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Primer and probe used for detecting EGFR TK1 sensitive mutant

A probe, sensitive technology, applied in the fields of biotechnology and medicine, can solve the problems of increased detection cost, stay, high complexity, etc., to achieve the effect of reducing detection sites, reducing detection cost, and reducing total time

Inactive Publication Date: 2018-09-07
SHANGHAI PERMED BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, digital PCR also has serious shortcomings, such as high equipment price, poor stability, and high requirements for the experimental environment.
In addition, digital PCR is generally more expensive than Sanger sequencing and ARMS PCR.
Therefore, the current detection of EGFR-TKI sensitive mutations by digital PCR is only limited to the detection of T790M mutations
The second-generation sequencing technology has the characteristics of high throughput and high sensitivity, but at the same time it also brings the disadvantages of high yield, high time-consuming, and high complexity
And for patients who have not been treated with EGFR-TKI, the incidence of T790M and exon 20 insertion resistance mutations is very low
Although the current detection of sensitive mutations of EGFR-TKI can include mutation detection of multiple gene sites, most of them are low-probability mutations, which invisibly increases the cost of detection, which may increase the treatment burden of patients

Method used

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  • Primer and probe used for detecting EGFR TK1 sensitive mutant
  • Primer and probe used for detecting EGFR TK1 sensitive mutant
  • Primer and probe used for detecting EGFR TK1 sensitive mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Detection limit, sensitivity and specificity of detection primers and probes

[0036] Construct plasmids containing 19 exon deletion mutations and L858R mutations respectively. The corresponding construction sequences in the plasmids are shown in Table 7, including 10 exon 19 exact mutations, L858R mutations, 19 exon wild type, and L858R wild type . Use the L858R wild-type plasmid sample with 19 exons to dilute the mutant plasmid by 1 fold, 10 fold, 100 fold, and 1000 fold, and then use the primers, probes and PCR kits designed above for detection to obtain each detection The lowest detection limit of the site. The result is figure 1 As shown, the minimum detection limit for each mutation site is 1%. When the number of cycles is 27-31, as shown in the figure, the mutation rate is between 0.1% and 1%. At this time, the mutation is suspected to be positive, and DNA needs to be extracted again for repeated testing.

[0037] Table 7 Plasmid detection sequence

[0038]

[0039]...

Embodiment 2

[0044] Detection of EGFR TKI sensitivity mutations in blood cfDNA of lung cancer patients. The experimental method is as follows:

[0045] 1. Take 5ml of lung cancer patient's blood. Centrifuge at 2500 rpm for 10 minutes and take 1 ml of upper plasma.

[0046] 2. Add 200μl of 5% SDS lysate to plasma.

[0047] 3. Add an equal volume of saturated phenol solution, gently shake for 5 minutes, centrifuge at 5000g for 15 minutes, and take the supernatant.

[0048] 4. Add an equal volume of chloroform to the supernatant, shake for 5 minutes, centrifuge at 3000g for 10 minutes, and take the supernatant.

[0049] 5. Add three times the volume of frozen ethanol to the supernatant and mix, centrifuge at 12,000 rpm for 2 minutes, and the DNA will sink to the bottom of the tube. Carefully discard the liquid.

[0050] 6. Drain the remaining liquid at the bottom of the tube and add 50μl TE to dissolve the DNA pellet.

[0051] 7. Use the above primers, probes and PCR kit to detect EGFR TKI sensitive mu...

Embodiment 3

[0054] Detection of EGFR TKI sensitivity mutations in tissue DNA of lung cancer patients. The experimental method is as follows:

[0055] 1. Take fresh tissue the size of soybeans, add 200μl of 10mg / ml proteinase K solution, and incubate at 70°C for 1h.

[0056] 3. Add an equal volume of saturated phenol solution, gently shake for 5 minutes, centrifuge at 5000g for 15 minutes, and take the supernatant.

[0057] 4. Add an equal volume of chloroform to the supernatant, shake for 5 minutes, centrifuge at 3000g for 10 minutes, and take the supernatant.

[0058] 5. Add three times the volume of frozen ethanol to the supernatant and mix.

[0059] 6. Add the mixed liquid to the DNA separation column, centrifuge at 12000 rpm for 1 min, and discard the lower liquid.

[0060] 7. Add 500 μl of 75% ethanol solution to the separation column, centrifuge at 12000 rpm for 1 min, and discard the lower liquid.

[0061] 8. Place the spin column in the open to evaporate the ethanol, add 50μl TE to elute the...

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Abstract

The invention discloses a primer and a probe used for detecting EGFR TK1 sensitive mutant. The nucleotide sequence of the primer disclosed by the invention is as shown in SEQ ID NO:1-13 and SEQ ID NO:16-17; and the nucleotide sequence of the probe disclosed by the invention is as shown in SEQ ID NO:14-15 and SEQ ID NO:18. The primer and the probe disclosed by the invention have the benefits that in detection, the mutation type with very low mutation rate is deleted, thus reducing total time required for experiment and reducing experiment cost under the condition of ensuring other ARMSPCR technical advantages; and the design locus still covers more than 90% of the EGFR TK1 sensitive mutant. In addition, relatively good detection efficiency can be obtained by brand-new designed detection primer and probe for 19 exon deletion mutation and L858R mutation.

Description

Technical field [0001] The invention relates to the fields of biotechnology and medical technology, in particular to a primer and probe for detecting sensitive mutations of EGFR TKI. Background technique [0002] Epidermal growth factor receptor EGFR (epidermal growth factor receptor) belongs to the epidermal growth factor receptor family (HER) and plays an important regulatory role in cell physiological processes. EGFR is anchored on the cell membrane to exert its biological effects, and it is composed of two parts: the inner membrane and the outer membrane. The outer part of the membrane mainly plays the role of receiving signals, and the inner part of the membrane mainly plays the role of transmitting signals downward through tyrosine kinase (TK). EGFR plays a vital and important role in the occurrence and development of tumors. EGFR-TK inhibitors (EGFR-TK inhibitor, EGFR-TKI) have been designed to block the signal transduction of tumor cells to achieve Inhibit tumor cell pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 雷豪志张群祝琳王煜蘅
Owner SHANGHAI PERMED BIOMEDICAL CO LTD
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