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Method for improving sensitivity of digital PCR and kit thereof

A kit and digital technology, applied in the method and its kit to improve the sensitivity of digital PCR, can solve the problems of limiting the value of ddPCR and difficult detection, so as to improve the success rate, enhance the value, and improve the low detection rate of trace amount. Effect

Active Publication Date: 2018-09-07
上海睿昂基因科技股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although ddPCR has very high sensitivity, it is often difficult to detect when encountering trace amounts of nucleic acids in samples
Greatly limit the value of ddPCR clinical application

Method used

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  • Method for improving sensitivity of digital PCR and kit thereof
  • Method for improving sensitivity of digital PCR and kit thereof
  • Method for improving sensitivity of digital PCR and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Preamplification Kit

[0108] The composition of the pre-amplification kit is shown in Table 9:

[0109] Table 9

[0110]

[0111]

[0112] The specific preparation method of the kit is:

[0113] ①Materials: primers and process water

[0114] ② Primer dilution:

[0115] Centrifuge all the primers instantaneously, add process water in proportion to make it dissolve, the concentration is 100 μmol, mix thoroughly, and set aside.

[0116] ③ Preparation:

[0117] Strictly follow the concentration and dosage in the above table, accurately draw the corresponding amount of each primer, prepare the pre-amplification reaction solution, 10 μL for each test, and use process water for the insufficient part. Mix well. Label the date of preparation, product name, volume, and batch number (all the raw materials used above must pass the inspection).

[0118] ④Storage conditions: The prepared solution is stored at 4°C and protected from light, and sent to quality i...

Embodiment 2

[0119] The usage method of embodiment 2 kit

[0120] 1. System preparation

[0121] Take out the corresponding reaction solution, melt and mix it at room temperature, centrifuge at 2000rpm for 10s, and prepare the PCR premix solution for each test as follows: 1.5 μL pre-amplification reaction solution + 7.5 μL S-MIX, and the PCR premix prepared above The mixed solution was dispensed into each PCR tube in an amount of 9 μL per tube.

[0122] Take 6 μL of cfDNA and the control substance in the kit and add them to the EP tubes containing the master mix.

[0123] 2. Pre-amplification procedure:

[0124] 42°C, 5min; 94°C, 5min; (94°C, 15sec; 60°C, 25sec; 72°C, 40sec) 8 cycles; 98°C, 10min; 4°C, 5min, the reaction system was set to 20μL.

[0125] 3. Take 1 μL of the pre-amplified product as a template for ddPCR reaction.

Embodiment 3

[0127] A kind of s-dd PCR kit that detects human EGFR gene mutation, its composition is as shown in table 10:

[0128] Table 10:

[0129]

[0130] The packaging and content of each component of the S-ddPCR kit are shown in Table 11:

[0131] Table 11:

[0132]

[0133]

[0134] The using method of kit of the present invention is:

[0135] 1. ddPCR detection

[0136] 1.1 Pre-amplification:

[0137] 1.1.1 Take out the pre-amplification reaction solution, thaw at room temperature and mix well, centrifuge at 2000rpm for 10s, prepare the PCR master mix for each test as follows: 1.5μL pre-amplification reaction solution + 7.5μL S-MIX, mix the above A good PCR master mix was dispensed into each PCR tube in an amount of 9 μL per tube.

[0138] Take 6 μL of cfDNA and the control substance in the kit and add them to the EP tubes containing the master mix.

[0139] 1.1.2. Pre-amplification procedure:

[0140] 42°C, 5min; 94°C, 5min; (94°C, 15sec; 60°C, 25sec; 72°C, 40se...

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Abstract

The invention relates to the field of gene detection, and in particular relates to a method for improving sensitivity of digital PCR and a kit thereof. The method for improving the sensitivity of thedigital PCR comprises at least the steps of: enriching a target nucleic acid sequence in a test sample or a mutant type gene sequence and a wild type gene sequence of a to-be-tested gene mutation sitein a same ratio in a linear range to obtain an enriched template for detecting the target nucleic acid sequence of the to-be-tested gene mutation site for the digital PCR detection. The method significantly improves the situations of a low detection rate and a high false positive of ddPCR (differential display PCR) for detecting the trace amount of the targeted nucleic acid sequence in the test sample, and greatly improves the sensitivity of the ddPCR and the value for use in clinical detection.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a method for improving the sensitivity of digital PCR and a kit thereof. Background technique [0002] Digital PCR quantitative analysis technology distributes single DNA molecules to independent reaction chambers, so that only a single DNA molecule exists in each unit. After PCR amplification reaction, the positive fluorescent signal is detected to realize single molecule quantification. Digital PCR technology gets rid of the dependence on the standard curve and has higher sensitivity and accuracy. It is an absolute quantitative analysis technology developed after real-time quantitative PCR. It has broad application prospects in gene mutation detection, copy number variation detection, and microbial detection. [0003] Digital PCR is mainly divided into three types: microfluidic digital PCR (Microfluidic digital PCR, mdPCR), droplet digital PCR (Droplet digital PCR, ddPCR) and chi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6876C12Q1/686
CPCC12Q1/686C12Q2563/159
Inventor 熊慧高尚先谢立群陶慧卿李云航
Owner 上海睿昂基因科技股份有限公司
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