Oligonucleotide, method and kit for detecting NonO-TFE3 fusion gene in sample

A NONO-TFE3, oligonucleotide technology, applied in the field of biological science and biology, can solve the problems of high cost and poor specificity, and achieve the effects of simple operation, convenient results, and avoiding hematological recurrence.

Inactive Publication Date: 2019-01-04
济南艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, because SYBR GreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

Method used

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  • Oligonucleotide, method and kit for detecting NonO-TFE3 fusion gene in sample
  • Oligonucleotide, method and kit for detecting NonO-TFE3 fusion gene in sample
  • Oligonucleotide, method and kit for detecting NonO-TFE3 fusion gene in sample

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Oligonucleotides for detecting the NONO-TFE3 fusion gene in the sample, the oligonucleotides include at least one of (1) and (2): (1) upstream and downstream primers for type 1 NONO-TFE3 and The base sequences of the probes are SEQ ID NO: 1, 2 and 3, as shown in Table 1. (2) For the upstream and downstream primers and probes of type 2 NONO-TFE3, their base sequences are SEQ ID NO, respectively : 4, 5 and 6, as shown in Table 1.

[0029] Further, the oligonucleotides also include upstream and downstream primers and probes for the β-actin internal reference gene, the base sequences of which are SEQ ID NO: 7, 8 and 9, respectively, as shown in Table 1.

[0030] Oligonucleotides used to detect the NONO-TFE3 fusion gene in a sample. The oligonucleotides include (1) and (2): (1) upstream and downstream primers and probes for type 1 NONO-TFE3, and their bases The base sequences are SEQ ID NO: 1, 2 and 3, as shown in Table 1. (2) For the upstream and downstream primers and probes o...

Embodiment 2

[0048] Operation process of the method of the present invention:

[0049] (1) Extract tissue RNA from blood samples: add 1ml of the red blood cell lysate in Example 1 to a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulant blood and mix well, let stand at room temperature for 10min; centrifuge at 5000rpm for 5min, Discard the supernatant and collect the cells at the bottom; add 0.5ml of the red blood cell lysate in Example 1 again, centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom; add 1ml of TRIzol to the cells and pipette repeatedly until the precipitate is completely dissolved, at room temperature Stand still for 5 minutes; add 0.2ml of chloroform and shake evenly; centrifuge at 14000rpm for 10 minutes at 4°C, transfer the supernatant layer to a new centrifuge tube (do not draw the white middle layer); add an equal volume of isopropanol, and mix thoroughly ,Stand for 10min at room temperature; Centrifuge at 14000rpm 4℃ for 10min...

Embodiment 3

[0063] Example 3 Positive plasmid detection and sensitivity detection

[0064] When preparing a positive plasmid, you can directly synthesize Type 1 NonO-TFE3cDNA and Type 2 NonO-TFE3cDNA, and then insert them into the pre-selected plasmids (here, take PMD18-T plasmid as an example for explanation), thus preparing PMD18 -T / NonO-TFE3-1 positive plasmid and PMD18-T / NonO-TFE3-2 positive plasmid.

[0065] Take the initial concentration as 1×10 11 The positive plasmid of copies / uL is used as a template, press 10 -1 , 10 -2 ……10 -10 Proportional dilution, respectively as a template for experiment, where the concentration is 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 The results are shown in Figure 1. The positive plasmid concentration corresponding to each amplification curve in Figure 1 is 10 from left to right. 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 copies / uL. It can be seen from Figure 1 that the primers and probes of the present invention can detect these two types of positi...

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Abstract

The invention discloses oligonucleotide, a method and a kit for detecting an NonO-TFE3 fusion gene in a sample. The oligonucleotide comprises upstream and downstream primers and a probe for 1-type NonO-TFE3, upstream and downstream primers and a probe for 2-type NonO-TFE3, and upstream and downstream primers and a probe for beta-actin internal reference genes. The oligonucleotide can quickly detect whether the sample contains the NonO-TFE3 fusion gene and an expression quantity of the fusion gene. A detection result completed by use of the method is accurate and sensitive and can assist in making an individual treatment scheme for an Xp11.2 translocation / TFE3 fusion gene-related kidney cancer patient.

Description

Technical field [0001] The present invention belongs to the field of biological science and biotechnology, and in particular relates to a method, oligonucleotides and kits for detecting NonO-TFE3 fusion genes in samples, using fluorescent PCR technology to detect human Xp11.2 translocation / TFE3 fusion gene related The expression level of NonO-TFE3 fusion gene in patients with primary renal cell carcinoma. Background technique [0002] Xp11.2 translocation / TFE3 gene fusion-associated renal cancer (referred to as Xp11.2 translocation renal cancer) is a newly classified by WHO in 2004 with a break in the TFE3 gene at the Xp11.2 locus, and is associated with PRCC, ASPL, PSF, CLTC, NonO and other related genes have balanced translocations to form an independent subtype of kidney cancer characterized by new fusion genes. At present, there are at least 8 types of gene fusions, but only 5 types have a clear site, which are the PRCC-TFE3 fusion gene caused by t(X; 1) (p11.2; q21), t(X; 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/158C12Q2561/113C12Q2563/107C12Q2561/101
Inventor 黄开新吴鹏飞王淑一
Owner 济南艾迪康医学检验中心有限公司
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