CN112226525B

CN112226525B - Reagent for diagnosing myasthenia gravis

Info

Publication number: CN112226525B
Application number: CN202010968305.7A
Authority: CN
Inventors: 乞国艳
Original assignee: Shijiazhuang People's Hospital Shijiazhuang First Hospital Shijiazhuang Tumor Hospital Hebei Myasthenia Gravis Hospital Shijiazhuang Cardiovascular Disease Hospital
Current assignee: Shijiazhuang People's Hospital Shijiazhuang First Hospital Shijiazhuang Tumor Hospital Hebei Myasthenia Gravis Hospital Shijiazhuang Cardiovascular Disease Hospital
Priority date: 2020-09-15
Filing date: 2020-09-15
Publication date: 2023-03-31
Anticipated expiration: 2040-09-15
Also published as: CN112226525A

Abstract

The invention discloses a reagent for diagnosing myasthenia gravis, and relates to application of a reagent for detecting the abundance of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46FAA, helicobacter _ cinaedi or Clostridium _ barrletii in preparation of a product for diagnosing myasthenia gravis, and also relates to a product for diagnosing myasthenia gravis, which is prepared on the basis that Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46FAA, helicobacter _ cinaedi or Clostridium _ barrletii shows a significant difference in myasthenia gravis.

Description

Reagent for diagnosing myasthenia gravis

Technical Field

The invention belongs to the technical field of biology, and relates to a reagent for diagnosing myasthenia gravis.

Background

Myasthenia Gravis (MG) is an autoantibody mediated, T cell dependent, complement and cytokine involvement, acquired autoimmune disease involving the postsynaptic membrane of the Neuromuscular junction (NMJ), with an annual average incidence of (8.0-20.0)/10 million people, and at all ages (Gilhus NE. Myastemia gravis. N Engl J Med.2016;375 (26): 2570-81.). The disease can affect skeletal muscles of the whole body, including extraocular muscles, pharyngeal muscles, muscles of four limbs and trunk, and has the problems of eyelid ptosis, double images of visual objects, dysphagia, limb weakness and even dyspnea, and serious patients can endanger life. The typical symptoms are easy fatigue of skeletal muscle, severe twilight morning or severe afternoon, and relieved rest, most of which are effective anticholinesterase inhibitors, and the pathogenesis of which is not clear (Dalakas MC. Novel future therapeutic options in myasethenia gravis. Autoimmun Rev.2013Ju1;12 (9): 936-41.). About 10% -20% of patients with ocular muscle type MG can heal themselves, about 20-30% of patients with ocular muscle type MG will remain confined to the extraocular muscles and not progress, most of the rest of patients may involve the medulla oblongata and the limb muscles within 3 years of onset, gradually develop into systemic type MG, about 2/3 of patients with MG will reach the peak of disease severity within 1 year after onset, and about 20% of patients with MG develop MG crisis within 1 year of onset (David G, norman B, tatsuji N, et al. In cases such as surgery, trauma, mental stress, infection, fever, pregnancy in women, childbirth, use of drugs affecting myasthenia gravis, or other autoimmune diseases, the muscle weakness symptoms in MG patients may be exacerbated. The immune pathogenesis of MG is complex, and possible factors include genetic predisposition, environmental factors, T cell and B cell synergy, T helper cell (Th)/regulatory T cell imbalance, abnormalities in cytokine and antibody secretion, and activation of the complement system. Early diagnosis of MG is of great significance for therapy.

The intestinal flora is an important component of the intestinal system, and human beings form a mutual-benefit symbiotic relationship with the intestinal flora in the long-term evolution process. When the species and amount of microorganisms in the intestine are changed due to changes in dietary habits, environmental factors, intestinal infections or other factors, the dynamic balance between the body and the intestinal flora is disturbed, resulting in intestinal micro-ecological disorders and dysfunction of various aspects of the body, which may lead to the development of diseases (Jandhyala S M, talukdar R, subramann C, et al., role of the normal gut microbiota [ J ]. World J Gastroenterol,2015,21 (29): 8787-8803.). Therefore, maintaining the dynamic balance of the intestinal flora is particularly important to the physiological health of the organism. The intestinal flora related to the myasthenia gravis is researched, the function of the intestinal flora in the occurrence and development process of the myasthenia gravis is researched, and a new thought is provided for diagnosis and treatment of the myasthenia gravis.

Disclosure of Invention

The invention aims to provide an intestinal flora associated with the occurrence and development of myasthenia gravis and application thereof in diagnosis and treatment of myasthenia gravis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of a reagent for detecting the abundance of flora selected from any one or more of intestinal bacteria Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46FAA, helicobacter _ cinaedi or Clostridium _ bartlettii in preparing a product for predicting myasthenia gravis.

Further, the flora is selected from any two or more of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46FAA, helicobacter _ cinaedi or Clostridium _ bartlettii.

Further, the reagent includes an oligonucleotide that specifically hybridizes to a target nucleotide sequence from Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46faa, helicobacter _ cinaedi, or Clostridium _ bartlettii.

Further, the target nucleotide sequence of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46faa, helicobacter _ cinaedi or Clostridium _ bartlettii, which is a fragment of the species-specific gene region, is a fragment of the species-specific gene region.

Further, the oligonucleotide is detectably labeled.

Further, the myasthenia gravis is childhood myasthenia gravis.

The invention provides a product for diagnosing myasthenia gravis, which comprises a reagent for detecting the abundance of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46FAA, helicobacter _ cinaedi or Clostridium _ bartlettii in a sample.

Further, the target nucleotide sequence of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46FAA, helicobacter _ cinaedi or Clostridium _ bartlettii is a fragment of the strain-specific gene region.

Further, the oligonucleotide is detectably labeled.

Further, the oligonucleotide includes a probe that specifically recognizes a target nucleotide sequence of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1 \46faa, helicobacter _ cinaedi, or Clostridium _ bartlettii or a primer that specifically amplifies a target nucleotide sequence of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46faa, helicobacter _ cinaedi, or Clostridium _ bartlettii.

Further, the amplification is PCR or RT-PCR, preferably, the amplification utilizes detectably labeled primers.

The invention provides application of a microbial marker in preparing a medicament for treating myasthenia gravis, wherein the microbial marker is any one or more selected from Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46FAA, helicobacter _ cinaedi or Clostridium _ bartlettii.

Further, the medicament includes an agent that increases the abundance of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46faa, helicobacter _ cinaedi, or Clostridium _ bartlettii.

Drawings

Fig. 1 is a violin diagram of the alpha and beta diversity distribution; wherein A-C is a distribution plot of alpha diversity at the phylum (A), genus (B) and species (C) levels based on the shannon index; D-F is a distribution plot of beta diversity at the phylum (D), genus (E) and species (F) levels based on the Bray-Curtis distance.

Figure 2 is PcoA of the relative abundance of all participants at different categorical levels, where panels a-F are PcoA of the relative abundance at phylum, class, order, family, genus, categorical levels, respectively, and the red and blue triangles represent MG and HC, respectively.

FIG. 3 is a graph showing the abundance of Pyramidobacter _ piscolens.

Detailed Description

In order to evaluate whether the composition of the intestinal symbiotic flora can be used as a prediction factor of myasthenia gravis, samples of myasthenia gravis patients and healthy people are collected, whole genome sequencing is carried out, statistics of sequencing data is carried out by using bioinformatics, intestinal flora related to diseases is found, the intestinal flora and the disease information are integrated, and the myasthenia gravis patients are predicted to the greatest extent. According to the invention, through whole genome sequencing, the remarkable difference of the Pyramidobacter _ piscolens, the Helicobacter _ cinaedi or the Clostridium _ bartlettii in myasthenia gravis patients and healthy people is found for the first time, and through the combined diagnosis analysis of different floras, the effect of the combined application of the Pyramidobacter _ piscolens and the Helicobacter _ cinaedi or the Clostridium _ bartlettii in the diagnosis of myasthenia gravis is higher.

In an embodiment of the invention, the present invention diagnoses myasthenia gravis by: detecting one or more nucleic acid fragments corresponding to a species of bacteria associated with diagnosis of myasthenia gravis in a nucleic acid sample from an individual. In a particular embodiment, a nucleic acid fragment corresponding to Pyramidobacter _ piscolens, helicobacter _ cinaedi or Clostridium _ bartlettii is detected. In practicing the methods described herein, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used, and are well known.

The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "probe" as used herein refers to a synthetic or biologically produced nucleic acid of 10 to 285 base pairs in length, which contains a specific nucleotide sequence that allows specific preferential hybridization to a target nucleic acid sequence under predetermined conditions, and optionally contains a moiety for detection or to enhance performance of the assay. A minimum of 10 nucleotides is usually required to obtain statistically specificity and form stable hybridization products, and a maximum of 285 nucleotides usually represents an upper limit on the length over which reaction parameters can be readily adjusted to determine mismatched sequences and preferential hybridization. The probe may optionally contain components that may help the probe function properly or optimally under certain test conditions. For example, the probe may be modified to increase its resistance to nuclease degradation (e.g., by capping), carry a detection ligand (e.g., fluorescein), or facilitate capture of the probe on a solid support (e.g., a polydeoxyadenosine "tail").

The term "primer" as used herein refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction, "PCR") to amplify a nucleotide sequence. Primers are designed based on the polynucleotide sequence of a particular target sequence (e.g., a specific 16S rDNA sequence). The design and validation of primers and probes is well known in the art.

The term "specific" as used herein means that a nucleotide sequence will hybridize to/amplify a predetermined target sequence and will not substantially hybridize to/amplify a non-target sequence under the conditions of the assay (typically stringent conditions are used).

The term "hybridization" as used herein refers to a method of: by this method, under predetermined reaction conditions, two partially or completely complementary nucleic acid strands are polymerized in an antiparallel manner to form a double-stranded nucleic acid having specificity and stability, which follows a definite rule that nucleic acid bases can pair with each other.

The term "stringent hybridization conditions" means about 35 ℃ to 65 ℃ and about 0.9mol of NaCl salt solution. Stringency can also be controlled by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the type and concentration of denaturants present, and the hybridization temperature. In general, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. In general, the stringency of the conditions under which hybridization occurs will determine certain characteristics of the preferred probes to be used.

Molecular biological methods for measuring the amount of a target nucleic acid sequence are well known in the art. These methods include, but are not limited to, endpoint PCR, competitive PCR, reverse transcriptase-PCR (RT-PCR), quantitative PCR (qPCR), reverse transcriptase qPCR (RT-qPCR), PCR-pyrosequencing, PCR-ELISA, DNA microarrays, in situ hybridization assays such as dot blot or fluorescence in situ hybridization assays (FISH), branched DNA, and multiplex versions of the methods.

Preferred primers and/or probes react in a predictable manner, typically by providing a direct linear response to an increase in bacterial nucleic acid sequence. The amount of a given nucleic acid sequence in a sample can be readily quantified by preparing and comparing appropriate standards. Preferably, said molecular method for gene quantification is selected from the group consisting of quantitative polymerase chain reaction (qPCR), PCR-pyrosequencing, fluorescence In Situ Hybridization (FISH), DNA microarray and PCR-ELISA.

One particularly preferred quantification method is FISH, which combines probe hybridization with fluorescence microscopy, confocal laser microscopy or flow cytometry to directly quantify individual bacterial sequences.

Another particularly preferred method of quantification is quantitative PCR (qPCR), also known as real-time PCR. Different instruments can be used, such as ABI Prism 7700SDS, geneAmp 5700 SDS, ABI Prism 7900 HT SDS from Applied Biosystems; iCycler iQ by Bio-Rad; smart Cycler from Cepheid; rotor-Gene of Corbett Research; lightCycler from Roche Molecular Light Biochemicals and Mx4000Multiplex from Stratagene. The qPCR process accurately quantifies PCR products in real time by measuring PCR product accumulation at a very early stage of the exponential phase of the reaction, thereby reducing PCR amplification efficiency-related quantitative deviations that occur in endpoint PCR.

qPCR may use different detection chemistries, all of which can be used in the qPCR instrument described above. The term "detection chemistry" refers to a method of reporting the amplification of a particular PCR product in real-time PCR, and may include hydrolysis or probes, molecular beacons, scorpions (scorpions), hybridization probes, and DNA binding dyes such as

Green I。

The methods of the invention may further comprise detecting and/or quantifying one or more biomarkers of myasthenia gravis. The term "biomarker" as used herein refers to a disease marker, typically a substance present in a body sample that can be readily measured. The body sample may be, for example, a blood, plasma or stool sample. Typically, the amount determined is related to the underlying disease pathophysiology (such as the presence or absence of a particular myasthenia gravis disease), making it useful for diagnosing and measuring the progression of the disease or therapeutic effect.

The methods of the invention can be used for screening or early detection of myasthenia gravis, for diagnosis of myasthenia gravis, for assays of disease activity, for monitoring the progression and/or activity of myasthenia gravis, for monitoring the recurrence of myasthenia gravis, and/or for determining the efficacy of a treatment for myasthenia gravis. The term "sample" or "test sample" as used herein refers to any liquid or solid material containing nucleic acids. In suitable embodiments, the test sample is obtained from a biological source (i.e., a "biological sample"), such as cells in culture, or is a tissue sample from an animal, and most preferably from a human. In an exemplary embodiment, the sample is stool.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out under conventional conditions.

Example 1 screening of intestinal flora associated with myasthenia gravis

1. Study subject and sample Collection

55 patients with myasthenia gravis of children and 36 Healthy Controls (HC) of the corresponding age and sex were collected at the myasthenia gravis treatment center of the first hospital, shijiazhuang, hebei province. Sample information is shown in table 1.

Diagnostic criteria: (1) clinical manifestations: drooping eyelids, diplopia, strabismus; (2) a neostigmine test positive; (3) positive for acetylcholine receptor antibody; (4) electromyography: the facial nerve has low frequency attenuation and no increase in high frequency. The compound (1) + (2) or (3) or (4) can be clearly diagnosed.

Typing: reference to the us myasthenia gravis association (MGFA) of 2000 suggests new clinical typing and quantitative myasthenia gravis score (QMG) standard types.

Inclusion criteria were: the patient is definitely diagnosed as eye muscle type myasthenia gravis and accords with the diagnosis standard.

Exclusion criteria: (1) age <2 years old 10 months or no age information; (2) Antibiotics except beta-lactams are used within 3 months; (3) other drugs/hormones for treating diseases; (4) use of anti-inflammatory drugs or unknown herbal medicines.

TABLE 1 clinical characteristics of samples

2. DNA extraction and sequencing

DNA was extracted from the sample using a DNA extraction kit and the procedure was as described in the instructions. The concentration of DNA was measured using a Fluorometer or a microplate reader (e.g., qubit Fluorometer, invitrogen), and the integrity and purity of the sample were measured using agarose gel electrophoresis (agarose gel concentration: 1% V, voltage: 150V, electrophoresis time: 40 min). Covaris was used to randomly break the genomic DNA and magnetic beads were used to screen for fragmented genomic DNA of average size 200-400 bp. The resulting DNA fragment was subjected to end repair, the 3 'end was adenylated, and a linker was ligated to the end of the 3' end adenylated fragment, followed by PCR amplification. The PCR product was purified using magnetic beads. Performing thermal deformation on the double-stranded PCR product, performing cyclization by using a splint oligonucleotide sequence, formatting single-stranded circular DNA (SsCir DNA) to construct a final library, and performing quality control on the library. The library was amplified with phi29 to yield DNA Nanospheres (DNB) with a molecular copy number of over 300. The obtained DNBs are added into reticular pores on a chip (fixed on an arrayed silicon chip), and a double-end sequence with the read length of 100bp/150bp is obtained by combining a probe anchoring polymerization technology (cPAS) and a double-end sequencing method (MDA-PE) of multiple displacement amplification.

3. Quality control

And performing quality control processing on the measured data to finally obtain high-quality data for subsequent analysis, wherein the quality control steps are as follows: 1) Filtering low-quality reads; 2) Decontaminate human genome sequences, screen for low quality reads and sequence adapters using FastP (REF 21) and its default parameters, align reads to the human genome (Hg 38) using Bowtie2 (REF 22), and screen for paired reads that cannot be aligned to the human genome using Samtools as clean reads for use in subsequent analyses.

4. Classification and functional Annotation

High quality reads were mapped to the mpa _ v20 marker gene database using metan 2, resulting in a class abundance map for different class levels for each sample. Py, combine the results of all samples using merge _ melan _ tables and obtain combined abundance spectra at different species levels using an internal script. On the other hand, high quality reads were mapped to uniref90 and chocoplan using humann2 to obtain gene abundance and pathway abundance maps. The abundances of all samples were then combined using the human 2_ Join _ Tables, human 2_ renorm _ table, and human 2_ Split _ stratefied _ table, respectively, and the abundances were normalized and hierarchically classified for annotation. In addition, KEGG and GO enrichment analyses were performed using humann2_ regroup _ table and humann 2.

5. Statistical analysis

All abundance results were analyzed for differences using wilcox. Test two. Side function in R, depending on the grouping of samples. The P value in each result will be corrected according to the BH method to obtain q values (FDR) for screening of species and pathways that exhibit significant differences. The α diversity of each sample was calculated using the Shannon index. At the same input, the Vegan packet in R with parameter 'method = dist _ method' was used to calculate the β diversity. ROC curves were also plotted using the pROC analysis of R and AUC areas were calculated.

And (3) carrying out Principal Component Analysis (PCA) on the classification map, calculating an eig result of the PCA by using an Ade4 software package of R, obtaining feature vectors of different PCs by using a dudi.

To correlate differential species with clinical phenotype of the sample, spearman correlation between features and clinical phenotype was calculated using corr. Tes method in R package, according to the parameters 'method = Spearman, use = pairwise, adjust = BH'.

6. Results

The different categorical levels of alpha and beta diversity based on Shannon index did not differ significantly between patients and healthy populations (figure 1).

The PCA and PcoA results show no significant aggregation profile in patients and healthy persons (fig. 2).

Species variability results analysis showed that there were 20 species exhibiting significant differences, of which 11 were ROC detection AUC values >0.7, as shown in table 2. The results of the combined diagnostic analysis of the 20 different bacterial populations are shown in table 3. Wherein, the abundances and diagnostic efficacies of Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46faa, helicobacter _ cinaedi or Clostridium _ bartlettii are shown in table 4, the combined diagnostic effects of the above-mentioned flora are shown in table 2, and the diagnostic efficacies are all significantly increased, which indicates that the diagnosis of myasthenia gravis by applying Pyramidobacter _ piscolens, lachnospiraceae _ bacterium _2_1_46faa, helicobacter _ cinalis or Clostridium _ bartlettii alone or in combination has a higher diagnostic efficacy, and can effectively distinguish myasthenia gravis from healthy persons.

TABLE 2 differential flora and AUC values

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TABLE 3 Combined diagnostic AUC values

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TABLE 4 content of intestinal flora and diagnostic efficacy

Example 2 verification of the accuracy of genomic sequencing

19 samples of myasthenia gravis and 13 samples of healthy persons were collected as in example 1, and the patient information is shown in table 5.

TABLE 5 sample clinical characteristics

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The differential bacteria Prevotella _ copri, clostridium _ bartlettii, fusobacterium _ mortierum and Helicobacter _ cinaedi were randomly selected for sequencing verification, and the diagnostic efficacy of the differential bacteria Prevotella _ copri, clostridium _ bartlettii and Helicobacter _ cinaedi in the application to myasthenia gravis was calculated.

The results show that the AUC values of Prevotella _ copri, clostridium _ bartlettii, fusobaterium _ mortierum and Helicobacter _ cinaedi are 0.736842105, 0.672064777, 0.821862348 and 0.615384615, respectively, which are equivalent to the results of the aforementioned detection, and indicate that the sequencing data of the metagenome is accurate.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that it would be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit of the invention, and these modifications and variations also fall within the scope of the claims of the present invention.

Claims

1. Use of an agent for detecting the abundance of a flora consisting of the intestinal species Helicobacter _ cinaedi and Clostridium _ bartlettii for the preparation of a product for predicting myasthenia gravis.

2. Use according to claim 1, characterized in that the agent comprises an oligonucleotide that specifically hybridizes to target nucleotide sequences from Helicobacter _ cinaedi and Clostridium _ bartlettii.

3. Use according to claim 2, characterized in that the target nucleotide sequences of Helicobacter _ cinaedi and Clostridium _ bartlettii are fragments of a species-specific gene region.

4. Use according to claim 2, wherein the oligonucleotide is detectably labelled.

5. Use according to any one of claims 1 to 4, wherein the myasthenia gravis is childhood myasthenia gravis.