Multiple visual nucleic acid detection method
A detection method and multiple technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high detection conditions, low sensitivity, high detection cost, etc., and achieve shortened detection time and high accuracy , the effect of reducing operating costs
Pending Publication Date: 2019-12-17
南京艾瑞谱生物技术有限公司
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[0007] Aiming at the problems of high detection cost, high requirements for detection conditions and low sensitivity in nucleic acid detection in the prior art, the present invention aims to provide a multiplex nucleic acid based on general primer hyperbranched rolling circle amplification and micro-nano particle aggregation effect Visual detection method, the detection cost of the method of the present invention is low, the operation steps are simple, the sensitivity is high, and the high-throughput detection of various nucleic acids can be realized
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Embodiment 1
[0039] The multiple visualization nucleic acid detection method of this embodiment, the specific steps include:
[0040] 1) Design corresponding padlock probe sequences and amplification universal primer sequences for each target nucleic acid molecule. This embodiment involves the detection of 8 target molecules, target molecules 1-8 (marked as #1, #2 , #3, #4, #5, #6, #7, #8) padlock probe sequences and amplification universal primer sequences are shown below respectively, the amplification universal primer sequences include two types respectively, which are recorded as sequence -1 and sequence-2:
[0041] Target Molecule #1 Padlock Probe Sequence: 5'-PO 3 -CTTACTGCTTGGTTATCTCG
[0042] GCTGAGAGCATGCCGTCTCTGGCTGAACACATGCCGTGCGTCCTGCAG TTC TTACGCTACTGTTCCAGTTCCATTAC-3';
[0043] Target molecule #1 amplification universal primer sequence: Sequence-1: 5'-CGACTCTCGTACGGCAGAG-3';
[0044] Sequence-2: 5'-GCAGGACGTCAAGAATGCG-3';
[0045] Target Molecule #2 Padlock Probe Sequenc...
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The invention belongs to the field of gene detection, and discloses a multiple visual nucleic acid detection method, which comprises the following steps: 1) respectively designing a corresponding padlock probe sequence and a universal primer sequence for different target nucleic acid molecules, wherein the padlock probe sequence contains a gene specific sequence and a universal primer recognitionsequence; 2) adding a padlock probe into a solution to be detected, simultaneously adding ligase and a reaction buffer solution, and carrying out a link reaction in a same reaction system; (3) distributing link reaction products into different reaction tubes which respectively contain universal primers matching the universal primer recognition sequence on the padlock probe, and carrying out a hyperbranched rolling circle amplification reaction in the reaction tubes to obtain amplification products; and 4) visual reaction: adding micro-nano particles, and observing the detection result according to whether agglomeration occurs or not. The method provided by the invention has the advantages of low detection cost, simple operation steps and high sensitivity, and can realize high-throughput detection of various nucleic acids.
Description
technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a multiple nucleic acid detection technology based on universal primer hyperbranched rolling circle amplification. Background technique [0002] The huge advantages of PCR and fluorescent quantitative PCR technology in terms of sensitivity, specificity and detection speed make it more and more widely used in the detection of nucleic acid target molecules. However, during the implementation of the nucleic acid detection technology of PCR and multiplex PCR, the requirements for hardware equipment and personnel are relatively high. Moreover, limited by the detection platform and detection cost, it is difficult to achieve high-throughput detection. At present, most products can only realize the detection of a single or limited target sequence. Although multiplex PCR technology or multiplex fluorescent PCR detection technology can realize multi-target molecular det...
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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2537/143C12Q2531/125C12Q2533/107C12Q2521/501C12Q2563/143C12Q2563/149
Inventor 汤从利李松何农跃陈柱
Owner 南京艾瑞谱生物技术有限公司