Molecular detecting method based on exonuclease and G tetramer and detecting kit

A technology for exonuclease and molecular detection, applied in the field of molecular detection methods and detection kits based on exonuclease and G tetramer, can solve the problems of increased cost and operation steps, low detection sensitivity of colorimetric method, enzyme Storage troubles and other problems, to achieve the effect of cheap, high sensitivity, simple operation

Active Publication Date: 2015-06-24
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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Problems solved by technology

However, the detection sensitivity of the colorimetric method based on G tetramer is low, and the sensitivity needs to be further improved.
The traditional signal amplification technology is mainly PCR and a series of signal amplification technologies relying o...

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  • Molecular detecting method based on exonuclease and G tetramer and detecting kit
  • Molecular detecting method based on exonuclease and G tetramer and detecting kit
  • Molecular detecting method based on exonuclease and G tetramer and detecting kit

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Embodiment Construction

[0037] A molecular detection kit based on exonuclease and G tetramer, including TMB chromogenic buffer, hemin, DNA hybridization buffer, also includes exonuclease and the following nucleic acid sequence:

[0038] DNA1: One end of the nucleic acid aptamer of the molecule to be tested is properly extended (both the 3' end and the 5' end) to form DNA1. Among the extended nucleic acids, the part near the end of the nucleic acid aptamer is the 1* region, and the rest is recorded as the 2* region ;

[0039] DNA2: DNA2 is continuously complementary to the 1* region of DNA1 and part of the nucleic acid aptamer to ensure the stability of the DNA1-DNA2 complex; at least 4 bases at the other end of DNA2 are not complementary to DNA1 to prevent being nucleic acid exonuclease cleavage;

[0040] DNA3: has a stem-loop structure, one end of which is region 1, the middle is sequentially region 2 and the connecting sequence, and the other end is a G tetramer sequence; the G tetramer has a nucl...

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Abstract

The invention discloses a molecular detecting method based on an exonuclease and a G tetramer and a detecting kit. Molecules are interacted with a nucleic acid aptamer, chain replacement reaction is started, a stem-loop structural DNA is opened, and then, an exonuclease mediated signal amplification technology is combined, so that a great number of G tetramer structures with activity can be generated. G tetramers are combined with hemin so as to have the catalytic activity of HRP and be capable of catalytically oxidizing TMB to generate blue substrates, a result can be seen by naked eyes, and the concentrations of molecules are directly related to the depth of blue. The molecular detecting method is simple in operation, capable of finishing the whole reaction at the room temperature, relatively high in sensitivity and favorable in specificity and can be used for rapidly detecting molecules on site; the detection limit for 17b-estradiol is 1 pM; and the detected result can be directly seen with naked eyes, but no detecting instruments are used.

Description

technical field [0001] The invention relates to a molecular detection method, in particular to a molecular detection method and a detection kit based on exonuclease and G tetramer. Background technique [0002] There are many harmful molecules in the environment, and it is very practical to detect these harmful small molecules quickly and at low cost. [0003] 17b-estradiol is considered to be the most potent and potentially influential class of endocrine disruptors, widely present in rivers, soil, atmosphere and agricultural products. It is mainly derived from pesticides, exhaust gas, food additives, human and animal excrement and domestic sewage. Even at extremely low concentrations, it will have a significant impact on organisms. Its content level is comparable to that of prostate cancer, breast cancer, ovarian cancer, There is a significant association between uterine cancer and male reproductive disorders. Traditional 17b-estradiol detection methods mainly include hig...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/44
CPCC12Q1/682C12Q2521/319C12Q2525/205
Inventor 陈俊华周顺桂温俊林
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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