Novel microrna for in vitro early diagnosis of mild cognitive impairment and alzheimer's dementia, and convergence diagnostic cartridge and diagnostic device using same

Abstract

The present invention relates to a novel microRNA for in vitro early diagnosis of mild cognitive impairment and Alzheimer's dementia, and a convergence diagnostic cartridge and diagnostic device using same. The novel miRNA of SEQ ID NO: 1 obtained by molecular analysis exhibits differences in expression levels in the plasma of a normal group, a mild cognitive impairment patient group, and an Alzheimer's dementia patient group, and has the effect of reducing the expression levels of mild cognitive impairment- and Alzheimer's dementia-related proteins. Therefore, by using a novel miRNA present in plasma as a molecular diagnostic index in a non-invasive manner, the present invention enables not only early diagnosis of Alzheimer's dementia but also screening for mild cognitive impairment and Alzheimer's dementia.

Description

Novel microRNA for in vitro early diagnosis of mild cognitive impairment and Alzheimer's dementia, and a convergence diagnostic cartridge and diagnostic device utilizing the same

[0001] The present invention relates to a novel microRNA for the in vitro early diagnosis of mild cognitive impairment and Alzheimer's dementia, and to a combined diagnostic cartridge and diagnostic device using the same.

[0002] This result was produced with the support of the project 'Development of source technology and device for early diagnosis of Alzheimer's disease incorporating convergence molecular diagnostic technology (Dementia Early Diagnosis Technology Development Research Group)' (Project No.: RS-2024-00441331), a biomedical technology development project of the Ministry of Science and ICT.

[0003] Although Alzheimer's dementia is currently diagnosed through neuropsychological testing and imaging based on cognitive impairment and the amyloid hypothesis, due to many limitations, active efforts are currently being made to develop diagnostic indicators for Alzheimer's dementia from blood, a relatively non-invasive sample.

[0004] miRNA is a small, single-stranded RNA with a sequence of about 20 nucleotides that can regulate proteins by binding to the 3'-UTR (3'-untranslated regions) of a specific target mRNA and inhibiting mRNA translation. To date, about 1,000 miRNAs have been identified in humans, but the functions of most miRNAs remain unknown.

[0005] Currently, dementia analysis using blood primarily relies on proteins; however, this approach suffers from significant patient-specific variability relative to structural stability and has the disadvantage of failing to fully reflect the pathology of Alzheimer's dementia in the blood. To derive more sensitive diagnostic indicators, smaller targets are required. Particularly in the case of Alzheimer's dementia, since abnormal protein aggregation is triggered by various environmental stimuli rather than solely by genetic factors, it is considered crucial to identify fundamental targets by analyzing changes in gene expression regulation prior to protein translation, rather than relying on protein results. When utilizing miRNA as a diagnostic indicator, it is a 21-25 nucleotide single-stranded RNA present in all biological fluids, including blood, urine, and saliva, that plays a role in directly controlling gene expression by inhibiting mRNA translation. Furthermore, it remains stable in the blood due to its resistance to degradation by RNA; defects in miRNA metabolism or function are closely linked to the onset of dementia, and miRNA is known to possess high specificity and sensitivity depending on the disease, making it the most optimal diagnostic indicator.

[0006] In diagnosing Alzheimer's dementia, obtaining an accurate medical history based on reports from caregivers, who know the patient best, is crucial. Physicians assess whether there have been changes in cognitive functions, including memory, compared to the past, and if so, when and how these changes manifested. Diagnosis is typically made through physical examinations, neurological examinations, mental status examinations, assessments of daily living function levels, biochemical tests such as blood tests, brain imaging, and neuropsychological tests; however, various limitations still exist in diagnosing Alzheimer's dementia, making early diagnosis difficult. Diagnosis is primarily based on cognitive impairment and the amyloid hypothesis, utilizing neuropsychological testing and imaging diagnostics. Recently, however, active efforts are being made to develop in vitro diagnostic indicators for Alzheimer's dementia using non-invasive blood samples.

[0007] Meanwhile, regarding diagnostic technologies using microRNA, Korean Registered Patent No. 2333797 discloses a novel microRNA biomarker for screening diagnosis of mild cognitive impairment and Alzheimer's dementia, and Korean Registered Patent No. 1992539 discloses a composition and method for diagnosing cognitive impairment diseases based on miRNA, but there has not yet been any disclosure of the novel microRNA for in vitro early diagnosis of mild cognitive impairment and Alzheimer's dementia according to the present invention, nor of a convergence diagnostic cartridge and diagnostic device using the same.

[0008] The present invention was derived from the above-mentioned requirements, and the present invention provides a novel microRNA for the in vitro early diagnosis of mild cognitive impairment and Alzheimer's dementia, a combined diagnostic cartridge and a diagnostic device using the same, and the present invention was completed by confirming that the novel miRNA of SEQ ID NO. 1 according to the present invention has a difference in expression levels in the plasma of normal, mild cognitive impairment patient groups and Alzheimer's dementia patient groups, and can reduce the expression levels of Alzheimer's dementia-related proteins.

[0009] To achieve the above objective, the present invention provides a biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising a novel microRNA consisting of the nucleotide sequence of SEQ ID NO. 1 as an active ingredient.

[0010] In addition, the present invention provides a biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising, as an active ingredient, a preparation capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO. 1.

[0011] In addition, the present invention provides a cartridge for the early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising a biomarker composition for the early diagnosis of Alzheimer's dementia.

[0012] In addition, the present invention provides a multiplexed POCT device equipped with a cartridge for the early diagnosis of mild cognitive impairment and Alzheimer's dementia.

[0013] In addition, the present invention comprises (1) a step of collecting blood from an individual;

[0014] (2) A step of separating plasma from the blood of step (1); and

[0015] (3) A step of confirming the expression level of miRNA of sequence number 1 in the plasma separated in step (2); thereby providing a method for early diagnosis of mild cognitive impairment and Alzheimer's dementia.

[0016] The present invention relates to a novel microRNA for the in vitro early diagnosis of mild cognitive impairment and Alzheimer's dementia, and to a convergence diagnostic cartridge and diagnostic device utilizing the same. The novel miRNA of SEQ ID NO. 1 according to the present invention is expressed in the plasma of a patient group with mild cognitive impairment and Alzheimer's dementia and has the effect of reducing the expression of proteins related to mild cognitive impairment and Alzheimer's dementia. Therefore, by utilizing the novel miRNA present in plasma, which has relatively low invasiveness, as a diagnostic indicator, the present invention has the effect of enabling not only the early diagnosis of Alzheimer's dementia but also the differentiation between mild cognitive impairment and Alzheimer's dementia.

[0017] Figure 1 shows the stem loop structure and sequence information of the novel miRNA11 identified in the present invention.

[0018] Figure 2 shows the synthetic sequences of miRNA11 mimic and inhibitor to confirm the novel miRNA function of the present invention.

[0019] Figure 3 shows the Western blot and immunofluorescence results confirming the change in expression levels of SNAP25 (Synaptosomal-Associated Protein, 25kDa), which is encoded by the target gene of the novel miRNA11 of the present invention. *** indicates that there is a statistically significant difference in the expression levels of the target protein between the control group that received no treatment and the group treated with the miRNA mimic or miRNA inhibitor of the present invention, with p<0.001.

[0020] Figure 4 shows the results of confirming whether there are differences in the expression levels of the novel miRNA of the present invention in the blood of actual patients by group, and represents the expression levels of the novel miRNA11 (Sequence No. 1) in the plasma of patients classified as Normal, MCI, and AD. * and *** indicate that the expression levels of miRNA11 in the mild cognitive impairment (MCI) group and the Alzheimer's dementia (AD) group increased statistically significantly compared to the normal group, with * being p<0.05 and *** being p<0.001. ### indicates that the expression levels of miRNA11 in the Alzheimer's dementia group increased statistically significantly more compared to the mild cognitive impairment group, with p<0.001.

[0021] Figure 5 is a POCT All in One PCR cartridge for diagnosing Alzheimer's dementia loaded with the novel miRNA11 of the present invention.

[0022] The present invention relates to a biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising a novel microRNA consisting of the nucleotide sequence of SEQ ID NO. 1 as an active ingredient.

[0023] In the present invention, microRNA (miRNA) refers to 21 to 23 non-coding RNAs that regulate gene expression after transcription by promoting the degradation of target RNA or inhibiting their translation. It includes not only miRNAs represented by specific nucleotide sequences, but also precursors of said miRNAs (pre-miRNA, pri-miRNA), miRNAs with biological functions equivalent to them, such as homologues (i.e., homologs or osolologs), variants such as genetic polymorphisms, and derivatives.

[0024] The novel microRNA composed of the nucleotide sequence of SEQ ID NO. 1 above is preferably derived from human plasma, but is not limited thereto.

[0025] The novel microRNA composed of the nucleotide sequence of SEQ ID NO. 1 is characterized by reducing the expression level of SNAP25 (Synaptosomal-Associated Protein, 25kDa) protein.

[0026] The concept of 'diagnosis' in the present invention includes determining the susceptibility of an object, namely a subject of examination, to a specific disease or condition, determining whether an object currently has a specific disease or condition, determining the prognosis of an object suffering from a specific disease or condition, or therametrics (e.g., monitoring the condition of an object to provide information on therapeutic efficacy). Furthermore, in the present invention, diagnosis refers not only to the early diagnosis of Alzheimer's dementia but also to distinguishing (screening) subjective cognitive impairment, mild cognitive impairment, and Alzheimer's dementia.

[0027] In addition, the present invention relates to a biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising, as an active ingredient, a preparation capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO. 1.

[0028] A preparation capable of detecting microRNA composed of the nucleotide sequence of SEQ ID NO. 1 is preferably selected from, but is not limited to, an antisense oligonucleotide complementary to the microRNA composed of the nucleotide sequence of SEQ ID NO. 1; a primer; a probe; and a loop-forming oligonucleotide to which a fluorescent agent or a quencher is connected.

[0029] In this invention, "mild cognitive impairment" refers to an intermediate stage between the decline in cognitive ability caused by normal aging and dementia. In other words, it describes a state where cognitive ability is lower than that of the same age group, and it differs from dementia in that daily life is still possible. This mild cognitive impairment is an intermediate stage between normality and dementia and is classified as a high-risk group for dementia.

[0030] In the present invention, 'Alzheimer's dementia' is a disease that is often accompanied by neuropsychiatric symptoms such as personality changes, agitation, depression, delusions, hallucinations, increased aggression, and sleep disorders, as well as neurological disorders such as rigidity and gait abnormalities, or physical complications such as fecal incontinence, infection, and bedsores in the late stages, and progresses very slowly in the early stages, making it difficult to recognize the onset.

[0031] In addition, the present invention relates to a cartridge for the early diagnosis of Alzheimer's dementia comprising a biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia.

[0032] In addition, the present invention relates to a multiplexed POCT device equipped with a cartridge for the early diagnosis of mild cognitive impairment and Alzheimer's dementia.

[0033] The above multiplexed POCT device may include, but is not limited to, one or more selected from a thermostat, a stirrer, and a disposable tip sample injection container.

[0034] The above multiplexed POCT device is preferably, but is not limited to, a device capable of performing any one of RT-PCR, real-time PCR, isothermal PCR, Northern blotting, and RNA protection analysis.

[0035] The device of the present invention may further include a nucleic acid capable of specifically binding to the microRNA of SEQ ID NO. 1, wherein the nucleic acid includes all of RNA, DNA, or RNA / DNA (chimera), and the DNA may include all of cDNA, genomic DNA, and synthetic DNA. The RNA may include all of total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA. In addition to the nucleic acid, the device of the present invention may include a known nucleic acid or a nucleic acid that may be discovered in the future that enables early diagnosis and screening of mild cognitive impairment and Alzheimer's dementia, and may also include an antibody for measuring known markers for early diagnosis of mild cognitive impairment and Alzheimer's dementia. The nucleic acids included in the device may be packaged individually or in any combination in different containers.

[0036] The device of the present invention may include a fluorescent substance for biosense labeling, an enzyme and culture medium for nucleic acid amplification, an instruction manual, etc. The cartridge of the present invention is a device for the early diagnosis of mild cognitive impairment and Alzheimer's dementia in which the nucleic acid is bound or attached, for example, to a solid phase. Examples of materials for the solid phase include plastic, paper, glass, silicone, etc., and plastic may be a preferred material for the solid phase due to ease of processing. The shape of the solid phase is arbitrary and may be, for example, square, circular, rectangular, film-shaped, etc.

[0037] The above multiplexed POCT device is preferably a high-throughput-screening (HTS) based multiplexed POCT device, but is not limited thereto.

[0038] The multiplexed POCT device described above may include an RNA extraction device, a thermal circulator, an automatic liquid handler, and a fluorescence detector. The multiplexed POCT device may be an all-in-one device with a built-in micro-whole blood separator, a fluorescence detector, and a liquid handler. The multiplexed POCT device is characterized by including a detection system that calculates a dementia risk index from human blood.

[0039] In addition, the present invention comprises (1) a step of collecting blood from an individual;

[0040] (2) A step of separating plasma from the blood of step (1); and

[0041] (3) A step of confirming the expression level of miRNA of sequence number 1 in the plasma separated in step (2) above; the present invention relates to a method for early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising: (3) a step of confirming the expression level of miRNA of sequence number 1 in the plasma separated in step (2) above.

[0042] The present invention will be described in more detail below using examples. These examples are solely for the purpose of more specifically explaining the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0043] 1. Securing samples from patients with mild cognitive impairment and Alzheimer's dementia

[0044] Blood from patients classified as normal, MCI (mild cognitive impairment), and AD (Alzheimer's disease) was collected in EDTA tubes, and plasma was separated and stored at -70℃ until use at the Neurobiology Laboratory of Gyeongsang National University.

[0045] 2. Total miRNA extraction

[0046] Total RNA in the plasma of each patient group and normal group obtained above was extracted using the QIAGEN miRNeasy serum / plasma separation kit, which can purify mainly miRNA and other small RNAs from small amounts of serum and plasma.

[0047] Subsequently, the Bioanalyzer RNA Small RNA chip was used to verify whether a sufficient amount of RNA had been secured for library preparation for NGS analysis.

[0048] 3. Next Generation Sequencing (NGS)

[0049] A small RNA-seq library was generated on the Illumina Platform using the SMARTer smRNA-Seq Kit, which enables the production of high-quality miRNA libraries even from small amounts of RNA. The prepared samples were sequenced using the NovaSeq 6000 sequencing platform according to the SMARTer smRNA-Seq Kit manual.

[0050] 4. Selection of miRNA data through analysis of raw data

[0051] From the raw data obtained by NGS analysis, contaminated or low-quantity data present in the plasma were removed in the order of Adapter, Quality, and Length trimming. miRNA Deep2* sequencing was performed on the clean data from which unnecessary information had been removed through this trimming process.

[0052] 5. miRNA Deep2* Sequencing

[0053] miRNA Deep2* is software capable of identifying novel miRNAs from purified, raw data. It can predict novel miRNAs by arranging miRBase (Pre-miRNA) libraries, which are cleaved into mature, star, and loop sequences, side by side according to the RNAfold algorithm. The RNAfold algorithm is derived by predicting the secondary structure where the RNA sequence has the minimum free energy using the most similar thermodynamic model. The RNA fold generation graphic includes the number of reads for each part of the hairpin, the score for minimum free energy, the score for the randfold, and the score for the conserved seed sequence.

[0054] The miRDeep2* system can detect known miRNAs and novel miRNAs through mapping utilizing an RNA database.

[0055] 6. Verification of novel miRNAs related to mild cognitive impairment and Alzheimer's dementia reflected in blood

[0056] To functionally verify the association of novel miRNAs with mild cognitive impairment and Alzheimer's disease, screening was performed using miRDB software, which scores target genes (miRNAs) based on miRNA sequences. Verification was then conducted by analyzing the expression levels of genes associated with mild cognitive impairment and Alzheimer's disease among the target genes with high gene scores.

[0057] SH-SY5Y cells, which are used as a neural function and differentiation model, were used. After transfecting SH-SY5Y cells with a miRNA mimic that exhibits the overexpression effect of a selected novel miRNA, a miRNA inhibitor that suppresses the expression of the novel miRNA, and a negative control miRNA, the protein expression of the target gene was confirmed by Western blotting and immunofluorescence.

[0058] Example 1. Sample Acquisition and Total RNA Extraction

[0059] Total RNA was extracted from each plasma sample collected from the patient group (MCI, AD) and the normal group using the QIAGEN miRNeasy Serum / Plasma Kit, which can purify miRNA and other small RNAs from small amounts of plasma. Subsequently, the RNA was quantified using the Bioanalyzer RNA Small RNA chip to confirm whether a sufficient amount of RNA had been secured for library production for NGS, and it was confirmed that a sufficient amount for NGS analysis had been obtained.

[0060] Example 2. Next Generation Sequencing (NGS)

[0061] (1) Library construction and extraction of raw data

[0062] To generate small RNA-seq libraries for sequencing on the Illumina Platform for NGS, the SMARTer smRNA-Seq Kit, which enables the production of high-quality miRNA libraries even from small amounts of RNA, was used. Libraries for the sequencing of total RNA from patient and normal groups obtained from plasma were constructed, and raw data was obtained by sequencing the constructed libraries using the NovaSeq 6000 sequencing platform.

[0063] (2) Selection of miRNA data from raw data

[0064] The raw data obtained from NGS analysis was trimmed in the order of Adapter, Quality, and Length to remove contaminated or insufficient data.

[0065] The presence of adapters (nonAdapter Read Count), appropriate length (Short Read Count), and low quality (Low Quality Read Count) were checked in the Total Read Count, which is unprocessed data obtained by sequencing. Then, miRNA Deep2* sequencing was performed using primary processed data from which unnecessary information was removed through this trimming selection process.

[0066] (3) miRNA Deep2* sequencing

[0067] miRNA Deep2* predicted novel miRNAs by arranging miRBase(Pre-miRNA) libraries, which are short-cut into mature, star, and loop sequences, side by side according to the RNAfold algorithm of the software capable of identifying newly known miRNAs from the primary processed data above. The RNAfold algorithm was derived by predicting the secondary structure in which the RNA sequence has the minimum free energy using the most similar thermodynamic model.

[0068] The RNA fold generation graphic includes the number of reads for each part of the hairpin, the score for minimum free energy, the score for the randfold, and the conserved seed sequence score. The miRDeep2* system detected known miRNAs and novel miRNAs through mapping utilizing an RNA database.

[0069] For each of the above samples, the final processed reads were classified into piRNA, tRNA, snRNA, snoRNA, known miRNA, and novel miRNA using miRBase v22.1 and a non-coding RNA database (RNAcentral release 14.0).

[0070] Genome mapping was performed using RSEM with Bowtie and STAR. Bowtie was subsequently used for miRDeep2 analysis using the genome sequence. Known / novel miRNAs predicted by miRDeep2 and other small RNAs matching RNAcentral were aligned using Bowtie (target small RNA, <50nt) and Bowtie2 (target smRNA, ≥50nt).

[0071] - RSEM (RNA-Seq by Expectation-Maximization): A tool for quantifying RNA-Seq transcripts

[0072] - Bowtie: Software commonly used for sequence alignment and sequence analysis

[0073] - STAR (Spliced ​​Transcripts Alignment to a Reference): fast RNA-seq read mapper

[0074] To predict novel miRNAs, uniquely clustered reads were aligned separately for the reference genome and precursor miRNAs. Mature star and loop sequences of miRNAs can be predicted according to the RNAfold algorithm using miRNA Deep2*. The RNAfold function predicted the minimum free energy second-order structure of RNA sequences using the nearest neighbor thermodynamic model. The graphics generated by RNAfold include actual in silico-folded hairpins, as well as the number of reads, minimum free energy score, randfold score, and score of the conserved seed sequence for each part of the hairpin.

[0075] novel miRNAs were derived using a score of -10 to 10 for miRDeep2*. The derived novel miRNAs are expressed in both the mild cognitive impairment group and the Alzheimer's dementia group, and among them, five miRNAs were selected that had a miRDeep2* score of 1 or higher, an RNA fold of "yes," and a number of mature reads of 10 or higher.

[0076] The stem loop structure of the selected novel miRNA11 is disclosed in Fig. 1, and sequence information is disclosed in Fig. 2.

[0077] Example 3. Confirmation of differences in gene expression levels of novel miRNAs for the early diagnosis of mild cognitive impairment and Alzheimer's dementia

[0078] To verify the association between novel miRNAs and Alzheimer's disease, we used miRDB software, which screens target genes based on miRNA sequences and scores. Among the target genes with high gene scores, genes associated with Alzheimer's disease were selected.

[0079] SH-SY5Y cells, which are used as a model for neural function and differentiation, were used. After transfecting SH-SY5Y cells with a miRNA mimic that exhibits the overexpression effect of a selected novel miRNA, a miRNA inhibitor that suppresses the expression of the novel miRNA, and a negative control miRNA, the protein expression of the target gene was confirmed by Western blotting and immunofluorescence.

[0080] miRNA mimic is a miRNA that can function like a novel miRNA actually found in vivo and was constructed with double bonds. miRNA inhibitor is a miRNA that can inhibit the corresponding miRNA by constructing it with single bonds as a sequence complementary to the derived miRNA. Meanwhile, negative control miRNA is a miRNA composed of miRNAs that do not regulate genes and serves as a standard for verification. The constructed novel miRNA11 and miRNA inhibitor are disclosed in Fig. 2.

[0081] [Genetic screening using miRDB software]

[0082] To select targets highly associated with Alzheimer's disease among the genes controlled by the novel miRNA, the novel miRNA11 mimic was screened by score using target prediction miRDB software based on the sequence 5'-AGGAAAGGGAGGGGAAAG-3' (Sequence No. 1). The miRNA11 inhibitor for the miRNA mimic of Sequence No. 1 is 5'-CUUUCCCCUCCCUUUCCU-3' (Sequence No. 2).

[0083] After setting the cut-off value of the gene score of the corresponding miRNA to 80, target genes related to mild cognitive impairment and Alzheimer's dementia were selected.

[0084] SNAP25 (Synaptosomal-Associated Protein, 25kDa) was selected as the target gene for miRNA11. SNAP25 is a multifunctional protein that forms the SNARE docking complex, which regulates extracellular secretion, along with syntaxin, synaptic vesicle protein VAMP, and synaptobrevin, and also regulates other types of voltage-gated calcium channels, playing an important role in various stages of neurotransmitter release.

[0085] miRNA11 inhibitors and miRNA11 mimics for the overexpression of novel miRNA11 were transfected into human neuroblastoma SH-SY5Y at concentrations of 1 nM, 10 nM, and 100 nM, respectively, for 48 hours, and immunofluorescence and proteins were isolated and Western blotting was performed. It was confirmed that the expression of the candidate target gene protein SNAP25 significantly decreased as miRNA11 binding to the 3'-UTR increased due to the overexpression of the novel miRNA11, and it was confirmed that the expression of the candidate target gene protein SNAP25 increased by removing miRNA11 binding to the 3'-UTR with a miRNA11 inhibitor (Fig. 3).

[0086] Meanwhile, to determine whether there were group-specific differences in the expression levels of the novel miRNA in the actual blood of patients, targeted sequencing was performed to analyze the expression levels of the novel miRNA11 in the plasma of patients classified as Normal, MCI, and AD. Significant differences in the expression levels of miRNA11 were confirmed between the mild cognitive impairment (MCI) group and the Alzheimer's dementia (AD) group (Fig. 4).

[0087] Example 4. Cartridge containing a novel miRNA for the early diagnosis of mild cognitive impairment and Alzheimer's disease, and diagnosis of Alzheimer's disease using the same

[0088] The dedicated PCR cartridge of LUKIN features a DNA / RNA extraction tube at the top. After the extraction process takes place at the top, the extracted sample is sent down through an interface tube, and the sample is injected into a PCR tube pre-loaded with target reagents using centrifugal force generated by rotation. The waste liquid used during the extraction process is transferred to a waste liquid tube located between the extraction tube and the PCR tube for safe storage. A characteristic feature of the waste liquid tube is that it is pre-filled with an absorbent agent that absorbs liquid and gels.

[0089] Pre-loaded PCR tubes are characterized by containing all the reagents necessary to perform PCR, such as primers, probes, and master mix.

[0090] When 25 µl of RNA extracted sample was loaded into a PCR tube, thermal cycling, fluorescence measurement, and real-time polymerase chain reaction (REAL TIME PCR) were performed simultaneously to diagnose Alzheimer's disease (Fig. 5).

Claims

1. A biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising a novel microRNA consisting of the nucleotide sequence of SEQ ID NO. 1 as an active ingredient.

2. A biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia, characterized in that, in claim 1, the novel microRNA composed of the nucleotide sequence of SEQ ID NO. 1 is derived from human plasma.

3. A biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia, characterized in that, in claim 1, the novel microRNA composed of the nucleotide sequence of SEQ ID NO. 1 reduces the expression level of SNAP25 (Synaptosomal-Associated Protein, 25kDa) protein.

4. A biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising, as an active ingredient, a preparation capable of detecting microRNA consisting of the nucleotide sequence of SEQ ID NO.

1.

5. A biomarker composition for the early diagnosis of mild cognitive impairment and Alzheimer's dementia, characterized in that, in claim 4, the preparation capable of detecting microRNA composed of the nucleotide sequence of SEQ ID NO. 1 is selected from one of: an antisense oligonucleotide complementary to the microRNA composed of the nucleotide sequence of SEQ ID NO. 1; a primer; a probe; and a loop-forming oligonucleotide to which a fluorescent agent or a quencher is connected.

6. A cartridge for the early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising the composition of claim 4.

7. A multiplexed POCT device equipped with a cartridge for the early diagnosis of mild cognitive impairment and Alzheimer's dementia as described in Paragraph 6.

8. A multiplexed POCT device according to claim 7, characterized in that the multiplexed POCT device comprises one or more selected from a thermostat, a stirrer, and a disposable tip sample injection container.

9. A multiplexed POCT device according to claim 7, characterized in that the multiplexed POCT device is capable of performing any one of RT-PCR, real-time PCR, isothermal PCR, Northern blotting, and RNA protection analysis.

10. A multiplexed POCT device according to claim 7, characterized in that the multiplexed POCT device is a high-throughput-screening (HTS) based multiplexed POCT device.

11. A multiplexed POCT device according to claim 7, characterized in that the multiplexed POCT device comprises an RNA extraction device, a thermal circulator, an automatic liquid handler, and a fluorescence detector.

12. A multiplexed POCT device according to claim 7, characterized in that the multiplexed POCT device is an all-in-one device with a built-in micro-sized whole blood separator, a fluorescence detector, and a liquid handler.

13. A multiplexed POCT device according to claim 7, characterized in that the multiplexed POCT device includes a detector that calculates a dementia risk index from human blood. 14.(1) A step of collecting blood from an individual; (2) A step of separating plasma from the blood of step (1); and (3) A step of confirming the expression level of miRNA of sequence number 1 in the plasma separated in step (2); a method for early diagnosis of mild cognitive impairment and Alzheimer's dementia comprising

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