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Primer composition used for detecting genome target zone and application thereof

A technology of primer composition and target region, applied in the field of primer composition of EGFR mutation site and detection kit, can solve the problems of false positive, unable to completely block the amplification of wild-type DNA, and high cost of reaction reagents, so as to improve the PCR efficiency, solving the effect of low template utilization

Inactive Publication Date: 2017-11-21
TIANJIN MEDICAL LAB BGI +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The average length of plasma free DNA is 170bp, and the DNA fragments released by tumor cell necrosis are less than 150bp. For fragmented plasma DNA templates, the shorter the length of the amplicon, the higher the coverage of the template. Currently, liquid biopsy is based on the second or third generation The amplicon length of the low-frequency mutation detection method of PCR is more than 70-130bp, and the coverage rate is only 23.5%-58.8%, while the total length of the upstream and downstream primers plus probe sequence is about 50-60bp. The reason for the longer amplicon is that there are High GC regions bring difficulties to the design of primers and probes. In order to avoid CG unbalanced regions, the length of the amplicon must be extended
[0007] CN 103923973 A discloses a method for detecting EGFR exon 19 deletion mutation based on a digital PCR platform. The principle of probe and primer design is: use peptide nucleic acids (peptide nucleic acids, PNA) to lock the wild-type detection site region , using two pairs of primer pairs and a pair of probes to detect the mutant type and the total DNA template that are not locked by PNA respectively, so as to calculate the mutation rate. This patent requires good specificity of PNA and high cost of reaction reagents
[0008] CN 103911427 A discloses a method and kit for detecting EGFR20 (T790M) and EGFR21 (L858R) gene point mutations based on a digital PCR platform. Actual testing needs
Using the amplification refraction mutation system (amplification refract tory mutation system, ARMS), PCR amplification is performed on the mutated target sequence, and the Taqman probe is used to detect the specific site of the amplified product. This method has a defect: the last base of the ARMS technology primer Mismatches do not completely block amplification of wild-type DNA, so there is a risk of false positives with this method

Method used

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  • Primer composition used for detecting genome target zone and application thereof
  • Primer composition used for detecting genome target zone and application thereof
  • Primer composition used for detecting genome target zone and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] The preparation of embodiment 1 detection kit

[0121] (1) Preparation of primer composition

[0122] Primer probes were designed to detect 23 hotspot mutations of the EGFR gene on the digital PCR platform, and the mutation sites of the EGFR gene are shown in Table 1 below:

[0123] Table 1

[0124]

[0125]

[0126] According to the hot spot mutation of GEFR, design primers such as figure 1 As shown, the specific primer sequences are shown in Table 2 below:

[0127] Table 2

[0128]

[0129] (2) Preparation of detection probes

[0130] According to the hot spot mutation of GEFR, the designed detection probes are shown in Table 3 below:

[0131] table 3

[0132]

[0133] Among them, MT indicates the mutant type, WT indicates the wild type, and bold letters indicate the mutation site;

[0134] (3) Assembly of the kit

[0135] Assemble the primer composition, detection probe and reagents related to the kit to prepare the detection kit.

Embodiment 2

[0136] Example 2 Detection of EGFR site mutation

[0137] The method for detecting EGFR site mutations comprises the steps of:

[0138] (1) Plasma separation: Use an anticoagulant tube to collect 6ml of fresh blood from the patient to separate the plasma within 4 hours, place the blood collection tube at 2000g at 4°C for 10min, take it out gently, transfer the supernatant to a new centrifuge tube, and centrifuge at 16000g at 4°C for 10min , pipette the supernatant into a new 2ml EP tube and store at -80°C;

[0139] (2) DNA extraction: Use 3ml of plasma to extract DNA using Qiagen’s Human Circulating Nucleic Acid Kit (QIAamp Circulating Nucleic Acid Kit) according to the instructions, and dissolve the DNA in 30 μl of AVE buffer or 30 μl of molecular biological water;

[0140] (3) Preparation system:

[0141] (a) The system composition of the EGFR 19del site is shown in Table 4 below:

[0142] Table 4

[0143] components

Final concentration

2×ddPCR Superm...

Embodiment 3

[0162] The EGFR positive standard HD734 purchased from Horizon was mixed with the EGFR negative standard HD709, fragmented by ultrasonic method, used as a simulated sample, and tested at different frequencies and concentrations by gradient dilution. After the DNA template was interrupted, the test results were as follows: Figure 5 shown, from Figure 5 It can be seen that the main peak of the fragment is located between 200bp and 300bp compared with the peak diagram of the known length fragment after the DNA template is interrupted;

[0163] Using the detection kit in Example 1 to detect with the method in Example 2, the results are shown in Table 9-11, and the two-dimensional diagram of some test results is as follows Figure 6(a)-Figure 6(c) Shown:

[0164] Table 9 EGFR gene exon 19 deletion site

[0165] Amount of DNA (ng)

20

30

50

80

100

120

0.04％

-

+

+

0.02％

+

-

+

0.01％

...

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Abstract

The invention relates to a primer composition used for detecting a genome target zone and application thereof and in particular relates to a primer composition for detecting EGFR (Epidermal Growth Factor Receptor) mutation sites of lung cancer by virtue of free plasma DNA and digital PCR (Polymerase Chain Reaction), as well as a detection kit and application thereof. The primer composition disclosed by the invention corresponds to the same template zone, the effect is achieved in the same amplified reaction, the amplification efficiency can be improved on premise of ensuring the specific amplification, and the primer composition is applicable to amplification of short fragment templates with low copy number. Moreover, the primer composition has high sensitivity, is capable of detecting 23 hotspot low frequency mutations of EGFR genes, has low sample amount, is applicable to plasma and other liquid samples, and is excellent in detection result and capable of taking complete genes into account.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a primer composition for detecting genome target regions and its application, in particular to a primer composition for detecting EGFR mutation sites of lung cancer through plasma free DNA and digital PCR, and detection of its composition Kits and applications. Background technique [0002] Tumor is a systemic and systemic disease involving multiple factors and multi-step development. Cancer-related gene abnormalities lead to abnormal biological behaviors of tumor cells such as evasion of apoptosis, infinite replication, angiogenesis, invasion and metastasis, and immune escape. root cause. Tumor-targeted drugs specifically act on specific mutation sites, and inhibit tumor cell growth, proliferation, and differentiation from further deterioration, with strong lethality and small side effects; the disadvantage is that drug-resistant mutations are prone to occur during treatment, so ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2563/159C12Q2563/107
Inventor 宋炎张乐橦刘磊李普均张盼王晶晶李志隆刘继龙叶明芝
Owner TIANJIN MEDICAL LAB BGI
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