CN115029436A - PCR kit for detecting RET gene M918T mutation and detection method thereof - Google Patents

Abstract

The invention relates to the field of gene mutation detection, and discloses a PCR kit for detecting RET gene M918T mutation, which comprises a nucleic acid amplification reagent and a reference substance. The nucleic acid amplification reagent comprises RET M918T reaction liquid, and the reaction liquid contains specific primers and fluorescent probes for detecting RET gene M918T mutation. The nucleic acid amplification reagent further comprises ddPCR Mix. The invention also discloses a detection method of the PCR kit for detecting RET gene M918T mutation. The kit of the application adopts a digital PCR technology, and can carry out quantitative detection on RET gene M918T mutation in a thyroid nodule fine needle biopsy sample, thereby assisting in diagnosing benign and malignant thyroid nodules.

Description

PCR kit for detecting RET gene M918T mutation and detection method thereof

Technical Field

The invention relates to the field of gene mutation detection, in particular to a PCR kit for detecting RET gene M918T mutation and a detection method thereof.

Background

The RET proto-oncogene encodes a single transmembrane glycoprotein receptor with tyrosine kinase activity, acting as an extracellular signaling molecule for glial cells. The occurrence and development of various tumors are closely related to the abnormal activation of RET gene. Among them, RET gene point mutation M918T is the most common mutation type in Medullary Thyroid Cancer (MTC). The M918T mutation may lead to drug resistance, particularly to the vascular endothelial growth factor inhibitor motesanib. Also, RETM918T may result in more aggressive MTC, with a poorer prognosis.

The method for detecting the mutation of the RET gene M918T mainly comprises the following steps: amplification and retardation mutation system method (ARMS); ② a Sanger sequencing method (Sanger sequencing); (iii) next-generation sequencing (NGS). The ARMS method is convenient and rapid and widely applied in clinic, but can only detect known mutation and cannot quantify the mutation; the first-generation sequencing flux is small, and the time consumption is long; NGS has a high flux, can simultaneously sequence millions to billions of DNA molecules at a time, but is insensitive to rare mutations and structural variations and expensive, and is still limited in its wide application.

Digital PCR (digital PCR) is a novel PCR technology, and can simultaneously and independently amplify and perform fluorescence detection on tens of thousands of micro-reaction wells. The kit has higher precision, repeatability and sensitivity, can quantitatively detect mutant genes, and is suitable for target spot quantification, Copy Number Variation (CNV) research and rare mutation detection. In the clinic, digital PCR enables absolute quantification. This is a relative quantification that is superior to qPCR methods. Especially, the method is more advantageous for detecting low-abundance mutation samples.

Disclosure of Invention

The present invention is directed to a PCR kit for detecting mutation of RET gene M918T, which solves the above problems of the background art.

In order to achieve the purpose, the invention provides the following technical scheme:

a PCR kit for detecting RET gene M918T mutation comprises nucleic acid amplification reagents and a reference substance. The nucleic acid amplification reagent comprises a reaction solution and ddPCRMix.

The reaction solution contains a specific primer for detecting RET gene M918T mutation, a wild type fluorescent probe and a mutant type fluorescent probe. The specific primers comprise a pair of upstream primers and downstream primers. The fluorescent probe is characterized in that a fluorescent reporter group is added at the 5 'end, and a fluorescent quenching group is added at the 3' end. Preferably, the fluorescent probe is an MGB probe. Preferably, FAM group is added at the 5 'end of the mutant type fluorescent probe, and MGB group is added at the 3' end; and a VIC group is added at the 5 'end of the wild type fluorescent probe, and an MGB group is added at the 3' end of the wild type fluorescent probe. Preferably, the sequences and modifications of the specific primers and fluorescent probes are as follows:

upstream primer 5'-CTCTTTAGGGTCGGATTCCAGTT-3'

Downstream primer 5'-TGCGTGGTGTAGATATGATCAAAA-3'

Wild type probe 5 '-FAM-ATGGATGGCAATTG-MGB-3'

Mutant probe 5 '-VIC-AATGGACGGCAATTG-MGB-3'.

The content of the specific primer contained in the reaction solution is 900nmol/L, and the content of the fluorescent probe is 250 nmol/L.

The ddPCRMix comprises dNTPs and buffers required for reaction. Preferably, the ddPCR Mix is Droplet PCR Supermix. More preferably, Droplet PCR Supermix is 2 × working solution, and is added in half of the total system amount during preparation.

The control includes a positive control and a negative control. The negative control is process water, and the positive control is a mixture of a mutant plasmid and a wild-type plasmid.

Preferably, the sequence of RET gene fragment contained in the mutant plasmid is shown in SEQ ID NO. 1. The sequence of RET gene fragment contained in the wild type plasmid is shown as SEQ ID NO.2

The second object of the present invention is to provide the method for detecting the PCR kit for detecting the mutation of RET gene M918T, which comprises the following steps:

(1) selecting corresponding commercial DNA extraction kits according to different sample types to extract DNA, and taking the extracted DNA as a detection template;

(2) preparing a RET M918T detection digital PCR reaction solution, which comprises:

(3) on the micro-drop generating chip, 70. mu.L of micro-drop generating oil and 20. mu.L of digital PCR reaction solution were added in this order. The droplet generation chip is placed in a sample preparation apparatus to generate droplets. The generated micro-droplets are transferred to a PCR amplification plate and membrane sealing is carried out by a membrane sealing instrument.

(4) And (3) placing the PCR amplification plate on a PCR instrument for amplification, wherein the amplification procedure is as follows:

(5) after amplification is completed, the PCR plate is placed in a flow-type droplet detector for data reading. After data is obtained, a threshold analysis result is defined.

Compared with the prior art, the invention has the beneficial effects that:

the kit of the application adopts a digital PCR technology, can carry out quantitative detection on RET M918T in a thyroid nodule fine needle biopsy sample, thereby assisting in diagnosing the benign and malignant thyroid nodules; compared with other detection means, the method adopted by the kit enables each template to be amplified in an independent system by dividing a traditional fluorescent PCR system into tens of thousands of micro-reaction units, thereby greatly improving the detection sensitivity; in addition, because the digital PCR is used for counting whether the corresponding template exists in the micro-reaction unit or not through the existence or nonexistence of an amplification signal, the initial quantity of each template can be absolutely quantified, and the quantification is more accurate without depending on a standard curve.

Drawings

FIG. 1 is a schematic diagram showing the principle of designing a mutant primer probe of the RET gene M918T in example 1.

FIG. 2 is a schematic diagram showing the principle of designing a wild-type primer probe for RET gene M918T in example 1.

FIG. 3 is a graph showing the results of detection of RET gene M918T mutant templates by the kit in example 2.

FIG. 4 is a graph showing the results of the test of the wild-type template of RET gene M918T by the kit in example 2.

FIG. 5 is a graph showing the results of the detection of partial mutant templates of RET gene M918T by the kit in example 2.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1

Referring to fig. 1-2, the reaction solution contains specific primers for detecting the mutation of RET gene M918T, wild-type fluorescent probes and mutant fluorescent probes. The specific primers comprise a pair of upstream primers and downstream primers. The fluorescent probe is characterized in that a fluorescent reporter group is added at the 5 'end, and a fluorescent quenching group is added at the 3' end. Preferably, the fluorescent probe is an MGB probe. Preferably, FAM group is added at the 5 'end of the mutant type fluorescent probe, and MGB group is added at the 3' end; and a VIC group is added at the 5 'end of the wild type fluorescent probe, and an MGB group is added at the 3' end of the wild type fluorescent probe. Preferably, the sequences and modifications of the specific primers and fluorescent probes are as follows:

upstream primer 5'-CTCTTTAGGGTCGGATTCCAGTT-3'

Downstream primer 5'-TGCGTGGTGTAGATATGATCAAAA-3'

Wild type probe 5 '-FAM-ATGGATGGCAATTG-MGB-3'

Mutant probe 5 '-VIC-AATGGACGGCAATTG-MGB-3';

the content of the specific primer contained in the reaction solution is 900nmol/L, and the content of the fluorescent probe is 250 nmol/L. The rest is supplemented with water.

The ddPCR Mix is 2 XDroplet PCR Supermix, and the amount added during the preparation is half of the total system amount.

The reference substance also comprises a positive control and a negative control, wherein the negative control is process water, the positive control is a mixture of a mutant plasmid and a wild plasmid, the molar ratio of the two sequences is about 10%, and then the mixed DNA is broken into fragments of about 300 bp. The EGFR gene fragment sequence contained in the mutant plasmid is shown as SEQ ID NO.1 and SEQ ID NO. 2.

The sample detected by the kit is a thyroid nodule fine needle aspiration biopsy sample; and purifying the DNA in the sample by using a nucleic acid extraction and purification reagent, and adding the DNA into the reaction system for detection.

The kit of the application adopts a digital PCR technology, can carry out quantitative detection on RET M918T in a thyroid nodule fine needle biopsy sample, thereby assisting in diagnosing the benign and malignant thyroid nodules; compared with other detection means, the method adopted by the kit can lead each template to be amplified in an independent system by dividing the traditional fluorescent PCR system into tens of thousands of micro-reaction units, thereby greatly improving the detection sensitivity; in addition, because the digital PCR counts whether the corresponding template exists in the micro-reaction unit or not through the existence or nonexistence of an amplification signal, the initial quantity of each template can be absolutely quantified, and the quantification is more accurate without depending on a standard curve.

Example 2

Referring to fig. 3 to 5, the method for detecting the PCR kit for detecting the mutation of RET gene M918T as described in example 1 includes the following steps:

(1) selecting corresponding commercial DNA extraction kits according to different sample types to extract DNA, and taking the extracted DNA as a detection template;

(2) preparing a RET M918T detection digital PCR reaction solution, which comprises:

(3) on the micro-drop generating chip, 70. mu.L of micro-drop generating oil and 20. mu.L of digital PCR reaction solution were added in this order. The droplet generation chip is placed in a sample preparation apparatus to generate droplets. The generated droplets are transferred to a PCR amplification plate and sealed by a membrane sealing instrument.

(4) And (3) placing the PCR amplification plate on a PCR instrument for amplification, wherein the amplification procedure is as follows:

(5) after amplification is completed, the PCR plate is placed in a flow-type droplet detector for data reading. After data is obtained, a threshold analysis result is defined.

The kit can detect the mutation with the level as low as 0.05 percent in the total amount of 5ng of DNA.

The primer sequences adopted by the invention are as follows:

SEQ ID NO.1

RET wild type sequence

TGCCCAGGAGTGTCTACAGCACTCCTCTGGTTACTGAAAGCTCAGGGATAGGGCCTGGCCTTCTCCTTTACCCCTCCTTCCTAGAGAGTTAGAGTAACTTCAATGTCTTTATTCCATCTTCTCTTTAGGGTCGGATTCCAGTTAAATGGACGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCAAAGTGATGTGTAAGTGTGGGTGTTGCTCTCTTGGGGTGGAGGTTACAGAAACACCCTTATACATGTAGTGGGGCCACGACGCCCGTCTGTGCAGCTTGGCCAGGGAATTG

SEQ ID NO.2

RET mutant sequences

TGCCCAGGAGTGTCTACAGCACTCCTCTGGTTACTGAAAGCTCAGGGATAGGGCCTGGCCTTCTCCTTTACCCCTCCTTCCTAGAGAGTTAGAGTAACTTCAATGTCTTTATTCCATCTTCTCTTTAGGGTCGGATTCCAGTTAAATGGATGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCAAAGTGATGTGTAAGTGTGGGTGTTGCTCTCTTGGGGTGGAGGTTACAGAAACACCCTTATACATGTAGTGGGGCCACGACGCCCGTCTGTGCAGCTTGGCCAGGGAATTG

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> Shanghai Rui JING Biotechnology GmbH

<120> PCR kit for detecting RET gene M918T mutation and detection method thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 300

<212> DNA

<213> Unknown (Unknown)

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tgcccaggag tgtctacagc actcctctgg ttactgaaag ctcagggata gggcctggcc 60

ttctccttta cccctccttc ctagagagtt agagtaactt caatgtcttt attccatctt 120

ctctttaggg tcggattcca gttaaatgga cggcaattga atcccttttt gatcatatct 180

acaccacgca aagtgatgtg taagtgtggg tgttgctctc ttggggtgga ggttacagaa 240

acacccttat acatgtagtg gggccacgac gcccgtctgt gcagcttggc cagggaattg 300

<210> 2

<211> 300

<212> DNA

<213> Unknown (Unknown)

<400> 2

tgcccaggag tgtctacagc actcctctgg ttactgaaag ctcagggata gggcctggcc 60

ttctccttta cccctccttc ctagagagtt agagtaactt caatgtcttt attccatctt 120

ctctttaggg tcggattcca gttaaatgga tggcaattga atcccttttt gatcatatct 180

acaccacgca aagtgatgtg taagtgtggg tgttgctctc ttggggtgga ggttacagaa 240

acacccttat acatgtagtg gggccacgac gcccgtctgt gcagcttggc cagggaattg 300

Claims (7)

1. A PCR kit for detecting RET gene M918T mutation is characterized in that a reaction solution contains a specific primer for detecting RET gene M918T mutation, a wild-type fluorescent probe and a mutant fluorescent probe. The specific primers comprise a pair of upstream primers and downstream primers. The fluorescent probe is characterized in that a fluorescent reporter group is added at the 5 'end of the fluorescent probe, and a fluorescent quenching group is added at the 3' end of the fluorescent probe. Preferably, the fluorescent probe is an MGB probe. Preferably, FAM group is added at the 5 'end of the mutant type fluorescent probe, and MGB group is added at the 3' end; and a VIC group is added at the 5 'end of the wild type fluorescent probe, and an MGB group is added at the 3' end of the wild type fluorescent probe. Preferably, the sequences and modifications of the specific primers and fluorescent probes are as follows:

upstream primer 5'-CTCTTTAGGGTCGGATTCCAGTT-3'

Downstream primer 5'-TGCGTGGTGTAGATATGATCAAAA-3'

Wild type probe 5 '-FAM-ATGGATGGCAATTG-MGB-3'

Mutant probe 5 '-VIC-AATGGACGGCAATTG-MGB-3';

the nucleic acid amplification reagent also comprises 2 x Droplet PCR Supermix.

2. The PCR kit for detecting the mutation of RET gene M918T as claimed in claim 1, wherein the reaction solution contains 900nmol/L of specific primers and 250nmol/L of fluorescent probes.

3. The PCR kit for detecting mutation of RET gene M918T as claimed in claim 1, wherein the ddPCRMix comprises dNTPs and buffer solution required for reaction. Preferably, the ddPCR Mix is Droplet PCR Supermix. More preferably, Droplet PCR Supermix is 2 × working solution, and is added in half of the total system amount during preparation.

4. The PCR kit for detecting mutation of RET gene M918T as claimed in any one of claims 1-3, wherein the control further comprises a positive control and a negative control.

5. The PCR kit for detecting mutation of RET gene M918T as claimed in claim 4, wherein the negative control is process water, and the positive control is a mixture of mutant plasmid and wild-type plasmid.

6. The PCR kit for detecting the mutation of RET gene M918T as claimed in claim 5, wherein the sequence of RET gene fragment contained in the mutant plasmid is shown in SEQ ID NO. 1. The sequence of RET gene fragment contained in the wild type plasmid is shown in SEQ ID NO. 2.

7. The method for detecting RET gene M918T mutation based on PCR kit as claimed in any one of claims 1-6, comprising the steps of:

(1) selecting corresponding commercial DNA extraction kits according to different sample types to extract DNA as a detection template;

(2) preparing a RET M918T detection digital PCR reaction solution, which comprises:

(3) on the micro-drop generating chip, 70. mu.L of micro-drop generating oil and 20. mu.L of digital PCR reaction solution were added in this order. The droplet generation chip is placed in a sample preparation apparatus to generate droplets. The generated droplets are transferred to a PCR amplification plate and sealed by a membrane sealing instrument.

(4) And (3) placing the PCR amplification plate on a PCR instrument for amplification, wherein the amplification procedure is as follows:

(5) after amplification is complete, the PCR plate is placed into a droplet analyzer for data reading. After data is obtained, a threshold analysis result is defined.

Images