LU503455B1

LU503455B1 - Application of Intestinal Flora Marker in Autism Diagnosis

Info

Publication number: LU503455B1
Application number: LU503455A
Authority: LU
Inventors: Jun Zhang
Original assignee: Jun Zhang
Priority date: 2023-02-09
Filing date: 2023-02-09
Publication date: 2023-08-09

Abstract

The invention provides an application of the reagent for detecting the intestinal flora abundance in preparing products for diagnosing autism, a product for diagnosing autism, a kit for diagnosing autism and a chip for diagnosing autism. According to the invention, the prokaryote Cyanobacterium is found to be related to autism for the first time, and its abundance is obviously increased in the population of autistic patients, and ROC curve analysis shows that it has high specificity and sensitivity as a detection variable, so the prokaryote Cyanobacterium can be used as a detection marker to be applied to the diagnosis of autistic patients. Prokaryote Cyanobacteria is used as the detection marker, which is completely non-invasive and has high accuracy.

Description

DESCRIPTION LUS03455

Application of Intestinal Flora Marker in Autism Diagnosis

TECHNICAL FIELD

The invention belongs to the field of biomedicine, in particular to the application of intestinal flora marker in autism.

BACKGROUND

Autism, also known as autistic disorder, is a representative disease of pervasive developmental disorder (PDD). DSM-IV-TR divides PDD into five types: autistic disorder, Retts syndrome, childhood disintegration disorder, Asperger syndrome and unspecified PDD. Among them, autistic disorder and Asperger's syndrome are common. There are different reports on the prevalence rate of autism, which is generally considered to be about 2~5/10,000 children, with a ratio of 3 ~ 4: 1 between men and women, and 3~4 times more boys than girls.

Autism can be divided into true autism and pseudo autism. True autism (organic autism) is the loss of brain thinking function, loss or serious loss of thinking function due to gene mutation.

Their faces are no different from those of ordinary people, but they are congenitally lacking in logical thinking abilities such as summarization, induction, analysis and judgment. And their lifelong intelligence is low. pseudo autism (functional autism) means that children have normal thinking ability without organic lesions in their brain thinking area, but their lack of intelligence is due to the unbalanced development of an certain acquired ability. There are essential differences between pseudoautism and true autism. True autism is caused by gene mutation.

According to the calculation, the proportion of human true autism should be close to one in 150,000. It is estimated that there should be no more than 120 children born with true autism every year in China and less than 36 patients born with true autism every year in the United

States. The true autism is a rare disease. There is no effective rehabilitation method at present.

Pseudo autism is due to the uneven development of several ability factors that affect human learning ability. That is, a certain ability is extremely weak or a certain ability is too strong, which may cause children to lose part or all of their thinking ability, resulting in lifelong mental deficiency and showing the characteristics of true autism. According to the diagnosis statistics of domestic hospitals, the incidence of autism is 2%o-3%o, of which the true autism accounts for less than 0.2% and the false autism accounts for more than 99.8%. LU503455

In 1980s, the research on autism entered a new stage. People began to abandon the so-called "improper parenting" hypothesis for cause of disease, explore the causes of autism from the biological field, and completely separate autism from schizophrenia in the identification and clinical diagnosis of clinical symptoms. Kolvin's research shows that autism has nothing to do with adult psychotic disorders, especially adult schizophrenia. DSM-III published in 1980 regarded childhood autism as a pervasive developmental disorder for the first time. Later, with the in-depth study of autism, people gradually realized that autism is a diffuse central nervous system developmental disorder caused by various environmental factors stimulation under the influence of certain genetic factors. On the basis of this understanding, many studies have been carried out from molecular inheritance to neuroimmunity, functional imaging, neuroanatomy and neurochemistry, and people try to find the cause of autism from these studies.

At present, there is still no hypothesis that can fundamentally and perfectly explain the cause of autism, so there is no good way to predict, diagnose and treat autism.

SUMMARY

In order to make up for the shortcomings of the prior art, the invention aims to provide an autism means and product.

We studied the abundance of intestinal microbial flora in autistic patients (caused by non-genetic factors) and normal children, and found that there was a significant correlation between the prokaryote Cyanobacteria in the intestinal microorganisms of the subjects and the occurrence of autism (P value was 0.008). This indicates that prokaryote Cyanobacteria is closely related to autism, which may be the main cause of pseudo autism.

The detection of prokaryote Cyanobacteria can effectively screen the subjects who have autism or are at risk of autism. (Note: Prokaryote Cyanobacterium is a very old bacterium in nature. Melainabacteria was discovered and named by Sara C Di Rienzi and others in 2013, and it is a branch of prokaryote Cyanobacterium in evolution. )

The purpose of the invention is to provide the application of a reagent for detecting the intestinal flora abundance in preparing products for diagnosing autism, and the microorganism includes prokaryote Cyanobacteria.

Preferably, the prokaryote Cyanobacteria includes Melainabacteria. LU503455

Preferably, the reagent comprises primer, probe, antisense oligonucleotide, aptamer or antibody for detecting the specificity of the microorganism.

Preferably, the specific primer is a primer capable of detecting 16SrRNA of the microorganism.

The purpose of the present invention is to provide a product for diagnosing autism, and the product comprises a reagent for detecting the abundance of prokaryote Cyanobacteria.

Preferably, the reagent comprises the primer for detecting the 16SrRNA of the prokaryote.

The purpose of the present invention is also to provides a kit for diagnosing autism, and the kit comprises a primer pair for specifically detecting 16SrRNA of prokaryote Cyanobacteria.

The purpose of the present invention is also to provides a chip for diagnosing autism, and it is characterized in that the chip comprises a solid-phase carrier and an oligonucleotide probe fixed on the solid-phase carrier, and the oligonucleotide probe specifically recognizes the 16SrRNA of prokaryote Cyanobacteria.

In the present invention, the term "probe" refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise specified, the term "probe" generally refers to a polynucleotide probe capable of binding to another polynucleotide (often referred to as a "target polynucleotide") through complementary base pairing. According to the stringency of hybridization conditions, the probe can bind to the target polynucleotide which lacks complete sequence complementarity with the probe. The probe can be labeled directly or indirectly, and its range includes primers. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase or in-situ hybridization assay.

Exemplary probes in the present invention include PCR primers and gene-specific DNA oligonucleotide probes, such as microarray probes fixed on microarray substrates, quantitative nuclease protection test probes, probes connected with molecular barcodes, and probes fixed on beads.

The probe has a base sequence complementary to a specific base sequence of the target gene. Here, the so-called "complementary to" can be used as long as it is hybridization, and it may not be completely complementary. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100%

with respect to this specific base sequence. These probes can be DNA or RNA, and in addition/°08488 they can be polynucleotides obtained by replacing some or all of their nucleotides with artificial nucleic acids such as polyamide nucleic acid (PNA), locked nucleic acid, bridged Nucleic Acid (LNA), 2°-0,4’-C-Ethylene-bridged nucleic acids (ENA), glycerol nucleic acid (GNA), threose nucleic acid (TNA).

As used herein, the term "sample" or "test sample" refers to any liquid or solid material containing nucleic acid. In a suitable embodiment, the test sample is obtained from a biological source (i.e., a "biological sample"), such as a cell in culture, or a tissue sample from an animal and most preferably from a human. In an exemplary embodiment, the sample is feces.

The term "abundance difference" means that higher or lower levels of microorganisms are obtained in patients with autism compared with the in vivo levels of normal or control targets.

In the present invention, the term "primer" means 7 to 50 nucleic acid sequences that can form a base pair complementary to the template strand and serve as a starting point for replicating the template strand. Primers are usually synthesized, but naturally occurring nucleic acids can also be used. The sequence of the primer does not necessarily need to be exactly the same as that of the template, as long as it is sufficiently complementary to hybridize with the template. Additional features that do not change the basic properties of primers can be mixed.

Examples of additional features that can be mixed include methylation, capping, substitution of more than one nucleic acid by homologues and modification between nucleic acids, but are not limited to this. In this application, the term "16s tRNA" refers to the rRNA that constitutes the 30S small subunit of prokaryotic ribosomes. On the one hand, most of the base sequence is highly preserved, on the other hand, some regions show high base sequence diversity. The difference is that there is almost no diversity among the same species, but there is diversity among the different species, so by comparing the sequences of 16SrRNA, prokaryotes can be effectively identified.

As an embodiment, in the present invention, the primer can be used to amplify the sequence of 16SrRNA retained in the corresponding microorganism, and after the sequence is amplified, the existence of microorganism or the level of microorganism can be detected by whether the expected product is generated or not. Various methods known in the art can be used for the sequence amplification method using primers. For example, polymerase chain reaction (PCR),

reverse transcription polymerase chain reaction (RT-PCR), multiplex PCR, touchdown PCR, hot/203455 start PCR, nested PCR, booster PCR, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction, vectorette PCR, Tail

PCR (thermal asymmetric interlaced PCR), ligase chain reaction, repair chain reaction, transcription-mediated amplification, self-sustainedsequence replication, and selective amplification reaction of target base sequence, but the scope of the present invention is not limited to this.

In addition, in the present invention, the preparation for detecting microorganism or determining microorganism level can be antibody, and the corresponding microorganism can be detected or microorganism level can be determined by using an immunological method based on antigen-antibody reaction. As analytical methods used for this, there are western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion,

Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitationassay, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, etc. The above methods are only for explaining the immune response of antibody-antigen, and the present invention is not limited to the above methods.

In addition, molecular immunology methods widely used in the field can be used to detect microorganisms or determine microbial levels in the present invention.

The invention has the advantages and beneficial effects that the prokaryote Cyanobacterium is found to be related to autism for the first time, and its abundance is obviously increased in the population of autistic patients, and ROC curve analysis shows that it has high specificity and sensitivity as a detection variable, so the prokaryote Cyanobacterium can be used as a detection marker to be applied to the diagnosis of autistic patients. Prokaryote Cyanobacteria is used as the detection marker, which is completely non-invasive and has high accuracy.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a comparative diagram of prokaryote Cyanobacteria flora abundance in autism group and control group in Embodiment 1 of the present invention;

Fig. 2 is a ROC curve with the prokaryote Cyanobacteria flora abundance as the detection variable in Embodiment 1 of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In order to evaluate whether the composition of intestinal symbiotic flora can be used as a predictor of autism, the invention collects samples of autistic patients and healthy people, performs 16SrRNA sequencing and uses bioinformatics to perform statistics of sequencing data, finds intestinal flora related to diseases, integrates intestinal flora with disease information, and predicts autistic patients to the greatest extent. According to the invention, through 16SrRNA sequencing, the correlation between prokaryote Cyanobacterium and autistic patients is discovered for the first time, and the prokaryote Cyanobacterium flora abundance in the autism group is obviously higher than that in the control group, indicating that prokaryote

Cyanobacterium can be used as a predictor of autism.

The present invention will be further described in detail with the attached figures and embodiments. The following embodiments are only used to illustrate the invention and are not used to limit the scope of the invention. The experimental methods without specific conditions in the embodiments usually follow the conventional conditions, such as those described in

Sambrook et al, Molecular Cloning: Laboratory Press (New York: Cold Spring Harbor

Laboratory Press, 1989), or those suggested by the manufacturers.

Embodiment 1

Screen the intestinal flora related to autism, detect, identify and verify. 1. Research object and sample collection

Fecal samples were collected from 10 patients with autism and 7 healthy people. Sample sources and selection criteria are as follows:

Autism group: from 10 families, aged 3-5 years. There are 10 patients diagnosed with autism. Exclusion criteria: (1) Suffering from other mental diseases (such as schizophrenia) and other neurodevelopmental disorders; (2) Suffering from hereditary metabolic diseases; (3)

Suffering from serious neurological diseases, history of craniocerebral injury and other major physical diseases; (4) Have a history of gastrointestinal infection or antibiotic use in the past one month.

Control group: 7 cases from 6 families (including 2 twins from the same family). Inclusion criteria: (1) Good health and no neurodevelopmental disorders; (2) Match with the age of the patient group. The exclusion criteria were the same as those in autism group. LU503455 2. 16S rRNA sequencing 2 .1. DNA extraction

The genomic DNA extraction kit was used to extract genomic DNA from the sample, and the steps were preformed by following the instructions. 2.2. Determination of DNA sample purity and concentration

Genomic DNA was detected by 1% agarose gel electrophoresis. 2 .3. PCR amplification

PCR amplification (or other primers) was carried out with V3~V4 variable region sequence of bacterial 16S rRNA gene as target and 338F-806R with barcode sequence as primer to obtain

PCR products; 2 4. 16S rRNA gene sequencing

After the PCR products were quantified and the library was constructed, high-throughput sequencing was performed by using Illumina MiSeq PE300 platform or novaseq PE250 to obtain the base sequence information of V3~V4 variable region of bacterial 16S rRNA gene (or the base sequence information of the region corresponding to other primers). 3. Data analysis 3 .1. The acquisition of the abundance information of flora.

The QIIME2 .0 software package was used to cluster the sequenced fragments by OTU (Taxonomic Operation Unit), and the OUT representative sequence is compared with the silva database (or the GREENGENES database) to obtain the taxonomic units (including phylum, class, order, family, genus and species) corresponding to OUT and their corresponding abundance information. 3.2. Analysis of species differences of intestinal flora

SPSS statistical software was used to analyze the Spearman correlation between flora abundance and autism, and the flora with significant correlation with autism was found out. At the same time, the ROC curve tool of SPSS statistical software is used to draw the ROC curve and calculate its AUC area. 4. Results

The prokaryote Cyanobacteria flora abundance in the autism group increased significantly,

and the average abundance of the prokaryote Cyanobacteria flora in the autism group was 10t3/903455 times that in the control group. The comparison of the flora abundance abundance is shown in

Fig. 1.

The results of correlation analysis between flora abundance and autism showed that there was a significant correlation between autism group and prokaryote Cyanobacteria flora abundance level (p=0.008). The analysis results are shown in Table 1.

Table 1 Correlation coefficient of the results of correlation analysis between autism group and prokaryotic Cyanobacteria flora abundance

T=

Graups abundance tho of Spearman Groups Cometation coefficient 15100 ASIE

Sig-{double sides} + RE

Cyarobacteria fora Cowelaton coefficient SIE” 100 abundance sig {double sides} S08 w | £7} Xi * * When the confidence (double test) is 0.01, the correlation is significant.

As shown in Fig. 2, the analysis of the receiver's operating characteristic curve (ROC curve) shows that the AUC is 0.857 and the P value is 0.015, which is statistically significant. The optimal critical value is 0.00006851, the corresponding specificity is 0.714, and the sensitivity is 1, which indicates that the application of prokaryote Cyanobacteria in the diagnosis of autism has high sensitivity.

Those skilled in the art can prepare probes, antisense oligonucleotides, aptamers or antibodies for detecting the specificity of the microorganisms through the above-mentioned embodiments and the well-known technologies in the art, which can also be used for detecting autism, so will not be repeated here.

At the same time, Those skilled in the art can also prepare reagents for detecting the intestinal flora abundance and products for diagnosing autism.

Embodiment 2 Preparation of autism diagnosis kit

According to the correlation between prokaryote Cyanobacterium and autism, autism can be diagnosed by detecting the abundance of prokaryote Cyanobacterium in a sample, and accordingly, the invention provides a kit for diagnosing autism based on detecting the abundan&&/ 503455 of prokaryote Cyanobacterium. The components of the kit are as follows: DNA extraction reagent, primer pair for specific detection of 16SrRNA of procaryote Cyanobacteria, reaction buffer, deoxynucleotide triphosphate (dNTPs), Taq-polymerase reverse transcriptase, DNase,

RNAse inhibitor, DEPC-water, sterile water and SYBR Green fluorescent dye.

Embodiment 3 Preparation of chip for diagnosing autism

This embodiment provides a chip for diagnosing autism, which comprises a solid-phase carrier and an oligonucleotide probe fixed on the solid-phase carrier. And the oligonucleotide probe specifically recognizes the 16SrRNA of the prokaryote Cyanobacteria. The specific operation method is the prior art, so it won't be repeated here.

The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be pointed out that for those skilled in the art, several improvements and modifications can be made to the invention without departing from the principle of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the invention.

Claims

CLAIMS LU503455

1. The application of a reagent for detecting the intestinal flora abundance in preparing products for diagnosing autism is characterized in that the microorganism includes prokaryote Cyanobacteria.

2. The application according to claim 1 is characterized in that the prokaryote Cyanobacteria includes Melainabacteria.

3. The application according to any of claims 1 or 2 is characterized in that the reagent comprises primer, probe, antisense oligonucleotide, aptamer or antibody for detecting the specificity of the microorganism.

4. The application according to claim 3 is characterized in that the specific primer is a primer capable of detecting 16SrRNA of the microorganism.

5. A product for diagnosing autism is characterized in that the product comprises a reagent for detecting the abundance of prokaryote Cyanobacteria.

6. The product according to claim 5 is characterized in that the reagent comprises the primer for detecting the 16SrRNA of the prokaryote.

7. A kit for diagnosing autism is characterized in that the kit comprises a primer pair for specifically detecting 16SrRNA of prokaryote Cyanobacteria.

8. A chip for diagnosing autism is characterized in that the chip comprises a solid-phase carrier and an oligonucleotide probe fixed on the solid-phase carrier, and the oligonucleotide probe specifically recognizes the 16SrRNA of prokaryote Cyanobacteria.