Method for detecting autism spectrum disorders using olfactory of nematodes

Abstract

A method for detecting autism spectrum disorders using the olfactory of nematodes, and a use of the olfactory of nematodes in detection and diagnosis of autism spectrum disorders. Nematodes are reared in the presence of an organism-related substance from a test subject or a treatment of the organism-related substance, and autism spectrum disorders are detected by using the chemotaxis of the olfactory of the nematodes as an indicator. The method for detecting autism spectrum disorders has high sensitivity and low costs; and the sample collection and analysis involved in the method are easy, and the costs are low.

Description

A method for detecting autism spectrum disorder using the sense of smell of nematodes Technical Field

[0001] The present invention relates to a method for detecting autism spectrum disorders using the sense of smell of nematodes. Background Art

[0002] Autism spectrum disorder (ASD) is a broad term that includes autism and several other autism-related disorders. These disorders all have similar symptoms, but the severity and type of symptoms can vary.

[0003] In DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition), disorders previously classified as Asperger syndrome, genetic autism, atypical autism, etc. have been integrated into the category of autism spectrum disorder.

[0004] Existing technologies for detecting and diagnosing autism spectrum disorders require doctors to conduct routine examinations of the child, such as physical examinations, hearing tests, lead poisoning tests, chromosome analysis, electroencephalograms, and magnetic resonance imaging. These examinations are then combined with the child's developmental status, daily behavior, and the diagnosis is made based on the child's medical history, clinical observations, diagnostic guidelines, and related examinations and assessments.

[0005] The above-mentioned diagnostic cycle is long and the diagnostic process is cumbersome, and is not applicable for large-scale screening of autism spectrum disorders.

[0006] There are related reports in existing patent literature on using the sense of smell of nematodes, especially Caenorhabditis elegans, for disease diagnosis, but these reports are only for cancer detection and are not involved in the detection of autism spectrum disorders. Summary of the Invention

[0007] The object of the present invention is to provide a method for detecting autism spectrum disorder using the sense of smell of nematodes.

[0008] The present inventors have discovered through research that the olfactory chemotaxis of nematodes or the response of their olfactory nerves can be used to detect patients with autism spectrum disorders.

[0009] The technical solutions of the present invention are as follows:

[0010] A use of nematodes in a kit for detecting autism spectrum disorders, wherein the kit is used for a method for detecting autism spectrum disorders using the nematodes' response to an odor of a test subject's organism-related substance or a processed product thereof as an indicator.

[0011] Preferably, the nematode is Caenorhabditis elegans.

[0012] Preferably, the nematode is a wild-type nematode, a mutant nematode or a transgenic nematode.

[0013] Preferably, when the nematode shows a positive response to the odor of the bio-related substance or a processed product thereof from the test subject, the test subject is determined to have an autism spectrum disorder or to be at risk of an autism spectrum disorder.

[0014] Preferably, when the nematode's olfactory nerve responds strongly to the odor of the test subject's organism-related substance or a processed product thereof, the test subject is determined to have an autism spectrum disorder or to be at risk of an autism spectrum disorder.

[0015] Preferably, the above-mentioned organism-related substance or processed product thereof is body fluid, cell, tissue, or culture or preservation fluid of cells or tissue.

[0016] Preferably, the above-mentioned body fluid is urine.

[0017] A detection kit for autism spectrum disorder, comprising nematodes.

[0018] Preferably, the nematode is Caenorhabditis elegans.

[0019] Preferably, the nematode is a wild-type nematode, a mutant nematode or a transgenic nematode.

[0020] A detection system for autism spectrum disorder, comprising:

[0021] Nematodes;

[0022] a storage portion that stores a living body-related substance or a processed substance thereof and the nematode; and

[0023] The detecting unit detects the reaction of the nematodes in the storage unit to the odor.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a method for detecting autism spectrum disorders using nematodes. This method allows for highly sensitive and low-cost detection of autism spectrum disorders. Furthermore, sample collection and analysis are simple and inexpensive. Furthermore, the method is capable of detecting autism spectrum disorders and is extremely useful for, for example, clinical evaluation of autism spectrum disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a diagram showing the arrangement of samples and nematodes in a culture dish according to an embodiment;

[0027] FIG2 is a data diagram of a urine sample 1:10 in the embodiment;

[0028] FIG3 is a data diagram of a urine sample at a ratio of 1:100 in the embodiment. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Example

[0030] 1. Urine sampling

[0031] The samples were collected from a children's hospital in Jiangsu Province. After delivery, they were placed in 10-mL polypropylene screw-cap tubes and stored at −20°C until use.

[0032] 2. Cultivation of OP50

[0033] Two bottles of LB liquid culture medium were sterilized at high temperature and high pressure. One bottle was used as a blank control. 100 μL of Escherichia coli OP50 culture medium was added to the other bottle. The culture was cultured on a shaker at 37°C and 170 rpm for 4 h until the culture medium became turbid. If the blank LB culture medium was clear, it could be placed at 4°C for later use.

[0034] 3. Cultivation of Caenorhabditis elegans

[0035] Inoculate a single clone of Escherichia coli (Escherichia coli strain OP50) into 50mL LB liquid culture medium and culture on a shaker at 37℃ for 4h. Cool the NGM culture medium after high-pressure sterilization to about 55℃, add the corresponding buffer, mix well and pour into the culture dish. Pipette 50μL OP50 bacterial droplet in the middle of the surface of the NGM culture medium, spread it evenly, air-dry, and take fresh, uncontaminated NGM culture medium coated with OP50 colonies for later use. Burn the platinum wire red on an alcohol lamp, let it cool, and then pick up Caenorhabditis elegans into the culture medium. Alternatively, you can also culture Caenorhabditis elegans in pieces, find the area with more nematodes with the naked eye and a microscope, and cut off a 1cm piece with tweezers burned by an alcohol lamp. 3 elegans culture medium and place it upside down on new NGM medium.

[0036] 4. Synchronization of Caenorhabditis elegans

[0037] Rinse a large number of well-grown, egg-laying C. elegans with 1 mL of M9 buffer. Transfer to an EP tube and allow to settle. Discard the supernatant. Add 500 μL of 10% NaClO and 500 μL of 1 M NaOH lysis buffer, mix by inversion, let stand for 3-5 minutes, centrifuge at 5000 rpm for 1 minute, and discard the supernatant. Rinse the remaining egg solution with M9 buffer twice. Finally, aspirate the eggs from the bottom layer and place them on NGM medium containing OP50. Incubate in a 20°C incubator.

[0038] 5. Preparation of experimental solutions and culture media

[0039] NGM solid medium: Weigh 3 g of sodium chloride, 2.5 g of peptone, and 17 g of agar, and add double-distilled water to 1 L; sterilize at 121°C for 20 min, cool to 55°C, add 1 mL of CaCl2 (1 M), 1 mL of MgSO4 (1 M), 1 mL of cholesterol solution (5 M), and 25 mL of KHPO4 buffer (1 M), mix well, and pour into a culture dish.

[0040] LB liquid medium: Weigh 10 g of tryptone, 5 g of sodium chloride, and 5 g of yeast extract, add double-distilled water to 1 L, and sterilize at 121°C for 20 min.

[0041] M9 buffer: 15.12 g Na2HPO4·12H2O, 3 g KH2PO4, 5 g NaCl, 0.25 g MgSO4·7H2O, add double-distilled water to 1 L, and sterilize at 121°C for 20 min.

[0042] 1M CaCl2: Weigh 11.10 g of anhydrous CaCl2 and add double-distilled water to 100 mL. Shake until the granules are dissolved. Place the solution in an autoclave at 121°C for 20 min. Allow the solution to cool to room temperature and store in a refrigerator at 4°C.

[0043] 1M MgSO4: Weigh 24.65 g of MgSO4·7H2O and add double-distilled water to 100 mL. Shake until the granules are dissolved. Place the solution in an autoclave at 121°C for 20 min. Allow the solution to cool to room temperature and store in a refrigerator at 4°C.

[0044] 1M KHPO 4 Buffer (pH = 6.0): Weigh 10.83 g KH2PO4 and 4.66 g K2HPO4·3H2O, add double-distilled water to 100 mL, shake until the granules are dissolved, place in an autoclave at 121°C for 20 min, cool the solution to room temperature, and store in a refrigerator at 4°C.

[0045] 5M cholesterol: Weigh 0.5 g of cholesterol and add 100 mL of anhydrous ethanol. Dissolve thoroughly, filter and sterilize, and store in a refrigerator at 4°C.

[0046] 10mL Levamisole: Weigh 0.12g of levamisole hydrochloride and add 50ml of double-distilled water. Store in a refrigerator at 4°C.

[0047] 6. Caenorhabditis elegans detection test

[0048] As shown in Figure 1, NGM medium was poured into a 90 mm culture dish and divided into three areas: A, B, and C. A dot was drawn in the center of the dish. Two lines were drawn 0.5 cm from each side of the dot to define area C. Two other dots were drawn 2 cm from each side to define areas A and B. 10 μl of sample diluted in double-distilled water was added to point A, and 10 μl of double-distilled water was added to point B. After both dots dried, 5 μl of levamisole was added. For the chemotaxis assay, L4 nematodes were washed and placed in area C. After 60 minutes, the number of worms present on the sample side (A) and the other side (B) was counted. The chemotaxis index was calculated using the following formula: Index = (A-B) / (A + B + C). Values ​​(-1 to 0) indicate repulsion of the urine sample; values ​​(0 to 1) indicate attraction to the urine sample.

[0049] As shown in Figures 2 and 3, the experimental results show that at both a 1:10 and 1:100 dilutions, urine from autistic patients is attractive to C. elegans, while urine from healthy individuals repels it. Across different dilution ratios, urine samples from autistic patients are more attractive to C. elegans at a 1:100 dilution, and the repetition rate between replicates is also higher. In actual testing, urine diluted 1:100 can be used as the test subject. For more accurate results, urine diluted 1:10 and 1:100 can be tested simultaneously. Patients who show attraction to C. elegans in both tests are considered autistic. A positive result in one test is considered suspected autism and is then repeated for confirmation. Patients diagnosed with autism by this method can be later confirmed using conventional autism testing methods.

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of nematodes in a kit for detecting autism spectrum disorders, the kit being used in a method for detecting autism spectrum disorders using the reaction of nematodes to an odor of a biologically related substance or a processed product thereof from a test subject as an indicator.

2. The use according to claim 1, characterized in that: The nematode is Caenorhabditis elegans.

3. The use according to claim 1, characterized in that The nematode is a wild-type nematode, a mutant nematode or a transgenic nematode.

4. The use according to any one of claims 1 to 3, characterized in that: When the nematode shows a positive response to the odor of the test subject's organism-related substance or a processed product thereof, the test subject is judged to have an autism spectrum disorder or to be at risk of an autism spectrum disorder.

5. The use according to any one of claims 1 to 3, characterized in that: When the olfactory nerve of the nematode responds strongly to the odor of the test subject's biological related substance or a processed product thereof, the test subject is determined to have an autism spectrum disorder or to be at risk of an autism spectrum disorder.

6. The use according to any one of claims 1 to 3, characterized in that: The bio-related substance or the processed product thereof is body fluid, cell, tissue, or culture or storage fluid of cells or tissue.

7. The use according to claim 6, characterized in that The body fluid is urine.

8. A test kit for autism spectrum disorder, characterized in that Contains nematodes.

9. The kit as claimed in claim 8, characterized in that The nematode is Caenorhabditis elegans.

10. The kit according to claim 9, characterized in that The nematode is a wild-type nematode, a mutant nematode or a transgenic nematode.

11. A detection system for autism spectrum disorder, characterized in that include: Nematodes; a storage section for storing the organism-related substance or its processed substance and the nematode; and, The detection unit detects the reaction of the nematodes in the storage unit to the odor.

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