A marker, kit and application for differentiating methamphetamine-induced psychosis from primary schizophrenia

Abstract

The application relates to a marker, a kit and an application for differentiating methamphetamine-induced psychosis from primary schizophrenia, and belongs to the technical field of biological medicine and judicial expertise. The application provides application of miRNA as a marker in preparation of a product for differentiating methamphetamine-induced psychosis from primary schizophrenia, wherein the miRNA comprises at least one of hsa-miR-11400, hsa-miR-1908-3p, hsa-miR-4433b-3p and nov-miR-chr20. The application takes nov-miR-chr20 as a forward specific marker of PS, and takes the other three miRNAs as forward indicating markers of MAP, effectively avoids false positive or false negative, improves the specificity and sensitivity of detection, ensures stability and repeatability, and solves the misdiagnosis problem caused by high overlap and similarity of MAP and PS.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and forensic identification technology, and in particular to a biomarker, reagent kit, and application for differentiating between methamphetamine-induced psychosis and primary schizophrenia. Background Technology

[0002] Methamphetamine (MET), commonly known as "ice," is one of the most widely used synthetic drugs globally. According to drug situation reports, synthetic drug abusers constitute a significant proportion of drug users. Long-term or high-dose abuse of MET has significant neurotoxicity, inducing a range of psychotic symptoms collectively known as MAP (Methamphetamine-associated Psychosis). Clinically, based on the duration of psychotic symptoms, MAP is divided into transient (MAP-T) and persistent (MAP-P) types. MAP-P patients continue to experience a wide range of psychotic symptoms, including hallucinations and delusions, even after prolonged abstinence (months or years).

[0003] Currently, the biggest challenge in clinical diagnosis and forensic psychiatric assessment lies in the high degree of overlap and similarity between MAP-P and primary schizophrenia (PS) in terms of psychotic symptoms. Both present with positive symptoms such as persecutory delusions, delusions of reference, and auditory hallucinations, and these symptoms are often chronic and difficult to cure. Existing international diagnostic criteria (such as DSM-5 or ICD-10) mainly differentiate between the two based on the history of substance use and the time point of symptom resolution (e.g., one month after abstinence). However, in actual clinical and judicial practice, drug use history is often obtained through patient self-reporting, which can lead to concealment or memory bias. More importantly, simple time boundaries are insufficient to explain why some MAP patients experience prolonged symptoms, making it almost impossible to distinguish between the two in cross-sectional psychiatric examinations. Since PS is usually considered an endogenous severe mental illness, patients may be assessed as having no or limited criminal responsibility when committing crimes; while MAP, as a drug-induced mental disorder, has a completely different legal characterization and assessment of criminal responsibility. Therefore, the lack of objective and accurate differential diagnostic techniques can easily lead to misdiagnosis and mistreatment, directly affecting the fairness of judicial trials.

[0004] Despite their highly similar clinical phenotypes, mounting neurobiological evidence suggests that MAP and PS may have distinctly different pathogenesis. PS is often considered an endogenous neurodevelopmental disorder with a strong genetic background, involving excessive synaptic pruning and abnormal neurodevelopment; while MAP is more often viewed as an exogenous neurotoxic injury state, closely related to oxidative stress, mitochondrial dysfunction, neuroinflammation, and damage to the monoamine neurotransmitter system. This fundamental difference in pathological mechanisms should theoretically project at the molecular level, providing a possibility for identifying differential biomarkers.

[0005] miRNAs are a class of endogenous non-coding RNAs approximately 22 nucleotides in length. They regulate gene expression post-transcriptionally and are widely involved in central nervous system development, plasticity regulation, and immune inflammatory responses. Studies have shown that brain-derived miRNAs can cross the blood-brain barrier and enter peripheral circulation via exosomes and other means, exhibiting extremely high stability in plasma. Therefore, plasma miRNAs are considered highly promising biomarkers for liquid biopsies of mental illnesses.

[0006] However, existing technologies have the following significant shortcomings: 1. Lack of targeted identification spectrum: Most existing transcriptomics studies focus on pairwise comparisons between PS patients and healthy controls, or between drug users and healthy controls. There is a lack of systematic studies that directly compare patients with persistent MAP-P symptoms with first-episode untreated PS patients in the same cohort.

[0007] 2. Insufficient biomarker specificity: Some reported biomarkers are often applicable to multiple mental illnesses (such as reflecting only general mental stress or inflammatory states), and cannot achieve the highly difficult identification and accurate differentiation of PS and MAP.

[0008] 3. Lack of clinical translation products: Currently, there are no mature in vitro diagnostic kits that can use specific miRNA combinations in peripheral blood to achieve objective differentiation between MAP (especially persistent type) and PS through quantitative detection.

[0009] In summary, there is an urgent need to develop a detection technology and kit based on specific plasma miRNA biomarkers. By capturing molecular signals that reflect the differences between endogenous developmental abnormalities and exogenous toxic damage, this technology can provide objective and auxiliary differential diagnostic evidence for clinicians and forensic experts. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a biomarker, reagent kit, and application for differentiating between methamphetamine-induced psychosis and primary schizophrenia.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of miRNA as a marker in the preparation of products for the differential diagnosis of methamphetamine-induced psychosis and primary schizophrenia, wherein the miRNA includes at least one of hsa-miR-11400, hsa-miR-1908-3p, hsa-miR-4433b-3p and nov-miR-chr20. The nucleotide sequence of hsa-miR-11400 is shown in SEQ ID NO: 1, the nucleotide sequence of hsa-miR-1908-3p is shown in SEQ ID NO: 2, the nucleotide sequence of hsa-miR-4433b-3p is shown in SEQ ID NO: 3, and the nucleotide sequence of nov-miR-chr20 is shown in SEQ ID NO: 4.

[0012] This invention found that hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p are highly expressed in the plasma of MAP patients, but are absent or significantly low (close to background values) in the plasma of PS patients. nov-miR-chr20 is specifically highly expressed in the plasma of PS patients, but absent or extremely low in MAP and healthy controls (HC). Therefore, nov-miR-chr20 can serve as a positive specific biomarker for PS, exhibiting high specificity in PS while remaining at a background level in MAP, effectively identifying endogenous PS characteristics. hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p can serve as positive indicator biomarkers for MAP (i.e., negative exclusion biomarkers for PS). This mutually exclusive and complementary expression pattern is equivalent to introducing double insurance in molecular diagnostics, effectively avoiding false positives or false negatives that may occur with a single biomarker, and solving the problem of misdiagnosis caused by the high overlap and similarity of psychotic symptoms between MAP-P and PS.

[0013] In a second aspect, the present invention provides the application of a reagent for detecting miRNA in the preparation of products for the differential diagnosis of methamphetamine-induced psychosis and primary schizophrenia, characterized in that the miRNA includes at least one of hsa-miR-11400, hsa-miR-1908-3p, hsa-miR-4433b-3p and nov-miR-chr20. The nucleotide sequence of hsa-miR-11400 is shown in SEQ ID NO: 1, the nucleotide sequence of hsa-miR-1908-3p is shown in SEQ ID NO: 2, the nucleotide sequence of hsa-miR-4433b-3p is shown in SEQ ID NO: 3, and the nucleotide sequence of nov-miR-chr20 is shown in SEQ ID NO: 4.

[0014] Thirdly, the present invention provides a primer set for detecting miRNA, the primer set including a forward primer and a reverse primer for amplifying the miRNA; The nucleotide sequence of the forward primer for amplifying hsa-miR-11400 is shown in SEQ ID NO: 6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 7. The nucleotide sequence of the forward primer for amplifying hsa-miR-1908-3p is shown in SEQ ID NO: 9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 7. The nucleotide sequence of the forward primer for amplifying hsa-miR-4433b-3p is shown in SEQ ID NO: 11, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 7. The nucleotide sequence of the forward primer for amplifying nov-miR-chr20 is shown in SEQ ID NO: 13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 7.

[0015] Furthermore, the primer set also includes reverse transcription primers for reverse transcription of the miRNA; The nucleotide sequences of the reverse transcription primers for reverse transcription of hsa-miR-11400 are shown in SEQ ID NO: 5; the nucleotide sequences of the reverse transcription primers for reverse transcription of hsa-miR-1908-3p are shown in SEQ ID NO: 8; the nucleotide sequences of the reverse transcription primers for reverse transcription of hsa-miR-4433b-3p are shown in SEQ ID NO: 10; and the nucleotide sequences of the reverse transcription primers for reverse transcription of nov-miR-chr20 are shown in SEQ ID NO: 12.

[0016] Furthermore, the primer set also includes primers for detecting an internal reference gene, which includes at least one of U6 snRNA and / or miR-16-5p. The nucleotide sequence of the U6 snRNA is shown in SEQ ID NO: 16, and the nucleotide sequence of the miR-16-5p is shown in SEQ ID NO: 17.

[0017] Furthermore, the primers for detecting the internal reference gene U6 snRNA include forward and reverse primers for amplifying U6 snRNA, and / or reverse transcription primers for reverse transcription of U6 snRNA. The nucleotide sequence of the forward primer for amplifying U6 snRNA is shown in SEQ ID NO: 15, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 14; the nucleotide sequence of the reverse transcription primer for reverse transcription of U6 snRNA is shown in SEQ ID NO: 14.

[0018] Given that the miRNA sequences are short and have large differences in GC content (especially nov-miR-chr20, which is a GC-rich repetitive sequence), this invention designs specific stem-loop reverse transcription primers and quantitative real-time PCR (qPCR) primer pairs to ensure the specificity and sensitivity of the detection.

[0019] Fourthly, the present invention provides a kit for differentiating between methamphetamine-induced psychosis and primary schizophrenia, the kit containing reagents for detecting the miRNA.

[0020] Furthermore, the kit contains the aforementioned primer set.

[0021] Furthermore, the kit also contains a reverse transcription reaction reagent, which includes at least one of reverse transcriptase, RNase inhibitor, dNTPs, and reverse transcription buffer.

[0022] In a specific embodiment of the present invention, the reverse transcription reaction reagents for reverse transcription include 5×RT Reaction Buffer, RNase Inhibitor, dNTP Mix, reverse transcription primers in the primer set, Reverse Transcriptase, and Nuclease-free Water.

[0023] Furthermore, the kit also contains real-time quantitative PCR reaction reagents, which include at least one of DNA polymerase, fluorescent dye, fluorescent probe, and real-time quantitative PCR buffer.

[0024] Furthermore, the real-time quantitative PCR reaction reagents for performing the real-time quantitative PCR reaction include 2×ChamQ SYBR qPCR Master Mix, the amplification primers in the primer set, 50×ROX Reference Dye 1, and Nuclease-free Water.

[0025] To achieve accurate differentiation between MAP and PS, this invention employs a "two-way verification" algorithm, establishing a classification logic based on the miRNA. Depending on the detection method (e.g., high-throughput sequencing or real-time quantitative PCR), corresponding expression cut-off values ​​are set: (1) Set detection threshold: If high-throughput sequencing (TPM) is used, set the TPM cutoff value (e.g., TPM < 0.1 is considered negative / not detected); if quantitative real-time PCR (qPCR) is used, set the Ct cutoff point (e.g., Ct > 31 is considered negative / not detected).

[0026] (2) PS Judgment Criteria: A test sample is judged as PS when it meets the following conditions. Condition A: The marker nov-miR-chr20 is detected positive (i.e., the relative expression level / TPM value is significantly higher than the cutoff value, or the Ct value is lower than the cutoff point); Condition B: The markers hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p are negative (i.e., the relative expression level / TPM value is lower than the cutoff value or close to 0, or the Ct value is higher than the cutoff point).

[0027] (3) MAP determination criteria: A test sample is determined to be MAP if it meets the following conditions: Condition C: The marker nov-miR-chr20 was negative (low expression or not detected); Condition D: At least one (preferably all) of the biomarkers hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p is detected as positive or shows high expression.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. A pioneering "two-way complementary" verification strategy significantly improves the accuracy of differential diagnosis. Existing technologies often search for differentially expressed genes in a single dimension, making it difficult to draw a clear line between MAP and PS. The biomarker combination screened in this invention constructs a unique two-way complementary identification logic: (1) Using nov-miR-chr20 as a positive specific marker of PS, it is highly expressed in PS, while it is at a background level in MAP, which can effectively identify endogenous PS characteristics. (2) Three other miRNAs (hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p) were used as positive indicator markers for MAP (i.e., negative exclusion markers for PS). These markers are rarely expressed in PS, but are significantly elevated in MAP. This mutually exclusive and complementary expression pattern is equivalent to introducing double insurance in molecular diagnosis, effectively avoiding false positives or false negatives that may be caused by a single marker, and solving the problem of misdiagnosis caused by the high overlap and similarity of psychotic symptoms between MAP-P and PS.

[0029] 2. This invention overcomes the limitations of relying on traditional psychiatric examination techniques, providing objective and quantitative evidence for forensic psychiatric evaluation: Currently, clinical and forensic evaluations primarily rely on patients' self-reported drug use history and psychiatric examination results, which are highly subjective and easily influenced by faking or memory bias. The kit provided by this invention is based on the quantitative detection of peripheral blood miRNA, independent of the patient's level of cooperation, and can provide objective Ct values ​​and relative expression levels. This provides independent biological evidence for determining the mental state and criminal responsibility at the time of the crime in forensic psychiatric evaluation, helping to resolve the core controversy in judicial practice regarding the classification of endogenous psychosis versus exogenous drug-induced psychosis.

[0030] 3. Stratified based on pathological mechanisms of neurodevelopmental abnormalities and exogenous neurotoxicity, demonstrating strong scientific rigor: The biomarker selection in this invention is not random screening, but rather based on profound differences in molecular biological mechanisms. Specifically, the PS-specific biomarker nov-miR-chr20 is an atypical miRNA containing CGG repeat sequences, directly associated with PS-specific genomic instability and neurodevelopmental abnormalities; while MAP-high expression group miRNAs are closely related to drug-induced neurotoxicity and immune metabolic stress responses. This pathological mechanism-based stratified detection ensures that the diagnostic results are not only statistically significant but also biologically sound, providing molecular guidance for subsequent precision treatment (such as differentiating between antipsychotic treatment and metabolic repair therapy).

[0031] 4. Overcame the technical challenges of detecting trace amounts of special sequences, achieving high detection sensitivity: Addressing the characteristics of nov-miR-chr20 being rich in GC repeat sequences (CG repeats) and other short, highly homologous miRNA sequences, this invention designed specific stem-loop reverse transcription primers. This design utilizes the three-dimensional spatial hindrance of the stem-loop structure to effectively prevent non-specific amplification of genomic DNA or precursor RNA, significantly improving the specificity and sensitivity of detecting trace amounts of mature miRNAs in plasma, and ensuring the stability and reproducibility of Ct values ​​in low-abundance samples.

[0032] 5. Non-invasive, rapid, and convenient, easily promoted and adapted for clinical and forensic scenarios: The test sample of this invention is peripheral venous blood, which has the advantages of being non-invasive, easy to collect, and having good patient compliance compared to cerebrospinal fluid testing or brain imaging examinations. The accompanying reagent kit adopts a mature qPCR technology platform, is easy to operate, has a short testing cycle (results are usually available within 4 hours), and is relatively inexpensive, making it suitable for promotion and use in mental health centers, drug rehabilitation institutions, and forensic identification centers at all levels. Attached Figure Description

[0033] Figure 1 Box scatter plots of different miRNA biomarkers in the four groups of subjects. The normalized expression level (TPM) in the PS group was significantly different compared to the HC, MAP-T, and MAP-P groups. A represents hsa-miR-11400; B represents hsa-miR-1908-3p; C represents hsa-miR-4433b-3p; and D represents nov-miR-chr20.

[0034] Figure 2 ROC curves for identifying MAP-P and PS using combined miRNA biomarkers.

[0035] Figure 3 A bar chart shows the relative expression levels of four miRNA markers in different clinical groups. A represents hsa-miR-11400; B represents hsa-miR-1908-3p; C represents hsa-miR-4433b-3p; and D represents nov-miR-chr20. Results showed that hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p were significantly overexpressed in the MAP group, while nov-miR-chr20 was specifically overexpressed in the PS group. ns indicates no significant difference ( P >0.05); express P <0.05; express P <0.01 (based on one-way ANOVA and multiple comparison tests).

[0036] Figure 4 Receiver operating characteristic (ROC) curves were plotted to differentiate PS and MAP-P using a combined miRNA biomarker combination in an independent validation cohort. Based on blinded qPCR data from the independent validation cohort (20 samples), a linear combination model was used to calculate the combined score, and ROC curves were then plotted. In the figure, the gray curve represents the diagnostic efficacy of the combined biomarker, and the red dashed line represents the random guessing reference line (AUC=0.5). The analysis showed that the AUC was 0.9600, indicating a highly statistically significant difference. P=0.0005), indicating that the biomarker combination provided by the present invention has extremely high identification accuracy and robustness in independent clinical samples. Detailed Implementation

[0037] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0038] Example 1: Subject Enrollment, Clinical Grouping, and Plasma Sample Collection 1. Study Subjects and Grouping: This study recruited 92 subjects, all of whom were Han Chinese aged 18-60. To reduce the potential interference of biological variables such as hormone levels in the biomarker screening stage, a representative subject group was selected for validation. Subjects were drawn from compulsory drug rehabilitation centers and mental health centers, and were diagnosed through structured clinical interviews conducted by two psychiatrists with associate chief physician or higher titles according to the DSM-5 criteria.

[0039] Based on clinical characteristics and substance use history, the subjects were divided into the following 4 groups: (1) PS group (n=23): The patients met the DSM-5 diagnostic criteria for schizophrenia. Key characteristics for inclusion were: first-episode, drug-naive patients who had never received antipsychotic medication before blood sample collection and had no history of drug or psychoactive substance abuse.

[0040] (2) MAP-P group (n=23): Meets the DSM-5 diagnostic criteria for "substance / drug-induced mental disorders"; key inclusion characteristic: psychotic symptoms (hallucinations, delusions, etc.) persist for more than 1 month after cessation of methamphetamine (METH) use (i.e., symptoms do not improve after 1 month of abstinence).

[0041] (3) MAP-T group (n=22): Meets the DSM-5 diagnostic criteria for "substance / drug-induced mental disorders"; key inclusion characteristic: psychotic symptoms appeared during or immediately after METH use, but completely disappeared within 1 month after discontinuation (spontaneous remission).

[0042] (4) Healthy control group (HC group, n=24): No personal or family history of mental illness; no history of substance abuse (excluding nicotine); no serious physical illness.

[0043] 2. Exclusion Criteria: To ensure the specificity of the biomarker, the following conditions were excluded from all groups: (1) Multiple substance abuse (mixed use of drugs other than tobacco and alcohol); (2) Secondary mental disorders caused by other physical illnesses (such as brain injury, epilepsy, encephalitis) or drugs; (3) Severe heart, liver, or kidney dysfunction or autoimmune diseases or infectious diseases (such as HIV positive).

[0044] 3. Sample Collection and Processing Blood collection: In the morning, all subjects had 10 mL of peripheral venous blood collected in a vacuum blood collection tube containing EDTA anticoagulant while fasting.

[0045] Plasma separation: Processing was performed within 2 hours after blood collection. First, the plasma was centrifuged at 3000 rpm for 10 min at 4°C. The upper plasma layer was carefully aspirated, avoiding the aspiration of leukocytes (buffy coat) and erythrocytes from the lower layer to prevent cell rupture and the release of intracellular miRNAs that could interfere with detection.

[0046] Storage: The isolated plasma samples were aliquoted into RNase-free cryovials, immediately flash-frozen in liquid nitrogen, and then transferred to an ultra-low temperature freezer at -80°C for storage until RNA extraction was performed.

[0047] Example 2: Total RNA extraction from plasma and design and synthesis of specific primers 1. Extraction of Total RNA from Plasma: In this example, TRIzol LS Reagent (Invitrogen, USA) was used to extract total RNA from the plasma samples collected in Example 1. The specific operating steps are as follows: (1) Thaw the plasma sample stored at -80℃ on ice and transfer 250 μL of plasma to a 1.5 mL RNase-free centrifuge tube.

[0048] (2) Add 750 μL TRIzol LS Reagent, vortex vigorously for 30 s to fully decompose it, and let it stand at room temperature for 5 min.

[0049] (3) Add 200 μL of chloroform, tighten the cap, shake vigorously for 15 s, and let stand at room temperature for 10 min.

[0050] (4) Centrifuge at 12,000 rpm for 15 min at 4℃. After centrifugation, the mixture will separate into three layers. Carefully aspirate the upper colorless aqueous phase (about 400~500 μL) into a new centrifuge tube.

[0051] (5) Add an equal volume of isopropanol to the colorless water in the upper layer, gently invert to mix, and let stand at room temperature for 10 min to precipitate RNA.

[0052] (6) Centrifuge at 12,000 rpm for 10 min at 4℃, discard the supernatant, and a small amount of gelatinous precipitate can be seen at the bottom of the tube.

[0053] (7) Add 1 mL of 75% (v / v) ethanol (prepared with DEPC water) to wash the precipitate, centrifuge at 7,500 rpm for 5 min at 4°C, and discard the supernatant.

[0054] (8) Air dry the precipitate at room temperature for 5-10 min (until the ethanol has completely evaporated), then add 20 μL of RNase-free water to dissolve the RNA.

[0055] (9) The concentration and purity (OD260 / 280 ratio) of RNA were detected using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Qualified samples were stored at -80℃ for later use.

[0056] 2. Design strategy for obtaining target miRNAs and specific primers: The extracted plasma total RNA was sequenced and analyzed. Differential expression analysis was performed on the obtained sequencing data. Differential expression analysis was performed between the MAP-T group and the PS group, and between the MAP-P group and the PS group. 34 and 15 differentially expressed miRNAs were obtained, respectively. Subsequently, Venn analysis was performed to select 4 miRNAs that were significantly different in both comparison groups, namely: hsa-miR-11400, hsa-miR-1908-3p, hsa-miR-4433b-3p, and nov-miR-chr20. Their nucleotide sequences are shown in Table 1.

[0057] Because the four target miRNA sequences mentioned above are short and lack a poly(A) tail, conventional reverse transcription methods are inefficient and have poor specificity. Furthermore, the target nov-miR-chr20 is a GC-rich repetitive sequence that readily forms secondary structures, making amplification difficult.

[0058] Therefore, this embodiment designed a specific stem-loop reverse transcription primer. This primer contains a universal stem-loop structure sequence and a specific sequence (6-8 bases) complementary to the 3' end of the target miRNA. During the PCR amplification stage, a specific forward primer consistent with the 5' end sequence of the target miRNA and a universal reverse primer complementary to the stem-loop structure were designed. Specifically, for nov-miR-chr20, a dedicated reverse primer was designed by optimizing the primer's Tm value (melting temperature) and 3' end anchoring sequence to overcome interference from high GC content. The nucleotide sequences of the primers are shown in Table 1. Furthermore, this invention also uses U6 snRNA as an internal reference gene and designs primers for reverse transcription and qPCR; miR-16-5p can also be used as an internal reference gene.

[0059] Table 1 3. Primer sequence synthesis: Based on the above design strategy, primer sequences were synthesized, and all primers were purified by PAGE.

[0060] Example 3: cDNA Synthesis and Real-Time Quantitative PCR Detection In this embodiment, total RNA extracted in Example 2 and synthesized specific primers were used to perform reverse transcription (RT) and real-time quantitative PCR (qPCR) amplification.

[0061] 1. Reverse transcription reaction (cDNA synthesis) Specific reverse transcription of miRNAs was performed using stem-loop primers.

[0062] (1) Preparation of reverse transcription reaction system: As shown in Table 2, prepare 20 μL of reverse transcription reaction system in an RNase-free PCR tube on ice. To ensure reverse transcription efficiency, multiplex reverse transcription mode (Multiplex RT) is used.

[0063] Table 2 Note 1: "Specific stem-loop RT primer mixture" contains reverse transcription stem-loop primers (SEQ ID NO: 5, 8, 10, 12 and 14) for hsa-miR-11400, hsa-miR-1908-3p, hsa-miR-4433b-3p, nov-miR-chr20 and internal reference U6 snRNA, with consistent final concentrations for each primer.

[0064] (2) Reverse transcription temperature program: Place the PCR tube in a thermal cycler and run a pulse reverse transcription program targeting the stem-loop primers for reverse transcription: Step 1: 16℃, 30 min (to promote the binding of stem-loop structure to short miRNA); Step 2: 42℃, 30 min (cDNA extension); Step 3: 85℃, 5 min (reverse transcriptase inactivation); Step 4: Store at 4℃.

[0065] After the reaction was completed, the reverse transcription product was diluted with 80 μL of nuclease-free water at a volume ratio of 1:5, and used as a qPCR template.

[0066] 2. Real-time quantitative PCR (qPCR) detection This step uses the ChamQ SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). This kit contains antibody-modified hot-start Taq DNA polymerase, and is detected using the specific fluorescent dye SYBR Green I. The detection instrument used is an ABI 7500 Real-Time PCR System.

[0067] (1) Preparation of qPCR reaction system: Under light-protected conditions, prepare 20 μL qPCR reaction system for each target of each sample. According to the recommended system of Vazyme kit and the optimized conditions for miRNA detection, the prepared qPCR reaction system is shown in Table 3.

[0068] Table 3 Note 2: Corresponds to SEQ ID NO: 6, 9, 11, 13 and 15 respectively. Note 3: The universal reverse primer SEQ ID NO: 7 is used for the four miRNAs. Note 4: For the ABI 7500 Real-Time PCR System, ROX Reference Dye 1 needs to be added to correct the fluorescence signal; if using instruments such as the Roche LightCycler 480 that do not require ROX correction, this component is not added, and water is used to make up the volume.

[0069] (2) Based on the hot-start characteristics of the ChamQ kit (only 30 s for activation), the qPCR amplification program is set as follows: Phase 1: Pre-denaturation (enzyme activation) 95℃, 30 s; Phase 2: Cyclic amplification (40 cycles) Denaturation: 95℃, 10 s; Annealing and extension: 60℃, 30 s (fluorescence signal is acquired at the end of this step); Phase 3: Melting curve analysis (MeltCurve) 95℃, 15 s; 60℃, 1 min; 95℃, 15 s (the instrument automatically performs continuous temperature increases and data acquisition).

[0070] (3) Data Acquisition and Processing: After the reaction, the melting curves are checked first. If each target well shows a single specific melting peak (without primer dimer impurities), the data is considered valid. The Ct values ​​of each reaction well are exported for subsequent statistical analysis. Samples with Ct values ​​greater than 31 or no amplification signal are considered undetectable.

[0071] Example 4: Detection Results and Statistical Analysis This embodiment processes the detection data obtained from the experiment, analyzes the differential expression of each target miRNA in different clinical groups, and evaluates its differential diagnostic efficacy.

[0072] 1. Data Calculation and Statistical Methods: High-throughput sequencing data were used for validation, and the normalized total expression level (TPM) of each target miRNA was calculated as an indicator of relative expression level. In practical applications of the kit, 2... -ΔΔCt The relative expression level was calculated based on qPCR data.

[0073] For samples that were not detected, the expression level was recorded as 0 in the statistical analysis. GraphPad Prism 9.5 software was used for data analysis and graphing. One-way ANOVA was used for comparisons among multiple groups, and independent samples t-tests or nonparametric tests were used for comparisons between two groups. P A value <0.05 is considered statistically significant.

[0074] 2. Results of PS-specific marker (nov-miR-chr20) expression detection: such as Figure 1 As shown in Figure D, nov-miR-chr20 exhibits a significant PS-specific high expression characteristic.

[0075] PS group: nov-miR-chr20 was specifically detected in PS group samples, with a mean relative expression level (Mean TPM) of 3.288.

[0076] In the non-PS group (MAP-T, MAP-P, HC): nov-miR-chr20 was expressed at extremely low levels or not detected in all four groups of samples, with an average relative expression level of only 0 to 0.005.

[0077] Compared with the PS group (3.288), the expression levels in both the MAP-T group and the MAP-P group showed highly statistically significant differences. P <0.001).

[0078] nov-miR-chr20 can serve as a "positive marker" for identifying PS, distinguishing between MAP and PS patients.

[0079] 3. Expression detection results of MAP-related biomarker combination (hsa-miR-11400, hsa-miR-1908-3p, hsa-miR-4433b-3p): (e.g.) Figure 1 A, Figure 1 B and Figure 1 As shown in C, these three miRNAs exhibited an expression trend that was completely opposite to that of nov-miR-chr20, showing specific "silence" or low expression in the PS group, while being widely detected in the methamphetamine-related group.

[0080] (1) hsa-miR-11400 PS group: Expression was extremely suppressed, with an average relative expression level of only 0.002. MAP-P group: Samples generally showed high expression, with an average relative expression level as high as 11.448. Intergroup comparison: The expression level of the MAP-P group was significantly higher than that of the PS group.

[0081] (2) hsa-miR-1908-3p PS group: showed a state of expression deficiency (mean 0). MAP-T group: expression level was significantly increased, with a relative expression level of 6.551. MAP-P group: although the expression level (mean 1.644) decreased compared to the acute phase, it still had significant differential significance compared to the "zero expression" state of the PS group.

[0082] (3) hsa-miR-4433b-3p PS group: Extremely low expression level (mean 0.002). HC group: Some basic expression was present (mean 7.290). MAP-related groups: Both MAP-T group (mean 10.838) and MAP-P group (mean 4.204) were effectively detected. Significance for differentiation: In the direct comparison between MAP-P and PS, hsa-miR-4433b-3p expression in the MAP-P group (4.204) was significantly higher than that in the PS group (0.002), demonstrating clear differentiation ability.

[0083] 4. Diagnostic efficacy assessment (ROC curve analysis): To assess the accuracy of the above biomarker combination in differentiating persistent MAP (MAP-P) from PS, receiver operating characteristic (ROC) curves were plotted and the area under the curve (AUC) was calculated.

[0084] Effectiveness of combined markers: such as Figure 2 As shown, a linear combination model was used to calculate the joint score using the four biomarkers mentioned above. The AUC value for distinguishing MAP-P from PS reached 0.8129 (95% CI: 0.593 - 0.881), and the difference was statistically significant. P = 0.0003). This indicates that the combination of biomarkers can effectively assist in the clinical differentiation between MAP and PS.

[0085] Example 5: Blind Validation of qPCR in Independent Small Sample Cohorts To further verify the stability and clinical diagnostic efficacy of the biomarker combination in different batches of samples and to eliminate possible sample selection bias in the discovery phase, this embodiment recruited an independent validation cohort for double-blind qPCR detection.

[0086] 1. Validation Cohort Overview: In strict accordance with the standards of Example 1, an additional 30 subjects were recruited and divided into three groups: PS group (n=10), MAP-P group (n=10), and HC group (n=10). The processing of all samples, RNA extraction, and qPCR detection procedures were consistent with those in Examples 2 and 3.

[0087] 2. Experimental Methods and Design Sample processing and testing: The collection, total RNA extraction, reverse transcription (cDNA synthesis) and qPCR testing of all plasma samples were strictly carried out in accordance with the standard operating procedures described in Examples 2 and 3.

[0088] Blinded design: During the testing process, the experimental operators are blinded to the clinical grouping information of the samples (BlindTest). The samples are classified and judged only according to the judgment logic set in Example 4 (such as setting specific Ct cutoff values ​​for different markers). After the experiment, the blinding is lifted and the accuracy is calculated.

[0089] (1) Set detection threshold: Set the cutoff point for Ct value. Ct>31 is considered negative / not detected.

[0090] (2) PS determination criteria: The test sample is determined to be PS when it meets the following conditions.

[0091] Condition A: The marker nov-miR-chr20 is detected positive (i.e., the Ct value is below the cutoff point); Condition B: The markers hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p are detected negative (i.e., the Ct value is higher than the cutoff point).

[0092] (3) MAP determination criteria: The test sample is determined to be MAP when it meets the following conditions.

[0093] Condition C: The marker nov-miR-chr20 is detected negative; Condition D: At least one of the biomarkers hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p must be positive.

[0094] 3. Test Results: (1) Detection results of PS-specific biomarker (nov-miR-chr20) Experimental results are as follows Figure 3As shown in D. PS group: In 7 out of 10 PS validation samples, expression of this biomarker was detected, with a mean Ct value of 23.597 (range: 22-27). Non-PS group (MAP-P and HC): In 20 non-PS samples, this biomarker was not detected or the Ct value was greater than the cutoff value (Ct>28), and was judged as negative. Using the Mann-Whitney U Test, there was a significant difference between the PS group and the non-PS group ( P = 0.0025).

[0095] (2) Detection results of MAP-related marker combination Experimental results are as follows Figure 3 A, Figure 3 B and Figure 3 As shown in C. MAP-P group: In 10 MAP-P validation samples, hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p showed high expression characteristics in multiple samples, with average Ct values ​​of 27.001 (range: 23–31), 21.630 (range: 17–27), and 11.368 (range: 7–31), respectively. PS group: In 10 PS validation samples, the expression of the three biomarkers was significantly suppressed in multiple samples. The expression levels of hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p in the MAP-P group were significantly higher than those in the PS group, effectively distinguishing MAP-P and PS samples.

[0096] 4. Validation Conclusions and Efficacy Evaluation: Based on the above qPCR test results, data from the independent validation cohort were analyzed using GraphPad Prism software. Identification Accuracy: The biomarker combination provided by this invention achieved a 90.0% accuracy rate in differentiating PS from MAP-P in the independent cohort. Sensitivity and Specificity: For PS identification, the sensitivity and specificity were both 90.0%; for MAP-P identification, the sensitivity and specificity were both 90.0%. ROC Analysis: The receiver operating characteristic (ROC) curve showed an AUC value of 0.9600 (…). P = 0.0005), indicating that the combination of biomarkers maintained extremely high robustness in independent samples and has good potential for clinical translation. Figure 4 ).

[0097] Example 6: A kit for differentiating between methamphetamine-induced psychosis and primary schizophrenia. I. Composition 1. Primer set, as shown in Table 1 of Example 2.

[0098] 2. Reverse transcription reaction reagents: 5×RT Reaction Buffer, RNase Inhibitor, dNTP Mix, reverse transcription primers from the primer set, Reverse Transcriptase, and Nuclease-free Water.

[0099] 3. Real-time quantitative PCR reaction reagents: 2×ChamQ SYBR qPCR Master Mix, the qRCR forward primer and qRCR reverse primer in the primer set, 50×ROX Reference Dye 1 and Nuclease-free Water.

[0100] II. Usage Method Reverse transcription and qPCR detection were performed according to the method in Example 3, and data were collected.

[0101] III. Judgment Criteria (1) Set detection threshold: Set the cutoff point for Ct value. Ct>31 is considered negative / not detected.

[0102] (2) PS determination criteria: The test sample is determined to be PS when it meets the following conditions.

[0103] Condition A: The marker nov-miR-chr20 is detected positive (i.e., the Ct value is below the cutoff point); Condition B: The markers hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p are detected negative (i.e., the Ct value is higher than the cutoff point).

[0104] (3) MAP determination criteria: The test sample is determined to be MAP when it meets the following conditions.

[0105] Condition C: The marker nov-miR-chr20 is detected negative; Condition D: At least one of the biomarkers hsa-miR-11400, hsa-miR-1908-3p, and hsa-miR-4433b-3p must be positive.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of miRNA as a biomarker in the preparation of products for differentiating between methamphetamine-induced psychosis and primary schizophrenia, characterized in that, The miRNA includes at least one of hsa-miR-11400, hsa-miR-1908-3p, hsa-miR-4433b-3p, and nov-miR-chr20; The nucleotide sequence of hsa-miR-11400 is shown in SEQ ID NO: 1, the nucleotide sequence of hsa-miR-1908-3p is shown in SEQ ID NO: 2, the nucleotide sequence of hsa-miR-4433b-3p is shown in SEQ ID NO: 3, and the nucleotide sequence of nov-miR-chr20 is shown in SEQ ID NO:

4.

2. The application of a reagent for detecting miRNA in the preparation of products for differentiating between methamphetamine-induced psychosis and primary schizophrenia, characterized in that... The miRNA includes at least one of hsa-miR-11400, hsa-miR-1908-3p, hsa-miR-4433b-3p, and nov-miR-chr20; The nucleotide sequence of hsa-miR-11400 is shown in SEQ ID NO: 1, the nucleotide sequence of hsa-miR-1908-3p is shown in SEQ ID NO: 2, the nucleotide sequence of hsa-miR-4433b-3p is shown in SEQ ID NO: 3, and the nucleotide sequence of nov-miR-chr20 is shown in SEQ ID NO:

4.

3. A primer set for detecting miRNA, characterized in that, The primer set includes a forward primer and a reverse primer for amplifying the miRNA of claim 1; The nucleotide sequence of the forward primer for amplifying hsa-miR-11400 is shown in SEQ ID NO: 6, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

7. The nucleotide sequence of the forward primer for amplifying hsa-miR-1908-3p is shown in SEQ ID NO: 9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

7. The nucleotide sequence of the forward primer for amplifying hsa-miR-4433b-3p is shown in SEQ ID NO: 11, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

7. The nucleotide sequence of the forward primer for amplifying nov-miR-chr20 is shown in SEQ ID NO: 13, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:

7.

4. The primer set as described in claim 3, characterized in that, The primer set also includes reverse transcription primers for reverse transcription of the miRNA of claim 1; The nucleotide sequences of the reverse transcription primers for reverse transcription of hsa-miR-11400 are shown in SEQ ID NO: 5; the nucleotide sequences of the reverse transcription primers for reverse transcription of hsa-miR-1908-3p are shown in SEQ ID NO: 8; the nucleotide sequences of the reverse transcription primers for reverse transcription of hsa-miR-4433b-3p are shown in SEQ ID NO: 10; and the nucleotide sequences of the reverse transcription primers for reverse transcription of nov-miR-chr20 are shown in SEQ ID NO:

12.

5. The primer set as described in claim 3, characterized in that, The primer set also includes primers for detecting an internal reference gene, which includes U6 snRNA and / or miR-16-5p.

6. The primer set as described in claim 5, characterized in that, The primers for detecting the internal reference gene U6 snRNA include forward and reverse primers for amplifying U6 snRNA, and / or reverse transcription primers for reverse transcription of U6 snRNA. The nucleotide sequence of the forward primer for amplifying U6 snRNA is shown in SEQ ID NO: 15, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 14; the nucleotide sequence of the reverse transcription primer for reverse transcription of U6 snRNA is shown in SEQ ID NO:

14.

7. A kit for differentiating between methamphetamine-induced psychosis and primary schizophrenia, characterized in that, The kit contains reagents for detecting the miRNA described in claim 1.

8. The kit according to claim 7, characterized in that, The kit contains the primer set as described in any one of claims 3 to 6.

9. The kit according to claim 7, characterized in that, The kit also contains reverse transcription reaction reagents, which include at least one of reverse transcriptase, RNase inhibitors, dNTPs, and reverse transcription buffer.

10. The kit according to claim 7, characterized in that, The kit also contains real-time quantitative PCR reaction reagents, which include at least one of DNA polymerase, fluorescent dye, fluorescent probe and real-time quantitative PCR buffer.

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