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Reagent kid for quantitatively testing mRNA (messenger ribonucleic acid) level of FIP1L1-PDGFRA (feline infectious peritonitis 1 like 1-platelet-derived growth factor receptor alpha) fusion genes

A technology of FIP1L1-PDGFRA and fusion gene is applied in the field of kits for quantitatively detecting the mRNA level of FIP1L1-PDGFRA fusion gene, which can solve the problems of cumbersome PCR process, unstable electrophoresis band size, and inability to meet the needs of minimal residual disease detection. , to ensure the effect of specific amplification

Inactive Publication Date: 2013-12-18
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the breakpoint of FIP1-like gene 1 (FIP1L1) at the DNA level is not fixed, it has been reported that exons 9-13 are involved, and the breakpoint of PDGFRA gene is basically on exon 12, but the breakpoints are often different, so electrophoresis The size of the band is not fixed. In order to identify whether it is a specific PCR product, it needs to be determined by sequencing, which makes the PCR process more cumbersome.
In addition, qualitative PCR cannot produce quantitative results, so it cannot meet the current needs of minimal residual disease detection

Method used

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  • Reagent kid for quantitatively testing mRNA (messenger ribonucleic acid) level of FIP1L1-PDGFRA (feline infectious peritonitis 1 like 1-platelet-derived growth factor receptor alpha) fusion genes
  • Reagent kid for quantitatively testing mRNA (messenger ribonucleic acid) level of FIP1L1-PDGFRA (feline infectious peritonitis 1 like 1-platelet-derived growth factor receptor alpha) fusion genes
  • Reagent kid for quantitatively testing mRNA (messenger ribonucleic acid) level of FIP1L1-PDGFRA (feline infectious peritonitis 1 like 1-platelet-derived growth factor receptor alpha) fusion genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, the kit for detecting the mRNA level of FIP1L1-PDGFRA fusion gene

[0026] 1. Composition of the RQ-PCR amplification system for FIP1L1-PDGFRA fusion gene mRNA

[0027] 1. Upstream primer Ⅰ: 5 primers FIe9, 10, 11, 12 and 13 respectively located on exons 9, 10, 11, 12 and 13 of FIP1L1;

[0028] 2. Downstream primer Ⅰ: 2 primers PDRi and PDRi2 located on exon 13 of PDGFRA;

[0029] 3. TaqMan probe Ⅰ: 2 TaqMan probes PDpro and PDpro2 located on exons 12 and 13 of PDGFRA respectively;

[0030] 4. Mastermix for fluorescent PCR.

[0031] The sequences and final concentrations of the above primers and probes are shown in Table 1.

[0032] Table 1. Primers and probes in the FIP1L1-PDGFRA public system

[0033]

[0034] Note: The 5' end of TaqMan probe Ⅰ is connected with the fluorescent reporter group FAM, and the 3' end is connected with the fluorescent quencher group TAMRA.

[0035] 2. Composition of the RQ-PCR amplification system II of the internal re...

Embodiment 2

[0063] Example 2, the specificity and accuracy of the kit for detecting the mRNA level of FIP1L1-PDGFRA fusion gene

[0064] 1. Samples to be tested: isolated bone marrow or peripheral blood mononuclear cells from 247 patients clinically diagnosed with eosinophilia;

[0065] 2. According to the karyotype analysis method in the literature "Qiu Jingying, Dang Hui, Ren Hanyun, etc. Autologous plasma culture system to improve the chromosomes of leukemia bone marrow cells. Journal of Beijing Medical University. 1993, 25: 249-251" for step 1 The test samples were identified for FIP1L1-PDGFRA fusion gene, and the results are shown in Table 1;

[0066] 3. Using the kit of Example 1 to detect the cDNA of the sample to be tested in step 1, the results were positive in 12 cases, and the FIP1L1-PDGFRAmRNA level is shown in Table 1;

[0067] 4. Directly sequence the positive PCR product in step 3, and compare it with the genomic DNA sequence of the gene FIP1L1 and PDGFRA. The sequencing m...

Embodiment 3

[0071] Example 3, Detection of FIP1L1-PDGFRA Fusion Gene mRNA Level Kit FIP1L1-PDGFRA Fusion Gene and Internal Reference Gene ABL Standard Curve

[0072] Take the sample cDNA numbered 1 in Example 2 and carry out 10-fold serial dilution (the initial concentration is recorded as 1, and the concentration after dilution is recorded as: 10 -1 、10 -2 、10 -3 ), using the kit in Example 1 to amplify the FIP1L1-PDGFRA fusion gene and the internal reference gene ABL in four different concentrations of samples, and do 2 repetitions for each concentration, the amplification curves of the FIP1L1-PDGFRA fusion gene and the internal reference gene ABL and a standard curve such as figure 2 with image 3 shown. image 3 The slopes of the standard curves of the FIP1L1-PDGFRA fusion gene and the internal reference gene ABL were -3.51 and -3.47, respectively, and the amplification efficiencies were consistent, indicating that a standard curve made with ABL plasmid standards could be used to...

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Abstract

The invention discloses a reagent kit for quantitatively testing mRNA (messenger ribonucleic acid) level of FIP1L1-PDGFRA (feline infectious peritonitis 1 like 1-platelet-derived growth factor receptor alpha) fusion genes. The test reagent contains upstream primers I, downstream primers I and TaqMan probes I which are used for real-time quantitative PCR (polymerase chain reaction) testing for mRNA of the FIP1L1-PDGFRA fusion genes. The upstream primers I include at least one of single-chain DNA (deoxyribose nucleic acid) shown as sequences 1, 2, 3, 4 and 5 in a sequence table, the downstream primers I include at least one of two single-chain DNA as shown in sequence 9 and 10 in the sequence table, and the TaqMan probes I include at least one of two single-chain DNA as shown in sequences 9 and 10 in the sequence table. The reagent kit has the advantages of speediness, simplicity, convenience and the like in testing, common types of FIP1L1-PDGFRA fusion genes can be covered in one experiment, and the mRNA level of the FIP1L1-PDGFRA fusion genes can be tested. The reagent kit can be used for FIP1L1-PDGFRA fusion gene screening and therapeutic evaluation for eosinophilia patients, and further can be used for monitoring minimal residual diseases.

Description

technical field [0001] The invention relates to a kit for quantitatively detecting the mRNA level of the FIP1L1-PDGFRA fusion gene. Background technique [0002] Many diseases have the manifestations of peripheral blood eosinophilia, most of which are due to increased reactivity caused by infection, allergy and autoimmune diseases. In some cases, they are clonal malignant diseases of the blood system. Diagnosis and subsequent treatment of patients are essential. A subset of myeloproliferative neoplasms (MPNs) with eosinophilic manifestations involve abnormalities in the platelet-derived growth factor receptor alpha (PDGFRA) gene, the most common type of abnormality being those with the FIP1-like gene 1-PDGFRA α(FIP1L1-PDGFRA) fusion gene. In addition to MPN, the FIP1L1-PDGFRA fusion gene is also found in individual acute myeloid leukemia and T-cell lymphoma with the manifestation of eosinophilia. The deletion of chromosome 4q12 leads to the formation of FIP1L1-PDGFRA fusi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 秦亚溱李金兰李玲娣江倩刘艳荣黄晓军
Owner PEOPLES HOSPITAL PEKING UNIV
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