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Nucleic acid detection method

A detection method and nucleic acid technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of multi-variety detection reagent obstacles, and achieve low detection cost, convenient operation, and cost reduction

Active Publication Date: 2014-09-03
CHANGZHOU FANGYUAN PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

JP technology is low in cost, easy to operate, and short in detection time. However, to detect a sequence, it is necessary to prepare a pair of modified probes designed according to the sequence, which brings obstacles to the large-scale development of multi-species detection reagents using its technology platform.

Method used

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Embodiment 1）

[0050] The nucleic acid detection method of this embodiment comprises the following steps:

[0051] ① Determine the target sequence to be tested. The target sequence for amplification and detection can be any nucleic acid, and the target sequence can be RNA, cDNA, genomic DNA, DNA from pathogenic microorganisms or viruses, or DNA treated with chemical reagents, various enzymes, and physical exposure. The target sequence can exist as single-stranded DNA or RNA, or as dissociated complementary strands.

[0052] The target sequence in this example is a synthetic DNA single strand with a length of 40 nucleotides (see SEQ ID NO 1 in the sequence listing for the DNA sequence 5' to 3'), in which there are 5 nucleotides at the 5' and 3' ends The T base and the target sequence of 30 base nucleotides in the middle can be hybridized with the padlock probe.

[0053] ② Design and synthesize padlock probes according to the target sequence.

[0054] The design principle of padlock probe i...

Embodiment 2）

[0073] The nucleic acid detection method of this example is different from Example 1 in that: this example completes the ligation reaction, rolling circle amplification (RCA) reaction and cross-linking probe detection of rolling circle amplification products in one step.

[0074] After completing the determination of the target sequence, the design and synthesis of padlock probes, the preparation of rolling circle amplification primers, and the preparation of cross-linking probes according to steps ① to ④ of Example 1, the padlock probes, rolling circle The mixture of amplification primers, cross-linking probe A, cross-linking probe B, buffer, ligase, polymerase, nuclease and the fragment to be tested is incubated at 20°C to 37°C for 4 to 21 hours and then the fluorescence signal value is monitored As the endpoint value, subtract the initial fluorescence value recorded before incubation without adding nuclease. If the difference before and after is greater than 3 times or more ...

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Abstract

The invention discloses a nucleic acid detection method comprising the following steps of determining an objective sequence to be detected and a target sequence to be detected; designing and synthesizing a padlock probe according to the objective sequence; preparing a rolling circle amplification primer; preparing a crosslinking probe; carrying out coupled reaction, and carrying out rolling circle amplification by utilizing the amplification primer through taking a coupled product as a template; after the rolling circle amplification is ended, detecting a rolling circle amplification product by using the crosslinking probe, and proving that the objective target sequence exists if a fluorescent signal is remarkably enhanced. A DNA (Deoxyribonucleic Acid) which can be detected by using a crosslinking probe detecting technology is amplified through the padlock probe and the rolling circle amplification reaction under the condition that a target to be detected exists, and no long-chain products can be detected by using the crosslinking probe detecting technology if no targets to be detected exist. One unit of sequence to be detected can be amplified to form hundreds of units of repetitive sequences through rolling circle amplification, and hundreds of times of signal amplification can be obtained on the basis of one unit of repetitive sequence by using the crosslinking probe detecting technology. The flexibility of the method disclosed by the invention is greatly enhanced.

Description

technical field [0001] The invention relates to a nucleic acid analysis and detection method, in particular to a method for detecting nucleic acid sequences by utilizing multiple enzymes under constant temperature conditions. Background technique [0002] Nucleic acid sequence detection occupies an extremely important position in modern biology and medicine. This field has grown exponentially since the 1990s, and it is likely to continue to develop in the next few decades. For example, detection of pathogenic bacteria or viruses in patient tissue samples, as well as specific cancer-related gene sequences, provides information and decision-making basis for disease diagnosis, treatment and rehabilitation. [0003] Studies have shown that single nucleotide polymorphisms (Single nucleotide polymorphisms, SNPs) and insertion / deletion of short gene segments are related to the pathogenesis of cancer and drug metabolism. Other studies have shown that certain RNAs play a key role in...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q2531/125C12Q2531/137C12Q2525/131
Inventor 晏雷赫尔曼辛蒂姆
Owner CHANGZHOU FANGYUAN PHARMA
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