Liquid-stage chip constant-temperature detection method for tiny RNA

A liquid chip and detection method technology, which is applied in the fields of life science and biology, can solve the problems of cumbersome detection steps, long time-consuming, and restrictions on the application of liquid chips

Inactive Publication Date: 2015-10-28
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When the liquid chip is applied to the detection of nucleic acid target molecules including miRNA, it often relies solely on the way of nucleic acid hybridization, the target molecule needs to be amplified in advance, and the amplification product or the hybridization of the target molecule and the microsp

Method used

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  • Liquid-stage chip constant-temperature detection method for tiny RNA
  • Liquid-stage chip constant-temperature detection method for tiny RNA
  • Liquid-stage chip constant-temperature detection method for tiny RNA

Examples

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Embodiment 1

[0094] Example 1: Each type of coding microsphere is immobilized with an upstream primer and / or a downstream primer corresponding to one miRNA target molecule, and six miRNA target molecules are simultaneously detected in a single detection system.

[0095] In this embodiment, the carboxyl-modified MicroPlex commercialized by U.S. Luminex Company is adopted. TM Fluorescence encoded microspheres, the encoded microspheres have 100 kinds of fluorescent encoded microspheres. In theory, when the upstream primer and / or downstream primer corresponding to a miRNA target molecule is immobilized on the surface of each coding microsphere, the upstream primer and / or downstream primer corresponding to each target molecule can label the same fluorescent reporter molecule, Simultaneous detection of 100 miRNA target molecules in a single reaction system. The present invention only uses the detection of six miRNA target molecules as an example to illustrate the detection effect of the present...

Embodiment 2

[0161] Embodiment 2: Each encoding microsphere surface immobilizes the upstream primers and / or downstream primers corresponding to two miRNA target molecules, and simultaneously detects six miRNA target molecules in a single detection system.

[0162] In this embodiment, the carboxyl-modified MicroPlex commercialized by U.S. Luminex Company is adopted. TM Fluorescence encoded microspheres, the encoded microspheres have 100 kinds of fluorescent encoded microspheres. In theory, when the upstream primers and / or downstream primers corresponding to two miRNA target molecules are immobilized on the surface of each encoding microsphere, the primers corresponding to the above two target molecules label two different fluorescent reporter molecules, but different encoding microspheres The fixed primers can be labeled with the same fluorescent reporter molecules, and 200 miRNA target molecules can be detected simultaneously in a single reaction system. The present invention only uses th...

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Abstract

The invention discloses a liquid-stage chip constant-temperature index amplification and detection method for tiny RNA target molecules. By fixing upstream and/or downstream primers to the surfaces of encoding microspheres and marking the two sides of notch agent recognition sequences of the primers fixed to the surfaces of the encoding microspheres with fluorescence report molecules which have the fluorescence resonance energy transfer effect and cancellation molecules, finally the high-throughput detection of various target molecules is achieved through target molecule specificity detectable fluorescence signals on the surfaces of the encoding microspheres. The design difficulty of miRNA inherent attributes on the amplification detection method for the target molecules is lowered, a liquid-state constant-temperature index amplification and detection system based on oligonucleotides primers is provided, and the method for achieving constant-temperature index amplification and absolute quantification under the constant-temperature condition can be achieved.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and relates to a liquid chip constant temperature detection method for qualitatively and quantitatively detecting various microRNAs (microRNA, miRNA) through liquid chip constant temperature exponential amplification. Background technique [0002] Compared with nucleic acid molecules such as genomic DNA, mRNA, and long noncoding RNA (lncRNA), microRNA (microRNA, miRNA) molecules have their particularities, such as: nucleic acid molecular fragments are extremely small (≤22nt), and GC content is widely distributed ( 5-95%), resulting in large differences in melting temperature (melting temperature, Tm), it is difficult to design matching amplification primers and detection probes; the proportion of total RNA is extremely low (≤0.01%), requiring a highly sensitive detection method ; Family molecular differences are small (may only have a single base difference), and the requirements for...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844
Inventor 黄庆府伟灵黄君富夏涵
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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