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CN-121992104-A - Biomarker detection kit and detection method for prognosis evaluation of multiple myeloma

CN121992104ACN 121992104 ACN121992104 ACN 121992104ACN-121992104-A

Abstract

The invention discloses a biomarker detection kit and a detection method for prognosis evaluation of multiple myeloma, and relates to the technical field of biological medicine detection, wherein the kit takes circMYC and circSETD double-ring RNA combination in serum exosomes as a core prognosis marker, contains an exosome solid-phase capture reagent, has sulfhydryl modified silica magnetic beads and the like; the invention selects serum exosome double-ring RNA as a core prognosis marker to construct a complete detection system, the dedicated reagent efficiently enriches and purifies exosomes, improves detection specificity and accuracy, provides reliable basis for prognosis layering, realizes integrated operation, simplifies flow, adapts to clinic, matches quantitative analysis with clear interpretation standard, can accurately distinguish patient risks, and has stable and convenient reagents, comparable results and improves clinical application value.

Inventors

  • ZHOU ZHENG
  • WU CHAOPING

Assignees

  • 周峥

Dates

Publication Date
20260508
Application Date
20260408

Claims (10)

  1. 1. The biomarker detection kit for prognosis evaluation of multiple myeloma is characterized by taking circMYC and circSETD double-loop RNA combination in serum exosomes as core prognosis markers, and comprises exosome solid-phase capture reagents, isothermal amplification detection reagents, double-quality control reagents, positive control and negative control, wherein the exosome solid-phase capture reagents comprise sulfhydryl modified silica magnetic beads, exosome binding buffer solution, first washing buffer solution and second washing buffer solution, the isothermal amplification detection reagents comprise a circMYC specific SDA primer pair, a circSETD2 specific SDA primer pair, a fluorescent quenching probe mixture, 10X isothermal reaction buffer solution, strand displacement DNA polymerase, dNTP mixed solution and RNase-free ultrapure water, and the double-quality control reagents comprise CD63 peptide fragment markers, circRPPH1 specific SDA primer pairs and circRPPH1 specific fluorescent probes.
  2. 2. The biomarker detection kit for prognosis evaluation of multiple myeloma according to claim 1, wherein the particle size of the sulfhydryl modified silica magnetic beads is 150-200 nm, the exosome binding buffer solution is a phosphate buffer system with pH of 7.4, the first washing buffer solution is Tris-HCl buffer solution with pH of 8.0 and contains Tween-20, and the second washing buffer solution is detergent-free PBS buffer solution.
  3. 3. The biomarker detection kit for prognosis evaluation of multiple myeloma according to claim 1, wherein the circMYC-specific SDA primer pair and the circSETD-specific SDA primer pair are designed across circular RNA reverse splice sites, wherein in the fluorescence quenching probe mixture, circMYC-specific probes label FAM fluorescent reporter groups, circSETD 2-specific probes label CY5 fluorescent reporter groups, and the hybridization regions of both probes cover reverse splice junction sequences corresponding to circular RNAs.
  4. 4. The biomarker detection kit for prognosis evaluation of multiple myeloma according to claim 1, wherein the CD63 peptide fragment marker is a biotin-labeled CD 63-specific antigen peptide fragment, the circRPPH 1-specific SDA primer pair is designed across the reverse splice site of circRPPH, and the circRPPH 1-specific fluorescent probe labels a HEX fluorescent reporter group.
  5. 5. The biomarker detection kit for prognosis evaluation of multiple myeloma according to claim 1, wherein the positive control is a mixed solution of circMYC and circSETD mimetics, the negative control is ultrapure water without exogenous nucleic acid pollution, and the components of the kit are packaged by independent freezing tubes.
  6. 6. A method for detecting a biomarker detection kit for prognosis evaluation of multiple myeloma, which is applicable to the biomarker detection kit for prognosis evaluation of multiple myeloma according to any one of claims 1 to 5, characterized in that the method comprises the following specific steps: s1, enriching and purifying exosomes, namely taking a serum sample to be detected, adding exosome binding buffer solution, a CD63 peptide fragment marker and sulfhydryl modified silica magnetic beads, incubating, removing a supernatant through magnetic separation, and sequentially adding a first washing buffer solution and a second washing buffer solution for washing to obtain exosome magnetic bead complexes; s2, exosome cracking and nucleic acid purification, namely adding exosome cracking liquid into exosome magnetic bead complex, adding purification reagent, and performing magnetic separation, washing and elution to obtain purified sample total annular RNA; S3, isothermal amplification and fluorescence detection, namely using total annular RNA as a template, adding circMYC and circSETD2 specific primer probe mixed solution, circRPPH1 specific primer probe mixed solution, 10 multiplied by constant temperature reaction buffer solution, strand displacement DNA polymerase and dNTP mixed solution, constructing an isothermal amplification reaction system, incubating at constant temperature, and collecting fluorescence signals in real time; S4, data correction and prognosis interpretation, namely correcting through CD63 peptide fragment marker signals and circRPPH amplification fluorescent signals, calculating the relative expression quantity of circMYC and circSETD2, and judging whether the patient is of a critical type or a high-risk type by combining a preset clinical cut-off value.
  7. 7. The method for detecting the biomarker detection kit for prognosis evaluation of multiple myeloma according to claim 6, wherein in the step S1, the amount of serum sample to be detected is 150-250 mu L, the amount of exosome binding buffer is 80-120 mu L, the amount of CD63 peptide fragment marker is 3-8 mu L, the amount of mercapto-modified silica magnetic beads is 5-15 mu L, the incubation time is 8-15 minutes at room temperature, the magnetic separation operation is that a centrifuge tube is placed on a magnetic separation frame for standing, the supernatant is discarded, and the first washing buffer and the second washing buffer are added during washing.
  8. 8. The detection method of the biomarker detection kit for prognosis evaluation of multiple myeloma according to claim 6, wherein in the step S2, the dosage of exosome lysate is 40-60 mu L, standing for 3-8 minutes at room temperature, the dosage of purification reagent is 40-60 mu L, standing for 1-5 minutes, washing the washing reagent for 2 times, the elution dosage of RNase-free ultrapure water is 15-25 mu L, and the washing reagent is placed on ice for standby.
  9. 9. The detection method of the biomarker detection kit for prognosis evaluation of multiple myeloma according to claim 6, wherein in the step S3, the total annular RNA template dosage is 3-8 mu L, the total volume of an isothermal amplification reaction system is 15-25 mu L, the incubation temperature at constant temperature is 35-39 ℃ for 50-70 minutes, and the fluorescent signal acquisition frequency is 1 time per 1 minute.
  10. 10. The method according to claim 6, wherein in the step S4, the specific operation of data correction is to use the fluorescence signal of the CD63 peptide fragment marker as an exosome extraction efficiency correction parameter and the amplified fluorescence signal of circRPPH1 as an isothermal amplification efficiency correction parameter, the relative expression amounts of circMYC and circSETD2 are calculated by adopting a delta Ct method, and the specific mode of prognosis risk stratification interpretation is that when the relative expression amounts of circMYC and circSETD2 are both lower than the corresponding cut-off values, the patient is judged to be at risk, otherwise the patient is judged to be at risk.

Description

Biomarker detection kit and detection method for prognosis evaluation of multiple myeloma Technical Field The invention relates to the technical field of biological medicine detection, in particular to a biomarker detection kit and a biomarker detection method for prognosis evaluation of multiple myeloma. Background Multiple myeloma is a malignant plasma cell disease, the disease progress of which has obvious individual variability and the prognosis effect of a patient has larger difference, so that accurate prognosis risk stratification is realized, the clinical treatment scheme of the disease is formulated, the disease progress is monitored, and the life quality of the patient is improved, the application of biomarkers in tumor prognosis evaluation is wider in recent years, annular RNA is used as a novel non-coding RNA, has stronger stability due to a unique annular structure, has obvious tissue and disease specificity, becomes an important research direction of tumor molecular markers, exosomes are used as extracellular vesicles, the carried annular RNA can be stably stored along with body fluid circulation, can be detected from body fluid samples which are easy to obtain such as serum, and provides possibility for tumor noninvasive prognosis evaluation, and isothermal amplification technology is gradually widened in application in nucleic acid detection due to the advantage of convenient operation, and combines the two to open up a novel technical path for noninvasive prognosis evaluation of multiple myeloma. The traditional multiple myeloma prognosis evaluation means depend on clinical pathological indexes and conventional laboratory detection results, risk judgment can be carried out only from a phenotype layer, accurate characterization on a disease molecular layer is lacking, the problems of insufficient specificity and sensitivity exist, early and accurate prognosis risk layering is difficult to realize, linear RNA is often selected as a marker for a detection method based on nucleic acid, the nucleic acid is easy to degrade in a body fluid sample, the stability and repeatability of the detection result are poor, meanwhile, the existing detection technology lacks efficient enrichment and purification means for exosomes, the interference of mixed nucleic acid and impurities in the sample is easy to occur, in addition, the existing circular RNA detection also has the problem of insufficient primer design pertinence, nonspecific amplification is easy to be caused, an effective quality control mechanism is not established in the whole detection process, the systematic errors in the exosome extraction and nucleic acid amplification processes are not considered, the accuracy of the detection result is further reduced, and the requirement of clinical accurate evaluation is difficult to meet. Disclosure of Invention The invention aims to make up the defects of the prior art, provides a biomarker detection kit and a detection method for prognosis evaluation of multiple myeloma, wherein circMYC and circSETD double-loop RNA in serum exosomes are used as prognosis markers, the kit integrates special reagents for exosome solid-phase capture, isothermal amplification detection, double quality control and the like, each component is independently packaged, a primer probe is designed in a targeted manner, the specificity and the stability of detection are ensured, a complete standardized system from exosome enrichment purification and nucleic acid extraction to isothermal amplification fluorescence detection, data correction and prognosis judgment is constructed by the matched detection method, double correction of extraction and amplification efficiency is realized through double quality control, and patient risk and high risk prognosis layering is accurately completed. The invention provides a biomarker detection kit for prognosis evaluation of multiple myeloma, which takes circMYC and circSETD double-loop RNA combination in serum exosomes as core prognosis markers and comprises exosome solid-phase capture reagents, isothermal amplification detection reagents, double-quality control reagents, positive control and negative control, wherein the exosome solid-phase capture reagents comprise sulfhydryl modified silica magnetic beads, exosome binding buffer solution, first washing buffer solution and second washing buffer solution, the isothermal amplification detection reagents comprise circMYC specific SDA primer pairs, circSETD specific SDA primer pairs, fluorescent quenching probe mixtures, 10X constant temperature reaction buffer solution, strand displacement DNA polymerase, dNTP mixed solution and RNase-free ultrapure water, and the double-quality control reagents comprise CD63 peptide section markers, circRPPH1 specific SDA primer pairs and circRPPH specific fluorescent probes. Further, the particle size of the sulfhydryl modified silica magnetic beads is 150-200 nm, the exosome binding buffer solution is a ph