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WO2023138266A1 - Biomarqueur associé à la maladie de parkinson et son application - Google Patents

Biomarqueur associé à la maladie de parkinson et son application Download PDF

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Publication number
WO2023138266A1
WO2023138266A1 PCT/CN2022/138797 CN2022138797W WO2023138266A1 WO 2023138266 A1 WO2023138266 A1 WO 2023138266A1 CN 2022138797 W CN2022138797 W CN 2022138797W WO 2023138266 A1 WO2023138266 A1 WO 2023138266A1
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saccharomyces cerevisiae
optionally
subject
fungi
biomarker
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Chinese (zh)
Inventor
张宝荣
夏丹丹
浦佳丽
张钰清
陈瑛
温轶
胡柳
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Zhejiang University ZJU
Zhejiang Yangshengtang Institute of Natural Medication Co Ltd
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Zhejiang University ZJU
Zhejiang Yangshengtang Institute of Natural Medication Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • the present invention relates to the field of biomedicine, in particular to biomarkers related to Parkinson's disease and their applications, related methods, corresponding kits, systems and the like.
  • Parkinson's disease is characterized by selective and progressive degeneration and loss of dopaminergic (DA) neurons in the substantia nigra compacta of the midbrain.
  • DA dopaminergic
  • PD is currently diagnosed mainly based on clinical typical motor symptoms and signs, and there are no other accurate and effective auxiliary bioinformatics indicators for early display of Parkinson's disease-related risks.
  • the course of PD includes preclinical PD and clinical PD (that is, PD is diagnosed based on typical clinical symptoms).
  • preclinical PD includes risk phase PD and prodromal phase PD.
  • motor symptoms have not yet appeared, and non-motor symptoms are the core manifestations, including constipation, hyposmia/loss, rapid eye movement sleep disorder (RBD), etc.
  • RBD rapid eye movement sleep disorder
  • PD in the clinical stage is divided into early, middle and late stages, among which grade 1-2 is early PD, grade 2.5-3 is middle-stage PD, and grade 4-5 is late PD.
  • the diagnosis of PD is mainly based on typical clinical symptoms. According to the current clinical diagnostic standards, in many cases, it may take 3-5 years of follow-up to make a clinical diagnosis of PD.
  • ET essential tremor
  • ET essential tremor
  • An object of the present invention is to provide a quick and simple biomarker for assisting in displaying biological information related to Parkinson's disease (PD), especially early PD.
  • PD Parkinson's disease
  • Another object of the present invention is to provide a quick and simple biomarker for assisting in distinguishing Parkinson's disease (PD) from essential tremor (ET).
  • PD Parkinson's disease
  • ET essential tremor
  • Yet another object of the present invention is to provide a quick and simple biomarker for identifying prodromal PD patients who are sleep behavior disorder (RBD) patients who may progress to PD.
  • RBD sleep behavior disorder
  • the present inventors found that the abundance of a fungus (Saccharomyces cerevisiae) was increased in the intestines of PD patients, whereas the abundance of this fungus was lower in the intestines of age-matched healthy people or ET patients. Furthermore, the inventors developed a detection method for this intestinal fungus, which can effectively distinguish PD patients, ET patients and healthy people through the detection of this fungus, and can be used for the discovery of prodromal PD patients (RBD progresses to PD), thereby realizing the auxiliary diagnosis of PD.
  • a fungus Sacharomyces cerevisiae
  • the present application finds that there is a fungus in the intestinal tract of PD patients, which can be used as a biomarker of PD, and the detection method of the fungus can be combined with the evaluation results of clinical scales to assist doctors in diagnosis.
  • the detection method includes but not limited to serum anti-Saccharomyces cerevisiae antibody (ASCA) IgG and IgA assay, fecal fungal ITS sequencing, fecal fungal fluorescent quantitative PCR assay, and the like.
  • ASCA serum anti-Saccharomyces cerevisiae antibody
  • IgG and IgA assay fecal fungal ITS sequencing
  • fecal fungal fluorescent quantitative PCR assay and the like.
  • the detection method of the present invention can be used independently as a clinical auxiliary diagnosis method, and can also be used as a combined composite index for clinical auxiliary diagnosis.
  • the present invention provides a biomarker to detect the presence and/or abundance of the fungus in the intestinal tract in the body of the subject's body.
  • PCR technology can detect a biomarker or the abundant biomarker of the selected winemaking yeast or the biomarker that can detect antibacterials in serum through ELISA to detect antifungal or selected wine yeast.
  • the present invention provides a biomarker, which is used to assist in displaying biological information related to Parkinson's disease (PD), especially early PD, or to assist in distinguishing Parkinson's disease (PD) from essential tremor (ET), or to identify prodromal PD patients, and the prodromal PD patients are sleep behavior disorder (RBD) patients who may progress to PD.
  • PD Parkinson's disease
  • ETD essential tremor
  • the present invention provides the use of biomarkers in the preparation of kits, wherein the kits are used to assist in the display of biological information related to Parkinson's disease (PD), especially early PD, or the kits are used to assist in distinguishing Parkinson's disease (PD) from essential tremor (ET), or the kits are used to identify patients with prodromal PD, wherein the biomarkers are used to detect the presence and/or abundance of fungi in the optional intestinal tract of a subject and/or the level of antibodies against the fungus in serum, optionally the fungus is Saccharomyces cerevisiae (Sac charomyces cerevisiae), wherein the biomarkers are selected from biomarkers capable of detecting the abundance of fungi or optionally Saccharomyces cerevisiae by ITS next-generation sequencing, PCR technology or biomarkers capable of detecting anti-fungal or optionally Saccharomyces cerevisiae antibody levels in serum by ELISA.
  • PD Parkinson's disease
  • ETD essential
  • the present invention provides a use of a biomarker in screening a candidate drug for treating Parkinson's disease (PD) or reducing the risk of early PD, wherein the biomarker is used to detect the presence and/or abundance of a fungus optionally in the intestinal tract of a subject and/or the antibody level against the fungus in serum, optionally the fungus is Saccharomyces cerevisiae, wherein the biomarker is selected from biomarkers capable of detecting the abundance of fungi or Saccharomyces cerevisiae through ITS next-generation sequencing, PCR technology or A biomarker capable of detecting the level of antibodies against the fungus or optionally Saccharomyces cerevisiae in serum by ELISA.
  • PD Parkinson's disease
  • the present invention provides a kit comprising reagents for detecting the presence and/or abundance of a fungus optionally in the intestinal tract of a subject and/or the level of antibodies against the fungus in serum, optionally the fungus is Saccharomyces cerevisiae, wherein the kit is used to assist in displaying biological information related to Parkinson's disease (PD), especially early PD, or the kit is used to assist in distinguishing Parkinson's disease (PD) from essential tremor (ET), or the kit is used to identify Phase PD patients.
  • PD Parkinson's disease
  • E essential tremor
  • the present invention provides a method for assisting in the screening of Parkinson's disease (PD), particularly early PD, comprising:
  • the abundance of the fungus or the level of antibodies against the fungus or optionally Saccharomyces cerevisiae is greater than the control indicates that the subject is at risk of developing PD or has early PD.
  • obtaining the subject sample includes selective isolation and enrichment of intestinal fungi, or optionally the sample is selected from blood and serum.
  • the present invention provides a method for assisting in displaying biological information related to Parkinson's disease (PD), especially early PD in a subject, comprising:
  • the abundance of the fungus or the level of antibodies against the fungus or optionally Saccharomyces cerevisiae is greater than the control indicates that the subject exhibits a risk of developing PD or has biological information associated with early PD.
  • obtaining the subject sample includes selective isolation and enrichment of intestinal fungi, or optionally the sample is selected from blood and serum.
  • the present invention provides a method for aiding in differentiating Parkinson's disease (PD) from essential tremor (ET), comprising:
  • obtaining the subject sample includes selective isolation and enrichment of intestinal fungi, or optionally the sample is selected from blood and serum.
  • the present invention provides a method for identifying a prodromal PD patient who is a sleep behavior disorder (RBD) patient who may progress to PD, the method comprising:
  • the abundance of the fungus or the level of antibodies against the fungus or optionally Saccharomyces cerevisiae is greater than the control indicates that the subject is a prodromal PD patient.
  • obtaining the subject sample includes selective isolation and enrichment of intestinal fungi, or optionally the sample is selected from blood and serum.
  • the present invention provides a system for assisting in the screening of Parkinson's disease (PD), particularly early PD, the system comprising:
  • a device for detecting the level of a biomarker according to the invention in a sample from a subject
  • PD Parkinson's disease
  • the detection device is a sequencing device, preferably an ITS next-generation sequencing device.
  • said detection device is a PCR device, preferably a real-time PCR device.
  • the detection device is an antibody level detection device, preferably an ELISA assay device.
  • said sample is selected from the group consisting of blood, serum, fluid from the digestive tract, and fecal extract.
  • biomarkers are selected from polynucleotides or antibodies.
  • said polynucleotide is a primer.
  • the antibody is an IgG antibody, an IgA antibody, or a combination thereof.
  • the present invention provides a system for assisting in the display of biological information related to Parkinson's disease (PD), particularly early PD, the system comprising:
  • a device for detecting the level of a biomarker according to the invention in a sample from a subject
  • PD Parkinson's disease
  • the detection device is a sequencing device, preferably an ITS next-generation sequencing device.
  • said detection device is a PCR device, preferably a real-time PCR device.
  • the detection device is an antibody level detection device, preferably an ELISA assay device.
  • said sample is selected from the group consisting of blood, serum, fluid from the digestive tract, and fecal extract.
  • biomarkers are selected from polynucleotides or antibodies.
  • said polynucleotide is a primer.
  • the antibody is an IgG antibody, an IgA antibody, or a combination thereof.
  • the present invention provides a system for aiding in differentiating Parkinson's disease (PD) from essential tremor (ET), the system comprising:
  • a device for detecting the level of a biomarker according to the invention in a sample from a subject
  • PD Parkinson's disease
  • ET essential tremor
  • the detection device is a sequencing device, preferably an ITS next-generation sequencing device.
  • said detection device is a PCR device, preferably a real-time PCR device.
  • the detection device is an antibody level detection device, preferably an ELISA assay device.
  • said sample is selected from the group consisting of blood, serum, fluid from the digestive tract, and fecal extract.
  • biomarkers are selected from polynucleotides or antibodies.
  • said polynucleotide is a primer.
  • the antibody is an IgG antibody, an IgA antibody, or a combination thereof.
  • the term “abundance” refers to the percentage of the amount of a certain fungus among all detected fungi.
  • the "abundance" of Saccharomyces cerevisiae refers to the percentage of the amount of Saccharomyces cerevisiae in all detected fungi;
  • Parkinson's disease patient or "PD patient” refers to a Parkinson's disease patient who has been diagnosed by at least one experienced neurologist according to the standard diagnostic criteria of the British Brain Bank.
  • essential tremor patient or "ET patient” refers to a patient with essential tremor diagnosed by at least one experienced neurologist according to the tremor consensus developed by the International Movement Disorders Association.
  • HC health control
  • ITS refers to the internal transcribed spacer (Internal Transcribed Spacer), located between fungal 18S, 5.8S and 28S rRNA genes, respectively ITS1 and ITS2.
  • 5.8S, 18S, and 28S rRNA genes are highly conserved, while ITS can tolerate more variation during evolution due to less natural selection pressure, and exhibits extremely extensive sequence polymorphism in most eukaryotes.
  • the conservative type of ITS is relatively consistent within the species, and the differences between the species are obvious, which can reflect the differences between species and even strains.
  • the ITS sequence fragments are relatively small (the lengths of ITS1 and ITS2 are 350bp and 400bp, respectively), which are easy to analyze and have been widely used in the phylogenetic analysis of different fungal species.
  • the present invention explored the relationship between intestinal fungi, intestinal inflammation-related serological antibodies and PD for the first time, and found that the abundance of intestinal fungi Saccharomyces cerevisiae, serum anti-Saccharomyces cerevisiae antibody (ASCA) IgG and IgA levels were significantly increased in PD patients.
  • the present invention finds for the first time that the relevant indicators of intestinal inflammation can be used as biomarkers for assisting early diagnosis of PD.
  • the intestinal fungus Saccharomyces cerevisiae can be detected by ITS next-generation sequencing or fluorescent quantitative PCR, and the serum anti-Saccharomyces antibody IgG and IgA composite indicators can be used to distinguish PD patients, healthy controls, HC and ET patients, and Saccharomyces indicators can also be used for the discovery of prodromal PD patients (RBD progresses to PD).
  • the present invention can assist in clinically diagnosing PD patients, realize rapid and accurate clinical diagnosis of early PD patients, and carry out early control of the disease.
  • Figure 1 shows the intestinal fungal composition and key differential fungi in the PD group and the HC group.
  • the left panel A is the PLSDA map based on the ASV abundance matrix, and the right panel B is the contribution of 25 differential ASVs to distinguish the two groups of samples.
  • Figure 2 shows the relative abundance of Saccharomyces cerevisiae Saccharomyces cerevisiae in PD group and HC group.
  • Figure 3 shows the intestinal fungal composition and key differential fungi in the PD group and the ET group.
  • the left panel A is the PLSDA map based on the ASV abundance matrix, and the right panel B is the contribution of 25 differential ASVs to distinguish the two groups of samples.
  • Figure 4 shows the relative abundance of Saccharomyces cerevisiae Saccharomyces cerevisiae in PD group and ET group.
  • Figure 5 shows the separation of yeast using mannan lectin-modified magnetic microspheres as an example, showing the schematic diagram of the magnetic separation of fungi.
  • Figure 6 shows the results of fluorescence detection showing magnetically specific capture.
  • the magnetic microsphere specific capture experiment was carried out (the emission wavelength of fungal fluorescence detection is 420nm, and the wavelength of bacterial fluorescence detection is 620nm). The results showed that only the fluorescent signal of fungi responded in the solution after magnetic separation, and there was no fluorescent signal of bacteria.
  • Figure 7 shows the results of magnetic separation and enrichment effects.
  • Figure 8 shows the real-time PCR quantitative results of Saccharomyces cerevisiae in PD patients and healthy controls HC.
  • Figure 9 shows the diagnostic accuracy of real-time PCR quantification of S. cerevisiae in distinguishing PD patients from healthy controls HC.
  • Figure 10 shows the real-time PCR quantitative results of Saccharomyces cerevisiae in PD patients and ET patients.
  • Figure 11 shows the diagnostic accuracy of real-time PCR quantification of S. cerevisiae in distinguishing PD patients from ET patients. This indicator can significantly distinguish PD patients from ET patients.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 12 shows the diagnostic accuracy of serum anti-S. cerevisiae antibody IgG levels in differentiating PD patients from healthy controls HC. Serum anti-S. cerevisiae IgG levels could significantly distinguish PD patients from HC.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 13 shows the diagnostic accuracy of serum anti-S. cerevisiae antibody IgA levels in differentiating PD patients from healthy controls HC.
  • Serum anti-S. cerevisiae antibody IgA levels can significantly distinguish PD patients from HC.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 14 shows the diagnostic accuracy of the composite index of serum anti-Saccharomyces cerevisiae antibody IgG and IgA levels to distinguish PD patients from healthy controls HC.
  • the composite index could significantly distinguish PD patients from healthy controls HC.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 15 shows the diagnostic accuracy of serum anti-S. cerevisiae IgG levels in differentiating PD patients from ET patients. Serum anti-S. cerevisiae IgG levels can significantly distinguish PD patients from ET patients.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 16 shows the diagnostic accuracy of serum anti-S. cerevisiae antibody IgA levels in differentiating PD patients from ET patients.
  • Serum anti-Saccharomyces cerevisiae antibody IgA levels can significantly distinguish PD patients from ET patients.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 17 shows the diagnostic accuracy of the composite index of serum anti-Saccharomyces cerevisiae antibody IgG and IgA levels to distinguish PD patients from ET patients.
  • the composite index can significantly distinguish PD patients from ET patients.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 18 shows the real-time PCR quantitative results of Saccharomyces cerevisiae in RBD patients.
  • Figure 19 shows the diagnostic accuracy of real-time PCR quantification of Saccharomyces cerevisiae in prodromal PD patients. This index can significantly distinguish RBD patients who progressed to PD from RBD patients who did not progress to PD.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 20 shows the diagnostic accuracy of serum anti-S. cerevisiae IgG levels in RBD patients who progressed to PD.
  • Serum anti-Saccharomyces cerevisiae IgG levels can identify patients with RBD who progress to PD.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 21 shows the diagnostic accuracy of serum anti-S. cerevisiae antibody IgA levels in RBD patients who progressed to PD.
  • Serum anti-Saccharomyces cerevisiae antibody IgA levels can identify patients with RBD who progress to PD.
  • ROC receiver operating characteristic curve
  • AUC area under the curve
  • 95% CI 95% confidence interval.
  • Figure 22 shows the diagnostic accuracy of the composite index of serum anti-Saccharomyces cerevisiae antibody IgG and IgA levels for RBD patients who progressed to PD.
  • the composite index identified RBD patients who progressed to PD.
  • ROC receiver operating characteristic curve; AUC: area under the curve; 95% CI: 95% confidence interval.
  • reagents, instruments, etc. used in the present invention are commercially available.
  • Example 1 Determination of the abundance of Saccharomyces cerevisiae by ITS next-generation sequencing to distinguish PD patients from healthy controls HC
  • the total DNA was extracted according to the instructions of QIAamp PowerFecal Pro DNA Kit (Qiagen, Cat No./ID: 51804), the DNA concentration and purity were detected by NanoDrop2000, and the quality of DNA extraction was detected by 1% agarose gel electrophoresis. Then use a universal primer pair for all fungi:
  • the PCR amplification products of the same sample were mixed and the PCR amplification products were recovered using 2% agarose gel, and the recovered products were purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). The recovered product was detected and quantified by 2% agarose gel electrophoresis and Quantus TM Fluorometer (Promega, USA).
  • NEXTFLEX Rapid DNA-Seq Kit use the NEXTFLEX Rapid DNA-Seq Kit to build a library, including the following steps: (1) adapter ligation; (2) use magnetic beads to screen and remove adapter self-ligating fragments; (3) use PCR amplification to enrich library templates; (4) magnetic beads to recover PCR amplification products to obtain the final library.
  • the taxonomic status of ASV was defined based on the SILVA database.
  • the number of sequences of all samples was normalized to 24781 (repeated 1000 times) to eliminate the differences between samples due to different sequencing depths, and then the ASV abundance matrix table of samples with the same number of sequences for each sample was obtained.
  • the sPLS-DA model When using the sPLS-DA model, we adopted the Centered Log Ratio transformation (CLR) to avoid spurious results. Based on the perf function and using leave-one-out cross-validation to test the misclassification rate of the model, the sPLS-DA model with the smallest error rate is the best classification model.
  • CLR Centered Log Ratio transformation
  • Example 2 Determination of the abundance of Saccharomyces cerevisiae by ITS next-generation sequencing to distinguish PD patients and ET patients
  • Example 2 We used the same method as in Example 1 to compare the intestinal fungal composition of the PD group and the ET group. Among the 25 differential ASVs, Saccharomyces cerevisiae Saccharomyces cerevisiae was again found to have the greatest contribution to distinguishing the two groups ( FIG. 3 ).
  • Example 3 Determination of Saccharomyces cerevisiae by fluorescent quantitative PCR to distinguish PD patients from healthy controls HC
  • fungi account for very little ( ⁇ 0.1%) in the intestinal flora, it is difficult to effectively obtain fungal information by direct sequencing methods, and it is necessary to selectively isolate and enrich fungi to facilitate subsequent research. Therefore, we first selectively isolate and enrich intestinal fungi, and then use primers for quantitative determination.
  • the separation and enrichment of microorganisms can improve the detection effect of target microorganisms.
  • the immunomagnetic separation method is a commonly used separation method in the detection of pathogenic microorganisms, and can be used for the detection of bacteria, fungi and other single-cell pathogenic sources (refer to, for example, BMC Microbiol., 2008, 22(8): 157-160, J. Clin. Microbiol., 2010, 48(4): 1126-1131).
  • the antibody that can specifically recognize the microorganism to be tested is connected to the surface of the magnetic microsphere as a capture probe for the microorganism to be tested.
  • the microorganism to be tested attached to the surface of the magnetic microsphere can be separated from the complex matrix sample by magnetic separation, and the influence of impurities and bacteria in the sample on the detection can be removed.
  • the magnetic separation process can reduce the volume of the liquid sample, which has the effect of enriching the microorganisms to be tested.
  • Bacteria are the biggest interference factor in the effective detection of fungi in samples. Since fungal cell wall components are very different from bacteria, specific components of fungal cell walls can be selected as capture sites to bind to capture probes.
  • the main component of the fungal cell wall is chitin
  • the main polysaccharide component of the yeast cell wall is mannan
  • the main polysaccharide component of the bacterial cell wall is peptidoglycan. Therefore, chitin and mannan can be selected as fungal-specific capture sites.
  • Recombinant chitinase can specifically recognize and bind to chitin. Therefore, a recombinant chitinase that can specifically bind to fungi was used as a capture probe.
  • mannan-binding protein can be selected as the recognition protein, specifically recognizes and binds to fungi of the genus Saccharomyces, and isolates yeasts in biological samples (refer to for example Proc.
  • Functionalized magnetic microspheres Two kinds are connected by designing and synthesizing, respectively coated with recombinant chitinase and mannan-binding protein (mannan lectin).
  • Functionalized magnetic microspheres can be attached to the fungal cell wall surface by binding to chitin and mannan on the fungal surface. Through magnetic separation, the fungi connected to the magnetic microspheres can be effectively separated from the complex system, and other substances in the system can be removed to achieve the effect of fungal separation and enrichment.
  • Figure 5 uses mannan lectin-modified magnetic microspheres to separate yeast as an example, demonstrating the principle of fungal magnetic separation.
  • Recombinant mannanboundin can be connected to the surface of TED-Ni-coated magnetic microspheres through His-tag, or can be modified by biotinylation (Biotin) to connect to the surface of streptavidin (SA)-coated magnetic microspheres. Both reactions can be used to make recombinant chitinase-coated magnetic microspheres.
  • the unique mannan component on the yeast cell wall will specifically bind to the recombinant mannan-boundin on the surface of the magnetic microspheres, thereby immobilizing on the surface of the magnetic microspheres, while the bacterial cell wall does not contain mannan components, so it will not specifically bind to the magnetic microspheres. Therefore, in the subsequent magnetic separation process, the bacteria and other free components not connected to the surface of the magnetic microspheres contained in the supernatant will be removed during the washing process, while the fungi fixed on the surface of the magnetic microspheres can be retained. And the magnetic separation process can reduce the volume of the final solution, so as to achieve the effect of fungal separation and enrichment.
  • the magnetic separation method using recombinant chitinase as a capture probe is similar to Figure 5, and fungi are captured by recognizing chitinase on the fungal cell wall.
  • Biotinylation kit (Abcam): Biotinylation Kit/Biotin Conjugation Kit (Fast, Type B)-Lightning-
  • Mannan-binding protein Recombinant Human Mannan Binding Lectin/MBL protein
  • Recombinant chitinase (Abcam): recombinant mouse chitinase 3-like protein 3;
  • Magnetic microspheres (Invitrogen): Dynabeads TM M-280 Streptavidin;
  • Magnetic stand (Invitrogen): DynaMag TM -2 magnetic stand;
  • biotinylation kit to biotinylate mannan lectin, and follow the instructions of the kit for specific operations;
  • the His-tag can be used to react with TED-Ni.
  • the reaction conditions and dissociation conditions refer to the product instructions of TED-Ni magnetic microspheres.
  • tissue cell disruptor to lyse the sample, setting 5, crush for 30 seconds, with an interval of 30 seconds, and repeat three times.
  • the SCoH sequence of the primer we designed for Saccharomyces cerevisiae is as follows:
  • Primer sequence F 5'-GTTAGATCCCAGGCGTAGAACAG-3'
  • Primer sequence R 5'-GCGAGTACTGGACCAAATCTTATG-3'
  • Annealing temperature is 58°C
  • the length of the amplified product is 400bp.
  • Example 4 Determination of Saccharomyces cerevisiae by fluorescent quantitative PCR to distinguish PD patients and ET patients
  • Saccharomyces cerevisiae was detected by real-time quantitative PCR, and 18 primary PD patients, 10 ET patients and 18 healthy controls HC were diagnosed in the Neurology Department of Zhejiang Second Hospital from February 01, 2021 to October 1, 2021.
  • Example 5 Serum anti-Saccharomyces cerevisiae antibody IgG or IgA and its composite index are used to distinguish PD patients and healthy controls HC
  • Serum anti-Saccharomyces cerevisiae antibody levels were measured on blood samples from volunteers.
  • Serum anti-Saccharomyces cerevisiae antibodies IgG and IgA were detected by enzyme-linked immunosorbent assay (ELISA). Serum anti-Saccharomyces cerevisiae IgG and IgA cut-off values were both 25 AU/ml.
  • Example 6 Serum anti-Saccharomyces cerevisiae antibody IgG or IgA and its composite index are used to distinguish PD patients from ET patients
  • Example 5 The same method as in Example 5 was used to detect the serum anti-Saccharomyces cerevisiae antibody levels of samples from PD patients and ET patients.
  • Example 7 Detection of Saccharomyces cerevisiae by fluorescent quantitative PCR to identify RBD patients who progressed to PD
  • measuring Saccharomyces cerevisiae by real-time PCR can identify patients with RBD who can progress to PD.
  • Example 8 Serum anti-Saccharomyces cerevisiae antibody IgG or IgA and its composite index can identify RBD patients who progressed to PD
  • intestinal inflammation-related serological antibodies we explored the relationship between intestinal fungi, intestinal inflammation-related serological antibodies and PD for the first time, and found that the abundance of intestinal fungi Saccharomyces cerevisiae, serum anti-Saccharomyces cerevisiae antibody (ASCA) IgG and IgA levels were significantly increased in PD patients.
  • ASCA serum anti-Saccharomyces cerevisiae antibody
  • intestinal inflammation-related indicators can be used as biomarkers for assisting early diagnosis of PD.
  • auxiliary early diagnosis our examples: 1. The intestinal fungus Saccharomyces cerevisiae is detected by ITS next-generation sequencing or fluorescent quantitative PCR; 2.
  • Serum anti-Saccharomyces cerevisiae antibody IgG and IgA composite indicators can be used to distinguish PD patients, healthy controls HC and ET patients; 3. Saccharomyces cerevisiae indicators can also be used for the discovery of prodromal PD patients (RBD progresses to PD).
  • the above-mentioned embodiments can assist in the clinical diagnosis of PD patients, realize rapid and accurate clinical diagnosis of early PD patients, and carry out early control of the disease.

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Abstract

La présente invention concerne un biomarqueur associé à la maladie de Parkinson (MP) et une application de celui-ci. Plus particulièrement, des champignons, en particulier Saccharomyces cerevisiae, peuvent être utilisés comme biomarqueurs pour le diagnostic de la MP. L'invention concerne également un procédé de détection de champignons, en particulier de Saccharomyces cerevisiae. Le biomarqueur et son procédé de détection peuvent être utilisés pour l'affichage subsidiaire d'informations biologiques relatives à la MP, plus particulièrement la MP précoce, peuvent être utilisés pour aider à distinguer la MP du tremblement essentiel (TE), et peuvent également être utilisés pour identifier un patient MP dans une phase prodromique, le patient MP dans la phase prodromique étant un patient souffrant d'un trouble du comportement du sommeil (TCS) présentant la possibilité d'évoluer vers la MP. L'invention concerne également un kit, un système et une application associée. Le procédé peut être utilisé indépendamment en tant que procédé de diagnostic subsidiaire clinique, et peut également être utilisé en tant qu'indice composite conjoint pour un diagnostic subsidiaire clinique.
PCT/CN2022/138797 2022-01-20 2022-12-13 Biomarqueur associé à la maladie de parkinson et son application Ceased WO2023138266A1 (fr)

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CN120064627B (zh) * 2025-01-22 2025-10-17 上海健康医学院 一组用于诊断帕金森病伴快速眼动睡眠行为障碍的生物标志物及其应用

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