CN116064741A - Application of guide RNA probe for detecting Demodex mites, kit and application thereof - Google Patents

Abstract

The invention discloses application of a guide RNA probe in preparation of a product for detecting Demodex mites, wherein the coding DNA sequence of the guide RNA probe is shown as at least one of SEQ ID No.1 to SEQ ID No. 10. The invention discloses a CRISPR-Cas13 system for detecting Demodex mites, which comprises the following components: cas13a nuclease, guide RNA probes and reporter molecules used in the above applications; the guide RNA probe aims at the demodex chitin synthase gene, and after the guide RNA probe recognizes the target gene, the Cas13a nuclease activates the enzyme activity and cuts the reporter molecule to release the detection signal. The invention discloses a kit, which comprises: isothermal amplification systems, CRISPR-Cas13 systems for detection of demodex mites as described above, and visualization systems.

Description

Application of guide RNA probe for detecting Demodex mites and kit and use thereof

Technical Field

The present invention relates to the field of molecular biotechnology, and in particular to an application of a guide RNA probe in the preparation of a product for detecting Demodex mites, a CRISPR-Cas13 system for detecting Demodex mites, and a kit and a method for using the kit.

Background Art

Demodicid mites are a type of mite that parasitizes the human body, mainly Demodex folliculorum and Demodex brevis. In the past, it was believed that Demodicid mites were non-pathogenic or low-pathogenic, because the vast majority of people infected with Demodicid mites have no symptoms, appearing as asymptomatic carriers, or only with slight itching or burning sensations. The clinical symptoms of Demodicid infection are related to factors such as the patient's immune status, nutritional status, parasitic worm species, and degree of infection. However, recent studies have found that when human Demodicid mites move in the skin, they cause mechanical damage to epithelial cells and glandular cells, causing hair follicles and sebaceous glands to lose their normal structure and function, causing hair follicle expansion and epithelial degeneration. When there are many parasites, it can cause lesions such as hyperkeratosis or incomplete keratinization, obstruction of sebaceous gland secretion, and proliferation and dilation of capillaries in the dermis; mechanical stimulation of the parasite and chemical stimulation of its secretions and metabolites can cause inflammatory reactions of skin tissue, leading to non-bacterial inflammatory reactions of the host's local skin. In addition, the metabolites of the parasite can cause allergic reactions, and the movement of the parasite in and out can carry other pathogens into the hair follicles or sebaceous glands, which can cause secondary infections, causing infiltration of cells around the hair follicles and proliferation of fibrous tissue. Common clinical symptoms include mild flushing and abnormal oiliness of the skin in the affected area, followed by diffuse flushing, congestion, secondary erythema eczema, or scattered red acne-like papules ranging in size from pinpoints to millets, pustules, crusts and desquamation, abnormal sebum exudation, enlarged hair follicle openings, rough surface, itching and burning sensations on the skin, etc. In addition, the infection rate and degree of Demodex in patients with skin diseases such as rosacea, folliculitis, acne, seborrheic dermatitis and blepharitis are significantly higher than those in healthy people and general skin disease patients, indicating that these phenomena may be related to the infection of Demodex.

Commonly used methods for detecting demodicosis include the cellophane tape method (CTP), the squeeze method, the eyelash microscopy method, and the external auditory canal demodicosis test.

Among them, the transparent tape method is currently an effective method for facial Demodex detection. It has the advantages of large detection area, high detection rate, and is painless and non-invasive. It is suitable for the investigation of Demodex infection in the population and obtaining adult worm sources, but it needs to be kept overnight and the patient must be sent for examination the next day. The compliance is poor and it is not suitable for rapid examination in clinical outpatient clinics. The squeeze method is a commonly used examination method in clinical outpatient clinics in my country. Microscopic examination can detect mites of all stages. The operation is simple and quick, but the inspection area is limited and the detection rate is low. Eyelash microscopy is a commonly used method for detecting ocular Demodex. When pulling out eyelashes under a slit lamp, it is best to choose eyelashes with cylindrical scales, which can detect Demodex of all stages; if the cylindrical scales are dense, add fluorescein solution to dissolve the dense scales and stimulate Demodex to migrate outward, thereby increasing the detection rate. The external auditory canal Demodex detection is to collect earwax and other external auditory canal secretions for microscopic examination of Demodex.

The above methods are all based on morphological methods, which require professional equipment and technical humans to identify Demodex mites. They can only be performed in professional institutions and are not suitable for patients to self-examine at home.

Summary of the invention

The purpose of the present invention is to solve the shortcomings of the prior art and to propose an application of a guide RNA probe in the preparation of a product for detecting Demodex mites, a CRISPR-Cas13 system for detecting Demodex mites, and a kit and a method for using the kit.

The present invention can solve the problem that the existing Demodex infection detection requires special equipment and professional technology, simplify the detection process, and patients can complete the detection at home. It is suitable for the diagnosis and screening of Demodex infection in any scenario and improves the sensitivity of detection.

One of the purposes of the present invention is to provide an application of a guide RNA probe in the preparation of a product for detecting Demodex mites, wherein the coding DNA sequence of the guide RNA probe is shown in at least one of SEQ ID No. 1 to SEQ ID No. 10.

The sequence of the guide RNA probe has been recorded in the applicant's prior application (application number 202210917814.6, application date August 1, 2022, invention name: Guide RNA probe and its application and kit for detecting dust mite allergens).

Preferably, the product is a medicine, a reagent or a kit.

Preferably, the target gene of the guide RNA probe is the Demodex chitin synthase gene. The Demodex chitin synthase gene is shown in SEQ ID No.13.

Preferably, the coding DNA sequence of the guide RNA probe includes: a nucleic acid sequence for Cas13a nuclease recognition, and a nucleic acid sequence complementary to the target gene.

The second object of the present invention is to provide a CRISPR-Cas13 system for detecting Demodex mites, comprising: Cas13a nuclease, the guide RNA probe used in the above application, and a reporter molecule; the guide RNA probe targets the Demodex mite chitin synthase gene, and the Cas13a nuclease activates the enzyme activity after the guide RNA probe recognizes the target gene, and cuts the reporter molecule to release a detection signal.

Preferably, the reporter molecule is a non-specific reporter molecule, whose sequence is /56-FAM/mArArUrGrGrCmAmArArUrGrGrCmA/3Bio/, wherein the 5' end is labeled with FAM and the 3' end is labeled with biotin.

Preferably, the method further comprises: a detection buffer, wherein the detection buffer comprises: T7 DNA polymerase, rNTPs and magnesium chloride.

Preferably, detection buffer C comprises: 2U T7 DNA polymerase, 10mM rNTPs and 3mM magnesium chloride.

The third object of the present invention is to provide a primer pair for the chitin synthase gene of Demodex mite, including: CHS-F and CHS-R, whose nucleic acid sequences are shown in SEQ ID No.11 and SEQ ID No.12 respectively.

The fourth object of the present invention is to provide the use of the above primer pair for the chitin synthase gene of Demodex mite in isothermal amplification.

The fifth object of the present invention is to provide a kit comprising: an isothermal amplification system, the above-mentioned CRISPR-Cas13 system for detecting Demodex mites, and a visualization system.

Preferably, the isothermal amplification system comprises: the above-mentioned primer pair targeting the Demodex chitin synthase gene.

Preferably, the isothermal amplification system further comprises: RPA recombinase, reaction buffer V, magnesium acetate solution, and nuclease-free water.

Preferably, the RPA recombinase comprises: T4 phage recombinase UvsX, auxiliary factor UvsY, DNA polymerase, single-stranded DNA binding protein and dNTPs.

Preferably, the reaction buffer V comprises: 40 mM Tris (pH=7.9), 64 mM potassium acetate, 8 mM magnesium acetate, 0.8 mM DTT, 2.4 mM ATP, 16 mM creatine phosphate, 80 ng/μL creatine kinase and polyethylene glycol with a mass concentration of 5%.

Preferably, the molar concentration of the magnesium acetate solution is 250 mM.

Preferably, the visualization system comprises: a lateral flow test strip and a detection chromatographic fluid.

Preferably, the front end of the lateral flow test strip is coated with gold nanoparticles with FAM antibodies, the detection line is coated with biotin antibodies, and the quality control line is coated with fixed antibodies.

The fifth object of the present invention is to provide a method for using the above-mentioned kit, comprising the following steps: amplifying the sample DNA in vitro through an isothermal amplification system; then specifically identifying the Demodex chitin synthase gene through a CRISPR-Cas13 system for detecting Demodex mites; and observing the detection signal through a visualization system to determine whether the sample to be tested contains Demodex mites.

The front end of the lateral flow test strip is coated with fluorescent microspheres with FAM antibodies. The fluorescent microspheres are made by a two-step process of (carboxyl) coupled antibodies, through the steps of dilution, activation, removal of residual EDC, coupled antibodies, blocking, and removal of unbound antibodies of the microspheres. The control line (C line) is at the rear end, on which sheep anti-mouse antibodies are coated; the test line (T line) is located in front of the control line, on which fixed antibodies are coated. The complete nonspecific reporter molecules are not restricted by the fixed antibodies on the test line, and will bind to the sheep anti-mouse antibodies and be retained on the control line for color development, while the nonspecific reporter molecules hydrolyzed by Cas13a can bind to the fixed antibodies on the test line for color development.

The present invention can detect Demodex mites, adopts a constant temperature amplification method and combines a lateral flow test strip, does not require complex temperature control, and has many technical advantages such as simple operation, high sensitivity, strong specificity, and fast detection speed. The present invention obtains hair follicle secretions by an extrusion method, and uses a guided RNA probe technology to detect the chitin synthase gene of Demodex mites in the hair follicle secretions. It has the characteristics of simple operation and high sensitivity, and does not require special equipment. Patients can complete the detection and result interpretation at home to understand whether they are infected with Demodex mites.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing that the hair follicle secretion extract of a suspected patient tested positive using the method of the present invention.

Figure 2 is a photograph of a suspected patient who tested positive under the transparent tape method.

DETAILED DESCRIPTION

The present invention will be further explained below in conjunction with specific embodiments.

Example 1

1.1 Primer and probe design

Primers were designed for the chitin synthase gene of Demodex mite, and the DNA sequence was synthesized as follows:

name Sequence (5' to 3') CHS-F GACCCGGATTATTATGAGT CHS-R TTAGCTTAATCTTACACTAA

Design the probe. The DNA sequence of the guide RNA probe is as follows:

1.2 Probe preparation

Synthesize the DNA sequence of the probe. Take 5 μL of the DNA sequence corresponding to the guide RNA probe (with T7) and add it to the transcription reaction system, mix well; place the reaction tube at 37°C for 16 hours. After the reaction is completed, purify the reaction product with magnetic beads to obtain the guide RNA sequence.

The transcription reaction system is:

Reagents Dosage Assay Buffer C 12μL DNA sequence corresponding to guide RNA (with T7) 5μL Nucleic acid-free water 3μL

1.3 Isothermal amplification

Take 5 μL of the sample to be tested and nucleic acid-free water and add them to the isothermal amplification reaction system respectively, and mix them evenly; place the reaction plate at 39° C. for 15 minutes. After the reaction is completed, the isothermal amplification reaction product of the sample to be tested is obtained.

Reagents Dosage Provide primer pairs for the chitin synthase gene of Demodex mite 2μL Buffer V and RNA Lyophilization Enzyme 25μL Magnesium acetate 2.5μL Nucleic acid-free water 15.5μL template 5μL

1.4 Targeted Detection

Take 1 μL of isothermal amplification product for detection. The detection reaction system is as follows:

Reagents content Cas13a nuclease 82.58nM Guide RNA probe 0.4μM Nonspecific reporter molecules 200nM Assay Buffer 8μL Isothermal amplification products 1μL

Non-specific reporter molecules are as follows:

After the reaction is completed, add 20 μL of test strip detection chromatography liquid to the product, mix and centrifuge, then insert the sample loading area of the lateral flow test strip into the detection liquid. After 2-5 minutes, it can be directly observed and interpreted by the naked eye based on the brightness difference of the detection line band.

Example 2

Patients suspected of being infected with Demodex were selected from the outpatient clinic, and their hair follicle secretions were obtained by squeezing. The samples were stored in Eppendorf tubes (EPs) at -20°C. No ethical approval was required for this study. The samples were placed in liquid nitrogen for freeze-thaw for 1 min, then ball-milled for 10 min, and repeated 4 times. gDNA was extracted using a DNA extraction kit (OMEGA, Georgia, USA) and stored in EPs at -20°C. The obtained extracts were tested according to the steps of Example 1, as shown in Figure 1.

At the same time, the transparent tape method was used for the above patients, and the results are shown in Figure 2.

The test results of the present invention and the cellophane tape method are compared as follows:

Although the detection of Demodex mites often uses the transparent tape method, the test results of this method are accurate and reliable, but it requires special equipment and professional technical knowledge, and the detection time is long and needs to be overnight.

By comparing with the traditional cellophane tape method, although the specificity of the method of the present invention is 78.0%, which is not as good as 91.4% of the cellophane tape method, the sensitivity of the method is 85.2%, which is significantly higher than 65.8% of the cellophane tape method; and the detection time is short, which can narrow the diagnosis range more quickly.

The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims (10)

1. The application of a guide RNA probe in preparing a product for detecting Demodex mites is characterized in that the coding DNA sequence of the guide RNA probe is shown in at least one of SEQ ID No.1 to SEQ ID No. 10.

2. The use according to claim 1, wherein the product is a medicament, a reagent or a kit.

3. The use according to claim 1, wherein the targeting gene of the guide RNA probe is a demodex chitin synthase gene.

4. The use according to claim 1, wherein the coding DNA sequence of the guide RNA probe comprises: a nucleic acid sequence for Cas13a nuclease recognition, and a nucleic acid sequence complementary to a target gene.

5. A CRISPR-Cas13 system for detecting demodex mites, comprising: cas13a nuclease, guide RNA probe for use in the use according to any one of claims 1-4, reporter molecule;

the guide RNA probe aims at the demodex chitin synthase gene, and after the guide RNA probe recognizes the target gene, the Cas13a nuclease activates the enzyme activity and cuts the reporter molecule to release the detection signal.

6. The CRISPR-Cas13 system for detecting demodex as recited in claim 5, further comprising: detection buffer, detection buffer includes: t7 DNA polymerase, rNTPs and magnesium chloride.

7. A primer set for a demodex chitin synthase gene, comprising: CHS-F and CHS-R have nucleic acid sequences shown in SEQ ID No.11 and SEQ ID No.12 in sequence.

8. A kit, comprising: an isothermal amplification system, CRISPR-Cas13 system for detecting demodex as claimed in claim 5 or 6, and a visualization system.

9. The kit of claim 8, wherein the isothermal amplification system comprises: a primer pair for the demodex chitin synthase gene as claimed in claim 7.

10. A method of using the kit of claim 8 or 9, comprising the steps of: carrying out in-vitro amplification on sample DNA by an isothermal amplification system; then, specificity recognition is carried out on the demodex chitin synthase gene through a CRISPR-Cas13 system for detecting demodex; and observing the detection signal through a visualization system to judge whether the sample to be detected contains Demodex.

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