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CN-121992131-A - Rapid detection kit and detection method for dermatophyte nucleic acid based on CRISPR/Cas12a technology

CN121992131ACN 121992131 ACN121992131 ACN 121992131ACN-121992131-A

Abstract

The invention discloses a dermatophyte nucleic acid rapid detection kit which comprises a CRISPR/Cas12a reaction system, a fluorescence report probe, an anti-FAM antibody-fluorescence quantum dot conjugate, a quantum dot immunochromatography test strip, a detection line and a control line, wherein the CRISPR/Cas12a reaction system comprises Cas12a protein and corresponding crRNA, the fluorescence report probe is provided with a 5 '-end FAM mark and a 3' -end biotin mark, the quantum dot immunochromatography test strip comprises a sample pad, a nitrocellulose membrane and an absorption pad, the detection line and the control line are arranged on the membrane, and the detection line is coated with streptavidin and sheep anti-mouse IgG. The invention also discloses a corresponding detection method. The invention can complete detection within 1h, has simple and convenient operation and higher detection sensitivity and specificity, and is suitable for the rapid detection and field application of dermatophytes.

Inventors

  • TAN FEI
  • HU XIAOPING
  • YANG LIANJUAN
  • TAN JINGWEN
  • XU PENG
  • CHAI XIAOYUN

Assignees

  • 上海市皮肤病医院

Dates

Publication Date
20260508
Application Date
20251229

Claims (10)

  1. 1. A rapid detection kit for dermatophyte nucleic acid based on CRISPR/Cas12a technology, which is characterized by comprising: A CRISPR/Cas12a reaction system comprising a Cas12a protein and a corresponding crRNA; A fluorescent reporter probe, ssDNA having a fluorescent group and a binding group at both ends, respectively; The primary antibody is an anti-fluorescent group antibody-fluorescent quantum dot conjugate; The quantum dot immunochromatography test strip comprises a sample pad, an adsorption reaction area and an absorption pad which are sequentially connected, wherein a test line and a control line are arranged on the adsorption reaction area, the control line is coated with a conjugate and is used for being combined with the conjugate group, the test line is coated with a secondary antibody and is used for being combined with the primary antibody, and the control line is closer to the sample pad than the test line.
  2. 2. The kit of claim 1, wherein the fluorescent moiety is selected from FAM, HEX, ROX, cy, cy5 and FITC, the binding moiety is selected from the group consisting of Biotin and Di goxin, the secondary antibody is goat anti-mouse IgG, the conjugate is streptavidin when the binding moiety is Biotin, and the conjugate is an anti-Digoxin antibody when the binding moiety is Digoxin.
  3. 3. The kit according to claim 2, wherein, The sequence of the fluorescent reporter probe is FAM-TTNTT-Biotin, wherein N represents 0-10 arbitrary bases, and FAM and Biotin are respectively positioned at the 5 'end and the 3' end or the 3 'end and the 5' end of ssDNA.
  4. 4. The kit according to claim 1, wherein the fungus is trichophyton rubrum and the crRNA has a sequence shown in SEQ ID NO. 5.
  5. 5. The kit according to claim 4, wherein the kit further comprises an isothermal amplification system having a specific primer pair for the ITS region of dermatophytes, wherein the nucleic acid sequences of the specific primer pair are shown in SEQ ID NOs 1 and 2 or SEQ ID NOs 3 and 4.
  6. 6. The kit according to claim 1, further comprising a nucleic acid rapid extraction component comprising a lysate and an extract, wherein the lysate comprises 30-70 mmol/L, preferably 50 mmol/L Tris-HCl, 20-30 mmol/L EDTA, preferably 25 mmol/L EDTA, 2-5%, preferably 3% SDS, 1-2%, preferably 1.2% PVP, pH 7.8-8.2, preferably 8.0; The extract contains 5-20 mmol/L, preferably 10 mmol/L, of Tris-HCl, pH 7.8-8.2, preferably 8.0, 0.5-2 mmol/L, preferably 1 mmol/L of EDTA, 0.2-0.5 mol/L, preferably 0.3-mol/L of NaAc, and 1-2%, preferably 1.2% PVP.
  7. 7. A rapid detection method of dermatophyte nucleic acid based on CRISPR/Cas12a technology is characterized by adopting the kit as claimed in claim 1, comprising the following steps: S1, mixing a sample to be detected with a lysate and an abrasive material, grinding the mixture, performing pyrolysis by adopting a microwave intermittent heating mode, adding an extracting solution, extracting the mixture by using a phenol-chloroform solvent to obtain an amplifiable DNA template, wherein the abrasive material is preferably pickled quartz sand, the lysate contains 30-70 mmol/L, preferably 50 mmol/L, of Tris-HCl with pH of 7.8-8.2, preferably 8.0, 20-30 mmol/L, preferably 25-mmol/L of EDTA, 2-5% of SDS, preferably 3% of SDS, 1-2% of PVP, preferably 1.2% of PVP, and the extracting solution contains 5-20 mmol/L, preferably 10 mmol/L, 5-2 mmol/L, preferably 1 mmol/L of EDTA, 0.2-0.5 mol/L, preferably 0.3-mol/L of Ac, and 1-2% of PVP; S2, adding the DNA template into an isothermal amplification reaction system, and amplifying for 25-50 minutes at a constant temperature of 30-42 ℃ to obtain an amplified product; S3, adding the amplification product into the fluorescent reporter probe, the anti-FAM antibody-fluorescent quantum dot conjugate and the CRISPR/Cas12a reaction system, and reacting for 20-40 minutes at 25-45 ℃; s4, adding the reaction liquid into the quantum dot immunochromatographic test strip, developing color within 5-15 minutes, and reading the result; Wherein a positive sample is represented by the test line developing, a negative sample is represented by the test line not developing but the control line developing, and neither the control line nor the test line developing is an invalid result.
  8. 8. The method according to claim 7, wherein the microwave batch heating is performed at a power of 200-800W W, preferably 400-600W, more preferably 600W, each heating for 10-30 seconds, cooling for 5-20 seconds at intervals, and a total treatment time of 5-15 minutes, wherein the isothermal amplification is a recombinase-assisted amplification at a temperature of 36-37 ℃ and an amplification time of 30-40 minutes.
  9. 9. The method of claim 7, wherein the molar ratio of Cas12a to crRNA in the CRISPR/Cas12a reaction system is 1:1-1:2 and the working concentration of the fluorescent reporter probe is 5-20 nM.
  10. 10. The method according to claim 7, wherein the crRNA has a guide sequence length of 20-22 nucleotides and a PAM site TTTN at the upstream of the target sequence, and the target fungus of the rapid detection method of dermatophyte nucleic acid based on CRISPR/Cas12a technology is trichophyton rubrum.

Description

Rapid detection kit and detection method for dermatophyte nucleic acid based on CRISPR/Cas12a technology Technical Field The invention relates to the field of molecular diagnosis and fungal infection detection, in particular to a rapid detection kit and a detection method for dermatophyte nucleic acid based on a CRISPR/Cas12a technology. Background Fungal dermatological diseases are common and frequently-seen infectious diseases in clinic, and the incidence rate of the fungal dermatological diseases is in an ascending trend along with the wide use of broad-spectrum antibiotics, glucocorticoids and immunosuppressants and the aggravation of population flow, and the proportion of drug-resistant strains is increased year by year. Especially after irregular treatment, rash is often atypical and increases the difficulty of diagnosis. Current clinical diagnosis relies mainly on characteristic rash and mycotic examination, and common methods include microscopy, culture, dermatology, woodlamp, serology, imaging, PCR, and the like. The direct microscopic examination is simple, convenient and quick (such as a KOH wet sheet method and a fluorescent staining method), but has the problems of high omission ratio, incapability of identifying strains and the like. The development of molecular diagnostic techniques has enabled early, rapid, specific detection of pathogens, common methods including PCR, FISH, probe hybridization, and the like. PCR has higher sensitivity and specificity, but is still limited by the problems of complicated operation, equipment dependence and the like. To solve these problems, various isothermal amplification techniques, including LAMP, RPA, RAA and the like, have been developed in recent years and have been used for detection of various fungi. The RAA technique can complete amplification in 5-20 minutes at 37℃isothermally, but still presents a risk of false positives. However, the introduction of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology provides higher specificity guarantee for an isothermal amplification system. Cas12a can recognize specific targets and activate cleavage of non-specific ssDNA under crRNA (CRISPR RNA) guidance, thereby achieving nucleic acid signal amplification, significantly reducing false positive rates. At present, the technology has been successfully applied to detection of human papilloma virus, new coronavirus, mycobacterium tuberculosis and cancer related genes, but the application in dermatomycosis has been rarely reported. On the other hand, the immunochromatography test paper is used as a novel detection platform based on the antigen-antibody specific binding and capillary flow principle, has the advantages of low cost, simplicity and convenience in operation, rapid result output and the like, and is widely used in detection scenes such as biological molecules, environmental pollutants, food safety and the like. The platform does not need complex instruments and professionals, and has the potential of on-site rapid screening. Point of care testing (POCT) emphasizes the rapid outcome near the patient, suitable for on-site decision making. The POCT detection method combining isothermal amplification and CRISPR technology not only has high sensitivity and specificity, but also can observe results through naked eyes, LEDs or UV. Further combined with immunochromatography test paper, more portable and readable signal display can be realized. At present, most test strips use colloidal gold nanoparticles (AuNP) for color development, but quantum dots (QuantumDots, QDs) are used as novel fluorescent marking materials, and have greater advantages due to high fluorescence intensity and photobleaching resistance, and compared with the traditional gold nanoparticles or organic dyes, the fluorescent signal is more sensitive and the background is lower. The existing dermatomycosis detection technology has the problems of long detection period (such as a few days for culture), insufficient sensitivity (such as strong subjectivity for direct microscopic examination), dependence on large instruments, inapplicability to basic scenes (such as PCR/fluorescent quantitative PCR), low specificity of partial immunological methods and the like, and is difficult to meet the diagnosis requirement of clinical 'quick, simple and convenient and on-site (POCT'). Disclosure of Invention In order to solve at least one of the defects in the prior art, the invention provides the following technical scheme that the kit comprises a kit and a detection method. In a first aspect, the invention provides a rapid dermatophyte nucleic acid detection kit based on CRISPR/Cas12a technology, comprising: A CRISPR/Cas12a reaction system comprising a Cas12a protein and a corresponding crRNA; A fluorescent reporter probe, ssDNA having a fluorescent group and a binding group at both ends, respectively; The primary antibody is an anti-fluorescent group antibody-fluorescent quantum d