AETCA (anchored-extension and telomeric complements amplification) detection reagent kit for telomerase and detection method

Abstract

The invention provides an AETCA (anchored-extension and telomeric complements amplification) detection reagent kit for telomerase and a detection method. The reagent kit comprises an anchored PCR (polymerase chain reaction) tube, a probe BT (SEQ ID No.2), PCR primer, denaturing lysis buffer, extension hybridization buffer, PCR buffer and washing buffer, wherein telomerase primer ATS (SEQ ID No.1) are fixed in the anchored PCR tube. By utilizing a part of probe sequence (the probe BT) complementary with a telomere repetitive sequence as a template, the probe is combined on the telomere repetitive sequence synthesized by the telomerase in a multicopy manner, and accordingly, amplification of the probe BT can be kept in efficient fixed length though the length of the telomere repetitive sequence is not constant, and the defects of the TRAP (tartrate resistant acid phosphatase) method are overcome. The detection method is convenient and easy to operate, sensitivity and specificity of activity detection of the telomerase can be improved, and the detection method is applicable to detection of various sources including cells or tissues of clinical samples such as sputum.

Description

(1) Technical field [0001] The invention relates to a telomerase anchored extension telomere sequence complementary amplification (Anchored-Extension and Telomeric Complements Amplification, AETCA) detection kit and detection method. (2) Background technology [0002] Telomerase (telomerase) is a special reverse transcriptase, which is a ribonucleoprotein (RNP) complex composed of RNA and protein. It contains 3 main components: telomerase RNA (hTR) template, telomerase catalytic subunit (hTERT) and telomerase-related protein (hTLP). hTR is the template for telomerase extension reaction, about 450 bases, which contains the template sequence of 5'-CUAACCCUAAC-3', hTERT is the protein catalytic subunit of telomerase reverse transcriptase, hTLP is telomerase adjustment unit. The main function of telomerase is to use the 3' end of the chromosome end (telomere) as a primer, and use its own RNA as a template to synthesize a telomere-TTAGGG-repeat sequence and add it to the end of...

AI Technical Summary

Problems solved by technology

[0004] The traditional TRAP method requires tedious polyacrylamide gel electrophoresis and staining steps

Since then, although many scholars have made improvements, especially the introduction of internal standards has enabled more accurate semi-quantitative determinations with the help of certain analyzers, these improvements are only in the post-PCR detection methods. The TRAP-PCR core reaction used are the same, thus the following inherent defects of the TRAP method cannot be improved:

[0005] (1) Low amplification efficiency

Because the TRAP method directly uses the product of the extension reaction of telomerase as the template for PCR, the obtained PCR products are a series of bands ranging in size from tens of bp to hundreds of bp, and the uncertainty of the amplified product leads to Amplification efficiency is greatly limited, thus reducing sensitivity;

[0006] (2) The problem of specificity

The problem derived from the uncertainty of TRAP amplification products is that the result analysis is easily interfered by non-specific PCR amplification;

[0007] (3) Poor repeatability / stability

Because the TRAP method directly adds the cell lysate to the PCR system, and the cell lysate often contains many components that inhibit the PCR reaction, so it is easy to cause the failure of the entire test;

[0008] (4) System limitations on sensitivity

The volume of cell lysate that can be added to the detection reaction system generally cannot exceed 4% of the total volume, which limits the sensitivity of the detection;

[0009] (5) The applicability is limited and it is not suitable for detecting the effect of telomerase inhibition

[0010] (6) It is difficult to combine the implementation of fluorescent real-time quantitative PCR

The main improvement of this method to the TRAP method is to change the indeterminate amplification products of different sizes in the TRAP method into a specific amplified fragment, but because it is also the coupling of telomerase extension reaction and PCR, it cannot be ruled out. Improved inhibition of PCR by cell lysates, therefore not as good in terms of reproducibility / stability etc.

In addition, reagents (dTTP and Taq enzyme) need to be replenished tube by tube in the middle of the reaction, which is also cumbersome

[0012] Non-PCR detection methods use isotopes, fluorescence, chemiluminescence, etc. to directly analyze and measure telomerase extension reaction products. These methods have not been widely used due to their limited sensitivity.

Images