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CN-120485369-B - Kit for detecting common fusion genes of myeloid leukemia based on multiplex digital PCR method

CN120485369BCN 120485369 BCN120485369 BCN 120485369BCN-120485369-B

Abstract

The invention discloses a kit for detecting common fusion genes of myeloid leukemia based on a multiplex digital PCR method, wherein specific primer probe combinations are adopted, and the kit can be used for rapid and accurate molecular detection of clinical 46 common fusion genes of myeloid leukemia by a preferable 7-color multiplex digital PCR technology platform. The preferred primer probe combination and detection system disclosed by the invention creatively adds a PCR enhancer into the PCR reaction liquid and creatively constructs a ROX+CY5 and Atto 425 (A425) +VIC dual-channel detection system, the PCR enhancer remarkably improves the efficiency and the specificity of a multiple amplification system, and the dual-channel system can realize the accurate detection of 46 fusion genes by 3 detection holes. The invention has the advantages of multiple accurate quantification, high sensitivity, strong specificity, strong anti-interference capability, good repeatability, simplicity, convenience, rapidness, high throughput, low cost and the like, has good clinical application prospect, and can provide reference for rapid and accurate diagnosis, treatment and medication selection, curative effect evaluation and prognosis judgment of clinical myeloid leukemia.

Inventors

  • YU RUNLIU
  • Hu Lingzhu
  • HE BEIBEI
  • YU PEI
  • REN XUYI

Assignees

  • 英维谱(浙江)生物科技有限公司

Dates

Publication Date
20260512
Application Date
20250610

Claims (8)

  1. 1. A kit for detecting common fusion genes of myeloid leukemia based on a multiplex digital PCR method is characterized by at least comprising a hole 1 primer probe mixed solution, a hole 2 primer probe mixed solution, a hole 3 primer probe mixed solution and a PCR reaction solution: The hole 1 primer probe mixed solution comprises nucleotides shown in SEQ ID NO. 001-SEQ ID NO.062, the hole 2 primer probe mixed solution comprises nucleotides shown in SEQ ID NO.020, SEQ ID NO.022, SEQ ID NO.027-028, SEQ ID NO.035-036 and SEQ ID NO.063-101, and the hole 3 primer probe mixed solution comprises nucleotides shown in ：SEQ ID NO.027-028、SEQ ID NO.035-036、SEQ ID NO.088、SEQ ID NO.090、SEQ ID NO.100-101、SEQ ID NO.102-136; The PCR reaction liquid of the kit comprises a PCR enhancer, wherein the PCR enhancer consists of betaine, DMSO, (NH 4) 2 SO 4 , BSA and gelatin, and the concentration of the PCR enhancer in the PCR reaction liquid is respectively 0.25M, 1.5wt%, 5mM, 0.05 mg/mL and 0.25 wt%; wherein, + represents the base modified locked nucleic acid LNA thereafter.
  2. 2. The kit of claim 1, wherein the final concentration of the primer in the well 1 primer probe mixture to the well 3 primer probe mixture is 250-900 nM and the final concentration of the probe is 250-500 nM.
  3. 3. The kit according to claim 1, wherein the probe has a fluorescent reporter group attached to the 5 'end and a fluorescent quenching group attached to the 3' end, and wherein the attached fluorescent reporter group is specifically: In the mixed solution of the primer probe of the hole 1, The probe shown in SEQ ID NO.11-12 is connected with a fluorescence report group VIC; the probe shown in SEQ ID NO.22-23 is connected with a fluorescence report group FAM; a probe shown in SEQ ID NO.26 is connected with a fluorescent reporter group ROX; the probe shown in SEQ ID NO.35-36 is connected with a fluorescence reporter group A425; The probe shown in SEQ ID NO.45-46 is connected with a fluorescence reporter group CY7; The probe shown in SEQ ID NO.52-53 is connected with a fluorescence reporter group CY5; The probe shown in SEQ ID NO.56 is connected with a fluorescence reporter group CY5.5; the probe shown in SEQ ID NO.59 is connected with fluorescent reporter groups ROX and CY5; The probe shown in SEQ ID NO.62 is connected with a fluorescence reporter group VIC and A425; hole 2 primer probe mixture: a probe shown in SEQ ID NO.65 is connected with a fluorescence reporter group VIC; the probe shown in SEQ ID NO.70-71 is connected with a fluorescence report group FAM; a probe shown in SEQ ID No.74 is connected with a fluorescent reporter group ROX; the probe shown in SEQ ID NO.35-36 is connected with a fluorescence reporter group A425; The probe shown in SEQ ID NO.22 is connected with a fluorescence reporter group CY7; The probe shown in SEQ ID NO.90-91 is connected with a fluorescence reporter group CY5; A probe shown in SEQ ID NO.94 is connected with a fluorescence reporter group CY5.5; The probe shown in SEQ ID NO. 97-98 is connected with fluorescent reporter groups ROX and CY5; The probe shown in SEQ ID NO.101 is connected with fluorescent reporter groups VIC and A425; hole 3 primer probe mix: a probe shown as SEQ ID NO.106 is connected with a fluorescence reporter group VIC; a probe shown in SEQ ID NO. 90 is connected with a fluorescence reporter group FAM; A probe shown in SEQ ID NO.112 is connected with a fluorescent reporter group ROX; the probe shown in SEQ ID NO.35-36 is connected with a fluorescence reporter group A425; the probe shown in SEQ ID NO.121-122 is connected with a fluorescence reporter group CY7; the probe shown in SEQ ID NO.125 is connected with a fluorescence reporter group CY5; the probe shown in SEQ ID NO.130-131 is connected with a fluorescence reporter group CY5.5; The probe shown in SEQ ID NO.135 is connected with fluorescent reporter groups ROX and CY5; A probe as shown in SEQ ID NO.101 is attached to the fluorescent reporter groups VIC and A425.
  4. 4. The kit according to claim 3, wherein the fluorescence quenching group is any one selected from the group consisting of BHQ1, BHQ2 and BHQ 3.
  5. 5. The kit of claim 1, wherein the PCR reaction solution further comprises DNA polymerase, mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP, a localization fluorescent dye.
  6. 6. The kit according to claim 1, the kit is characterized by further comprising: (1) Reverse transcription reagent comprising RT enzyme, RNase Inhibitor, dNTP, oligo (dT) 18 Primer, random 6 mers Primer, and reaction Buffer; (2) A digital PCR microfluidic chip and a microdroplet generating oil; (3) Positive control No.1 comprises NUP 98:HOXA 11, BCR:ABL 1, RUNX 1:RUNX 1T1, MLL:AF 17, FIP1L 1:PDGFRA, KAT6 A:CREBBP fusion gene variant plasmid and ABL1 reference gene plasmid; (4) Positive reference substance No. 2 is a mixed solution containing FUS:: ERG and NPM1:: ALK fusion gene variant plasmid and ABL1 reference gene plasmid; (5) The positive control No. 3 comprises a mixed solution of AFDN, KMT2A, MLLT, KMT2A, AFF1, KMT2A, MLLT1, ETV6, ABL1, PLZF, RARα, ETV6, PDGFRB fusion gene variant plasmid and ABL1 reference gene plasmid; (6) No. 4 positive control comprises KMT2A MLLT and SET MLLT, NUP214 fusion gene variant plasmid and ABL1 reference gene plasmid; (7) No. 5 positive control comprises mixed solution of CBFB, MYH11, PML, RARA, NPM1, MLF1, KMT2A, ELL, ETV6, JAK2, RUNX1, RPL22, RUNX1, MECOM fusion gene variant plasmid and ABL1 reference gene plasmid; (8) No. 6 positive control comprises RUNX1, CBFA2T3 and DEK, NUP214 fusion gene variant plasmid and ABL1 reference gene plasmid; (9) NC, mixed solution containing ABL1 reference gene plasmid.
  7. 7. The kit of claim 6, wherein the positive control has a concentration of about 75 copies/. Mu.L of each fusion gene variation plasmid and the ABL1 reference plasmid has a concentration of about 1500 copies/. Mu.L.
  8. 8. The kit according to claim 1, wherein the amplification system comprises 7.5. Mu.L of PCR reaction solution, 2.5. Mu.L of well 1 primer probe mixture or well 2 primer probe mixture or well 3 primer probe mixture, 5. Mu.L of cDNA of the detected sample, 15. Mu.L of the total system, and the PCR amplification conditions comprise 95 ℃ for 10min, 98 ℃ for 15s,62 ℃ for 1min,40 cycles, 28 ℃ for 5 min and 28 ℃ hold.

Description

Kit for detecting common fusion genes of myeloid leukemia based on multiplex digital PCR method Technical Field The invention belongs to the field of molecular biology gene detection, and in particular relates to a kit for detecting common fusion genes of myeloid leukemia based on a multiplex digital PCR method. Background Myeloid leukemia (Myeloid Leukemia) is a group of malignant tumors affecting the bone marrow and blood, mainly involving abnormal proliferation and maturation disorders of myeloid cells. Myeloid leukemia is largely divided into two major classes, acute myeloid leukemia (Acute Myeloid Leukemia, AML) and chronic myeloid leukemia (Chronic Myeloid Leukemia, CML) depending on the rate of progression of the disease. Treatment of AML typically involves intensive chemotherapy and hematopoietic stem cell transplantation, while treatment of CML relies on tyrosine kinase inhibitors targeting BCR-ABL fusion genes, such as Imatinib (Imatinib) and the like. The detection of the fusion gene has important values in the aspects of rapid and accurate diagnosis, risk and prognosis evaluation, treatment selection, curative effect monitoring and the like of AML and CML. In recent years, with the continuous progress of molecular diagnosis technology, the technology and method for detecting leukemia fusion genes are also continuously developed and perfected, so that the fusion genes can be rapidly and accurately detected, and the detection method is particularly critical for realizing accurate clinical diagnosis and treatment in early stage of diseases. The RT-qPCR technology method can greatly shorten the length of a target product and solve the problem that nucleic acid fragmentation cannot be detected, and the closed system can solve the problem of pollution of RT-PCR amplified products, and has the characteristics of high specificity, wide quantitative range, high sensitivity and the like. At present, fusion gene detection products based on the technical platform are more developed and occupy the mainstream of clinical application, and related products and technologies are reported in partial literature and patents. Based on the platform, two technical systems of a one-step method and a two-step method exist, but the one-step method RT-qPCR technology can only be used for detecting a single fusion gene, the technology needs to determine the copy number of the fusion gene by means of a standard curve, the 1-tube can only detect a single target, the target gene and the internal standard gene need to be respectively manufactured into the standard curve, and the target gene and the internal standard gene are greatly influenced by amplification efficiency and PCR inhibitors, and have the advantages of limited relative quantitative accuracy, low flux, complex operation, high cost and high requirement on an RNA template. The simultaneous detection reagent for multiple fusion genes is a two-step detection product generally, and is a qualitative product because different targets cannot be accurately quantified due to mutual competitive inhibition, and the existing PCR reaction solution mainly has the problems that (1) the amplification efficiency is low, when primers or probes for multiple targets (such as BCR: ABL1, PML: RARA, RUNX1: RUNX1T 1) exist simultaneously, primer dimers or heterohybridization (such as combination of BCR primers and RUNX1 probes) can be formed due to sequence similarity, the reaction reagent is consumed, the effective concentration is reduced, meanwhile, multiple pairs of primers compete for limited Taq DNA polymerase and dNTPs, targets with low amplification efficiency (such as fusion genes with high GC content) can be pressed by targets with high efficiency (such as genes with low GC content), so that amplification deviation is caused, (2) the specificity is poor, the primers can be combined with templates in a non-perfect matching way, a mixed band or false positive result is generated, and a trace inhibitor can be limited in sensitivity, and templates with low concentration can not be detected in a general sample. The platform channel is generally 4 channels, 3 targets can be detected in each hole, the number of holes of various fusion gene screening products is generally more (8-16 holes), the requirements on the number and quality of samples are higher, the flux is lower, the unit cost is high, the multiple detection competition interference is serious, the sensitivity is lower (the detection limit is more than 1000 copies/reaction), and the clinical high-sensitivity accurate diagnosis and treatment requirement cannot be met. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a primer probe combination and a detection kit which have high sensitivity, high accuracy and high specificity, are simple and convenient and quick to operate, can simultaneously realize absolute quantification of a plurality of fusion genes, and particularly, the invention can