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Universal primer single fluorescence molecule detection technique and kit

A universal primer and molecular detection technology, applied in the field of nucleic acid detection, can solve problems such as false positives and interference of experimental results

Inactive Publication Date: 2018-08-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the application of this technology, there are often many pairs of different primers that interfere with each other and inhibit each other, which will interfere with the experimental results and often produce false positive results.

Method used

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  • Universal primer single fluorescence molecule detection technique and kit
  • Universal primer single fluorescence molecule detection technique and kit
  • Universal primer single fluorescence molecule detection technique and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Embodiment 1, the establishment of the nucleic acid detection method based on the multiple digital PCR of universal primer

[0131] In this example, transgenic corn was used as a sample, four transgenic screening elements, P-35S, T-NOS, NPTII, and PAT, and the internal reference adh1 of corn were selected as the target gene, and a multiplex nucleic acid detection method based on digital PCR was established.

[0132] 1. Grinding of the sample to be tested

[0133] (1) Dry all non-transgenic negative samples and transgenic samples in hot air at a lower temperature to reduce the moisture content in crop seeds and protect the genome from degradation;

[0134] (2) Put the dried sample into a ball mill for pulverization, the pulverization procedure is pulverization for 50 seconds, and place it at room temperature for 1 min, and lower the pulverized sample to room temperature to reduce the degradation of the genome during the pulverization process;

[0135] (3) Collect the po...

Embodiment 2

[0162]Example 2, Optimization of the nucleic acid detection method of multiplex digital PCR based on universal primers

[0163] (1) Optimization of universal primers and probes

[0164] 1. Design of primers

[0165] The universal primer modifies a common sequence with the same nucleotide sequence at the 5' end of each pair of primers, and any specific primer modified at the 5' end by the universal primer is called a modified specific primer. When there are modified specific primers and universal primers in the reaction system at the same time, in the first stage of the first ten cycles of the PCR process, the modified specific primers play a role in amplifying the target sequence with universal primers at both ends, and the universal primers play a huge role in the second stage. To ensure that each target sequence has a uniform amplification efficiency. Universal primers can effectively reduce the interference between multiple pairs of primers, so that multiple pairs of prim...

Embodiment 3

[0222] Embodiment 3, the sensitivity experiment of the nucleic acid detection method based on the multiplex digital PCR of universal primer

[0223] (1) Absolute limit of quantitation aLOD and absolute limit of detection aLOQ of the method

[0224] In order to explore the detection limit of this method, the genomes of transgenic maize MON863 and BT11 (gifted by Monsanto) (50ng / μl, solvent is water) and BT11 (50ng / μl, solvent is water) were uniformly mixed at a volume ratio of 1:1 , and then dilute the mixture with water to prepare a series of samples to be tested, which are recorded as 5x, 10x, 50x, 100x, 500x, 1000x, 5000x and 10000x respectively.

[0225] Carry out the sensitivity test according to the multiplex digital PCR amplification method described in Step 4 of Example 1, the difference is that the above-mentioned diluted series of samples to be tested are respectively replaced successively with the genomic DNA components in the reaction system shown in Table 2, the re...

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Abstract

The invention provides a multi-digital PCR (polymerase chain reaction) detection method, a primer, a probe and a kit. By combined application of a universal primer and multi-digital PCR, a novel nucleic acid detection method is developed, and at least trace, sensitive and high-precision high-flux multi-nucleic-acid detection especially transgenic detection is realized. In addition, by optimizationof factors including universal primer types, concentration, modification specific primer concentrations and the like, a high-flux and high-specificity multiplex transgenic ingredient screening detection system or kit is established. The system is subjected to specificity verification to guarantee that the detection system meets various transgenic ingredient detection requirements of import portsin China.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and in particular relates to a universal primer single fluorescent molecule detection technology and a kit. Background technique [0002] With the development of transgenic technology, more and more genetically modified crop lines have been approved. As of 2017, 488 GM events have been approved worldwide. In the literature that has been reported, most of the detection methods for genetically modified ingredients are specific detection methods directly targeting a certain genetically modified event. The increasing number of approved transgenic events poses great challenges to event-specific detection methods. At present, the screening method is used to preliminarily screen which samples contain transgenes, and then perform event-specific detection on these pre-screened samples. However, the screening process and the specific detection process are not efficiently and organically combined, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11C12Q1/6895
CPCC12Q1/6851C12Q1/6895C12Q2600/16C12Q2531/113C12Q2537/143
Inventor 许文涛罗云波牛晨启黄昆仑徐媛聪杜再慧贺晓云
Owner CHINA AGRI UNIV
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