Method for detecting multiple trace targets based on long probe

A technology of long probes and targets, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of increased detection costs, false positives of chip hybridization, etc., and achieve rapid detection, low cost, and saving experimental synthesis cost effect

Inactive Publication Date: 2014-03-19
ZHEJIANG FORESTRY UNIVERSITY
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Problems solved by technology

The above method is widely favored by researchers due to its advantages of good multiplicity, high sensitivity, and strong specificity. However, because chip hybridization is prone to false positive

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  • Method for detecting multiple trace targets based on long probe
  • Method for detecting multiple trace targets based on long probe
  • Method for detecting multiple trace targets based on long probe

Examples

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Embodiment 1

[0041] Example 1: A method based on a novel long probe to simultaneously detect multiple trace targets, respectively detect 50 leaf samples of potato virus suspected to be infected, and determine the types of silencing inhibitors in the infected samples. There are 6 commonly known species, respectively tobacco rattle virus 16kDa gene (16kDa TRV ), cucumber mosaic virus 2b gene (2b CMV ), sugar beet western yellow virus P0 gene (P0 BWYV ), Potato virus Y HcPro gene (HcPro PVY ), Potato virus X P25 gene (P25 PVX ), Potato leafroll virus P0 gene (P0 PLRV ); The inventive method specifically carries out following steps successively:

[0042] 1) Download the nucleic acid sequences of the 6 inhibitors to be detected and internal reference 18S rRNA from the NCBI website, clone the corresponding full-length sequences, see Table 1 for the full-length primers, and use DNAstar software to analyze each strain of each inhibitor and internal reference The nucleic acid sequences were co...

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Abstract

The invention discloses a method for detecting multiple targets based on a long probe. The method is characterized by comprising the steps of: 1) obtaining targets to be detected and the nucleotide sequences of internal references 18S rRNA (Ribosomal Ribose Nucleic Acid) thereof, and designing identification probes for two oligonucleotides closely connected together in the conserved region of each target to be detected and the internal reference 18S rRNA thereof; 2) designing universal primers and selecting a filler long sequence, connecting the end 3' of the universal sequence Tag2 with the end 5' of the upstream identification probe to form an upstream LDR (Ligase Detection Reaction) probe, and connecting the filler long sequence with the end 5' of the universal sequence Tag1 and the end 3' of the downstream identification probe to form a downstream LDR long probe; 3) carrying out the ligase detection reaction between the upstream LDR probe and the downstream LDR long probe, and a target sample to be detected; 4) amplifying the connected product by using the upstream and downstream universal primers, obtaining stripe information through agarose nucleic acid electrophoresis, and thus obtaining the infection condition of the mixed targets.

Description

technical field [0001] The invention relates to a multiplex trace detection and analysis method, in particular to a novel long probe trace nucleic acid electrophoresis detection method based on ligase reaction. Background technique [0002] Pathogens, as an important class of disease microorganisms, seriously affect many aspects such as clinical diagnosis, water and environmental analysis, food safety and biological control. Especially for the H7N9 highly pathogenic avian influenza that broke out in central China in 2013, it has become a very necessary and urgent task to control and detect pathogens to humans again. Therefore, the establishment of effective diagnostic methods is of great significance for the prevention and control of pathogens and the improvement of the quality of animals and plants. [0003] Potato, as one of the world's food crops, has been infected by various potato viruses for a long time, which seriously affects the yield and quality of potato. During...

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
CPCC12Q1/6862C12Q2545/101C12Q2565/125C12Q2537/143
Inventor 汪平徐英武吕洪玉
Owner ZHEJIANG FORESTRY UNIVERSITY
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