid, economical, highly sensitive and highly specific MP detection method.

[0164] (3) The present invention has established a one-tube detection system with higher sensitivity, which combines ERA amplification and Cas12a detection, obtaining a higher fluorescence value than the traditional two-tube system. This method helps to quickly detect MP in one tube without the need for additional primer or crRNA design, nor physical isolation methods. We simply added the Cas12a activator containing crRNA and F-Q to the tube cap, and the remaining components were mixed at the bottom of the tube. This method effectively solves the possible contamination problem during the secondary lid-opening process. The MP-ERA-Cas12a system exhibits high sensitivity, with a detection limit of LOD for MP as low as 1 copy / μL. When used in conjunction with the LFA strip, the sensitivity can reach 10 2 copies / μL, thus overcoming the limitations of large instruments and improving the accessibility of detection in remote areas. In addition, the MP-ERA-Cas12a system has been proven effective in clinical sample testing.

[0165] (4) The present invention has successfully combined the specific detection ability of ERA and CRISPR / Cas12a. Based on the previously established two-tube system, a one-tube system and an LFA system were developed for MP detection. During the reaction process of the MP-ERA-Cas12a system, there is no need to open the lid, thus reducing aerosol contamination and minimizing the risk of false positives. In addition, the method produces results within 1 h. The detection sensitivity of this system is 1 copy / μL, while the detection sensitivity of the LFA is 10 2 copies / μL, without the need to use advanced instruments or equipment. The system exhibits strong specificity and is not affected by other pathogens. As a new method for MP detection, the MP-ERA-Cas12a technology has important practical application prospects.

[0166] The following will further elaborate on the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, they are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0167] The experimental materials involved in the present invention can be obtained from commercial channels without special instructions.

[0168] 1. Materials and Methods

[0169] 1.1 Materials and Reagents

[0170] The recombinant plasmids, primers, and crRNAs used in this study were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences of the primers and crRNAs are shown in Table 1. The enzyme recombinase isothermal amplification kit was purchased from GenDx Biotech Co., Ltd. (Suzhou, China). The materials required for CRISPR / Cas12a-based detection were all purchased from Tolo Biotech Co., Ltd. (Shanghai, China), including: Cas12a High-yield crRNA Synthesis and Purification Kit, LbCas12a (Cpf1) nuclease, 10×HOLMES Buffer, HOLMES ssDNA Reporter Gene (FAM), CRISPR-LFA ssDNA Reporter Gene, and CRISPR Single System Detection Strip (FAM / FITC).

[0171] Table 1 Inserted Fragments in Primers, crRNAs, and Recombinant Plasmids

[0172]

[0173]

[0174] * indicates the finally used primers / crRNAs.

[0175] 1.2 Strains and Clinical Samples

[0176] The strains used in this study (obtained from Huaibei People's Hospital) are shown in Table 2. 34 nasopharyngeal swabs were collected from 34 patients diagnosed with MP infection in Huaibei People's Hospital for clinical feasibility evaluation. Nucleic acids were extracted from these clinical samples using the GenePure 96 system.

[0177] Table 2 Strains Used in This Study

[0178]

[0179]

[0180] a P: Positive, N: Negative

[0181] 1.3 Construction of Recombinant Plasmids

[0182] According to the sequence of the MP conserved gene P1 (NCBI accession number: PP908509.1), three segments of sequences (positions 725 - 1325 of the P1 sequence, positions 1698 - 2152 of the P1 sequence, positions 3778 - 4238 of the P1 sequence) were selected and cloned onto the pUC19 vector (purchased from Sangon Biotech, Shanghai) to construct recombinant plasmids. The schematic diagram of the structure of the finally used recombinant plasmid is as Figure 1As shown. The nucleic acid concentration of the synthetic plasmid was measured using an ultraviolet spectrophotometer, and the concentration was converted to the copy number. The calculation formula is: C (copy number / μL) = [6.02×10 23 ×C (ng / μL)×10 -9 / [DNA length (bp)×660]. Then the DNA sample was resuspended in ddH 2 O and stored at -20 °C for future use.

[0183] 1.4 Design of isothermal amplification primers and crRNA

[0184] According to the ERA primer design principle, using the highly conserved MP P1 gene as the target, ERA primers were designed using Primer Premier 5.0 software. The specificity of the primers was evaluated by BLAST analysis. Using the PAM site (TTTV) of Cas12a, the CRISPR RGEN tool was used to determine the target position. The finally obtained crRNA sequence was 5'-AAUUUCUACUAAGUGUAGAU NN NNNNNNNNNNNNNNNNNN -3', where the underlined part represents the specific target sequence complementary to the spacer region. The positions of the ERA primers and crRNA are as Figure 2 shown, and the schematic diagram of MP-ERA-Cas12a is as Figure 3 shown.

[0185] 1.5 Screening of the best primer and crRNA combination

[0186] In the initial stage of the study, the best primer and crRNA combination was screened. The best combination varies for different experimental systems. Therefore, before conducting subsequent experiments, it is necessary to try and determine the best combination. Throughout the experiment, the environmental conditions and dosages of each combination were kept consistent to ensure that any differences in the results were only attributed to differences in the combination efficacy. For different target sequences, different primer pairs were designed, and the synthesized upstream and downstream candidate primers (F1, F2, F3, R1, R2, R3) were paired to form F1R1, F2R2, and F3R3 primer pairs (the sequences are shown in Table 1 respectively). Considering that different crRNAs have different cleavage efficiencies for different target sequences, we synthesized 5 crRNAs with high expected cleavage efficiencies. The combined primers and crRNAs were introduced into the MP-ERA-Cas12a fluorescence reaction system for detection (F1R1-crRNA1, F2R2-crRNA2-1, F2R2-crRNA2-2, F3R3-crRNA3-1, F3R3-crRNA3-2, the sequences are shown in Table 1 respectively). The template concentration was set at 10 7copies / μL, with ddH2O as the negative control. Each sample was repeated three times. The optimal primers were determined by analyzing the fluorescence generation time and the trend of the fluorescence curve.

[0187] 1.6 Enzymatic recombinase amplification

[0188] ERA amplification was performed using an isothermal amplification kit (purchased from GenDx Biotech Co., Ltd.) according to the manufacturer's instructions. The total ERA reaction system was 50 μL, which included 20 μL of the lysing agent (as shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.), 2.5 μL each of 10 μM forward primer F2 and reverse primer R2 (the specific primer sequences are shown in Table 1), and 10 7 copies of the plasmid template ( Figure 1 the plasmid shown). An appropriate amount of ddH2O was added to make the total volume reach 48 μL. After thoroughly mixing the mixture, it was transferred into the basic amplification reagent provided by the kit (the basic amplification reagent as shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.). Subsequently, 2 μL of the activator (as shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.) was added to the tube cap, and the activator was introduced into the premix by brief centrifugation, followed by brief oscillation and rapid centrifugation. The reaction system was incubated at 37 °C in a water bath tube for 20 min. After the reaction, the amplification products were purified for subsequent experiments.

[0189] 1.7 Establishment and optimization of the MP-ERA-Cas12a two-tube detection system

[0190] System optimization was carried out in the initial stage of establishing the MP detection system that binds to Cas12a. By changing the concentration of the F-Q fluorescent probe and the ratio of Cas12a to crRNA (Cas12a / crRNA), the optimal composition ratio of the detection system was determined. Specifically, using a positive recombinant plasmid ( 7 the plasmid shown) containing 10 Figure 1 copies as the template, the concentration of the F-Q reporter gene (purchased from Tolo Biotech Co., Ltd., FAM-TTTTT-BHQ1) was tested, including 100 nM, 200 nM, 300 nM, 400 nM, and 500 nM. At the same time, the ratio of Cas12a / crRNA was evaluated, including 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, and 1:4. Finally, the optimal component ratio (1:2.5) was selected based on the fluorescence values in the experimental results.

[0191] 1.8 Establishment and optimization of the MP-ERA-Cas12a one-tube detection system

[0192] Based on the two - tube MP - ERA - Cas12a system, a one - tube reaction system was established, which can perform isothermal amplification and detection in the same tube. Five sample - adding methods were attempted on the basis of the two - tube system ( Figure 4 ), and the plasmid template used was 400 copies ( Figure 1 the plasmid shown), 4 μL of lysing agent, 0.5 μL each of 10 μM forward primer F2 and reverse primer R2 (final concentration 250 nM) (as shown in Table 1), 0.4 μL of activator (the activator shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.), 1 / 5 of the freeze - dried microspheres (the freeze - dried microspheres shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.), 0.25 μL of 10 μM LbCas12a (i.e., final concentration 125 nM) and 1 μL of 10×HOLMES Buffer. The Cas12a activator consisted of 1.562 μL of 4 μM crRNA (i.e., final concentration 312.5 nM) and 1 μL of 3 μM HOLMES ssDNA reporter (FAM) fluorescent probe (i.e., final concentration 150 nM), the ratio of Cas12 protein to guide RNA was 1:2.5, and water was added to 20 μL (the system before optimization).

[0193] The system of the finally determined sample - adding method (i.e., the optimized system) requires three premixes: the ERA amplification system, the Cas12a detection system, and the Cas12a activator, and the Cas12a activator is added to the reaction tube cap. The amplification system includes 4 μL of lysing agent, 6.85 μL of ddH2O, 10 7 copies of plasmid template ( Figure 1The plasmid shown), 0.5 μL each of 10 μM forward primer F2 and reverse primer R2 (final concentration 250 nM) (as shown in Table 1), 2 μL of activator (such as the activator shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.), and 1 / 5 of the lyophilized microspheres (such as the lyophilized microspheres shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.). The detection system contains 0.25 μL of 10 μM LbCas12a (i.e., final concentration 125 nM) and 1 μL of 10×HOLMES Buffer. The Cas12a activator consists of 1.562 μL of 4 μM crRNA (i.e., final concentration 312.5 nM) and 2 μL of 3 μM HOLMES ssDNA reporter (FAM) fluorescent probe (i.e., final concentration 300 nM), and the ratio of Cas12 protein to guide RNA is 1:2.5. The fluorescent probe was purchased from Tolo Biotech Co., Ltd., with the 5′ end labeled with the FAM fluorescent reporter group and the 3′ end labeled with BHQ1, and the specific sequence is FAM-TTTTT-BHQ1). Before the reaction, 15.25 μL of the amplification system and 1.25 μL of the detection system were added to the bottom of the tube, while 3.5 μL of the Cas12a activator was placed in the tube cap. There is no need to open the lid during the experiment. The system was then incubated at 37 °C for 20 min to promote amplification. After that, the Cas12a activator was brought to the bottom of the tube by brief centrifugation. Finally, at The reaction was carried out at 37 °C for 35 min in a

[0194] Establishment and optimization of the 1.9MP-ERA-Cas12a LFA system (one-tube method)

[0195] Based on the optimized one-tube method, we combined the trans-cleavage function of LbCas12a protein with the ERA isothermal amplification technology and introduced a biotin-conjugated CRISPR-LFA ssDNA reporter (F-B) (the fluorescent reporter was synthesized by Sangon Biotech (Shanghai) Co., Ltd., with the FAM fluorescent reporter group labeled at the 5′ end and Biotin labeled at the 3′ end, and the specific sequence is FAM-TTTTTTTTATT-Biotin) to achieve visual observation of the detection results. This method aims to develop a new method for detecting MP, called the MP-ERA-Cas12a LFA system. During the establishment of the LFA system, the ERA amplification and Cas12a detection systems were kept consistent with the above, and the only change was to replace the reporter probe F-Q (purchased from Tolo Biotech Co., Ltd., with the FAM fluorescent reporter group labeled at the 5′ end and the fluorescent quenching group BHQ1 labeled at the 3′ end. The specific sequence is FAM-TTTTT-BHQ1) with the reporter gene F-B modified with FAM and biotin (the fluorescent reporter was synthesized by Sangon Biotech (Shanghai) Co., Ltd., with the FAM fluorescent reporter group labeled at the 5′ end and Biotin labeled at the 3′ end, and the specific sequence is FAM-TTTTTTTTATT-Biotin).

[0196] The F-B probe functions equivalently to an antigen in LFA. Both too high or too low antigen concentration may lead to the occurrence of the "hook effect". Therefore, this study first needed to determine the optimal concentration of the probe. The specific operation was to dissolve the probe in water or diluent to prepare a series of dilution gradients (10 μM, 1 μM, 100 nM, 10 nM, and 1 nM), and then directly insert the blank test strip into the mixed probe diluent to observe the resulting bands. The situation with the lowest probe concentration and an invisible T line was determined as the optimal reaction condition.

[0197] Although the fluorescence system could produce preliminary results within 10 min, the test strips used in this study showed a slight possibility of false positives, and there were significant differences between the two systems. To reduce the confusion caused by these differences, we systematically optimized the incubation time of the CRISPR system in the test strip system. The reaction tubes were incubated at 37 °C for 5, 10, 15, 20, 30, and 60 min respectively. After incubation, the reaction products were transferred to the sample absorption pad of the test strip. The template concentration was 10 7 copies / μL, ddH 2 O was used as a negative control, and each sample was repeated three times. After completion, the amplification products and test strips were placed in a sealed bag for proper disposal.

[0198] 1.10 Sensitivity and Specificity of the MP-ERA-Cas12a System

[0199] To evaluate the sensitivity of the above three methods, we detected nucleic acids at different concentrations. The MP nucleic acid target was serially diluted from 10 7 copies to 1 copy, with a dilution factor of 10 each time for sensitivity analysis. In addition, to evaluate the specificity of the MP-ERA-Cas12a system, we detected MP and six non-MP strains, with ddH 2 O as a blank control. Nucleic acids from all bacterial samples were extracted using a rapid bacterial genomic DNA isolation kit (purchased from Sangon Biotech, Shanghai), while fungal nucleic acids were extracted using the Bead Beating method. In both analyses, the MP-ERA-Cas12a system was performed according to the above three methods, and each system was repeated three times.

[0200] 1.11 Verification of the clinical feasibility of the MP-ERA-Cas12a system

[0201] To demonstrate the clinical feasibility of the MP-ERA-Cas12a system, a total of 34 clinical samples were collected from Huaibei People's Hospital. These samples were all nasopharyngeal swabs from confirmed MP patients. DNA was extracted from these clinical samples using the GenePure 96 system. The extracted DNA was evaluated using the above three MP-ERA-Cas12a systems.

[0202] 2. Results

[0203] 2.1 Principle of the MP-ERA-Cas12a detection system

[0204] The schematic diagram based on MP-ERA-Cas12a is as Figure 3 shown. First, through the ERA technique, the DNA recombinase binds to the primer (the primer shown in Table 1) to form a protein-DNA complex, which can recognize the homologous sequence in the MP double-stranded DNA and initiate DNA synthesis, thus achieving exponential amplification of the target gene. Subsequently, guided by crRNA, Cas12a recognizes the target sequence through the PAM site. Finally, the trans-cleavage function of Cas12a is activated, resulting in random cleavage of ssDNA in the environment. The ends of these ssDNAs are modified fluorescent groups and quenching groups, which can be detected by GenePure 96. If the detection result is negative, it means that the ssDNA reporter probe has not been digested, so no fluorescence signal can be detected.

[0205] In the one-tube assay, the amplification system at the bottom of the test tube is activated at 37 °C, and exponential amplification of the target gene is achieved through the ERA system. Due to the lack of crRNA, the activity of Cas12a cannot be activated. After the ERA reaction is completed, crRNA and the F-Q probe are added to the system, and a brief centrifugation is performed to activate the detection activity related to trans-cleavage. Cas12a recognizes and binds to the amplification product by pairing with crRNA, thereby activating its cis-cleavage and trans-cleavage activities. Importantly, 3.5 μL of the Cas12a activator on the test tube cap remains in place due to surface tension, and the one-tube system eliminates the need to open the cap, thus reducing the risk of aerosol contamination. This method not only enables specific detection through crRNA guidance, solving the false positive problem caused by non-specific amplification, but also promotes signal amplification and output. Therefore, this platform has significant application potential as a simple and sensitive nucleic acid detection tool.

[0206] The reporter probe of MP-ERA-Cas12a LFA is labeled with biotin at one end and FAM (purchased from ToloBiotech Co., Ltd.) at the other end. The lateral flow strip contains two lines: the lower control line (C line) and the upper test line (T line). Streptavidin is coated on the C line, while goat anti-mouse secondary antibody is coated on the T line, and the anti-FAM monoclonal antibody is labeled with colloidal gold. The intact CRISPR probe captures all the colloidal gold on the C line. When the probe is cleaved by the Cas enzyme, the colloidal gold-bound fragment cannot be captured on the C line but forms a band on the T line. The presence or absence of the T line band determines whether the Cas enzyme is activated. A visible T line indicates a positive result, while an invisible T line indicates a negative result. The presence of the C line confirms that the strip functions properly. Compared with other methods that use qPCR to observe the fluorescence reaction results, this method is more convenient because it does not require large instruments or equipment and can effectively meet the rapid detection needs in primary medical and health settings.

[0207] 2.2 Optimal primer and crRNA combinations for the MP-ERA-Cas12a system

[0208] In this experiment, a total of 3 recombinant plasmids were designed (containing three inserted fragments at positions 725 - 1325, 1698 - 2152, and 3778 - 4238 of the P1 sequence, 3 pairs of ERA amplification primers, and 5 crRNAs (as shown in Table 1)), and their specific combinations are as Figure 2 shown. The results showed ( Figure 5 ) that after adding the combined primer pairs and crRNAs to the system, the fluorescence curves generated by the five groups of reactions all showed a linearly increasing trend. The reaction results using F1R1-crRNA1, F3R3-crRNA3-1, and F3R3-crRNA3-2 showed that using ddH2 The fluorescence signal of the control group of O increased, indicating that the effects of these three combinations lacked specificity for MP detection. Therefore, the results of the test group failed to provide strong evidence for MP detection ( Figure 5 A, D, and E). Regarding F2R2-crRNA2-1 and F2R2-crRNA2-2, the fluorescence curve generated by the F2R2-crRNA2-1 reaction showed a relatively steep upward trend and reached saturation at 20 min. In contrast, the fluorescence curve of the F2R2-crRNA2-2 reaction had a gentler slope and did not reach saturation even after 35 min. In addition, the fluorescence value generated by F2R2-crRNA2-1 at 35 min was 1.3 times that of F2R2-crRNA2-2 and approximately 35 times that of the negative control ( Figure 5 B and C). Therefore, due to its higher fluorescence intensity, F2R2-crRNA2-1 was finally selected.

[0209] Using primer F2R2 reached the reaction plateau with a high fluorescence value and was considered the most effective primer. In addition, the results showed that crRNA2-1 was an ideal guide RNA targeting the P1 gene. Therefore, the complementary DNA sequence of crRNA1 was considered the most suitable target sequence (positions 1698-2152 of P1).

[0210] 2.3 Establishment and optimization of the MP-ERA-Cas12a two-tube detection system

[0211] The system was optimized by adding different concentrations of F-Q probes to the reaction. As Figure 6 shown in A, when the F-Q concentration was 100 nM and 200 nM, the fluorescence curve leveled off after approximately 30 min; while when the F-Q concentration was 300 nM, 400 nM, and 500 nM, the fluorescence curve leveled off after approximately 35 min. It is worth noting that within the concentration range of 300 nM to 500 nM, the fluorescence intensity at the F-Q probe plateau exceeded that of other concentrations, as Figure 6 shown in B. Considering that there was no significant difference in the fluorescence values at concentrations of 300 nM, 400 nM, and 500 nM, in order to save materials, we selected 300 nM of the F-Q probe as the optimal reaction condition for the MP-ERA-Cas12a fluorescence system.

[0212] The ratio of Cas12a to crRNA significantly affected the trans-cleavage performance of the CRISPR / Cas12a system. When the Cas12a / crRNA ratio was 1:2.5, the fluorescence value of the reaction system (4.651±0.616) was higher than other ratios. In addition, the fluorescence curve showed a steep linear upward trend and reached the plateau after 35 min ( Figure 6C and D). Therefore, the ratio of Cas12a / crRNA was determined to be 1:2.5.

[0213] Establishment and optimization of the 2.4MP-ERA-Cas12a one-tube detection system

[0214] During the development of the one-tube system, five sampling methods were evaluated. The results showed ( Figure 7 A), three reactions with 15% glycerol added ( Figure 4 C, D, and E) had no obvious fluorescence values. In contrast, the results of the other two addition methods showed that the fluorescence value of the reaction with only the Cas12a activator (crRNA and F-Q probe) added to the cap was higher than that of the reaction with the entire detection system added to the cap ( Figure 4 A, B, and 7A). Therefore, the next step in system optimization will be based on Figure 4 the sample addition method shown in B.

[0215] The amplification system included 4 μL of lysing agent, 6.85 μL of ddH2O, 10 7 copies of plasmid template ( Figure 1 the plasmid shown), 0.5 μL each of 10 μM forward primer F2 and reverse primer R2 (as shown in Table 1), 2 μL of activator (as shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.), and 1 / 5 of the lyophilized microspheres (as shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.). The detection system contained 0.25 μL of 10 μM LbCas12a and 1 μL of 10× HOLMES Buffer. The Cas12a activator consisted of 1.5 μL of 4 μM crRNA and 2 μL of 3 μM F-Q fluorescent probe (purchased from Tolo Biotech Co., Ltd., labeled with FAM fluorescent reporter group at the 5′ end and fluorescent quenching group BHQ1 at the 3′ end, and the specific sequence was FAM-TTTTT-BHQ1). Before the reaction, 15.25 μL of the amplification system and 1.25 μL of the detection system were added to the bottom of the tube, while 3.5 μL of the Cas12a activator was placed in the tube cap. There was no need to open the lid during the experiment. The system was then incubated at 37 °C for 20 min to promote amplification. After that, the Cas12a activator was dropped to the bottom of the tube by brief centrifugation. The finally determined final concentration of the fluorescent probe was 300 nM, the final concentration of Cas12a was 125 nM, the final concentrations of forward primer F2 and reverse primer R2 were 250 nM, the final concentration of guide RNA was 312.5 nM, and the ratio of Cas12a to guide RNA was 1:2.5.

[0216] As Figure 7 shown in B, the unoptimized one-tube system ( Figure 4The sample addition method shown in B, with the system unoptimized, had a relatively low fluorescence value (0.826241741 ± 0.040). In contrast, the optimized one-tube system ( Figure 4 The sample addition method shown in B, with the system optimized) had a fluorescence value increased by approximately 6.24 times. This finding emphasizes the importance of system optimization in promoting the establishment of the one-tube method. A comparison between the optimized one-tube system and the optimized two-tube system clearly shows that, with the same template DNA concentration, the one-tube system reaches the plateau earlier than the two-tube system, and the fluorescence intensity is increased by 1.6 times. This method not only simplifies the operation steps but also improves the fluorescence value of the results.

[0217] Establishment and optimization of the 2.5MP-ERA-Cas12a LFA system

[0218] To minimize the intensity of the T line, the optimal concentration of the F-B probe for the MP-ERA-Cas12a LFA system must be determined. As observed from the test strip, the negative control produced a band only on the T line. As the concentration of the F-B probe decreased, the intensity of the band on the T line increased, while the band on the C line gradually became lighter. Notably, when the F-B probe concentration was 40 nM, the intensity of the T line was comparable to that observed at 100 nM, and the intensity of the T line was significantly lower than that of the C line ( Figure 8 A). Therefore, to save experimental materials, 40 nM was used as the optimal concentration of the F-B probe for the MP-ERA-Cas12a LFA system.

[0219] As an end-point monitoring system, the incubation time of the MP-ERA-Cas12a LFA system is crucial for the results and needs to be optimized and explored. As Figure 8 shown in B, starting from 20 min, the increase in the incubation time was associated with an increase in the intensity of the T line and a decrease in the intensity of the C line. After incubating for 25 min, the color difference between the positive and negative results was already significant. Therefore, 25 min with an obvious color difference and a short time was selected as the optimal incubation time.

[0220] Sensitivity and specificity of the 2.5MP-ERA-Cas12a system

[0221] To evaluate the sensitivity of the MP-ERA-Cas12a system, we detected nucleic acids at different concentrations ( Figure 9 ). The template concentration gradually decreased from 10 7 copies / μL to 1 copy / μL, resulting in a gradual decrease in the fluorescence intensity. The fluorescence intensity was statistically analyzed at 35 min, and Figure 9 B and Figure 9The data shown in D. The actual detection limits of the two-tube method and the single-tube method are as low as 1 copy / μL. It is worth noting that significant differences can be observed within 20 min after Cas12a activation, indicating that the MP-ERA-Cas12a system has high sensitivity for detecting MP. Compared with the system with a lower copy number, the systems with higher copy numbers (10 7 、10 6 and 10 5 ) produce fluorescence curves that reach the plateau earlier ( Figure 9 A and C). In addition, the fluorescence value generated by the one-tube method system is about 1.5 times that of the two-tube method system. As for the LFA system, it can also detect templates with different copy numbers, as shown in Figure 9 E. When the copy number is 10 1 , the T line band is weak; when the copy number exceeds 10 1 copies / μL, the T line band becomes stronger. The actual detection limit of LFA is as low as 10 2 copies / μL.

[0222] Specificity is a key feature of the detection system. The specificity of the MP-ERA-Cas12a system was verified by using nucleic acids of various pathogens such as Ureaplasma urealyticum, Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Candida albicans, Candida tropicalis, and Aspergillus fumigatus (UU, E. coli, SE, SAU, CAL, CTR, and AF) (provided by Huaibei People's Hospital). As shown in Figure 9 F, only MP showed obvious fluorescence signals, while other pathogens did not show fluorescence. The MP detection was performed using the LFA system ( Figure 9 G), and the results were consistent with the fluorescence analysis results in Figure 9 F. Only MP showed an obvious band on the T line, while other pathogens only showed bands on the C line, indicating that the MP-ERA-Cas12a system has strong specificity for MP.

[0223] 2.6 Verification of the clinical feasibility of the MP-ERA-Cas12a system

[0224] In this study, we evaluated the performance of the above-mentioned MP-ERA-Cas12a one-tube fluorescence detection system and the test strip detection system using 34 known positive samples. The results showed that the MP-ERA-Cas12a fluorescence detection system successfully detected 34 positive samples, and the test strip detection system successfully detected 33 positive samples ( Figure 10)。Taking the fluorescence result as the gold standard, the positive prediction rate of the MP-ERA-Cas12a test strip system was calculated to be 97.06%, reflecting a high degree of consistency between the two methods in MP detection. This finding further confirmed the reliability and effectiveness of the MP-ERA-Cas12a system in the detection of clinical samples, highlighting its strong clinical applicability in practical applications.

[0225] Discussion

[0226] Mycoplasma pneumoniae (MP) is a common respiratory pathogen that can cause symptoms such as pharyngitis, tracheitis, and bronchitis. Although most infections are self-limiting, a small proportion of infections may progress to pneumonia. There are currently various methods for detecting MP. However, these methods usually require specialized procedures and advanced laboratory equipment, coupled with high costs, making them unsuitable for point-of-care testing. The CRISPR system reduces the non-specific signals caused by off-target amplification of ERA, while the ERA system enhances the sensitivity of the CRISPR system. Both systems can work at 37°C, simplifying the experimental procedures. However, if the amplification system and the CRISPR system are pre-mixed in the reaction tube before the reaction, at very low template concentrations, Cas12a will continuously digest the amplification products and primers, resulting in a decrease in amplification efficiency. To solve this problem, most methods use the widely used two-tube system, separating the amplification reaction and the detection reaction. However, manual transfer of the amplification products will complicate the process and increase the risk of contamination. Many studies have also adopted the "physical isolation" strategy to solve this problem. However, these solutions may make the operation process more cumbersome.

[0227] In this study, we established a one-tube detection system with higher sensitivity, combining ERA amplification and Cas12a detection, and obtained higher fluorescence values than the traditional two-tube system. This method helps to quickly detect MP in one tube, without the need for additional primer or crRNA design, nor physical isolation methods. We simply added the Cas12a activator containing crRNA and F-Q to the tube cap, and the remaining components were mixed at the bottom of the tube. This method effectively solved the possible contamination problem during the secondary tube-opening process. The MP-ERA-Cas12a system showed high sensitivity, with a detection limit (LOD) for MP as low as 1 copy / μL. When used in conjunction with the LFA strip, the sensitivity can reach 10 2 copies / μL, thus overcoming the limitations of large instruments and improving the accessibility of detection in remote areas. In addition, the MP-ERA-Cas12a system has been proven to be effective in the testing of clinical samples.

[0228] The system established in this study was used to analyze 34 hospital clinical positive samples. The results of the LFA were consistent with those of qPCR, indicating a strong consistency between the two methods. In addition, the detection results of the system for other pathogens were all negative, demonstrating its high specificity. However, this study has certain limitations: First, when evaluating the specificity of the system, only 8 pathogens including MP were selected for evaluation, which may not provide a comprehensive assessment. Subsequent experiments will test other pathogens related to respiratory tract infections to further study the specificity of the detection system. Second, only 34 clinical samples were used in this analysis, which may introduce bias and limit the universality of the research results.

[0229] In summary, this study successfully combined the specific detection ability of ERA and CRISPR / Cas12a. Based on the previously established two-tube system, a one-tube system and an LFA system were developed for MP detection. During the reaction process of the MP-ERA-Cas12a system, there is no need to open the lid, thus reducing aerosol contamination and minimizing the risk of false positives. In addition, the method produces results within 1 h. The detection sensitivity of the system is 1 copy / μL, while the detection sensitivity of the LFA is 10 2 copies / μL, without the need to use advanced instruments or equipment. The system exhibits strong specificity and is not affected by other pathogens. As a new method for MP detection, the MP-ERA-Cas12a technology has important practical application prospects.

[0230] All documents mentioned in this invention are cited herein by reference as if each individual document was specifically and individually cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for detecting whether Mycoplasma pneumoniae exists in a sample, characterized in that: The detection method comprises: (a) providing a reaction system, the reaction system comprising: a detection system, an amplification system, and a Cas12 activator, wherein the detection system, the amplification system, and the Cas12 activator are located in the same tube, the Cas12 activator is located on the tube cover, the detection system and the amplification system are located at the bottom of the tube, and the Cas12 activator contains a guide RNA and a nucleic acid probe, the detection system contains a Cas12 protein, and the amplification system contains a sample-derived nucleic acid molecule of Mycoplasma pneumoniae to be detected and an amplification primer for isothermal amplification of the nucleic acid molecule of Mycoplasma pneumoniae to be detected, wherein the guide RNA guides the Cas12 protein to specifically bind to the nucleic acid molecule of Mycoplasma pneumoniae to be detected; (b) performing nucleic acid amplification on the Mycoplasma pneumoniae nucleic acid molecule in the amplification system at the bottom of the same tube to obtain an amplification product of the Mycoplasma pneumoniae nucleic acid molecule; (c) in the same tube, adding the Cas12 activator on the tube cover to the detection system at the bottom of the tube and the amplification system containing the amplification product, and detecting the detectable signal emitted by the nucleic acid probe; Among them, if the nucleic acid probe is cut by the Cas12 protein, it means that Mycoplasma pneumoniae is present in the sample; if the nucleic acid probe is not cut by the Cas12 protein, it means that Mycoplasma pneumoniae is not present in the sample.

2. The method according to claim 1, characterized in that The detection includes: qualitative detection or quantitative detection.

3. The method according to claim 1, characterized in that The detection includes fluorescence detection method and colloidal gold detection method.

4. The method according to claim 1, characterized in that The nucleic acid molecule of Mycoplasma pneumoniae to be detected is selected from the following group: single-stranded DNA, double-stranded DNA, or a combination thereof.

5. The method according to claim 1, characterized in that The length of the guide RNA is 55-60nt.

6. The method according to claim 1, characterized in that The final concentration of the guide RNA is 250-400 nM, preferably 300-350 nM.

7. The method according to claim 1, characterized in that The nucleic acid probe is detectably labeled.

8. The method according to claim 1, characterized in that The nucleic acid probe is a single-stranded DNA or single-stranded RNA labeled with a fluorescent group and biotin.

9. The method according to claim 1, characterized in that The nucleic acid probe is a single-stranded DNA or single-stranded RNA labeled with a fluorescent group and a quenching group.

10. A kit for detecting Mycoplasma pneumoniae, characterized in that: The kit comprises: (i) a first container and a detection system located in the first container, wherein the detection system contains a Cas12 protein; (ii) a second container and an amplification system located in the second container, wherein the amplification system contains amplification primers for amplifying nucleic acid molecules of Mycoplasma pneumoniae to be detected; (iii) a third container and a Cas12 activator located in the third container, wherein the Cas12 activator contains a guide RNA and a nucleic acid probe; and labels or instructions.

Images