Establishment and application of rapid detection method of aspergillus fumigatus era / crispr-cas12a

Abstract

The application provides a rapid detection method of Aspergillus fumigatus ERA / CRISPR-Cas12a, and application thereof, and further provides a corresponding kit and a method for detecting Aspergillus fumigatus. The application first establishes a rapid detection system of ERA (Enzymatic Recombinase Amplification) combined with CRISPR / Cas12a, optimizes the reaction system, and improves the sensitivity of one-pot method, the fluorescence detection limit is 1 fg / μL, the detection sensitivity of the test strip can reach 10 fg / μL, and no cross reaction occurs with other bacteria and fungi. The detection of Aspergillus fumigatus can be completed within 60 min, and the detection result is directly reflected through fluorescence and nucleic acid test strips.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to the establishment and application of a rapid detection method for Aspergillus fumigatus ERA / CRISPR-Cas12a. Background Technology

[0002] Fungi are widely distributed in nature. Under normal circumstances, due to the presence of the human immune system, they generally do not cause infection. However, when immunity is low, the risk of fungal infection increases significantly. Epidemiological statistics show that more than 1 billion people worldwide are affected by fungal infections, and invasive fungal infections cause approximately 1.5 million deaths annually. Compared to equally serious infectious diseases such as tuberculosis or malaria, the number of deaths from invasive fungal diseases may be even greater. Aspergillus fumigatus is a saprophytic filamentous fungus found in soil and compost. It is also the pathogen of many lung diseases in humans, birds, and other mammals. It spreads through asexual spore formation and is the main pathogen causing invasive aspergillosis, which usually occurs in immunocompromised populations such as newborns and the elderly. It is reported that the global incidence of invasive aspergillosis exceeds 300,000 cases annually, but due to inadequate diagnostic techniques, the reported data may only represent 50% to 65% of the actual cases. Even with timely diagnosis and treatment, the related mortality rate can still reach 30% to 80%. Statistics show that aspergillosis remains the most common fungal infection in patients undergoing hematopoietic stem cell transplantation (HSCT). In lung transplant patients, 10%–15% will develop aspergillosis, and the mortality rate is extremely high if early diagnosis and treatment are delayed. Currently, hospitals use a combination of pure fungal culture and microscopic examination to diagnose aspergillosis. While this method has high specificity, it is time-consuming. The GM test detects galactomannan, and in clinical applications, it is often performed on the blood and bronchoalveolar lavage fluid of patients suspected of having active disease. BAL testing has higher sensitivity than serum testing, but it has a high false-positive rate. Molecular biological diagnostic methods, including polymerase chain reaction (PCR) and real-time quantitative PCR, have high specificity. However, PCR technology requires specialized equipment and skilled personnel, making it difficult to implement in remote areas with insufficient medical equipment. Therefore, developing a rapid, accurate, and suitable method for detecting Aspergillus fumigatus in primary healthcare institutions is of significant clinical importance.

[0003] In recent years, isothermal amplification techniques such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase isothermal amplification (RPA) have emerged, demonstrating great potential in the field of pathogen detection due to their advantages such as not requiring complex instruments. ERA technology utilizes a recombinase-primer complex to specifically recognize target sequences at room temperature, combined with single-stranded DNA binding protein (SSB) and DNA polymerase to achieve rapid amplification, completing nucleic acid amplification within 20 minutes, significantly shortening detection time.

[0004] The CRISPR / Cas system is widely used in molecular diagnostics due to its highly specific target recognition capabilities. After recognizing target DNA under the guidance of crRNA, the Cas12a protein activates its non-specific cleavage activity, enabling it to indiscriminately cleave surrounding single-stranded DNA reporter molecules. This characteristic makes it an ideal tool for high-sensitivity fluorescence or test strip detection.

[0005] Therefore, there is an urgent need in this field to develop a visualization detection method that combines high sensitivity and high specificity. Summary of the Invention

[0006] One object of the present invention is to provide a visualization detection method that combines high sensitivity and high specificity.

[0007] In a first aspect of the present invention, a two-step detection system for detecting Aspergillus fumigatus is provided, the detection system comprising:

[0008] (a) Cas12 protein, wherein the Cas12 protein is Cas12 or a Cas protein having similar bypass single-stranded DNA cleavage activity to Cas12;

[0009] (b) A guide RNA that guides the Cas12 protein to specifically bind to the nucleic acid molecules of Aspergillus fumigatus; and

[0010] (c) A nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA;

[0011] The ratio (molar ratio) of the Cas12 protein to the guide RNA is 1-3:1-3, preferably 1:1-3.

[0012] In another preferred embodiment, the concentration of Cas12 protein is 150-500 nM, more preferably 200-400 nM, and even more preferably 200-300 nM.

[0013] In another preferred embodiment, the working temperature of the Cas12 protein is 35-40°C, preferably 35-38°C.

[0014] In another preferred embodiment, the detection includes qualitative detection or quantitative detection.

[0015] In another preferred embodiment, the detection includes fluorescence detection and colloidal gold detection.

[0016] In another preferred embodiment, the fluorescence detection method is performed using an enzyme-linked immunosorbent assay (ELISA) reader or a fluorescence spectrophotometer.

[0017] In another preferred embodiment, the detection system further contains (d) buffer solution.

[0018] In another preferred embodiment, the inspection system also contains nucleic acid molecules of Aspergillus fumigatus to be detected.

[0019] In another preferred embodiment, the nucleic acid molecules of Aspergillus fumigatus are nucleic acid molecules that have undergone isothermal amplification.

[0020] In another preferred embodiment, the nucleic acid molecule of the Aspergillus fumigatus to be detected is selected from the group consisting of single-stranded DNA, double-stranded DNA, or combinations thereof.

[0021] In another preferred embodiment, the nucleic acid molecule of the Aspergillus fumigatus to be detected is artificially synthesized DNA.

[0022] In another preferred embodiment, the nucleic acid molecules of the Aspergillus fumigatus to be detected are derived from a non-cultured sample or a sample obtained by a culture method selected from the group consisting of: cell culture, bacterial culture, viral culture, fungal culture, microbial culture, organoid culture, in vivo enrichment culture of animals, and plant culture.

[0023] In another preferred embodiment, the nucleic acid molecules of the Aspergillus fumigatus to be detected include wild-type or mutant DNA.

[0024] In another preferred embodiment, the nucleic acid molecule of the Aspergillus fumigatus to be detected includes DNA, such as cDNA, obtained by reverse transcription or amplification of RNA.

[0025] In another preferred embodiment, the sample is an in vitro or ex vivo sample.

[0026] In another preferred embodiment, the isothermal amplification includes ERA, RPA, and RAA, with ERA being preferred.

[0027] In another preferred embodiment, the concentration of the Aspergillus fumigatus nucleic acid molecules to be detected before amplification is 10. 0 -10 6 aM, better location, 10 3 -10 6 aM.

[0028] In another preferred embodiment, the concentration of the nucleic acid molecules of Aspergillus fumigatus to be detected before amplification is 100 pg / µL - 1 fg / µL, more preferably, 100 pg / µL - 10 fg / µL, and even more preferably, 100 pg / µL - 100 fg / µL.

[0029] In another preferred embodiment, the nucleic acid molecules of Aspergillus fumigatus were amplified using the amplification primers shown in SEQ ID NO.3 and SEQ ID NO.4.

[0030] In another preferred embodiment, the guide RNA includes a direct repeat (DR) sequence capable of binding to the Cas12 protein and a guide sequence capable of targeting the target sequence.

[0031] In another preferred embodiment, the guide RNA comprises any of the sequences shown in SEQ ID NO. 1-2.

[0032] In another preferred embodiment, the concentration of the guide RNA is 100-500 nM, more preferably 200-400 nM.

[0033] In another preferred embodiment, the nucleic acid probe is labeled with a detectable tag.

[0034] In another preferred embodiment, the detectable marker includes a fluorescent group and a quenching group.

[0035] 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 combinations thereof.

[0036] In another preferred embodiment, the quenching group is selected from the group consisting of: TAMARA, BHQ1, BHQ2, BHQ3, DABSYL, Dabcyl, eclipse, MGB, or combinations thereof.

[0037] In another preferred embodiment, the fluorescent group and the quenching group are each independently located at the 5' end, 3' end, and middle of the nucleic acid of the nucleic acid probe.

[0038] In another preferred embodiment, the length of the nucleic acid probe is 5-50 nt, more preferably 5-30 nt, more preferably 5-20 nt, more preferably 5-15 nt, and even more preferably 5-10 nt.

[0039] In another preferred embodiment, the nucleic acid probe comprises single-stranded DNA.

[0040] In another preferred embodiment, the nucleic acid probe comprises single-stranded DNA with a detectable label.

[0041] In another preferred embodiment, the nucleic acid probe is a single-stranded DNA labeled with a fluorescent group and a quencher group.

[0042] In another preferred embodiment, when the detection is a fluorescence detection method, the concentration of the nucleic acid probe is 50-500 nM, preferably 300-500 nM.

[0043] In another preferred embodiment, when the detection is a colloidal gold detection method, the concentration of the nucleic acid probe is 100-800 nM, more preferably 200-800 nM or 200-400 nM.

[0044] In another preferred embodiment, when the detection is a colloidal gold detection method, the CRISPR cutting time is 15-40 minutes, preferably 15-30 minutes.

[0045] 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 combinations thereof.

[0046] 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 combinations thereof.

[0047] In another preferred embodiment, the source of Cas12a is selected from the group consisting of: *Ciliobacterium*, *Listeria*, *Corynebacterium*, *Sartreus*, *Legionella*, *Treponema*, *Aggregatibacter*, *Eubacterium*, *Streptococcus*, *Lactobacillus*, *Mycoplasma*, *Bacteroides*, *Flaviivola*, *Flavobacterium*, *Azotobacter*, *Sphaerochaeta*, *Glucosidobacterium*, *Neisseria*, *Rochetomyces*, *Parvibaculum*, *Staphylococcus*, *Nitratifractor*, *Mycoplasma*, *Camptotheca*, *Trichophyton*, or combinations thereof.

[0048] In another preferred embodiment, the source of the Cas12a is selected from the group consisting of: *Francisellatularensis* (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), and *Bacteroidales* KA00251. Bacterium 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), and Smithella sp. SC_K08D17.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), Leptospirainadaiserovar Lyme (LiCas12a), Lachnospiraceae bacterium MA2020 *Lachnospiraceae bacterium MA2020* (Lb2Cas12a), *Porphyromonas macaca* (PmCas12a), *Moraxella bovoculi 237* (MbCas12a), *Eubacterium eligens* (EeCas12a), *Candidatus Saccharibacteria bacterium* (CsbCas12a), *Eubacterium rectale* (ErCas12a), *Agathobacter rectalisstrain* (ArCas12a), *Butyrivibrio sp. NC3005* (BsCas12a), *Arcobacter butzleri* (AbCas12a), or combinations thereof.

[0049] In another preferred embodiment, when the nucleic acid molecules of Aspergillus fumigatus are not present in the system, the nucleic acid probe is not cleaved by the Cas12 protein; while when the nucleic acid molecules of Aspergillus fumigatus are present in the system, the nucleic acid probe is cleaved by the Cas12 protein.

[0050] A second aspect of the present invention provides a two-step method for detecting Aspergillus fumigatus in a single tube. The system includes a detection system and an amplification system, wherein the detection system is located at the tube cap and the amplification system is located at the bottom of the tube. The detection system comprises:

[0051] (a) Cas12 protein, wherein the Cas12 protein is Cas12 or a Cas protein having similar bypass single-stranded DNA cleavage activity to Cas12;

[0052] (b) A guide RNA that guides the Cas12 protein to specifically bind to the nucleic acid molecules of Aspergillus fumigatus; and

[0053] (c) A nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA;

[0054] The concentration of the Cas12 protein is 250 nM-1000 nM, preferably 500 nM-1000 nM;

[0055] The amplification system includes an isothermal amplification system for amplifying Aspergillus fumigatus nucleic acid molecules.

[0056] In another preferred embodiment, the concentration of the guide RNA is 30-800 nM, more preferably 80-600 nM, and even more preferably 200-400 nM.

[0057] In another preferred embodiment, the working temperature of the Cas12 protein is 35-40°C, preferably 35-38°C.

[0058] In another preferred embodiment, the detection includes qualitative detection or quantitative detection.

[0059] In another preferred embodiment, the detection includes fluorescence detection and colloidal gold detection.

[0060] In another preferred embodiment, the fluorescence detection method is performed using an enzyme-linked immunosorbent assay (ELISA) reader or a fluorescence spectrophotometer.

[0061] In another preferred embodiment, the detection system further contains (d) buffer solution.

[0062] In another preferred embodiment, the amplification system contains nucleic acid molecules of Aspergillus fumigatus to be detected.

[0063] In another preferred embodiment, the nucleic acid molecule of the Aspergillus fumigatus to be detected is selected from the group consisting of single-stranded DNA, double-stranded DNA, or combinations thereof.

[0064] In another preferred embodiment, the amplification system further comprises:

[0065] (e1) Polymerase used to amplify nucleic acid molecules of Aspergillus fumigatus;

[0066] (e2) Reverse transcriptase used for reverse transcription;

[0067] (e3) dNTPs used for amplification and / or reverse transcription reactions.

[0068] In another preferred embodiment, the amplification system further contains reagents for isothermal amplification reactions.

[0069] In another preferred embodiment, the isothermal amplification includes ERA, RPA, and RAA, with ERA being preferred.

[0070] In another preferred embodiment, the amplification system further contains specific nucleic acid amplification primers.

[0071] In another preferred embodiment, the amplification primers include SEQ ID NO.3 and SEQ ID NO.4.

[0072] In another preferred embodiment, the concentration of the amplification primers is 8-15 μM, preferably 10 μM.

[0073] In another preferred embodiment, the amplification time is 15-30 minutes.

[0074] In another preferred embodiment, the concentration of the nucleic acid molecules of Aspergillus fumigatus to be detected in the system (the concentration of Aspergillus fumigatus nucleic acid molecules before amplification) is 100 pg / µL-1fg / µL, more preferably 100 pg / µL-100fg / µL, and even more preferably 10 pg / µL-100 pg / µL.

[0075] In another preferred embodiment, the guide RNA includes a direct repeat (DR) sequence capable of binding to the Cas12 protein and a guide sequence capable of targeting a target sequence.

[0076] In another preferred embodiment, the guide RNA comprises any of the sequences shown in SEQ ID NO. 1-2.

[0077] In another preferred embodiment, the length of the nucleic acid probe is 5-50 nt, more preferably 5-30 nt, more preferably 5-20 nt, more preferably 5-15 nt, and even more preferably 5-10 nt.

[0078] In another preferred embodiment, the concentration of the nucleic acid probe is 200-400 nM.

[0079] A third aspect of the present invention provides a two-step kit for detecting Aspergillus fumigatus, the kit comprising:

[0080] (i) A first container and a Cas12 protein located within the first container, the Cas12 protein being Cas12 or a Cas protein having similar bypass single-stranded DNA cleavage activity to Cas12;

[0081] (ii) A second container and a guide RNA located within the second container, the guide RNA guiding the Cas12 protein to specifically bind to the nucleic acid molecules of Aspergillus fumigatus;

[0082] (iii) A third container and a nucleic acid probe located within the third container, wherein the nucleic acid probe is single-stranded DNA;

[0083] (iv) The fourth container and the amplification primers located within the fourth container for amplifying the nucleic acid molecules of Aspergillus fumigatus to be detected;

[0084] (v) The fifth container and the reagents located within the fifth container for the isothermal amplification reaction of the nucleic acid molecules of Aspergillus fumigatus to be detected;

[0085] And labels or instructions.

[0086] In another preferred embodiment, the guide RNA comprises any of the sequences shown in SEQ ID NO. 1-2.

[0087] In another preferred embodiment, the ratio (molar ratio) of the Cas12 protein to the guide RNA is 1-3:1-3, more preferably 1:1-3.

[0088] In another preferred embodiment, when the detection is a fluorescence detection method, the concentration of the nucleic acid probe is 50-500 nM, preferably 300-500 nM.

[0089] In another preferred embodiment, when the detection is a colloidal gold detection method, the concentration of the nucleic acid probe is 100-800 nM, more preferably 200-800 nM, and even more preferably 300-600 nM.

[0090] In another preferred embodiment, the amplification primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

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

[0092] (vii) The sixth container and the nucleic acid molecules of Aspergillus fumigatus to be detected within the sixth container.

[0093] In another preferred embodiment, one or more of the first container, second container, third container, fourth container, fifth container, and sixth container are different containers or the same (same) container.

[0094] In another preferred embodiment, the first container, the second container, and the third container are the same container.

[0095] In another preferred embodiment, the fourth container, the fifth container, and the optional sixth container are the same container.

[0096] A fourth aspect of the present invention provides a kit for detecting Aspergillus fumigatus using a one-tube two-step method, the kit comprising:

[0097] (i) A first container and a Cas12 protein located within the first container, the Cas12 protein being Cas12 or a Cas protein having similar bypass single-stranded DNA cleavage activity to Cas12, the concentration of the Cas12 protein being 250 nM to 1000 nM, preferably 500 nM to 1000 nM.

[0098] (ii) Optionally, a second container and a guide RNA located within the second container, the guide RNA guiding the Cas12 protein to specifically bind to the nucleic acid molecules of Aspergillus fumigatus;

[0099] (iii) A third container and a nucleic acid probe located within the third container, wherein the nucleic acid probe is single-stranded DNA;

[0100] (iv) The fourth container and the amplification primers located within the fourth container for amplifying the nucleic acid molecules of Aspergillus fumigatus to be detected;

[0101] (v) The fifth container and the reagents located within the fifth container for the isothermal amplification reaction of the nucleic acid molecules of Aspergillus fumigatus to be detected;

[0102] (vi) PCR tube;

[0103] And labels or instructions;

[0104] The components in the first container, the optional second container, and the third container are located in the cap of the PCR tube, while the components in the fourth container and the fifth container are located in the bottom of the PCR tube.

[0105] In another preferred embodiment, the guide RNA comprises any of the sequences shown in SEQ ID NO. 1-2.

[0106] In another preferred embodiment, the concentration of the guide RNA is 30-800 nM, more preferably 80-600 nM, and even more preferably 200-400 nM.

[0107] In another preferred embodiment, the kit further includes:

[0108] (vii) The sixth container and the nucleic acid molecules of Aspergillus fumigatus to be detected within the sixth container.

[0109] In another preferred embodiment, the components in the sixth container are located at the bottom of the PCR tube.

[0110] The fifth aspect of this invention provides a two-step method for detecting the presence of Aspergillus fumigatus in a sample, the detection method comprising:

[0111] (a) A reaction system is provided, the reaction system comprising: the detection system and the amplification system described in the first aspect of the present invention, the amplification system containing nucleic acid molecules of Aspergillus fumigatus to be detected from a sample source and amplification primers for isothermal amplification reaction to amplify the nucleic acid molecules of Aspergillus fumigatus to be detected;

[0112] (b) Perform nucleic acid amplification on the Aspergillus fumigatus nucleic acid molecules in the amplification system to obtain the amplification product of Aspergillus fumigatus nucleic acid molecules;

[0113] (c) Mix the detection system with an amplification system containing the amplification product, and detect the detectable signal emitted by the nucleic acid probe;

[0114] If the nucleic acid probe is cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is present in the sample; if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is not present in the sample.

[0115] In another preferred embodiment, the ratio (molar ratio) of the Cas12 protein to the guide RNA is 1-3:1-3, more preferably 1:1-3.

[0116] In another preferred embodiment, the concentration of Cas12 protein is 150-500 nM, more preferably 200-400 nM, and even more preferably 200-300 nM.

[0117] In another preferred embodiment, the working temperature of the Cas12 protein is 35-40°C, preferably 35-38°C.

[0118] In another preferred embodiment, the detection includes qualitative detection or quantitative detection.

[0119] In another preferred embodiment, the detection includes fluorescence detection and colloidal gold detection.

[0120] In another preferred embodiment, the fluorescence detection method is performed using an enzyme-linked immunosorbent assay (ELISA) reader or a fluorescence spectrophotometer.

[0121] In another preferred embodiment, the detection system further contains (d) buffer solution.

[0122] In another preferred embodiment, the amplification system further comprises:

[0123] (e1) Polymerase used to amplify nucleic acid molecules of Aspergillus fumigatus;

[0124] (e2) Reverse transcriptase used for reverse transcription;

[0125] (e3) dNTPs used for amplification and / or reverse transcription reactions.

[0126] In another preferred embodiment, the concentration of the nucleic acid molecules of the Aspergillus fumigatus to be detected is 10. 0 -10 6 aM, better location, 10 3 -10 6 aM.

[0127] In another preferred embodiment, the concentration of the nucleic acid molecules of Aspergillus fumigatus to be detected is 100 pg / µL - 1 fg / µL, more preferably, 100 pg / µL - 10 fg / µL, and even more preferably, 100 pg / µL - 100 fg / µL.

[0128] In another preferred embodiment, the guide RNA comprises any of the sequences shown in SEQ ID NO. 1-2.

[0129] In another preferred embodiment, the concentration of the guide RNA is 100-500 nM, more preferably 200-400 nM.

[0130] In another preferred embodiment, the length of the nucleic acid probe is 5-50 nt, more preferably 5-30 nt, more preferably 5-20 nt, more preferably 5-15 nt, and even more preferably 5-10 nt.

[0131] In another preferred embodiment, when the detection is a fluorescence detection method, the concentration of the nucleic acid probe is 50-500 nM, preferably 300-500 nM.

[0132] In another preferred embodiment, when the detection is a colloidal gold detection method, the concentration of the nucleic acid probe is 100-800 nM, more preferably 200-800 nM, and even more preferably 300-600 nM.

[0133] In another preferred embodiment, the sequences of the amplification primers are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0134] The sixth aspect of this invention provides a two-step method for detecting the presence of Aspergillus fumigatus in a sample using a single tube, the detection method comprising:

[0135] (a) Provide a system according to a second aspect of the present invention, wherein the detection system and the amplification system in the system are located in the same tube, the detection system is located at the tube cap, the amplification system is located at the tube bottom, and the amplification system contains nucleic acid molecules of Aspergillus fumigatus to be detected from a sample source and amplification primers for isothermal amplification reaction to amplify the nucleic acid molecules of Aspergillus fumigatus to be detected;

[0136] (b) At the bottom of the same tube, perform nucleic acid amplification on the Aspergillus fumigatus nucleic acid molecules in the amplification system to obtain the amplification product of the Aspergillus fumigatus nucleic acid molecules;

[0137] (c) In the same tube, the detection system of the tube cap is added to the amplification system containing the amplification product at the bottom of the tube, and the detectable signal emitted by the nucleic acid probe is detected;

[0138] If the nucleic acid probe is cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is present in the sample; if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is not present in the sample.

[0139] In another preferred embodiment, the working temperature of the Cas12 protein is 35-40°C, preferably 35-38°C.

[0140] In another preferred embodiment, the detection includes qualitative detection or quantitative detection.

[0141] In another preferred embodiment, the detection includes fluorescence detection and colloidal gold detection.

[0142] In another preferred embodiment, the fluorescence detection method is performed using an enzyme-linked immunosorbent assay (ELISA) reader or a fluorescence spectrophotometer.

[0143] In another preferred embodiment, the detection system further contains (d) buffer solution.

[0144] In another preferred embodiment, the amplification system further comprises:

[0145] (e1) Polymerase used to amplify nucleic acid molecules of Aspergillus fumigatus;

[0146] (e2) Reverse transcriptase used for reverse transcription;

[0147] (e3) dNTPs used for amplification and / or reverse transcription reactions.

[0148] In another preferred embodiment, the amplification system further contains reagents for isothermal amplification reactions.

[0149] In another preferred embodiment, the isothermal amplification includes ERA, RPA, and RAA, with ERA being preferred.

[0150] In another preferred embodiment, the concentration of the guide RNA is 30-800 nM, more preferably 80-600 nM, and even more preferably 200-400 nM.

[0151] In another preferred embodiment, the amplification primers include SEQ ID NO.3 and SEQ ID NO.4.

[0152] In another preferred embodiment, the concentration of the amplification primers is 8-15 μM, preferably 10 μM.

[0153] In another preferred embodiment, the amplification time is 15-25 minutes.

[0154] In another preferred embodiment, the concentration of the nucleic acid molecules of Aspergillus fumigatus to be detected in the system (the concentration of nucleic acid molecules before amplification) is 100 pg / µL-1fg / µL, more preferably, 100 pg / µL-100fg / µL, and even more preferably, 100 pg / µL-10pg / µL.

[0155] In another preferred embodiment, the length of the nucleic acid probe is 5-50 nt, more preferably 5-30 nt, more preferably 5-20 nt, more preferably 5-15 nt, and even more preferably 5-10 nt.

[0156] In another preferred embodiment, the concentration of the nucleic acid probe is 200-400 nM.

[0157] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0158] Figure 1 The diagram illustrates a one-pot nucleic acid detection process using CRISPR / Cas12a combined with ERA amplification. (A) Schematic diagram of a one-pot rapid nucleic acid detection platform combining the CRISPR / Cas12a system with ERA, fluorescence, and visualization lateral flow test strips. (B) The 15% glycerol and Cas12 detection system is placed at the bottom of the tube, with the ERA amplification mixture added on top. (C) The ERA amplification mixture is dissolved and mixed with sucrose and placed at the bottom of the tube, while the Cas12a mixture is added to the top layer. (D) The ERA amplification mixture and the Cas12a mixture are mixed simultaneously at the bottom of the tube.

[0159] Figure 2This study demonstrates the optimization of the ERA / CRISPR-Cas12a fluorescence system for detecting Aspergillus fumigatus. (A) Screening for optimal ERA primers for Aspergillus fumigatus amplification, followed by gel electrophoresis of the amplified fragments, with M: 2000 bp DNA Marker; (B) Screening for the optimal ssDNA-FQ concentration, and fluorescence curves at different concentrations; NTC served as a negative control (amplification using enzyme-free water as a template). Error bars represent the mean ± standard deviation (SD) of three replicates. (C) Screening for optimal crRNA, and real-time fluorescence detection curves for different crRNAs; (D) Comparison of fluorescence intensity of different crRNAs at 45 min of fluorescence detection; (E) Optimization of the ERA / CRISPR-Cas12a detection system, comparing the effect of different Cas12a / crRNA ratios on the fluorescence curve; (F) Comparison of fluorescence intensity of crRNAs at 45 min of fluorescence detection. (G) Optimization of the lateral flow test strip detection system. The optimal ssDNA-FB concentration was selected, and the effect of different ssDNA-FB concentrations on the test strip detection system without target addition was investigated. T represents the detection limit, and C represents the control line. (H) The effect of different CRISPR / Cas12a incubation times on the appearance of the T line in the test strip detection system.

[0160] Figure 3 The sensitivity analysis of the two-step CRISPR / Cas12a fluorescence method is shown. (AC) at 10 6 -10 0 (a) 1% agarose gel electrophoresis of PCR amplification products using aM target DNA as template, CRISPR / Cas12a fluorescence curves of PCR amplification products at different gradients, and fluorescence values ​​at the end of the detection. (DF) Gel electrophoresis of ERA amplification products at different gradients, fluorescence curves, and final fluorescence intensity. (B) Sensitivity of CRISPR / Cas12a-LFA detection of 100 pg-0.1 fg genomic DNA ERA amplification products. (H, I) Fluorescence curves and fluorescence values ​​of 100 pg-0.1 fg genomic DNA ERA amplification products detected by CRISPR / Cas12a.

[0161] Figure 4This paper demonstrates the establishment and optimization of a one-pot ERA-CRISPR / Cas12a detection system. (A, B) Establishment of the one-pot method. ① Fluorescence curve and fluorescence value of the one-pot method after separating the tube cap; ② Fluorescence curve and fluorescence value of the one-pot method after adding glycerol to the Cas12a detection system; ③ Fluorescence curve and fluorescence value of the one-pot method after adding sucrose to the Cas12a detection system; ④ Fluorescence curve and fluorescence value of the one-pot method during amplification and detection. (C) Optimization of LbCas12a concentration in the one-pot method. (D) Fluorescence curve of the optimized one-pot method. (G) Optimization of incubation time for amplification of ERA products in the one-pot method. (E, H) Sensitivity analysis of the one-pot ERA-CRISPR / Cas12a detection system, using 100 pg-0.1 fg genomic DNA and the sensitivity of the one-pot ERA-CRISPR / Cas12a fluorescence and test strip detection system. (F, I) Specificity analysis of the ERA / CRISPR-Cas12a fluorescence and test strip detection system. Aspergillus fumigatus (AF) Candida albicans (CA), Candida tropicalis (CT) Aspergillus niger (AN) Penicillium chrysogenum (PC) Alternaria alternata (AA) Botrytis cinerea (BC) Aspergillus cristatus (AC). (J) Clinical sample testing, 1-32 are positive clinical samples obtained from the hospital, and 33-62 are negative clinical samples. LFA:+ indicates a positive test result; LFA:- indicates a negative test result. Detailed Implementation

[0162] Through extensive and in-depth research, the inventors have developed for the first time a two-step or one-tube two-step (one-pot) system for detecting Aspergillus fumigatus. Specifically, this invention establishes for the first time a rapid detection system combining ERA (Enzymatic Recombinase Amplification) with CRISPR / Cas12a, which is 100 times more sensitive than the LAMP direct amplification nucleic acid detection system. In this one-pot method, the detection system and amplification system are placed at a constant temperature of 37°C, with the detection system and amplification system respectively placed on the top and bottom of the centrifuge tube, thus solving the problem of aerosol contamination caused by product transfer. By optimizing the reaction system, the sensitivity of the one-pot method is improved, with a fluorescence detection limit of 1 fg / µL and a test strip detection sensitivity of up to 10 fg / µL, without cross-reactivity with other bacteria or fungi. Detection of Aspergillus fumigatus can be completed within 60 minutes, and the results are visually reflected through fluorescence and nucleic acid test strips. Furthermore, clinical applicability was assessed by detecting clinical samples of aspergillosis, demonstrating good performance. Pure culture confirmed 32 positive and 30 negative samples out of 62 sputum samples. Using a one-pot ERA-CRISPR / Cas12a system to detect these 62 clinical samples, the fluorescence detection sensitivity was 93.75% and specificity was 93.33%, while the test strip detection sensitivity was 90.63% and specificity was 96.67%. Moreover, this ERA combined with CRISPR / Cas12a detection is the first application to *Aspergillus fumigatus*, providing a rapid method for real-time and rapid detection of aspergillosis. Based on this, the inventors completed this invention.

[0163] the term

[0164] The term "PCR" stands for Polymerase Chain Reaction, a technique suitable for amplifying target nucleic acids.

[0165] As used herein, “CRISPR” refers to clustered regularly interspaced short palindromic repeats derived from the immune system of microorganisms.

[0166] CRISPR-Cas: A unique genomic element derived from bacteria and archaea, serving as an adaptive immune defense system to defend against invading bacteriophages or foreign nucleic acids. This system consists of clusters of regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas proteins, or Cas for short).

[0167] The term "Cas protein" refers to CRISPR-associated proteins, which are related proteins in the CRISPR system.

[0168] The term "Cas12a" (formerly "Cpf1") refers to a crRNA-dependent endonuclease, which is a type VA enzyme in the CRISPR system.

[0169] The terms "Cas12b" and "C2c1" are used interchangeably and refer to sgRNA-dependent endonucleases, which are type VB enzymes in the CRISPR system.

[0170] The term "PAM" refers to the protospacer-adjacent motif, which is a short DNA sequence that is directly adjacent to the DNA sequence targeted by CRISPR effector proteins. It is essential for Cas12a or Cas12b to cleave double-stranded DNA. For example, the PAM of Cas12a is TTTV.

[0171] The term "target DNA or RNA molecule" refers to the DNA or RNA to be tested or a specific portion thereof when the molecule to be tested is a nucleic acid molecule; when the molecule to be tested is a non-nucleic acid molecule, the target DNA or RNA molecule is a pre-designed nucleic acid sequence.

[0172] Cas protein

[0173] The "Cas protein" mentioned in this article refers to CRISPR-related proteins (sometimes translated as CRISPR-Cas effector proteins, CRISPR / Cas effector proteins, CRISPR-Cas effectors, or CRISPR / Cas effectors), which can be either type V or type VI Cas proteins. Type V Cas proteins, once bound to a cis-cleavage substrate under the guidance of guide RNA to form a ternary complex of Cas protein-guide RNA-cis-cleavage substrate, can induce their trans-cleavage activity, i.e., randomly cleaving single-stranded nucleic acids and their equivalents (nucleic acid equivalents such as nucleic acid analogs).

[0174] The Cas protein described in this specific embodiment is a protein with trans-cleavage activity. In particular, it is a Cas protein that retains its activity, especially trans-cleavage activity, at temperatures higher than the system temperature at which the isothermal amplification reaction is performed.

[0175] The Cas protein described in this specific embodiment can be a type V Cas protein; the Cas protein is selected from the following group: type VA Cas protein, type VB Cas protein, type VC Cas protein, type VD Cas protein, type VE Cas protein, type VF Cas protein, type VG Cas protein, type VH Cas protein, type VI Cas protein, type VJ Cas protein, type VL Cas protein, type VM Cas protein or combinations 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 combinations thereof.

[0176] In specific embodiments, the term "Cas protein" as used herein refers to proteins with trans-cleavage activity, such as Cas12, and also encompasses functional variants of Cas proteins or their homologs or orthologs. As used herein, a "functional variant" of a protein means a variant of a protein that at least partially retains the trans-cleavage activity of that protein. Functional variants may include mutants (which may be insertion, deletion, or substitution mutants), including polymorphs, etc. Functional variants also include fusion products of such proteins with another generally unrelated nucleic acid, protein, polypeptide, or peptide. Functional variants may be naturally occurring or artificial. Advantageous embodiments may involve engineered or non-naturally occurring type V DNA-targeting effector proteins.

[0177] In one embodiment, the V-type Cas protein or its orthologs or homologs may contain one or more mutations, and therefore the nucleic acid molecule encoding it may have one or more mutations. The mutations may be artificially introduced and may include, but are not limited to, one or more mutations in the catalytic domain.

[0178] In one embodiment, the V-type Cas protein may be derived from: *Trichophyton*, *Listeria*, *Corynebacterium*, *Sartreus*, *Legionella*, *Treponema*, *Aggregatibacter*, *Eubacterium*, *Streptococcus*, *Lactobacillus*, *Mycoplasma*, *Bacteroides*, *Flaaviivola*, *Flavobacterium*, *Azotobacter*, *Sphaerochaeta*, *Glucosidobacterium*, *Neisseria*, *Rhodotorula*, *Parvibaculum*, *Staphylococcus*, *Nitratifractor*, *Mycoplasma*, *Camptotheca*, *Trichophyton*, or combinations thereof.

[0179] Table 1. Sub-attributes of the V-type family effect (Source: doi: 10.3389 / fcell.2020.622103)

[0180]

[0181] a V represents A, C, and G.

[0182] b R represents A and G

[0183] C B represents C, G, and T.

[0184] Guide RNA

[0185] As used herein, the “guide RNA” is a fusion of mature crRNA and tracrRNA, or a fusion of mature crRNA and scoutRNA, or crRNA alone as a guide RNA.

[0186] Generally, guide RNA can contain direct repeat sequences (DR sequences) and a guide sequence, or consist primarily of or composed of direct repeat sequences and a guide sequence (also called a spacer sequence in the context of endogenous CRISPR systems). In different CRISPR systems, depending on the Cas protein it relies on, gRNA can include crRNA and tracrRNA, crRNA and scoutRNA, or only crRNA. crRNA and tracrRNA can be artificially fused to form single guide RNA (sgRNA). In some cases, the guide sequence is a polynucleotide sequence, typically 15-28 nt in length, that is sufficiently complementary to the cis-cleaved substrate DNA to hybridize with it and guide the specific binding of the CRISPR / Cas protein-guide RNA complex to the cis-cleaved substrate DNA. The direct repeat sequences can fold into specific structures (such as stem-loop structures) for Cas protein recognition to form the complex. The guide sequence does not need to be 100% complementary to the cis-cleaved substrate DNA. The guide sequence is not complementary to the nucleic acid in the trans-cleavage reporter molecule.

[0187] In some implementations, when optimal alignment is achieved, the complementarity (match) between the guide sequence and its corresponding cis-cleaved 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 capabilities of a person skilled in the art. For example, publicly available and commercially available alignment algorithms and programs exist, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in MATLAB, Bowtie, Geneious, Biopython, and SeqMan.

[0188] The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably and include DNA, RNA, or their hybrids, which can be double-stranded or single-stranded.

[0189] The term "homology" or "identity" is used to refer to the sequence similarity between two polypeptides or two nucleic acids.

[0190] Matching conditions. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (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. Typically, two sequences are compared to produce the greatest possible identity. Such an alignment can be determined by using, for example, the identity of amino acid sequences through conventional methods, referring to, for example, the teachings of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, Pearson & Lipman, 1988, Pro. Natl. Acad. Sci. USA85:244, Thompson et al., 1994, Nucleic Acids Res 22:467380, etc., by computerized execution of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group). Alternatively, the BLAST algorithm, available from the National Center for Biotechnology Information (NCBI www.ncbi.nlm.nih.gov / ), can be used with default parameters.

[0191] Target nucleic acid molecules (nucleic acid molecules of Aspergillus fumigatus to be detected)

[0192] As used herein, when the molecule to be detected is a nucleic acid molecule, the term "target nucleic acid molecule" refers to a polynucleotide molecule or its amplification product, transcription product, or reverse transcription product extracted from a biological sample (the sample to be tested). When the molecule to be detected is a non-nucleic acid molecule, the term "target nucleic acid molecule" is a pre-designed nucleic acid sequence. 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 amoebas, and multicellular organisms (e.g., 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 caused by pathogenic microorganisms such as pathogenic bacteria or viruses). For example, a 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, seroma, saliva, cerebrospinal fluid, aqueous or vitreous fluid, or any bodily secretion, exudate, biological fluid (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab from the surface of the skin or mucous membrane. The sample can also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens, such as tumor biopsies) or may contain cells (primary cells or cultured cells) or a culture medium conditioning 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).

[0193] In other embodiments, the biological sample may be plant cells, callus, tissue or organ (such as root, stem, leaf, flower, seed, fruit), etc.

[0194] In this 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. Alternatively, the target nucleic acid molecule is amplified using a technique known in the art, wherein the amplification technique is isothermal amplification, which may be ERA, RPA, or RAA, with ERA being preferred.

[0195] In this invention, the target nucleic acid molecules are amplified using isothermal amplification reagents, all of which are from isothermal amplification kits of Suzhou Xianda Gene Technology Co., Ltd.

[0196] A two-step amplification and detection method using a single tube without opening the cap.

[0197] This specific embodiment discloses a two-step method for amplifying and detecting target nucleic acid molecules (nucleic acid molecules of Aspergillus fumigatus to be detected) without opening the tube.

[0198] In a preferred embodiment, this specific embodiment provides a two-step method for detecting the presence of Aspergillus fumigatus in a sample using a single tube, the detection method comprising:

[0199] (a) Provide a system according to a second aspect of the present invention, wherein the detection system and the amplification system in the system are located in the same tube, the detection system is located at the tube cap, the amplification system is located at the tube bottom, and the amplification system contains nucleic acid molecules of Aspergillus fumigatus to be detected from a sample source and amplification primers for isothermal amplification reaction to amplify the nucleic acid molecules of Aspergillus fumigatus to be detected;

[0200] (b) At the bottom of the same tube, perform nucleic acid amplification on the Aspergillus fumigatus nucleic acid molecules in the amplification system to obtain the amplification product of the Aspergillus fumigatus nucleic acid molecules;

[0201] (c) In the same tube, the detection system of the tube cap is added to the amplification system containing the amplification product at the bottom of the tube, and the detectable signal emitted by the nucleic acid probe is detected;

[0202] If the nucleic acid probe is cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is present in the sample; if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is not present in the sample.

[0203] In this invention, a representative nucleic acid probe is a single-stranded DNA or single-stranded RNA with a luminescent group and a quencher group attached to both ends, or a single-stranded DNA or single-stranded RNA with a luminescent group and biotin attached to both ends. Therefore, once the probe is cut, the luminescent group can emit light or form a band on the T line.

[0204] In this specific embodiment, the presence of target nucleic acid molecules, such as nucleic acid molecules from Aspergillus fumigatus, in a sample can be determined by detecting fluorescence.

[0205] In this specific embodiment, the suitable Cas protein is a V-type Cas protein with trans-cleavage activity, preferably Cas12a or Cas12b, and more preferably FnCas12a, LbCas12a, ErCas12a, or Evcas12a. , Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a, CeCas12a, PrCas12a, CsbCas12a, BhCas12a, SsCas12a, Lb3Cas12a, BpCas12 a, PdCas12a, BfCas12a, PcCas12a, cMtCas12a, PeCas12a, LiCas12a, Lb2Cas12a, PmCas12a, MbCas12a, EeCas12a, CsbCas12a, ArCas12a, BsCas12a, AbCas12a , AsCas12a, or combinations thereof.

[0206] The method described in this specific embodiment can rapidly detect the presence of target nucleic acid molecules (Aspergillus fumigatus) in a sample. Furthermore, by combining it with isothermal amplification techniques (such as ERA, RPA, or RAA, preferably ERA), the sensitivity and specificity of this detection method can be significantly improved. Various isothermal amplification techniques in the prior art can theoretically be used in this invention; this specific embodiment only lists preferred embodiments. The components used in the various amplification techniques in this application, such as:

[0207] NTPs, buffer, and Mg required for RNA amplification 2+ And so on, as well as RNase H required when reverse transcriptase is unable to digest single-stranded RNA;

[0208] dNTPs, buffer, and Mg required for DNA amplification 2+ All of these are provided for the purchase of reagent kits;

[0209] These contents are common knowledge in this field, so they are not specifically described in this application.

[0210] Two-step amplification and detection method

[0211] This specific embodiment discloses a two-step method for amplifying and detecting target nucleic acid molecules (nucleic acid molecules of Aspergillus fumigatus to be detected).

[0212] In a preferred embodiment, this specific embodiment provides a two-step detection method for target nucleic acid molecules (nucleic acid molecules of Aspergillus fumigatus to be detected), the detection method comprising:

[0213] (a) A reaction system is provided, the reaction system comprising: the detection system of claim 1 and an amplification system, the amplification system containing nucleic acid molecules of Aspergillus fumigatus to be detected from a sample source and amplification primers for isothermal amplification reaction to amplify the nucleic acid molecules of Aspergillus fumigatus to be detected;

[0214] (b) Perform nucleic acid amplification on the Aspergillus fumigatus nucleic acid molecules in the amplification system to obtain the amplification product of Aspergillus fumigatus nucleic acid molecules;

[0215] (c) Mix the detection system with an amplification system containing the amplification product, and detect the detectable signal emitted by the nucleic acid probe;

[0216] If the nucleic acid probe is cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is present in the sample; if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is not present in the sample.

[0217] This invention significantly improves the sensitivity and specificity of the one-tube two-step method for detecting Aspergillus fumigatus by optimizing the concentrations of Cas12, guide RNA, and probes, and by screening amplification primers (the fluorescence value of the optimized one-tube two-step method is 3 times higher than that of the unoptimized method).

[0218] The main advantages of this invention include:

[0219] (1) The inventors have developed a two-step or one-tube two-step (one-pot) system for detecting Aspergillus fumigatus for the first time. Specifically, this invention establishes a rapid detection system combining ERA (Enzymatic Recombinase Amplification) with CRISPR / Cas12a, which is 100 times more sensitive than the LAMP direct amplification of Aspergillus fumigatus nucleic acid detection system. In this invention's one-pot method, the detection system and amplification system are placed at a constant temperature of 37°C, with the detection system and amplification system respectively placed on the top and bottom of the centrifuge tube, thus solving the problem of aerosol contamination caused by product transfer. By optimizing the reaction system, the sensitivity of the one-pot method is improved, with a fluorescence detection limit of 1 fg / µL and a test strip detection sensitivity of up to 10 fg / µL, without cross-reactivity with other bacteria or fungi. Detection of Aspergillus fumigatus can be completed within 60 minutes, and the detection results are visually reflected through fluorescence and nucleic acid test strips. Furthermore, the clinical applicability was assessed by detecting clinical samples of aspergillosis, demonstrating good performance. Pure culture confirmed 32 positive and 30 negative samples out of 62 sputum samples. Using a one-pot ERA-CRISPR / Cas12a system to detect these 62 clinical samples, the fluorescence method showed a sensitivity of 93.75% and a specificity of 93.33%, while the test strip method showed a sensitivity of 90.63% and a specificity of 96.67%. Moreover, this ERA combined with CRISPR / Cas12a detection is the first application of this method to *Aspergillus fumigatus*, providing a rapid method for real-time and rapid detection of aspergillosis.

[0220] (2) In this invention, the ERA-CRISPR / Cas12a system was applied to the rapid detection of Aspergillus fumigatus. By targeting the conserved gene anxC4 in Aspergillus fumigatus, highly specific primers and crRNA were designed and combined with lateral flow test strips to establish a visual detection method with both high sensitivity and high specificity. By placing the reaction system and the detection system on the top and bottom of the centrifuge tube respectively, the one-pot reaction system was optimized, solving the problems of aerosol contamination and low amplification efficiency in traditional methods. The reliability of the ERA-CRISPR / Cas12a technology was verified through clinical samples. The development of this technology not only provides a new tool for the early diagnosis of Aspergillus fumigatus infection, but also provides a technical reference for the rapid detection of other fungal pathogens.

[0221] (3) Based on the anxC4 gene of Aspergillus fumigatus, this invention establishes a rapid detection system combining ERA (Enzymatic Recombinase Amplification) with CRISPR / Cas12a. Under a constant temperature of 37℃, the reaction system and detection system are placed on the top and bottom of a centrifuge tube, respectively, thus solving the problem of aerosol contamination caused by product transfer. By optimizing the reaction system, the sensitivity of the one-pot method is improved, with a fluorescence detection limit of 1 fg / µL and a test strip detection sensitivity of up to 10 fg / µL, without cross-reactivity with other bacteria or fungi. Detection of Aspergillus fumigatus can be completed within 60 min, and the detection results are visually reflected through fluorescence and nucleic acid test strips. Furthermore, the clinical applicability was assessed by detecting clinical samples of aspergillosis, demonstrating good performance. Pure culture confirmed 32 positive and 30 negative samples out of 62 sputum samples. Using a one-pot ERA-CRISPR / Cas12a system to detect these 62 clinical samples, the fluorescence method showed a sensitivity of 93.75% and a specificity of 93.33%, while the test strip method showed a sensitivity of 90.63% and a specificity of 96.67%. Moreover, this ERA combined with CRISPR / Cas12a detection is the first application of this method to *Aspergillus fumigatus*, providing a rapid method for real-time and rapid detection of aspergillosis.

[0222] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0223] Unless otherwise specified, all experimental materials involved in this invention can be obtained from commercially available sources.

[0224] 1. Materials and Methods

[0225] 1.1 Materials and Instruments

[0226] The *Aspergillus fumigatus* strain used in this study (ATCC MYA-4609) was purchased from ATCC. All routine primers used in the experiments were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and all reagents used in the culture media were from Sangon Biotech (Shanghai) Co., Ltd. The P6 High-Fridelity Primix, LbCas12a, Cas12a High Yield crRNA Synthesis and Purification Kit, modified FAM and BHQ1 reporter genes (ssDNA), and lateral flow test strips were all purchased from Shanghai Tulugang Biotechnology Co., Ltd. The DNA product purification kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the ERA nucleic acid amplification kit was purchased from Suzhou Xianda Biotechnology Co., Ltd.; the sputum extraction kit was purchased from Hangzhou Dilan Biotechnology Co., Ltd.; the real-time PCR instrument was Roche Lightcycler96; and the gel imaging system was purchased from Shanghai Qinxiang Scientific Instruments Co., Ltd. The strain used in this study was obtained from Huaibei Normal University. *Aspergillus fumigatus* sputum samples were provided by Huaibei People's Hospital, and the sample collection was approved by the hospital's ethics committee (No.: 2024-052).

[0227] 1.2 Culture and genome extraction of Aspergillus fumigatus strains

[0228] The Aspergillus fumigatus strain was cultured on potato dextrose agar (PDA) plates at 30°C for 4 days. Spores were inoculated into 5 mL of PDA liquid medium and cultured for 24 h. Mycelial balls and spores were collected and processed according to the fungal genome extraction procedure.

[0229] 1.3 Design of specific primers and crRNA

[0230] The *Aspergillus fumigatus* anxC4 gene (GenBank accession number AY598940) was located. Based on the selected anxC4-specific amplification fragment (SEQ ID NO.5), specific crRNA sequences were designed using the online tool CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). Two high-scoring crRNA sequences, each 41 nt in length, were selected based on the software's rating and PAM position. crRNA 1 and crRNA 2 were obtained using the Cas12a High Yield crRNA Synthesis and Purification Kit. Using NCBI BLAST and Primer Premier 5, one set of PCR primers (PCR-F / R) and three sets of ERA primers (ERA-F1 / 1R, ERA-F2 / R2, and ERA-F3 / R3) were designed in the crRNA 1 and crRNA 2 gene sequence regions; all were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Table 2).

[0231]

[0232] Table 2. Primers and crRNAs used in this study

[0233]

[0234] 1.4 PCR and ERA amplification system

[0235] The PCR reaction system consisted of 25 μL P6 High-Fridelity Primix, 2 µL each of forward and reverse primers (10 µmol / L), and 2 μL of genomic DNA template, with enzyme-free water added to a final volume of 50 μL. The PCR reaction was performed using an Eppendorf PCR instrument, following the following amplification steps: pre-denaturation at 95°C for 5 min, followed by denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, for 35 cycles, and a final extension at 72°C for 10 min.

[0236] ERA amplification system (50 µL): Prepare a pre-mixed solution of 20 µL solvent, 2.5 µL each of 10 µM forward and reverse primers, and 2 µL genomic DNA template with 23.5 µL enzyme-free water. Transfer the mixture to the basic reaction unit, ensuring the lyophilized powder in the basic reaction unit of the ERA nucleic acid amplification kit is fully dissolved. Add 2 µL of activator to the inside of the reaction tube cap, centrifuge briefly, and then incubate at 37°C for amplification. After PCR and RPA amplification, perform agarose gel electrophoresis on 2.5 µL of the amplification product to confirm the amplification results.

[0237] 1.5 CRISPR-Cas12a Detection System

[0238] In a 20 μL CRISPR-Cas12a reaction mixture, 10×HOLMES Buffer, 0.5 µL Cas12a, 2 µL crRNA, and 2 µL ssDNA reporter probe (FAM-TTTTT-BHQ1) were added. The CRISPR-Cas12a fluorescence detection reaction was performed on a Roche LightCycler® 96 real-time PCR instrument at 37°C. Fluorescence signals were collected every 30 seconds for 45 minutes (90 cycles at 37°C for 30 seconds each).

[0239] 1.6 ERA primers and crRNA screening:

[0240] Genomic DNA from *Aspergillus fumigatus* was amplified using three pairs of ERA primers (Table 2), and primers with single-specific amplification products were screened. Two designed crRNAs (Table 2) were added to the CRISPR / Cas12a system, and the optimal combination of ERA amplification primers and crRNAs was selected based on the endpoint fluorescence value and amplification curve. Enzyme-free water was used as a control, and the experiment was performed in triplicate.

[0241] 1.7 Establishment and Optimization of a Two-Step CRISPR / Cas12a Detection System

[0242] CRISPR / Cas12a detection is available in two methods: fluorescence and lateral flow strip (LFA). Fluorescence detection involves placing the mixture in a quantitative real-time PCR instrument at 37°C to measure fluorescence. Lateral flow strip detection involves placing the mixture in a 37°C water bath, adding enzyme-free water to a final volume of 50 µL, mixing thoroughly, and then immersing the sample detection end of the strip in the reaction solution to observe the results.

[0243] Optimization of the fluorescence detection system: The template DNA concentration was fixed at 1 nM, and the concentrations of LbCas12a, crRNA, and ssDNA were optimized. Six molar ratios (Cas12a / crRNA) were set: 2.5:1, 1.25:1, 1:1, 1:1.2, 1:1.6, and 1:2. Five ssDNA concentrations were set: 50, 100, 200, 300, 400, and 500 nM, with other conditions remaining unchanged. Enzyme-free water was used as a negative control, and fluorescence values ​​were observed under different conditions.

[0244] Optimization of the test strip detection system: The ssDNA probe concentration in the test strip detection solution was diluted to a series of gradients: 0 nM, 100 nM, 200 nM, 300 nM, 400 nM, 600 nM, and 800 nM. Empty test strips were directly inserted into the mixed test strip detection solution, and the bands were observed. The lowest probe concentration at which the T line disappeared was considered the optimal concentration. Optimization of CRISPR / Cas12a cutting time: CRISPR cutting times were set to 5, 10, 15, 20, 25, and 30 min. Fluorescence detection and test strip detection were performed. Based on the color development of the test strip detection line under different reaction times, the optimal cutting time was selected.

[0245] 1.8 Sensitivity Evaluation of the Two-Step CRISPR / Cas12a Detection System

[0246] To compare the detection sensitivity of the two-step PCR-CRISPR / Cas12a and ERA-CRISPR / Cas12a methods, the template was diluted to 10⁻⁶. 6 10 5 10 4 103 10 2 10 1 10 0 aM (concentration before amplification) was used for PCR and ERA amplification, respectively. 2 µL of the amplification product was added to the Cas12a detection system, and the fluorescence curve and fluorescence value were recorded using a quantitative real-time PCR instrument. Genomic DNA was diluted to 100 pg / µL, 10 pg / µL, 1 pg / µL, 100 fg / µL, 10 fg / µL, 1 fg / µL, and 0.1 fg / µL (nucleic acid concentration before amplification) to further verify its detection performance.

[0247] 1.9 Establishment of a one-pot ERA-CRISPR / Cas12a detection system

[0248] The ERA mixture consisted of 20 μL of enzyme-free water, 20 μL of solvent, and 2.5 μL each of 10 μM forward and reverse primers (ERA / F2 and ERA / R2 in Table 2), completely dissolving the lyophilized reagent powder. The Cas12a detection system consisted of 0.5 μL of 1 μL of 1 μL of bCas12a, 2 μL of 300 nM crRNA, 2 μL of 10×HOLMES Buffe, and 2 μL of 300 nM ssDNA reporter gene (FAM-TTTTT-BHQ1). Before the reaction, the sample mixture was premixed with 0.4 μL of 100 pg / µL template and 1 μL of activator. Four methods were used for one-pot detection. ① First, 8.6 μL of the prepared ERA mixture was added to the bottom of the centrifuge tube. Then, 10 μL of the prepared Cas12a detection system was added to the cap. Then, quickly add 1.4 μL of the prepared sample mixture to the ERA mixture and immediately tighten the tube cap. Incubate the PCR tube at 37°C for 20 minutes. After incubation, thoroughly mix the two systems by simple rotation and monitor using a real-time PCR instrument. Figure 1 A). ② Add 15% glycerol between the ERA amplification system and the Cas12a detection system ( Figure 1 B). ③ Add 1.7 mg of sucrose to the Cas12a detection system ( Figure 1 C). ④ Directly mix the ERA amplification system with the Cas12a detection system, and perform amplification and detection simultaneously. Figure 1 D).

[0249] 1.10 Optimization of the One-Pot ERA-CRISPR / Cas12a Detection System

[0250] The amount of genomic DNA sample added to the one-pot ERA amplification system was increased from 0.4 μL to 1 μL, the final concentration of ssDNA was 300 nM, and the final concentrations of 10 µM Cas 12a protein in the system were 125 nM, 250 nM, 500 nM, 750 nM, and 1000 nM. The ERA amplification time was set to 5, 10, 15, 20, 25, 30, and 60 min, and the detection time was 30 min.

[0251] 1.11 Sensitivity Evaluation of the One-Pot ERA-CRISPR / Cas12a Detection System

[0252] To compare the sensitivity of the one-pot ERA-CRISPR / Cas12a method for detecting Aspergillus fumigatus, Aspergillus fumigatus DNA was extracted and serially diluted 10-fold. Genomic DNA at concentrations of 100 pg / µL, 10 pg / µL, 1 pg / µL, 100 fg / µL, 10 fg / µL, 1 fg / µL, and 0.1 fg / µL (pre-amplification nucleic acid concentrations) was used as templates and detected using the one-pot ERA-CRISPR / Cas12a detection system. Enzyme-free water was used as a control. The detection limit of the system was determined based on the intensity of the fluorescence signal and whether the test line on the test strip developed color.

[0253] 1.12 Specificity Evaluation of the One-Pot ERA-CRISPR / Cas12a Detection System

[0254] Using the extracted DNA from all tested strains (Table 3) as templates, the optimized one-pot ERA-CRISPR / Cas12a system was employed for detection, with enzyme-free water as a control. The specificity of the system was determined based on the fluorescence signal intensity and whether the test strip detection line developed color.

[0255] Table 3. Fungal strains used in this study

[0256]

[0257] 1.13 Clinical evaluation of one-pot ERA-CRISPR / Cas12a

[0258] Sixty-two sputum samples were collected from Huaibei People's Hospital, including 32 positive sputum samples diagnosed with Aspergillus fumigatus and 30 negative sputum samples that did not contain Aspergillus fumigatus (ethical approval: the sample collection protocol of this study was approved by the hospital's ethics committee). These samples were tested for diagnosis using pure culture, qPCR, and one-pot ERA-CRISPR / Cas12a.

[0259] 2. Results and Discussion

[0260] 2.1 Screening of ERA, PCR primers and crRNA

[0261] Using 1 nM Aspergillus fumigatus genomic DNA as a template, three sets of designed ERA primers (Table 2) were used for ERA amplification followed by electrophoresis. All three sets of ERA primers amplified a single band, with ERA primer 2 (ERA / F2, ERA / R2) producing a brighter band. Figure 2 A) Primers were further screened based on the brightness of the bands and the results of the optimal crRNA screening experiment. The fluorescence curves of crRNA 1 and crRNA 2 were compared in terms of time and fluorescence intensity after ERA-CRISPR / Cas12a detection. Figure 2 (C, D) crRNA 1 was more effective than crRNA 2. Although both reached the detection plateau at 25 min, the overall fluorescence intensity of crRNA 1 was 1.5 times that of crRNA 2. Therefore, the optimal primers for ERA amplification were determined to be F2 / R2, with an amplification product of 252 bp. crRNA 1 was also identified as the optimal crRNA sequence and used in subsequent reaction system optimization experiments.

[0262] 3.2 Establishment of a two-step ERA-CRISPR / Cas12a detection system

[0263] To optimize the Cas12a / crRNA reaction system, six molar ratios (2.5:1, 1.25:1, 1:1, 1:1.2, 1:1.6, and 1:2) of Cas12a / crRNA were set. Experimental results showed that the accumulation of fluorescence signal reached its peak when the Cas12a to crRNA ratio reached 1:1.2. Therefore, 1:1.2 was selected as the optimal ratio for subsequent experiments. At this ratio, the Cas12a concentration was 250 nM and the crRNA concentration was 300 nM. Figure 2 E, F).

[0264] CRISPR / Cas12a fluorescence detection reporter molecule (ssDNA-FQ) concentration optimization, reporter molecule concentration optimization results ( Figure 2 (B) The higher the concentration of the fluorescent reporter molecule, the stronger the fluorescence intensity. Within the concentration range of 50-300 nM, the fluorescence value increases with increasing concentration. However, at 300 nM, as the reporter probe concentration continues to increase, the fluorescence value tends to saturate, and the amplification results are essentially the same as at 300 nM. Considering both detection cost and experimental results, 300 nM was ultimately chosen as the optimal fluorescent probe concentration for instrumental detection.

[0265] Optimization results of reporter molecule (ssDNA-FB) concentration (e.g., FAM-TTATTATT-Biotin) in the test strip detection system. Figure 2 (G) At low concentrations, ssDNA-FB produces a red band on the test line, indicating a false positive. As the ssDNA-FB concentration increases, the T-line becomes lighter, while the C-line gradually darkens. At ssDNA-FB concentrations of 300 nM and 400 nM, the T-line intensity is significantly lower than the C-line, eliminating the false positive effect caused by the T-line. Considering all factors, 300 nM ssDNA-FB was used for subsequent CRISPR / Cas12a test strip detection. The test strip showed two visible red lines after 15 minutes, and clear C and T lines after 30 minutes. The subsequent test strip detection time was set to 30 minutes. Figure 2 H).

[0266] 3.3 Two-step CRISPR / Cas12a sensitivity detection

[0267] The sensitivity of the two-step ERA-CRISPR / Cas12a assay was compared with that of the two-step PCR-CRISPR / Cas12a assay. Aspergillus fumigatus DNA was diluted to 10... 6 10 5 10 4 10 3 10 2 10 1 10 0 aM was used for sensitivity assessment. Results showed that for PCR amplification, concentrations below 10... 2 At aM, almost no amplification bands can be observed. Figure 3 A), while combining PCR with CRISPR / Cas12a provides a sensitivity one gradient higher than electrophoresis, reaching 10. 1 aM ( Figure 3 B, C). 1% agarose gel electrophoresis images of ERA amplification products at 10 0 Amplification bands can still be seen at aM ( Figure 3 D), 10 -1 At aM, ERA-CRISPR / Cas12a showed no fluorescence, therefore the detection limit for ERA-CRISPR / Cas12a is 10. 0 aM ( Figure 3E, F). To further verify the detection performance of this method, the extracted Aspergillus fumigatus genomic DNA was serially diluted 10-fold for sensitivity evaluation. At higher Aspergillus fumigatus template concentrations, the ERA / CRISPR-Cas12a fluorescence system reacted rapidly, with the fluorescence curve rising quickly, reaching saturation within 20 min. At lower template concentrations, the reaction rate was relatively slow, remaining unsaturated even after 45 min, and the resulting fluorescence value was also lower; however, compared to the negative control, the low template concentration still showed a significant amplification curve, and the fluorescence intensity after 45 min was significantly higher than that of the negative control. The template detection limit was 1 fg / μL, indicating high sensitivity. Figure 3 H, I). These results indicate that the two-step ERA-CRISPR / Cas12a detection method developed in this study has better sensitivity than the conventional PCR-CRISPR / Cas12a detection method. ERA amplification can be completed quickly without temperature fluctuations, resulting in higher sensitivity and better visualization when combined with lateral flow test strips. The detection limit of the two-step ERA-CRISPR / Cas12a method using lateral flow test strips is the same as that of the fluorescence method, both reaching 1 fg / µL (H, I). Figure 3 G).

[0268] 3.4 Establishment of a one-pot ERA-CRISPR / Cas12a detection system

[0269] In the two-step ERA-CRISPR / Cas12a detection platform, ERA amplification and Cas12a detection are separated into two independent steps, requiring the transfer of the amplification product into the Cas12a detection system. However, ERA amplification is highly efficient, and aerosol contamination is easily generated during the transfer process, leading to false positives. Directly mixing the ERA amplification system and the Cas12a detection system in one pot for fluorescence detection results in significantly reduced sensitivity and fluorescence intensity. Figure 4 (A, B) Since Cas12a cleavage and ERA amplification share the same DNA substrate, mixing them directly in one pot leads to competition between the two, resulting in decreased sensitivity. The ERA amplification system is added to the bottom of the reaction tube, and the Cas12a detection system is added to the top. After ERA amplification, the Cas12a detection system is centrifuged to the bottom of the tube and mixed with the ERA product, achieving detection of Aspergillus fumigatus with a clear fluorescence curve. While adding glycerol and sucrose can improve detection sensitivity, the effect is not as significant as bottom separation, and the addition of these two substances makes the detection system more viscous, hindering the migration of the detection solution on the test strip and affecting the visualization of the test strip detection.

[0270] 3.5 Optimization of the One-Pot ERA-CRISPR / Cas12a Detection System

[0271] By comparing several one-pot methods described above, the ERA amplification system was ultimately chosen to be added to the bottom of the reaction tube, while the Cas12a detection system was added to the top. After ERA amplification, the Cas12a detection system was centrifuged to the bottom of the tube and mixed with the ERA product, thus enabling the detection of Aspergillus fumigatus. The one-pot ERA-CRISPR / Cas12a detection method avoids aerosol contamination caused by amplification product transfer and eliminates the sensitivity reduction problem caused by DNA substrate competition between Cas12a cleavage and ERA amplification. To further improve sensitivity, the component ratios of the ERA amplification system and the Cas12a detection system were adjusted. The template concentration in the amplification system was increased from 0.4 µL to 1 µL, and the crRNA sequence was crRNA1 as shown in the table. The amplification primers were ERA / F2 and ERA / R2, with a primer concentration of 10 µM. The probe was FAM-TTTTT-BHQ1 with a concentration of 300 nM. The optimal concentration of Cas12a in the detection system was 500 nM. Figure 4 C), thus making the sensitivity of the one-pot method comparable to that of the two-step method. Figure 4 D), all were 1 fg / µL ( Figure 4 E), under the same conditions, the detection limit of the test strip is 10 fg / µL ( Figure 4 H), which is one gradient lower than the fluorescence method. Optimizing the amplification time revealed two clearly visible red lines after 15 minutes of ERA amplification, but the T-line weakened significantly after 25 minutes, indicating that the amplification time should not be too long. Figure 4 G).

[0272] 3.6 Specificity evaluation of the one-pot ERA-CRISPR / Cas12a detection system

[0273] Using genomic DNA from all strains in Table 3 as templates, a one-pot ERA-CRISPR / Cas12a system was employed for detection to determine the specificity of the method. The results showed that ( Figure 4 (F, I) Aspergillus fumigatus can produce obvious fluorescent signals, and the test strip shows two clearly visible red lines, which is a positive phenomenon. The fluorescence intensity of the other groups is not obvious, and only C line is shown, which is a negative result, indicating that the method has high specificity.

[0274] 3.7 Clinical evaluation of one-pot ERA-CRISPR / Cas12a

[0275] Sixty-two sputum samples were tested using traditional culture, one-pot ERA-CRISPR / Cas12a, and quantitative real-time PCR. Results showed 32 positive samples and 30 negative samples. The results of the ERA-CRISPR / Cas12a method were largely consistent with those of pure culture. Figure 4 Compared with qPCR results, the sensitivity of the fluorescence method for detecting positive samples was 93.75% and the specificity was 93.33% (Table 4), while the sensitivity of the test strip was 90.63% and the specificity was 96.67% (Table 5).

[0276] Table 4 Comparison of results between one-pot ERA-CRISPR / Cas12a fluorescence detection system and qPCR detection system

[0277]

[0278] Table 5 Comparison of results between the one-pot ERA-CRISPR / Cas12a test strip detection system and qPCR detection system.

[0279]

[0280] 4. Discussion

[0281] Aspergillus, a group of opportunistic pathogenic fungi widely distributed in nature, is an important pathogenic microorganism causing human respiratory infections. Invasive aspergillosis is mainly caused by Aspergillus fumigatus, most commonly occurring in immunocompromised populations. Early detection of Aspergillus fumigatus is considered a challenging diagnosis. Currently, various detection methods each have their advantages and disadvantages, and none can simultaneously achieve high sensitivity, high specificity, short detection time, and simple operation of equipment. Several CRISPR / Cas12a combined detection systems have been established for the detection of various pathogens, such as SARS-CoV-2, African swine fever virus, Mycoplasma pneumoniae, and Candida albicans. Since its advent, PCR technology has greatly promoted the development of modern molecular biology, becoming an indispensable tool in modern molecular biological research and clinical diagnosis. ERA amplification is an isothermal amplification technique that does not require temperature changes, making it more convenient to operate. Comparing conventional PCR amplification with ERA amplification combined with CRISPR / Cas12a, the ERA-CRISPR / Cas12a technology has a sensitivity 10 times higher than the PCR-CRISPR / Cas12a technology, demonstrating high sensitivity. However, fluorescence methods still require the use of large instruments. To address this issue, a test strip probe is introduced into the detection system instead of the fluorescence probe. Results are observed directly with the naked eye via the side-flow test strip, reducing the need for large instruments and truly meeting the requirements for on-site testing. This process requires manual opening of the cap after amplification to add the product to the CRISPR detection system. Opening the cap can easily generate aerosols, leading to false positives.

[0282] If Cas12a comes into contact with a small amount of target DNA in the early stages of amplification, it will immediately cleave the template or primers, inhibiting subsequent amplification and leading to false negatives. The ERA amplification system and the CRISPR / Cas12a detection system are physically separated using the tube cap and bottom. The core principle is that the spatial separation of the reaction tubes allows amplification and detection to proceed in stages. Finally, the two systems are mixed by centrifugation, achieving a single-tube detection method. Glycerol and sucrose are used to increase the viscosity of the reaction system, slowing down molecular diffusion and thus delaying the premature binding of the Cas12a protein to the amplification product, preventing amplification inhibition and achieving the single-tube method.

[0283] By comparing several one-pot methods, this study successfully constructed a rapid visual detection system for Aspergillus fumigatus based on a one-pot ERA-CRISPR / Cas12a method. Optimal ERA primers and crRNA were designed and screened based on the specific gene anxC4. Comparing four one-pot methods, the method with the ERA amplification system and Cas12a detection system placed at the tube cap and bottom, respectively, was selected as the most effective, successfully resolving false positives caused by product transfer. Furthermore, by optimizing the components in the one-pot reaction system, the sensitivity of the one-pot method was successfully comparable to that of the two-step method, achieving a fluorescence sensitivity of 1 fg / µL and a test strip sensitivity of 10 fg / µL, demonstrating high sensitivity. In clinical sample testing, the one-pot ERA-CRISPR / Cas12a fluorescence method showed a specificity of 93.3%, and the test strip detection showed a specificity of 93.75%, exhibiting high specificity. In terms of detection time, compared with the traditional culture method, this method is shorter, with an amplification time of 20 minutes, a detection time of 30 minutes, and a result display time of 5 minutes, completing the entire detection process within 55 minutes. Regarding result reading, both the fluorescence method and the test strip method offer clear and intuitive results. Therefore, the one-pot ERA-CRISPR / Cas12a detection system exhibits high sensitivity and specificity, enabling rapid, accurate, and intuitive identification of Aspergillus fumigatus in sputum samples. Based on these results, the one-pot ERA-CRISPR / Cas12a method presented in this study can be considered a more advantageous tool for Aspergillus fumigatus detection.

[0284] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A two-step detection system for detecting Aspergillus fumigatus, characterized in that, The detection system includes: (a) Cas12 protein, wherein the Cas12 protein is Cas12; the concentration of the Cas12 protein is 200-400 nM; (b) A guide RNA that guides the Cas12 protein to specifically bind to the nucleic acid molecules of Aspergillus fumigatus, the guide RNA being at a concentration of 200-400 nM, and the guide RNA having the sequence shown in SEQ ID NO. 1; and (c) A nucleic acid probe, wherein the nucleic acid probe is a single-stranded DNA; The molar ratio of the Cas12 protein to the guide RNA is 1:1-2. The detection system also contains nucleic acid molecules of Aspergillus fumigatus to be detected. The nucleic acid molecules of Aspergillus fumigatus are nucleic acid molecules that have undergone isothermal amplification. The isothermal amplification is ERA, and the nucleic acid molecules of Aspergillus fumigatus are amplified using the amplification primers shown in SEQ ID NO.3 and SEQ ID NO.

4. The detection methods include fluorescence detection and colloidal gold detection. When the detection is a fluorescence detection method, the concentration of the nucleic acid probe is 300-500 nM; When the detection is performed using colloidal gold detection, the concentration of the nucleic acid probe is 200-400 nM.

2. The detection system as described in claim 1, characterized in that, The concentration of Cas12 protein is 200-300 nM.

3. The detection system as described in claim 1, characterized in that, The working temperature of the Cas12 protein is 35-40℃.

4. The detection system as described in claim 3, characterized in that, The working temperature of the Cas12 protein is 35-38℃.

5. A two-step method for detecting Aspergillus fumigatus, characterized in that, The system includes a detection system and an amplification system, wherein the detection system is located at the tube cap and the amplification system is located at the bottom of the tube, and wherein the detection system includes: (a) Cas12 protein, wherein the Cas12 protein is Cas12; (b) A guide RNA that guides the Cas12 protein to specifically bind to the nucleic acid molecules of Aspergillus fumigatus; and (c) Nucleic acid probe, wherein the nucleic acid probe is single-stranded DNA and the concentration of the nucleic acid probe is 200-400 nM; The concentration of the Cas12 protein is 500 nM-1000 nM; the concentration of the guide RNA is 200-400 nM; and the guide RNA is the sequence shown in SEQ ID NO.

1. The amplification system includes an isothermal amplification system for amplifying Aspergillus fumigatus nucleic acid molecules, the amplification system containing the nucleic acid molecules of Aspergillus fumigatus to be detected, and the isothermal amplification is ERA; In the system, the concentration of the nucleic acid molecules of Aspergillus fumigatus to be detected in the system is 100 pg / µL-1fg / µL; Furthermore, the amplification system also contains specific nucleic acid amplification primers, namely SEQ ID NO.3 and SEQ ID NO.

4.

6. A kit for detecting Aspergillus fumigatus using a two-step method in one tube, characterized in that, The kit includes: (i) A first container and a Cas12 protein located within the first container, wherein the Cas12 protein is Cas12 and the concentration of the Cas12 protein is 500 nM-1000 nM; (ii) A second container and a guide RNA located within the second container, the guide RNA guiding the Cas12 protein to specifically bind to the nucleic acid molecules of Aspergillus fumigatus, the concentration of the guide RNA being 200-400 nM, the guide RNA being the sequence shown in SEQ ID NO.1; (iii) A third container and a nucleic acid probe located within the third container, wherein the nucleic acid probe is single-stranded DNA and the concentration of the nucleic acid probe is 200-400 nM; (iv) A fourth container and amplification primers located within the fourth container for amplifying the nucleic acid molecules of Aspergillus fumigatus to be detected, said amplification primers being shown in SEQ ID NO.3 and SEQ ID NO.4; (v) A fifth container and reagents located within the fifth container for isothermal amplification of nucleic acid molecules of Aspergillus fumigatus to be detected, wherein the isothermal amplification is ERA; (vi) PCR tube; And labels or instructions; The kit also includes: (vii) The sixth container and the nucleic acid molecules of Aspergillus fumigatus to be detected located in the sixth container, the concentration of the nucleic acid molecules of Aspergillus fumigatus to be detected is 100 pg / µL-1fg / µL; The components in the first container, the optional second container, and the third container are located in the cap of the PCR tube, while the components in the fourth container, the fifth container, and the sixth container are located in the bottom of the PCR tube.

7. A two-step method for detecting the presence of Aspergillus fumigatus in a sample, which is neither a disease diagnosis nor a treatment, characterized in that, The detection method includes: (a) Providing a reaction system comprising: a detection system and an amplification system, wherein the amplification system contains nucleic acid molecules of Aspergillus fumigatus to be detected from a sample source and amplification primers for isothermal amplification reaction to amplify the nucleic acid molecules of Aspergillus fumigatus to be detected, wherein the isothermal amplification is ERA, and the sequences of the amplification primers are shown in SEQ ID NO.3 and SEQ ID NO.4; (b) Perform nucleic acid amplification on the Aspergillus fumigatus nucleic acid molecules in the amplification system to obtain the amplification product of Aspergillus fumigatus nucleic acid molecules; (c) Mix the detection system with an amplification system containing the amplification product, and detect the detectable signal emitted by the nucleic acid probe; If the nucleic acid probe is cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is present in the sample; if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is not present in the sample. The detection system includes: (a1) Cas12 protein, wherein the Cas12 protein is Cas12; the concentration of the Cas12 protein is 200-400 nM; (b1) A guide RNA that guides the Cas12 protein to specifically bind to the nucleic acid molecules of Aspergillus fumigatus, the guide RNA being at a concentration of 200-400 nM, and the guide RNA having the sequence shown in SEQ ID NO. 1; and (c1) Nucleic acid probe, wherein the nucleic acid probe is single-stranded DNA; The molar ratio of the Cas12 protein to the guide RNA is 1:1-2. When the detection is a fluorescence detection method, the concentration of the nucleic acid probe is 300-500 nM; When the detection is performed using colloidal gold detection, the concentration of the nucleic acid probe is 200-400 nM.

8. A two-step, non-disease-diagnostic, and non-treatment method for detecting the presence of Aspergillus fumigatus in a sample, characterized in that, The detection method includes: (a) A system according to claim 5, wherein the detection system and the amplification system are located in the same tube, the detection system is located at the tube cap, the amplification system is located at the bottom of the tube, the amplification system contains nucleic acid molecules of Aspergillus fumigatus to be detected from the sample and amplification primers for isothermal amplification reaction to amplify the nucleic acid molecules of Aspergillus fumigatus to be detected, the isothermal amplification is ERA, and the concentration of the nucleic acid molecules of Aspergillus fumigatus to be detected in the system is 100 pg / µL-1fg / µL, and the amplification primers are SEQ ID NO.3 and SEQ ID NO.4; (b) At the bottom of the same tube, perform nucleic acid amplification on the Aspergillus fumigatus nucleic acid molecules in the amplification system to obtain the amplification product of the Aspergillus fumigatus nucleic acid molecules; (c) In the same tube, the detection system of the tube cap is added to the amplification system containing the amplification product at the bottom of the tube, and the detectable signal emitted by the nucleic acid probe is detected; If the nucleic acid probe is cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is present in the sample; if the nucleic acid probe is not cleaved by the Cas12 protein, it indicates that Aspergillus fumigatus is not present in the sample. Furthermore, the concentration of Cas12 protein in the detection system is 500nM-1000nM; the concentration of guide RNA in the detection system is 200-400nM; the concentration of nucleic acid probe in the detection system is 200-400nM, and the guide RNA is the sequence shown in SEQ ID NO.1.

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