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Telomerase activity detecting probe, reagent kit and method

A detection reagent and detection probe technology, applied in the field of molecular detection, can solve the problems of being fixed on a special support, low detection sensitivity, poor specificity, etc., and achieve low price, low reagent cost and broad application value. Effect

Inactive Publication Date: 2014-03-26
SHENZHEN INST OF ADVANCED TECH
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Problems solved by technology

The traditional technology for detecting telomerase activity is mainly based on polymerase chain reaction (PCR) to amplify telomerase repeat segment (TTAGGG) (TRAP) to detect telomerase activity, but these methods are time-consuming and expensive. The operation is cumbersome and the specificity is not good
In order to overcome the shortcomings of polymerase chain reaction (PCR)-based methods, some detection methods that do not require polymerase chain reaction (PCR) have also been developed in recent years, such as fiber optic sensors, surface plasmon resonance technology, fluorescence, electrochemical, etc. , but these methods have the disadvantages of low detection sensitivity, cumbersome operation, time-consuming or the need to immobilize telomerase-binding primers on special supports

Method used

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  • Telomerase activity detecting probe, reagent kit and method
  • Telomerase activity detecting probe, reagent kit and method
  • Telomerase activity detecting probe, reagent kit and method

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Embodiment 1

[0113] figure 1 A schematic diagram of the structure of the telomerase-binding primer provided in the embodiment of the present invention, the telomerase-binding primer includes A, B, and C in sequence. Wherein, the nucleotide sequence of A is a recognition site of a nicking endonuclease added to the nucleotide sequence of C, and the B has a DNase sequence, and the DNase sequence has a hemin-binding The poly-G nucleotide sequence of the site, the two ends of the DNase sequence have the recognition site of the nicking endonuclease (as shown by X' in the figure), the nucleotide sequence A, the distance A The 15-24 bases at the 5' end have a recognition site for a nicking endonuclease (as shown by X in the figure); the nucleotide sequence C has a sequence recognized by telomerase;

[0114] combine figure 1 , the present embodiment provides a primer for detecting telomerase activity, comprising the steps of:

[0115] (1) In vitro synthesis of telomerase-binding primer (TS), t...

Embodiment 2

[0129] This embodiment provides a flow chart of a method for detecting telomerase activity, such as figure 2 shown. Depend on figure 2 It can be seen that this detection method can be roughly divided into three steps. The first step: the telomerase-binding primer (TS) with a nicking endonuclease site. A poly-G sequence (TTAGGG) is continuously added to the 3′ end. Step 2: The reverse primer is complementary to the extension product of telomerase - the poly-G sequence (TTAGGG), which is copied and extended under the action of the polymerase. After forming a double strand, the new amplification template and the nicking endonuclease are digested Site formation; the new amplification template is cleaved by a nicking endonuclease, and then the polymerase uses the newly formed amplification template as a template for strand displacement (SDA) replication, thereby generating a telomerase product-polyG sequence (TTAGGG ), DNase and a primer (trigger) that initiates the following ...

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Abstract

The invention provides a telomerase activity detecting probe, a telomerase activity detecting reagent kit and a telomerase activity detecting method. The design of telomerase combination primers (TS) and reverse primers is simple, and TS only needs to comprise DNA (deoxyribonucleic acid) enzyme sequences and restriction enzyme cutting sites of cutting enzyme and does not need any modification; the reverse primers only need to comprise sequences complemented with multiple-G sequences extending from telomerase, so the primers are easy to design, and the feasibility is high. The telomerase activity detecting method provided by the invention has the advantages that the reverse primers are only combined with TS primers extending from result telomerase, the nonspecific amplification is reduced, in addition, in the chemical luminous reaction, only amplified telomerase multiple-G sequences and DNA enzyme can be combined with hemin to form a tetramer, and chemical luminous signals are generated, so the telomerase activity detecting method provided by the invention has higher specificity and higher sensitivity. In addition, the telomerase activity detecting reagent kit provided by the invention has the advantages that the cost is low, the operation is simple, and the time is saved.

Description

technical field [0001] The invention relates to the field of molecular detection, in particular to a probe, kit and method for detecting telomerase activity. Background technique [0002] Highly sensitive detection of telomerase activity is of great significance for early diagnosis and drug screening of cancer and other related diseases. The traditional technology for detecting telomerase activity is mainly based on polymerase chain reaction (PCR) to amplify telomerase repeat segment (TTAGGG) (TRAP) to detect telomerase activity, but these methods are time-consuming and expensive. The operation is cumbersome and the specificity is not good. In order to overcome the shortcomings of polymerase chain reaction (PCR)-based methods, some detection methods that do not require polymerase chain reaction (PCR) have also been developed in recent years, such as fiber optic sensors, surface plasmon resonance technology, fluorescence, electrochemical, etc. , but these methods have the d...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48C12N15/11
CPCC12Q1/6848
Inventor 王黎娟张春阳张艳
Owner SHENZHEN INST OF ADVANCED TECH
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