Method for establishing and optimizing mycoplasma pneumoniae detection system based on era-crisspr / cas12a

Abstract

The application provides a method for establishing and optimizing a mycoplasma pneumoniae detection system based on ERA-CRISPR / Cas12a. The application provides a method for detecting whether mycoplasma pneumoniae exists in a sample and a corresponding kit. The method and the kit of the application can quickly, economically, highly sensitively and highly specifically detect mycoplasma pneumoniae.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and in particular, the present application relates to the establishment and optimization of a method for detecting mycoplasma pneumoniae based on ERA-CRISPR / Cas12a. BACKGROUND

[0002] Mycoplasma pneumoniae (MP) was first identified in 1944 by Eaton et al. from sputum samples of patients with atypical pneumonia. MP appears as short and thin filamentous structures of 2-5 pm in length, which are difficult to stain in Gram staining, but appear light purple in Giemsa staining. The source of MP infection is from patients or carriers, and the pathogen is transmitted through its nasal, pharyngeal, laryngeal and tracheal secretions, mainly through droplets or aerosols. The infection is contagious during both the incubation and treatment periods. MP can cause a variety of respiratory diseases and lead to a variety of extrapulmonary complications. Preschool children and adolescents with immature immune systems are particularly susceptible to clinical symptoms and are the main affected population of MP. Although MP infection is usually considered to be mild and self-limiting, its severity is often overlooked, and more than 10% of children hospitalized for MP infection need to enter the intensive care unit. The clinical symptoms of MP infection are diverse and often similar to those of other respiratory pathogens, making clinical diagnosis complex. Therefore, laboratory diagnosis is essential for effective clinical treatment. The laboratory diagnosis of MP includes isolation and culture, serological detection, antibody detection and molecular biology techniques, etc. Although isolation and culture is considered the "gold standard" for diagnosing MP infection, the culture and morphological observation of MP are challenging, and the culture process is time-consuming. In order to achieve the best serological detection of MP infection, at least two serum samples are usually collected every two weeks, which is a time-consuming procedure. In addition, the limited immune response observed in infants and the elderly, as well as the 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, which 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 test, are usually simple and fast, but have low sensitivity. These methods can also produce high background levels of interfering antibodies in healthy individuals and cause non-specific reactions, resulting in false positive results. Therefore, they are not suitable for early screening of MP, but are more suitable for retrospective investigation of MP infection. Molecular biology techniques are the main methods for detecting MP nucleic acids, mainly including PCR techniques and isothermal amplification techniques. However, various PCR-based detection methods are not suitable for rapid detection and widespread use in resource-limited areas due to complex operation procedures, long detection periods, and dependence on professional equipment and electricity. Isothermal amplification techniques cover a class of emerging molecular biology techniques, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) rapid detection methods. 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 rapid diagnosis of MP.

[0003] Enzymatic Recombinase Amplification (ERA) is a patented technology developed by GenDx Biotech Co., Ltd. in 2019. This technology is an enhanced version of RPA, which uses enzymes from different species to accelerate the reaction. The modified DNA recombinase interacts with the primer to form a protein-DNA complex, which then locates the homologous sequence in the double-stranded DNA and initiates DNA synthesis, thereby starting 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 sensitivity in detection, enabling the amplification of trace amounts of nucleic acid templates to a detectable level. At a constant temperature of 35 to 42°C, specific genes can be amplified and qualitatively detected, and the results can be observed by agarose gel electrophoresis and other methods. In most cases, trace amounts of nucleic acid samples can be amplified to a detectable level within 15 minutes. This technology is characterized by high stability and high sensitivity, with a detection limit of 10 1 -10 2 copies per reaction. The entire process is simple, does not require specialized equipment, and is easy to operate without the need for professional skill training.

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are specific DNA sequences identified in prokaryotes, and CRISPRs and their associated proteins (Cas proteins) play a key role in the adaptive immune system of archaea and bacteria, providing defense against exogenous plasmid and bacteriophage invasive nucleic acids. In 2015, Zhang Feng and his team identified the V-type 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 his team discovered that Cas12 protein has a "transcleavage" effect, thereby 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 form a ternary complex, the complex exhibits significant "transcleavage" activity. Taking advantage of 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, high-sensitivity, and high-specificity MP detection method. SUMMARY

[0006] The purpose of the present application is to provide a rapid, economical, high-sensitivity, and high-specificity MP detection method.

[0007] In the first aspect of the present application, a method for detecting the presence or absence of Mycoplasma pneumoniae in a sample is provided, the detection method comprising:

[0008] (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 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 a Cas12 protein, the amplification system contains a nucleic acid molecule of the Mycoplasma pneumoniae to be detected from the sample and amplification primers for isothermal amplification of the nucleic acid molecule of the Mycoplasma pneumoniae to be detected, wherein the guide RNA guides the Cas12 protein to specifically bind to the nucleic acid molecule of the Mycoplasma pneumoniae to be detected;

[0009] (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;

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

[0011] wherein if the nucleic acid probe is cleaved by the Cas12 protein, it indicates that the sample contains Mycoplasma pneumoniae; and if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that the sample does not contain Mycoplasma pneumoniae.

[0012] In another preferred embodiment, the detection comprises qualitative detection or quantitative detection.

[0013] In another preferred embodiment, the detection comprises fluorescence detection or colloidal gold detection.

[0014] In another preferred embodiment, the fluorescence detection is performed using an enzyme label instrument or a fluorescence spectrophotometer.

[0015] In another preferred embodiment, the nucleic acid molecule of the 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 embodiment, the nucleic acid molecule of the Mycoplasma pneumoniae to be detected is naturally occurring DNA or artificially synthesized DNA.

[0017] In another preferred embodiment, the nucleic acid molecule of 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, microorganism culture, organoid culture, in vivo enrichment culture in animals and plant culture.

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

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

[0020] In another preferred embodiment, the sample is a nucleic acid sample prepared from a sample selected from the group consisting of throat swab, alveolar lavage, nasal swab.

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

[0022] In another preferred embodiment, the guide RNA comprises a sequence as set forth in SEQ ID NO. 1-5.

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

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

[0025] In another preferred embodiment, the detectable label comprises a fluorescent group, a quencher group.

[0026] In another preferred embodiment, the fluorescent group 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 embodiment, 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 embodiment, the fluorescent group and the quencher group are each independently located at the 5' end, the 3' end and the middle of the nucleic acid of the nucleic acid probe.

[0029] In another preferred embodiment, the nucleic acid probe has a length of 5-50 nt, preferably 5-30 nt, more preferably 5-20 nt, 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, the final concentration of the nucleic acid probe is 200-400 nM, preferably 250-350 nM, such as 300 nM, when detected by fluorescence detection.

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

[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 comprises a Cas12 protein and a buffer.

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

[0040] In another preferred embodiment, the final concentration of the Cas12 protein in the detection system is 100-250 nM, preferably 110-200 nM, more 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 embodiment, the Cas12a is selected from the group consisting of 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 embodiment, the Cas12a is selected from the group consisting of 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.

[0045] In another preferred embodiment, 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_K08D17 (SmCas12a), and Lachnospiraceae bacterium YP2017 (Lb3Cas12a).SC_K08D17) (SsCas12a), Lachnospiraceae bacterium MC2017 (Lb3Cas12a), Bytyrivibrio proteoclasticus (BpCas12a), Prevotella disens (PdCas12a), Butyrivibrio fibrisolvens MD2001 (BfCas12a), Porphyromonas crevioricanis PcCas12a, Candidatus Methanoplasma termitum (CMtCas12a), Peregrinibacteria bacterium (PeCas12a), Leptospira inada iserovar 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 embodiment, 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 embodiment, the amplification primer comprises:

[0048] (P1) F1R1 primer of Mycoplasma pneumoniae P1 gene: sequence as shown in SEQ ID NO: 6 and 7;

[0049] (P2) F2R2 primer of Mycoplasma pneumoniae P1 gene: sequence as shown in SEQ ID NO: 8 and 9;

[0050] (P3) F3R3 primer of Mycoplasma pneumoniae P1 gene: sequence as shown in SEQ ID NO: 10 and 11.

[0051] In another preferred embodiment, the combination of the amplification primer and the guide RNA is selected from the group consisting of:

[0052] (Z1) F1R1 primer of Mycoplasma pneumoniae P1 gene: sequence as shown in SEQ ID NO. 6 and 7, guide RNA crRNA1, sequence as shown in SEQ ID NO: 1;

[0053] (Z2) F2R2 primer of Mycoplasma pneumoniae P1 gene: sequence as shown in SEQ ID NO: 8 and 9, guide RNA crRNA2-1, sequence as shown in SEQ ID NO: 2;

[0054] (Z3) F2R2 primer of Mycoplasma pneumoniae P1 gene: sequence as shown in SEQ ID NO: 8 and 9, guide RNA crRNA2-2, sequence as shown in SEQ ID NO: 3;

[0055] (Z4) F3R3 primer of Mycoplasma pneumoniae P1 gene: sequence as shown in SEQ ID NO: 10 and 11, guide RNA crRNA3-1, sequence as shown in SEQ ID NO: 4.

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

[0057] In another preferred embodiment, the amplification system further comprises an activator.

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

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

[0060] In another preferred embodiment, the isothermal amplification is selected from the group consisting of ERA (Enzymatic Replikation Isotherm Amplification), RPA (Recombinase Polymerase Amplification), RAA (Recombinase Aided Amplification).

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

[0062] In another preferred embodiment, the reagents for isothermal amplification comprise:

[0063] (a1) DNA polymerase from ERA isothermal amplification kit from GenDx Biotech Co., Ltd. for amplifying nucleic acid molecules of Mycoplasma pneumoniae to be detected;

[0064] (a2) freeze-dried microspheres for amplification reaction (ERA isothermal amplification kit from GenDx Biotech Co., Ltd.;

[0065] (a3) activator MC for activating amplification reaction (ERA isothermal amplification kit from GenDx Biotech Co., Ltd.);

[0066] (a4) 6*Loading Buffer for amplification reaction (ERA isothermal amplification kit from GenDx Biotech Co., Ltd.).

[0067] In another preferred embodiment, in step (b), the nucleic acid amplification of the Mycoplasma pneumoniae nucleic acid molecules in the amplification system is carried out at 37-42°C, preferably 37-40°C.

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

[0069] In another preferred embodiment, step (c) further comprises a centrifugation step to make the Cas12a activator drop to the bottom of the tube.

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

[0071] In another preferred embodiment, the method is a tube method.

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

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

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

[0075] (ii) a second container and an amplification system in 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 in the third container, the Cas12 activator containing a guide RNA and a nucleic acid probe;

[0077] and a label or an instruction.

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

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

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

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

[0082] (P3) F3R3 primers for Mycoplasma pneumoniae P1 gene: sequences 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 group consisting of:

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

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

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

[0087] (Z4) F3R3 primers of Mycoplasma pneumoniae P1 gene: sequences as shown in SEQ ID NOs: 10 and 11, guide RNA crRNA3-1, sequence as shown in SEQ ID NO: 4.

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

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

[0090] (iv) a fourth container and nucleic acid molecules of Mycoplasma pneumoniae to be detected in the fourth container.

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

[0092] (v) a fifth container and reagents for isothermal amplification of nucleic acid molecules of Mycoplasma pneumoniae to be detected in 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 a PCR tube, the Cas12 activator is located in the tube cap, and the detection system and the amplification system are located in the tube bottom.

[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, multiple, 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 a PCR tube, the Cas12 activator is located in the tube cap, and the detection system and the amplification system are located in the tube bottom.

[0100] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0101] Figure 1 The three target fragments selected from the P1 gene were integrated into three different recombinant plasmids. The image shows the positive recombinant plasmid finally used.

[0102] Figure 2 The combination of 3 targets, 3 pairs of primers and 5 crRNAs is shown. crRNA1 is used to recognize Target 1, 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 The principle diagram of the MP-ERA-Cas12a system for detecting MP is shown. After DNA extraction from the sample, the target sequence is subjected to 20 to 30 min of ERA amplification. Before the amplification process starts, crRNA and ssDNA are added to the reaction tube cap. After the completion of the ERA amplification, the LbCas12a-crRNA complex recognizes the amplification product, triggering "transcleavage" to cut the ssDNA reporter probe within about 35 min. Subsequent analysis can be performed by observing the fluorescence signal or using LFA test strips. T: test line, C: control line.

[0104] Figure 4 Different sampling methods were tried when establishing the MP-ERA-Cas12a one-tube 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 The fluorescence curves generated by the positive samples in each combination are shown. NTC: no template control.

[0106] Figure 6 The fluorescence curves generated by using different concentrations of F-Q and different Cas12a / crRNA ratios are shown. A: fluorescence curves generated by using different concentrations of F-Q. B: fluorescence values generated by different concentrations of F-Q at 50 min. C: fluorescence curves generated by using different Cas12a / crRNA ratios. D: fluorescence values generated by different ratios of Cas12a / crRNA at 35 min. NTC: no template control.

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

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

[0109] Figure 9 The sensitivity analysis of the MP-ERA-Cas12a system is shown. A: Fluorescence curves generated by the two-tube method system under different copy number templates. B: Fluorescence curves generated by the two-tube method system at 35 min under different copy number templates. C: Fluorescence curves generated by the one-tube method system under different copy number templates. D: Fluorescence curves generated by the one-tube method system at 35 min under different copy number templates. E: Fluorescence curves generated by the LFA system under different copy number templates. F: Specific 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: Specific analysis of the MP-ERA-Cas12a LFA system.

[0110] Figure 10 The results of the MP-ERA-Cas12a system detecting 34 known positive samples are shown. +: positive, -: negative. DETAILED DESCRIPTION

[0111] The inventors first developed a rapid, economical, high-sensitivity and high-specificity MP detection method through extensive and in-depth research. The present application first puts the detection system, the amplification system and the Cas12 activator into the same tube, and the Cas12 activator is located in the tube cover, and the detection system and the amplification system are located at the bottom of the tube, which integrates the amplification and detection steps in one reaction container, effectively reducing the pollution and false positive risk caused by handling multiple test tubes, and the present application can be detected by fluorescence detection method and colloidal gold detection method. And the experimental results of the present application show that the one-tube method of the present application can produce a fluorescence signal in 1h, 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 application can realize rapid on-site screening, and within 5min, a visible band will appear on the test paper, and the detection limit is 10 2copies / μL. All methods showed high specificity to MP. The MP-ERA-Cas12a detection system of the present application has significant advantages, including fast processing speed, no need for complex instrument equipment, and simple operation, which is particularly suitable for resource-limited clinical environments. The system is an efficient tool for early diagnosis of MP, which has important public health and clinical significance. On this basis, the present application is completed.

[0112] The term

[0113] The term "PCR" is a polymerase chain reaction technology, which is a technology suitable for target nucleic acid amplification.

[0114] As used herein, "CRISPR" refers to Clustered Regularly Interspaced Short Palindromic Repeats, which is 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 strong affinity of biotin can be used to amplify or enhance the detection signal in the detection system. As biotin is easy to bind to proteins (such as antibodies, etc.) with a covalent bond, and the avidin molecule combined with the enzyme reacts with the biotin molecule combined with the specific antibody, which not only plays a multi-level amplification role, but also shows color due to the catalytic effect of the enzyme when encountering the corresponding substrate, achieving the purpose of detecting unknown antigen (or antibody) molecules.

[0116] CRISPR-Cas: a unique genomic element derived from bacteria and archaea, as an adaptive immune defense system to resist invading phages or foreign nucleic acids. The system is composed 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 "Cpf1") refers to a crRNA-dependent endonuclease, which is a V-A type enzyme in the classification of the CRISPR system.

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

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

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

[0122] Cas protein

[0123] The "Cas protein" described herein refers to a CRISPR-associated protein (sometimes translated as CRISPR-Cas effector protein, CRISPR / Cas effector protein, CRISPR-Cas effector, CRISPR / Cas effector), which can be a type V Cas protein or a type VI Cas protein. The type V Cas protein, once binds 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, can induce its trans-cleavage activity, i.e. random cleavage of single-stranded nucleic acid and its equivalents (nucleic acid equivalents such as nucleic acid analogs).

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

[0125] The Cas protein described in the present specific embodiment can be a type V Cas protein; the Cas protein is selected from the following group: 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 the present specific embodiment includes Cas12, for example Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12l, Cas12m or a combination thereof.

[0126] In a specific embodiment, Cas proteins, as referred to herein, having trans-cleavage activity, such as Cas12, also encompass functional variants of Cas proteins or homologs or orthologs thereof. A “functional variant” of a protein, as used herein, refers to a variant of such a protein that at least partially retains the trans-cleavage activity of the protein. Functional variants can include mutants (which can be insertion, deletion, or substitution mutants), including polymorphs, and the like. Also included in functional variants are fusion products of such a protein with another, generally unrelated, nucleic acid, protein, polypeptide, or peptide. Functional variants can be naturally occurring or can be man-made. Advantageous embodiments can involve engineered or non-naturally occurring V-type DNA-targeting effector proteins.

[0127] In an embodiment, the V-type Cas protein or orthologs or homologs thereof can comprise one or more mutations, and thus the nucleic acid molecule encoding the same 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 an embodiment, the V-type Cas protein can be from: Leptospira, Listeria, Corynebacterium, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Azospira, Sphaerochaeta, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter, Lachnospira, or a combination thereof.

[0129] Table I V-type family effector attributes (derived 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 either a mature crRNA fused to a tracrRNA as a guide RNA, or a mature crRNA fused to a scout RNA as a guide RNA, or a crRNA alone as a guide RNA.

[0136] Generally, a guide RNA can comprise, or essentially consist of, or consist of, direct repeat sequences (also referred to as DR sequences) and a guide sequence. The gRNA can include crRNA and tracrRNA, or crRNA and scoutRNA, or only crRNA, in different CRISPR systems depending on the Cas protein it relies on. The crRNA and tracrRNA can be artificially engineered to form a single guide RNA (sgRNA). In some cases, the guide sequence is a polynucleotide sequence that has sufficient complementarity to a cis-cleavage substrate DNA to hybridize to the cis-cleavage substrate DNA and direct specific binding of the CRISPR / Cas protein-guide RNA complex to the cis-cleavage substrate DNA, typically having a sequence length of 15-28 nt. The direct repeat sequences can fold to form a specific structure (e.g., stem-loop structure) for recognition by the Cas protein 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 some embodiments, the degree of complementarity (match) between the guide sequence and its corresponding cis-cleavage substrate DNA, when optimally aligned, is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining optimal alignment is within the capabilities of a person of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman 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 hybrids thereof, which can be double-stranded or single-stranded.

[0139] The terms "homology" or "identity" are used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules occupied by adenine, or a position in each of two polypeptides occupied by lysine), then the molecules are identical at that position. Generally, comparisons are made using the complete sequences aligning the two sequences to produce the maximum identity. Such alignments can be determined by computerized running of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group) using, for example, the default parameters of the programs. BLAST algorithms available from the National Center for Biotechnology Information (NCBI www.ncbi.nlm.nih.gov / ) can also be used, using the default parameters.

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

[0141] As used herein, when the nucleic acid molecule to be detected is a nucleic acid molecule, the "target nucleic acid molecule" refers to a polynucleotide molecule extracted from a biological sample (a sample to be detected) or an amplification product, a transcription product, a reverse transcription product thereof. When the non-nucleic acid molecule to be detected is a nucleic acid molecule, 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 unicellular organisms such as bacteria, yeast, protozoa and amoeba, and multicellular organisms (for example, plants or animals, including samples from healthy or apparently healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, for example, infection with a pathogenic microorganism such as a pathogenic bacterium or virus). For example, the biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, serum tumor, saliva, cerebrospinal fluid, aqueous or vitreous fluid, or any body secretion, exudate, exudate (for example, fluid obtained from an abscess or any other infected or inflamed site), or fluid obtained from a joint (for example, 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 a biopsy or autopsy specimen, such as a tumor biopsy) or can comprise cells (primary cells or cultured cells) or a culture medium conditioned by any cell, tissue or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cell centrifugation preparations, cytological smears, body fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and / or tissue sections (e.g., cryostat tissue sections and / or paraffin-embedded tissue sections).

[0142] In other embodiments, the biological sample can be a plant cell, callus, tissue or organ (such as roots, stems, leaves, flowers, seeds, fruits), etc.

[0143] In the present application, the target nucleic acid molecule includes a DNA molecule, also includes an RNA molecule or a DNA molecule formed by reverse transcription of RNA, or further, the target nucleic acid molecule is amplified by a technology known in the art, and the amplification technology is isothermal amplification technology, which can be ERA, RPA, RAA.

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

[0145] One-tube amplification and detection method without opening the cap

[0146] The present embodiment discloses a one-tube amplification and detection method without opening the cap for detecting the target nucleic acid molecule (the nucleic acid molecule of Mycoplasma pneumoniae to be detected).

[0147] In a preferred embodiment, the present embodiment provides a method for detecting whether Mycoplasma pneumoniae exists in a sample, which comprises:

[0148] (a) providing a reaction system, which comprises 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 cap of the tube, 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, the amplification system contains a sample-derived nucleic acid molecule of Mycoplasma pneumoniae to be detected and amplification primers for isothermal amplification of the nucleic acid molecule of Mycoplasma pneumoniae to be detected, wherein the guide RNA guides the specific binding of the Cas12 protein to the nucleic acid molecule of Mycoplasma pneumoniae to be detected;

[0149] (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;

[0150] (c) adding the Cas12 activator in the cap of the tube to the detection system at the bottom of the tube and the amplification system containing the amplification product to detect a detectable signal emitted by the nucleic acid probe in the same tube;

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

[0152] In a preferred embodiment, the present application combines the ERA isothermal amplification technology with the CRISPR / Cas12a system, develops and evaluates a new method for rapid detection of MP, called MP-ERA-Cas12a system. By taking advantage of the "transcleavage" effect generated after the target gene is recognized and cut by the CRISPR / Cas12a system, the present application introduces two types of reporter probes (fluorescent probes and LFA probes), and accordingly establishes the MP-ERA-Cas12a fluorescent system and the LFA test strip system. In addition, the present application develops a one-tube detection method, which separates the ERA amplification and the CRISPR detection without the need to open the reaction tube for liquid transfer. This design not only improves the sensitivity, 37℃-42℃, but also simplifies the operation process, significantly reduces the possibility of false positive results caused by aerosol pollution, and thus ensures the accuracy of the detection results.

[0153] In the present application, the representative nucleic acid probe is a single-stranded DNA or single-stranded RNA with a luminescent group and a quenching group at both ends, and a single-stranded DNA or single-stranded RNA with a luminescent group and biotin at both ends, so that once the probe is cut, the luminescent group can emit light or form a band on the T line.

[0154] In the present embodiment, the presence or absence of the target nucleic acid molecule, such as the nucleic acid molecule of Mycoplasma pneumoniae, in the system to be detected can be determined by detecting fluorescence.

[0155] In the present embodiment, the presence or absence of the target nucleic acid molecule, such as the nucleic acid molecule of Mycoplasma pneumoniae, in the system to be detected can be determined by detecting fluorescence.

[0156] In the present embodiment, the suitable Cas protein is a type V Cas protein with trans-cleavage activity, preferably Cas12a, 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 the present embodiment can be used to quickly detect whether the sample contains the target nucleic acid molecule (Mycoplasma pneumoniae). In addition, by combining with an isothermal amplification technique (such as any one of ERA, RPA, RAA), the sensitivity of the detection method can be greatly improved. Various isothermal amplification techniques in the prior art can be used in the present application in theory, and the present embodiment only lists the preferred embodiments. The components used in various amplification techniques in the present application, such as:

[0158] NTP, buffer, Mg required for RNA amplification 2+ and RNase H required when the reverse transcriptase has the function of non-digestion of single-stranded RNA;

[0159] dNTP, buffer, Mg required for DNA amplification 2+ and the like;

[0160] These contents are well known in the art, so the present application does not specifically describe them.

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

[0162] (1) The present application first develops a rapid, economical, high sensitivity and high specificity method for detecting MP. The present application first puts the detection system, the amplification system and the Cas12 activator into the same tube, and the Cas12 activator is located in the tube cover, and the detection system and the amplification system are located at the bottom of the tube, and the amplification and detection steps are integrated in one reaction container, which effectively reduces the pollution and false positive risk caused by handling multiple test tubes, and the present application can be detected by fluorescence detection method and colloidal gold detection method. And the experimental results of the present application show that the one-tube method of the present application can produce a fluorescence signal in 1h, 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 application can realize rapid on-site screening, and within 5min, a visible band will appear on the test paper, and the detection limit is 10 2 copies / μL. All methods show high specificity for MP. The MP-ERA-Cas12a detection system of the present application has significant advantages, including fast processing speed, no need for complex instrument equipment and simple operation, especially suitable for resource-limited clinical environment. The system is a high-efficiency tool for early diagnosis of MP, and has important public health and clinical significance.

[0163] (2) The present application combines ERA isothermal amplification technology with CRISPR / Cas12a system, develops and evaluates a new type of rapid MP detection method, called MP-ERA-Cas12a system. By using the "trans cleavage" effect produced after the target gene is recognized and cut by the CRISPR / Cas12a system, we introduce two types of reporter probes (fluorescent probes and LFA probes), and accordingly establish MP-ERA-Cas12a fluorescence system and LFA test strip system. In addition, we develop a one-tube detection method, which separates ERA amplification and CRISPR detection without opening the reaction tube for liquid transfer. This design not only improves the sensitivity, 37℃-42℃, but also simplifies the operation process, significantly reduces the possibility of false positive results caused by aerosol pollution, thereby ensuring the accuracy of the detection results. Our goal is to create a rapid, economical, high sensitivity and high specificity MP detection method.

[0164] (3) The present application establishes a one-tube detection system with higher sensitivity, which combines ERA amplification with 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 or physical isolation method. We simply add the Cas12a activator containing crRNA and F-Q to the tube cap, and mix the remaining components at the bottom of the tube. This method effectively solves the pollution problem that may occur during the second opening process. The MP-ERA-Cas12a system shows high sensitivity, with a detection limit LOD as low as 1 copy / μL. When used with LFA strips, 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 clinical sample testing.

[0165] (4) The present application successfully combines the specificity of ERA and CRISPR / Cas12a detection. Based on the previously established two-tube system, a one-tube system and LFA system are developed for MP detection. The MP-ERA-Cas12a system does not need to open the cap during the reaction process, thereby reducing aerosol pollution and minimizing the risk of false positives. In addition, the method produces results within 1h. The detection sensitivity of the system is 1 copy / μL, while the detection sensitivity of LFA is 10 2 copies / μL, without the need for advanced instruments or equipment. The system shows strong specificity and is not affected by other pathogens. The MP-ERA-Cas12a technology as a new method for MP detection has important practical application prospects.

[0166] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, and the conditions are generally according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0167] The experimental materials involved in the present application can be obtained from commercial channels unless otherwise specified.

[0168] 1. Materials and methods

[0169] 1.1 Materials and reagents

[0170] The recombinant plasmids, primers and crRNA used in this study were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. The sequences of the primers and crRNA are shown in Table 1. The enzyme recombinant isothermal amplification kit was purchased from GenDx Biotech Co., Ltd. (Suzhou, China). The materials required for CRISPR / Cas12a-based detection were purchased from Tolo Biotech Co., Ltd. (Shanghai, China), including: Cas12a high-yield crRNA synthesis and purification kit, LbCas12a (Cpf1) nuclease, 10x HOLMES Buffer, HOLMES ssDNA reporter gene (FAM), CRISPR-LFA ssDNA reporter gene, and CRISPR single system detection strip (FAM / FITC).

[0171] Table 1 Primers, crRNA and inserted fragments in recombinant plasmids

[0172]

[0173]

[0174] * indicates the final primer / crRNA used.

[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. Thirty-four nasopharyngeal swabs were collected from 34 patients diagnosed with MP infection at Huaibei People's Hospital for clinical feasibility evaluation. The GenePure 96 system was used to extract nucleic acids from these clinical samples.

[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 sequences (positions 725-1325 of the P1 sequence, positions 1698-2152 of the P1 sequence, and positions 3778-4238 of the P1 sequence) were selected and cloned into the pUC19 vector (purchased from Shanghai Shengong) to construct recombinant plasmids. The structure of the final recombinant plasmid is shown in Figure 1The nucleic acid concentration of the synthesized plasmid was determined by ultraviolet spectrophotometry, and the concentration was converted to copy number, with the formula: C (copy number / μL) = [6.02 x 10 23 x C (ng / μL) x 10 -9 ] / [DNA length (bp) x 660]. The DNA sample was then resuspended in ddH2O and stored at -20°C for future use.

[0183] 1.4 Design of isothermal amplification primers and crRNA

[0184] According to the design principles of ERA primers, the highly conserved MP P1 gene was targeted, and the 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 target position was determined using the CRISPR RGEN tool. The final crRNA sequence was 5'-AAUUUCUACUAAGUGUAGAU NN NNNNNNNNNNNNNNNNNN -3', where the underlined part represents the specific target sequence complementary to the spacer. The positions of the ERA primers and crRNA are shown in Figure 2 , and the schematic diagram of MP-ERA-Cas12a is shown in Figure 3 .

[0185] 1.5 Screening of optimal primer and crRNA combination

[0186] In the initial stage of the study, the optimal primer and crRNA combination was screened. The optimal combination varies for different experimental systems, so before proceeding with subsequent experiments, the optimal combination must be tried and determined. Throughout the experiment, the environmental conditions and amounts of each combination were kept consistent to ensure that any differences in results are solely due to the different effectiveness of the combinations. Different primer pairs were designed for different target sequences, and the synthesized upstream and downstream candidate primers (F1, F2, F3, R1, R2, R3) were paired to form F1R1, F2R2, and F3R3 primer pairs (sequences shown in Table 1, respectively). Considering that different crRNAs have different cutting efficiencies for different target sequences, we synthesized five crRNAs with high expected cutting efficiency. 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, sequences shown in Table 1, respectively). The template concentration was set to 10 7Copies / μL, with ddH2O as a negative control. Each sample was tested 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 the isothermal amplification kit (purchased from GenDx Biotech Ltd.) according to the manufacturer's instructions. The total ERA reaction volume was 50 μL, including 20 μL of solvent (as shown in the ERA isothermal amplification kit from GenDx Biotech Ltd.), 2.5 μL each of 10 μM forward primer F2 and reverse primer R2 (specific primer sequences are shown in Table 1), and 10 μM... 7 Copy of plasmid template ( Figure 1 (The plasmid shown). Add an appropriate amount of ddH2O to bring the total volume to 48 μL. After thoroughly mixing the mixture, transfer it to the basic amplification reagent provided in the kit (such as the basic amplification reagent shown in the ERA isothermal amplification kit from GenDx Biotech). Then, add 2 μL of activator (such as the activator shown in the ERA isothermal amplification kit from GenDx Biotech) to the tube cap, introduce the activator into the premix by brief centrifugation, briefly vortex to mix, and then centrifuge rapidly. Incubate the reaction system in a water bath at 37°C for 20 min. After the reaction, purify the amplification product for subsequent experiments.

[0189] 1.7 Establishment and Optimization of the MP-ERA-Cas12a Two-Pipe Method Detection System

[0190] System optimization was performed in the initial stages of establishing the MP detection system binding to Cas12a. The optimal composition of the detection system was determined by varying the concentration of the FQ fluorescent probe and the Cas12a / crRNA ratio. Specifically, a system containing 10... 7 Copy of positive recombinant plasmid ( Figure 1 Using the plasmid shown as a template, the concentrations of the FQ reporter gene (purchased from Tolo Biotech, FAM-TTTTT-BHQ1) were tested at 100 nM, 200 nM, 300 nM, 400 nM, and 500 nM. The Cas12a / crRNA ratio was also evaluated at 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 ​​observed in the experimental results.

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

[0192] A single-tube reaction system was established based on the two-tube MP-ERA-Cas12a system, enabling isothermal amplification and detection in the same tube. Five sample loading methods were tested based on the two-tube system. Figure 4 The plasmid template used has 400 copies. Figure 1 The following ingredients were prepared: plasmid (as shown in Table 1), 4 μL of solvent, 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 (as shown in the ERA isothermal amplification kit from GenDx Biotech), 1 / 5 of lyophilized microspheres (as shown in the ERA isothermal amplification kit from GenDx Biotech), 0.25 μL of 10 μM LbCas12a (final concentration 125 nM), and 1 μL of 10×HOLMES Buffer. The Cas12a activator consisted of 1.562 μL of 4 μM crRNA (final concentration 312.5 nM) and 1 μL of 3 μM HOLMESssDNA reporter (FAM) fluorescent probe (final concentration 150 nM), with the ratio of Cas12 protein to guide RNA being 1:2.5. Water was added to a final volume of 20 μL (the original system).

[0193] The finalized sample loading method (i.e., the optimized system) requires three premixed solutions: the ERA amplification system, the Cas12a detection system, and the Cas12a activator. The Cas12a activator is added to the reaction tube cap. The amplification system includes 4 μL of solvent, 6.85 μL of ddH2O, and 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 (as shown in the ERA isothermal amplification kit purchased from GenDx Biotech Co., Ltd.), and 1 / 5 of lyophilized microspheres (as 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 10x 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), the ratio of Cas12 protein to guide RNA is 1:2.5. The fluorescent probe is purchased from Tolo Biotech Co., Ltd., labeled with FAM fluorescent reporter group at the 5' end and BHQ1 at the 3' end, 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 are added to the bottom of the tube, and 3.5 μL of the Cas12a activator is placed in the tube cap. There is no need to open the cap during the experiment. The system is then incubated at 37°C for 20 min to promote amplification. After that, the Cas12a activator is dropped to the bottom of the tube by brief centrifugation. Finally, the reaction is carried out in the 96 instrument at 37°C for 35 min. 96 instrument at 37°C for 35 min.

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

[0195] On the basis of the optimized one-tube method, we combined the trans -cleavage function of LbCas12a protein with the isothermal amplification technology of ERA, and introduced a CRISPR-LFA ssDNA reporter combined with biotin (F-B) (the fluorescent reporter group was synthesized by Sheng Wu Bioengineering (Shanghai) Co., Ltd., with FAM fluorescent reporter group labeled at the 5' end and Biotin labeled at the 3' end, and the specific sequence was FAM-TTTTTTTTATT-Biotin) to realize the naked-eye observation of the detection results. This method aims to develop a new method for detecting MP, which is called MP-ERA-Cas12a LFA system. During the establishment of the LFA system, the ERA amplification and Cas12a detection system remain the same as above, and the only change is that the reporter probe F-Q (purchased from Tolo Biotech Co., Ltd., with FAM fluorescent reporter group labeled at the 5' end and fluorescent quencher BHQ1 labeled at the 3' end. The specific sequence is FAM-TTTTT-BHQ1) is replaced by the FAM and biotin modified reporter gene F-B (the fluorescent reporter group was synthesized by Sheng Wu Bioengineering (Shanghai) Co., Ltd., with FAM fluorescent reporter group labeled at the 5' end and Biotin labeled at the 3' end. The specific sequence is FAM-TTTTTTTTATT-Biotin).

[0196] The F-B probe in LFA is functionally equivalent to the antigen. Both too high or too low concentrations of antigens can lead to the occurrence of "hook effect". Therefore, the first thing we need to do in this study is to determine the optimal concentration of the probe. The specific operation is 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 insert the blank test strip directly into the mixed probe diluent to observe the bands produced. The case where the lowest concentration of the probe and the T line cannot be seen is determined as the optimal reaction condition.

[0197] Although the fluorescent system can produce preliminary results within 10 min, the test strip used in this study shows a slight possibility of false positive, and there are significant differences between the two systems. In order 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 tube was incubated at 37°C for 5, 10, 15, 20, 30 and 60 min, respectively. After incubation, the reaction product was transferred to the sample absorption pad of the test strip. The template concentration was 10 7 copies / μL, ddH2O as negative control, and each sample was repeated three times. After completion, the amplification product and test strip were properly disposed of in a sealed bag.

[0198] 1.10 Sensitivity and specificity of MP-ERA-Cas12a system

[0199] To evaluate the sensitivity of the above three methods, we detected nucleic acids at different concentrations. MP nucleic acid targets were diluted from 10 7 Serial dilutions were performed 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 ddH2O as a blank control. The nucleic acids of all bacterial samples were extracted by the rapid bacterial genomic DNA isolation kit (purchased from Shanghai Biotechnology), and the fungal nucleic acids were extracted by 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 Clinical feasibility verification of the MP-ERA-Cas12a system

[0201] To demonstrate the clinical feasibility of the MP-ERA-Cas12a system, a total of 34 clinical samples from the People's Hospital of Huaibei City were collected. These samples were all nasopharyngeal swabs from patients diagnosed with MP. DNA extraction was performed on 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 of the MP-ERA-Cas12a is shown in Figure 3 . First, through the ERA technology, the DNA recombinase combines with 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, thereby achieving exponential amplification of the target gene. Subsequently, under the guidance of crRNA, Cas12a recognizes the target sequence through the PAM site. Finally, the transcleavage function of Cas12a is activated, leading to the random cleavage of ssDNA in the environment. The ends of these ssDNA are modified fluorescent groups and quencher groups, which can be detected by the GenePure 96. If the detection result is negative, it indicates that the ssDNA reporter probe has not been digested, and therefore no fluorescence signal can be detected.

[0205] In a tube method experiment, the amplification system at the bottom of the test tube was activated at 37°C, and the target gene was exponentially amplified through the ERA system. Due to the lack of crRNA, the activity of Cas12a cannot be activated. After the completion of the ERA reaction, crRNA and F-Q probes were added to the system, and a brief centrifugation was performed to activate the detection activity related to the transcleavage. Cas12a recognizes and binds to the amplification product by pairing with crRNA, thereby activating its cis-cleavage and trans-cleavage activity. Importantly, 3.5 μL of Cas12a activator on the test tube cap remains in place due to surface tension, and the tube method system eliminates the need to open the cap, thereby reducing the risk of aerosol contamination. This method not only achieves specific detection through crRNA guidance, but also solves the problem of false positives caused by non-specific amplification, and promotes the amplification and output of signals. 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 test strip contains two lines: the lower control line (C line) and the upper detection line (T line). The C line is coated with avidin, while the T line is coated with a goat anti-mouse secondary antibody, and the anti-FAM monoclonal antibody is labeled with colloidal gold. The complete CRISPR probe captures all colloidal gold on the C line. When the probe is cut by Cas enzyme, the colloidal gold-bound fragments cannot be captured on the C line, but form a band on the T line. The presence or absence of the T line band determines whether the Cas enzyme is activated. Visible T line indicates a positive result, while invisible T line indicates a negative result. The presence of C line confirms the normal function of the test strip. Compared with other methods that use qPCR to observe 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 combination of MP-ERA-Cas12a system

[0208] In this experiment, three recombinant plasmids were designed (containing three insert fragments of 725-1325 sites of P1 sequence, 1698-2152 sites of P1 sequence, and 3778-4238 sites of P1 sequence, three pairs of ERA amplification primers, and five crRNAs (as shown in Table 1), and the specific combination is as shown in Table 2. Figure 2 Figure 5 ​), the fluorescence curves of the five groups of reactions showed a linear upward trend after the combined primer pairs and crRNA were added to the system. The results of the reactions using F1R1-crRNA1, F3R3-crRNA3-1, and F3R3-crRNA3-2 showed that the fluorescence signal of the control group using ddH2O was enhanced, indicating that the effects of these three combinations lacked specificity for MP detection. Therefore, the results of the test groups failed to provide strong evidence of MP detection Figure 5 A, D, and E). As for F2R2-crRNA2-1 and F2R2-crRNA2-2, the fluorescence curve generated by the F2R2-crRNA2-1 reaction showed a relatively steep upward trend, reaching saturation at 20 min. In contrast, the fluorescence curve of the F2R2-crRNA2-2 reaction had a gentler slope, and saturation was not reached 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 about 35 times that of the negative control Figure 5 B and C). Therefore, F2R2-crRNA2-1 was ultimately selected due to its higher fluorescence intensity.

[0209] The use of primer F2R2 to reach the reaction plateau and the high fluorescence value were considered to be the most effective primers. 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 to be the most suitable target sequence (positions 1698-2152 of P1).

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

[0211] Different concentrations of F-Q probes were added to the reaction to optimize the system. As shown in Figure 6 A, when the concentration of F-Q was 100 nM and 200 nM, the fluorescence curve tended to be stable after about 30 min; when the concentration of F-Q was 300 nM, 400 nM, and 500 nM, the fluorescence curve tended to be stable after about 35 min. Notably, the fluorescence intensity of the F-Q probe plateau in the concentration range of 300 nM to 500 nM exceeded that of other concentrations, as shown in Figure 6 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 F-Q probe as the optimal reaction condition for the MP-ERA-Cas12a fluorescence system.

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

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

[0214] During the development of the one-tube system, five sampling methods were evaluated. The results show that Figure 7 A), three reactions Figure 4 C, D and E) do not have obvious fluorescence values. In contrast, the results of the other two addition methods show that the fluorescence value of the reaction in which only the Cas12a activator (crRNA and F-Q probe) is added to the cap is higher than that of the reaction in which the entire detection system is added to the cap Figure 4 A, B and 7A). Therefore, the next step of system optimization will be based on the sample addition method shown in Figure 4 B.

[0215] The amplification system includes 4 μL of dissolving agent, 6.85 μL of ddH2O, 10 7 copies of plasmid template Figure 1The assay contained 0.5 μL of the plasmid shown in Table 1, 0.5 μL of each of the 10 μM forward primer F2 and reverse primer R2, 2 μL of activator (as shown in the ERA isothermal amplification kit from GenDxBiotech), and 1 / 5 of the lyophilized microspheres (as shown in the ERA isothermal amplification kit from GenDxBiotech). The assay 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 FQ fluorescent probe (purchased from Tolo Biotech, labeled with the FAM fluorescent reporter group at the 5′ end and the BHQ1 fluorescent quencher group at the 3′ end, the specific sequence being FAM-TTTTT-BHQ1). Before the reaction, 15.25 μL of amplification system and 1.25 μL of detection system were added to the bottom of the tube, while 3.5 μL of Cas12a activator was placed in the cap. The cap did not need to be opened during the experiment. The system was then incubated at 37°C for 20 min to promote amplification. Afterwards, the Cas12a activator was briefly centrifuged to allow it to settle to the bottom of the tube. The final determined concentrations were: fluorescent probe 300 nM, Cas12a 125 nM, forward primer F2 and reverse primer R2 250 nM, guide RNA 312.5 nM, and the Cas12a to guide RNA ratio 1:2.5.

[0216] like Figure 7 As shown in B, the unoptimized one-tube system ( Figure 4 The sample addition method shown in B (unoptimized system) resulted in a low fluorescence value (0.826241741 ± 0.040). In contrast, the optimized one-tube method system... Figure 4 The fluorescence value of the sample addition method shown in B (with the system optimized) increased by approximately 6.24 times. This finding highlights the importance of system optimization in advancing the establishment of the one-tube method. Comparing the optimized one-tube method with the optimized two-tube method clearly shows that, with the same template DNA concentration, the one-tube method reaches the plateau phase earlier and exhibits a 1.6-fold increase in fluorescence intensity. This method not only simplifies the operational steps but also improves the resulting fluorescence value.

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

[0218] To minimize the intensity of the T-line, the optimal F-B probe concentration of the MP-ERA-Cas12a LFA system must be determined. It was observed from the test strip that the negative control only produced a band on the T-line. As the F-B probe concentration decreased, the band intensity on the T-line increased, while the band on the C-line gradually faded. Notably, at an F-B probe concentration of 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 chosen as the optimal F-B probe concentration of the MP-ERA-Cas12a LFA system.

[0219] As an endpoint monitoring system, the incubation time of the MP-ERA-Cas12a LFA system is crucial and needs to be optimized and explored. As shown in Figure 8 B, starting from 20 min, the increase in incubation time was associated with an increase in T-line intensity and a decrease in C-line intensity. After 25 min of incubation, the color difference between positive and negative results was already very significant. Therefore, 25 min, which had a clear color difference and took less time, was chosen as the optimal incubation time.

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

[0221] To evaluate the sensitivity of the MP-ERA-Cas12a system, we detected nucleic acids Figure 9 ) of different concentrations. The template concentration was gradually reduced from 10 7 copies / μL to 1 copy / μL, resulting in a gradual decrease in fluorescence intensity. The fluorescence intensity was statistically analyzed at 35 min, and the data are shown in Figure 9 B and Figure 9 D. The actual detection limit of the two-tube and one-tube methods was as low as 1 copy / μL. Notably, significant differences were observed within 20 min after Cas12a activation, indicating that the MP-ERA-Cas12a system had high sensitivity in detecting MP. Compared with systems with lower copy numbers, systems with higher copy numbers (10 7 , 10 6 , and 10 5 ) produced fluorescence curves that reached the plateau earlier Figure 9 A and C). In addition, the fluorescence value produced by the one-tube system was about 1.5 times that of the two-tube system. As for the LFA system, it could also detect templates of different copies, as shown in Figure 9 E. When the copy number was 10 1 , the T-line band was weak; when the copy number exceeded 10 1 copies / μL, the T-line band became stronger. The actual detection limit of the LFA was as low as 10 2copies / 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 Fig. Figure 9 F, only MP showed a significant fluorescence signal, while the other pathogens showed no fluorescence. The MP detection was performed using the LFA system (Fig. Figure 9 G), and the results were consistent with the fluorescence analysis results of Fig. Figure 9 F. Only MP showed a significant band on the T line, while the other pathogens showed a band only on the C line, indicating that the MP-ERA-Cas12a system had strong specificity for MP.

[0223] 2.6 Verification of 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 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 (Fig. Figure 10 ). Taking the fluorescence results as the gold standard, the positive predictive rate of the MP-ERA-Cas12a test strip system was calculated to be 97.06%, reflecting the high consistency of the two methods in MP detection. This finding further confirmed the reliability and effectiveness of the MP-ERA-Cas12a system in clinical sample detection, emphasizing its strong clinical applicability in practical application.

[0225] Discussion

[0226] Mycoplasma pneumoniae (MP) is a common respiratory pathogen that can cause symptoms such as pharyngitis, tracheitis, and bronchitis. While most infections are self-limiting, a small percentage can progress to pneumonia. There are currently multiple methods for detecting MP, however, these methods often require specialized procedures and advanced laboratory equipment, coupled with high costs, making them unsuitable for field testing. The CRISPR system reduces the non-specific signal brought by non-target amplification of the ERA, while the ERA system enhances the sensitivity of the CRISPR system. Both systems can work at 37°C, simplifying the experimental steps. However, if the amplification system and the CRISPR system are premixed in the reaction tube before the reaction, Cas12a will continuously digest the amplification products and primers at very low template concentrations, leading to reduced amplification efficiency. To solve this problem, most methods use the widely used two-tube system, which separates the amplification reaction and the detection reaction. However, manually transferring the amplification products complicates the process and increases the risk of contamination. Many studies have also adopted a "physical isolation" strategy to solve this problem, however, these solutions can make the operation process more cumbersome.

[0227] In this study, we established a one-tube detection system with higher sensitivity, combining ERA amplification with Cas12a detection, which 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 or physical isolation methods. We simply added the Cas12a activator containing crRNA and F-Q to the tube cap, and mixed the remaining components at the bottom of the tube. This method effectively solves the problem of contamination that may occur during the secondary opening process. The MP-ERA-Cas12a system showed high sensitivity, with a detection limit LOD as low as 1 copy / μL for MP. When used in conjunction with LFA strips, 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 was proven to be effective in clinical sample testing.

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

[0229] In summary, the present study successfully combined the specificity of ERA with the detection ability of CRISPR / Cas12a. Based on the previously established two-tube system, a one-tube system and LFA system were developed for MP detection. The MP-ERA-Cas12a system reaction process does not need to open the cover, thereby reducing the risk of aerosol pollution and 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 LFA is 10 2 copies / μL without the use of advanced instruments or equipment. The system shows strong specificity and is not affected by other pathogens. The MP-ERA-Cas12a technology as a new method for MP detection has important practical application prospects.

[0230] All documents mentioned in this application are incorporated herein by reference as if each individual document were incorporated by reference. Furthermore, it is to be understood that various modifications can be made in substituting and modifying the application as described above and also many changes can be made in the implementation and combinations of the applications' components as would be understood by those skilled in the art, while still accomplishing the objectives of the application.

Claims

1. A method for detecting the presence or absence of Mycoplasma pneumoniae in a sample, which is a one-tube method, and which is a non-diagnostic and non-therapeutic method, characterized in that, The detection method comprises: (a) providing a 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 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 a Cas12 protein, the amplification system contains a nucleic acid molecule of a Mycoplasma pneumoniae to be detected from a sample and an amplification primer for isothermal amplification of the nucleic acid molecule of the Mycoplasma pneumoniae to be detected, wherein the guide RNA guides the Cas12 protein to specifically bind to the nucleic acid molecule of the 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) adding the Cas12 activator in the tube cap to the detection system and the amplification system containing the amplification product at the bottom of the tube in the same tube, and detecting a detectable signal emitted by the nucleic acid probe; and the amplification primer is a F2R2 primer of a Mycoplasma pneumoniae P1 gene, the sequences of which are shown in SEQ ID NO: 8 and 9; the guide RNA is crRNA2-1, the sequence of which is shown in SEQ ID NO: 2, and the nucleic acid probe is a single-stranded DNA; wherein if the nucleic acid probe is cut by the Cas12 protein, it indicates that the sample contains Mycoplasma pneumoniae; and if the nucleic acid probe is not cut by the Cas12 protein, it indicates that the sample does not contain Mycoplasma pneumoniae.

2. The method of claim 1, wherein, The detection comprises qualitative detection or quantitative detection.

3. The method of claim 1, wherein, The detection comprises fluorescence detection or colloidal gold detection.

4. The method of claim 1, wherein, When the fluorescence detection is used for detection, the final concentration of the nucleic acid probe is 200-400 nM.

5. The method of claim 4, wherein, When the fluorescence detection is used for detection, the final concentration of the nucleic acid probe is 250-350 nM.

6. The method of claim 1, wherein, When the colloidal gold detection is used for detection, the concentration of the nucleic acid probe is 20-60 nM.

7. The method of claim 6, wherein, When the colloidal gold detection is used for detection, the concentration of the nucleic acid probe is 30-50 nM.

8. The method of claim 1, wherein, The final concentration of the guide RNA is 250-400 nM.

9. The method of claim 8, wherein, The final concentration of the guide RNA is 300-350 nM.

10. The method of claim 1, wherein, The nucleic acid probe is labeled with a detectable label.

11. The method of claim 1, wherein, The nucleic acid probe is labeled with a fluorescent group and a biotin label.

12. The method of claim 1, wherein, The nucleic acid probe is labeled with a fluorescent group and a quencher group.

13. The method of claim 1, wherein, The isothermal amplification is performed at 37-42℃.

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