CN112322744A - Composition and kit for breast cancer molecular typing detection based on RT-PCR - Google Patents

Composition and kit for breast cancer molecular typing detection based on RT-PCR Download PDF

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CN112322744A
CN112322744A CN202011438465.7A CN202011438465A CN112322744A CN 112322744 A CN112322744 A CN 112322744A CN 202011438465 A CN202011438465 A CN 202011438465A CN 112322744 A CN112322744 A CN 112322744A
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刘沛
王林海
王紫芸
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Beijing Originpoly Bio Tec Co Ltd
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Abstract

The invention discloses a composition and a kit for breast cancer molecular typing detection based on RT-PCR, wherein the composition comprises detection primers of four target genes ER, PR, HER2 and KI-67 and detection primers of four reference genes CALM2, RPL37A, OAZ1 and B2M, and the kit also comprises PCR reaction liquid and the like. By adopting the primer group, a polygene multiple fluorescence channel detection means can be adopted, reference genes are used as a reference, the expression levels of ER, PR, HER2 and KI-67mRNA are calculated in a relatively quantitative mode, and according to the expression levels of the four genes, the breast cancer molecule typing which the patient belongs to is judged, so that the primer group has the advantages of high detection sensitivity, high accuracy, better detection efficiency on a template with a low copy level and the like.

Description

Composition and kit for breast cancer molecular typing detection based on RT-PCR
Technical Field
The invention relates to the technical field of breast cancer molecular typing detection, in particular to a composition and a kit for breast cancer molecular typing detection based on RT-PCR.
Background
The incidence of breast cancer worldwide has been on the rise since the end of the 70 s of the 20 th century. In the United states, 1 woman will have breast cancer in their lifetime. China is not a high-incidence country of breast cancer, but is not optimistic, and the growth rate of the incidence of breast cancer in China is 1-2% higher than that of the high-incidence country in recent years. According to 2009 breast cancer onset data published by the national cancer center and health department disease prevention and control agency 2012, it is shown that: the incidence of breast cancer of women in the national tumor registration area is 1 st of malignant tumors of women, the incidence (thickness) of breast cancer of women is 42.55/10 ten thousand in total nationwide, 51.91/10 ten thousand in cities and 23.12/10 ten thousand in rural areas.
Breast cancer is a highly heterogeneous tumor, and traditional pathomorphic typing has gradually shown its imperfection in current clinical practice. The 1999 national cancer institute proposed the concept of molecular typing of tumors, i.e., the integrated molecular analysis technology, shifted the classification basis of tumors from morphology to a new tumor classification system based on molecular features. Perou et al, in 2000, proposed molecular typing of breast cancer, which was classified into type 5 by the 3 immunophenotypic genes ER, PR, HER 2: luminal a, luminal B, HER2 overexpression, basal cell type, and normal breast type. A consensus was reached in 2011 by a panel of experts at st. gallen conference: according to the detection result of IHC on ER, PR, HER2 and Ki-67 gene indexes, the breast cancer can be divided into 4 types of Luminal A type, Luminal B type, HER2 positive and Triple Negative Breast (TNBC), and the breast cancer is particularly called clinical pathological typing as a simple approximate alternative method. The st.gallen conference in 2013 proposes that clinical pathological substitute molecules are classified into 4 types, namely a Luminal A type, a Luminal B type, a HER2 positive type and a basal cell type (triple negative breast cancer belongs to the type), and all molecular subtypes have differences in gene characteristics, disease age, clinical characteristics, malignancy, treatment sensitivity, prognosis and the like. The phenotype and clinical pathology characteristics of each molecular subtype gene are as follows:
Figure BDA0002829262640000011
Figure BDA0002829262640000021
breast cancers, which are pathologically identical, are highly heterogeneous at the molecular level due to molecular genetic alterations, leading to a very different prognosis of the tumor and response to treatment. The breast cancer genotyping provided based on the gene expression profile can reflect the biological behavior of the tumor more accurately, judge prognosis and is beneficial to selecting and researching more targeted personalized treatment methods. An individualized, systemic treatment regimen should be tailored to each patient's molecular profile and other relevant factors. Accurate breast cancer typing is the basis of clinical treatment, and the current breast cancer typing method commonly used in clinic is a semi-quantitative method using Immunohistochemistry (IHC). The method has a great influence on interpretation of results due to subjective and objective factors such as different laboratories and professional experience of pathologists, which are limited by the technology. The results of a joint survey of the american society of pathology (CAP) and the American Society of Clinical Oncology (ASCO) show: the error rate of ER and PR in breast cancer IHC detection carried out in the American pathology laboratory can reach 20 percent, while Ki-67 Immunohistochemistry (IHC) detection cannot carry out effective laboratory quality control, even though considerable inconsistency exists among the best pathology laboratories all over the world and even slides of the same pathologist at different times, the repeatability is poor, and the accuracy and clinical reference significance of the current immunohistochemistry, particularly the Ki-67 detection are greatly reduced. If the typing is wrong, the doctor is forced to select the wrong treatment scheme.
In recent years, the diagnosis, staging, treatment selection, prognosis judgment and the like of breast cancer enter the molecular pathology stage from histopathology and cytopathology, and the accurate automatic quantitative detection is carried out on the gene expression of related markers in a tumor paraffin-embedded tissue by adopting a real-time fluorescent quantitative gene amplification technology, so that subjective factors can be avoided, the quality control can be better carried out, and the consistency, repeatability and accuracy of the detection are greatly improved.
For real-time fluorescent quantitative PCR detection, the design of primers is particularly important, and the design is directly related to the sensitivity and specificity of detection and the accuracy of detection results and is the key of success or failure of detection.
Disclosure of Invention
The invention aims to provide a breast cancer molecular typing detection composition which has the advantages of high sensitivity and good specificity, can be used for simultaneously detecting only by subpackaging two tubes, can produce a result at one time, and is simple and convenient.
The technical scheme of the invention is detailed as follows:
a composition for breast cancer molecular typing detection based on RT-PCR comprises a target gene detection primer group and an internal reference gene detection primer group, wherein,
the nucleotide sequence of the target gene detection primer group is as follows:
ER Gene RT-PCR Forward primer (ER-F1): GAGGAGTTTGTGTGCCTCA (SEQ ID NO:1),
ER Gene RT-PCR reverse primer (ER-R1): GGATATGGTCCTTCTCTTCC (SEQ ID NO: 2);
PR Gene RT-PCR Forward primer (PR-F1): AGATGCTGTATTTTGCACCTGA (SEQ ID NO:3),
PR Gene RT-PCR reverse primer (PR-R1): CAAACTCCTGTGGGATCTGC (SEQ ID NO: 4);
HER2 gene RT-PCR forward primer (HER 2-F1): CTCCACACTGCCAACCG (SEQ ID NO:5),
HER2 gene RT-PCR reverse primer (HER 2-R1): CTGGACCCCAGCAGTGC (SEQ ID NO: 6);
KI-67 Gene RT-PCR Forward primer (KI-67-F1): GGTAATGCACACTCCACCT (SEQ ID NO:7),
KI-67 gene RT-PCR reverse primer (KI-67-R1): ACATGGATTTCTGAACCTGAC (SEQ ID NO: 8);
the nucleotide sequence of the reference gene detection primer group is as follows:
CALM2 gene RT-PCR forward primer (CALM 2-F1): GCAGAATCCCACAGAAGCA (SEQ ID NO:9),
CALM2 gene RT-PCR reverse primer (CALM 2-R1): TTCTTGCCATCATTGTCAG (SEQ ID NO: 10);
RT-PCR forward primer of RPL37A gene (RPL 37A-F1): TGTGGTTCCTGCATGAA (SEQ ID NO:11),
RT-PCR reverse primer for RPL37A gene (RPL 37A-R1): GACAGCGGAAGTGGTAT (SEQ ID NO: 12);
OAZ1 Gene RT-PCR Forward primer (OAZ 1-F1): CTCCTAAGCCTGCACAGC (SEQ ID NO:13),
OAZ1 Gene RT-PCR reverse primer (OAZ 1-R1): CCCGGACCCGGGTTA (SEQ ID NO: 14);
B2M Gene RT-PCR Forward primer (B2M-F1): CTGTGCTCGCGCTACTCT (SEQ ID NO:15),
B2M gene RT-PCR reverse primer (B2M-R1): CTTTCCATTCTCTGCTGGAT (SEQ ID NO: 16).
The four target genes and the four reference genes comprise a plurality of exons, a plurality of mRNA spliceosomes can be formed, the selected target sequence position is an expression region closely related to the occurrence and development of breast cancer, the four target genes can be placed in a tube for detection, the four reference genes can also be placed in a tube for detection, and when the four reference genes are placed in a tube for use, different fluorescent markers are needed to be carried out among different genes. The primers are obtained by screening a large number of related primers, the sensitivity is high, the specificity is good, the amplification of a plurality of genes in one tube can be ensured not to be mutually inhibited, the respective amplification efficiency is not influenced, and the amplification efficiency of multiple genes in one tube is consistent with the single amplification efficiency.
Preferably, the composition further comprises MGB probes corresponding to the target gene and the reference gene,
the MGB probe of the target gene has the following specific nucleotide sequence:
ER Gene RT-PCR Probe (ER-FP 1): FAM-ATTCTGGAGTGTACACA-MGB (SEQ ID NO: 17);
PR Gene RT-PCR Probe (PR-FP 1): HEX-ATACTAAATGAACAGCGGATG-MGB (SEQ ID NO: 18);
HER2 gene RT-PCR probe (HER2-FP 1): CY5-ACGAGTGTGTGGGCGAG-MGB (SEQ ID NO: 19);
KI-67 Gene RT-PCR Probe (KI-67-FP 1): ROX-ATCATCAAGGAACAGCCT-MGB (SEQ ID NO: 20);
the MGB probe of the internal reference gene has the following specific nucleotide sequence:
CALM2 gene RT-PCR probe (CALM2-FP 1): FAM-GATGCTGATGGTAATGGC-MGB (SEQ ID NO: 21);
RT-PCR probe for RPL37A gene (RPL37A-FP 1): HEX-TGGCTGGCGGTGC-MGB (SEQ ID NO: 22);
OAZ1 Gene RT-PCR Probe (OAZ1-FP 1): CY5-AGTGAGAGTTCCAGG-MGB (SEQ ID NO: 23);
B2M Gene RT-PCR Probe (B2M-FP 1): ROX-CTATCCAGCGTACTCCAA-MGB (SEQ ID NO: 24);
carrying out different fluorescence channel marking modification among different target genes; different fluorescence channel marking modification is carried out among different reference genes.
The probe sequence can be specifically combined with a region related to breast cancer molecular typing; MGB modification is introduced into a probe sequence, so that the sequence of the probe can be shortened, and the recognition of the probe on a template sequence is improved; the probe sequence is basically in a region spanning two exons, so that amplification of a genome DNA template can be reduced, capture of an RNA template can be enhanced, and detection accuracy is improved. For fluorescent channel marker modification, the main points of screening are to observe the trend of whether the amplification efficiency of four genes in standard samples with different copy numbers is optimal, whether a fluorescent curve is a standard S-shaped amplification curve and whether the fluorescent curve is consistent compared with each gene single amplification.
The invention also provides a breast cancer molecular typing detection kit which comprises any one of the compositions.
Preferably, in the kit, the target gene detection primer set and the MGB probe of the target gene are placed in one tube, and the internal reference gene detection primer set and the MGB probe of the internal reference gene are placed in one tube.
Preferably, the kit further comprises PCR reaction solution, wherein each PCR reaction solution comprises 0.5. mu.L of reverse transcriptase 1U/. mu.L, 0.5. mu.L of Taq DNA polymerase 1U/. mu.L, 3. mu.L of dNTPs 25mM, and Mg 1.5mM2+mu.L of 10 XDNA polymerase buffer 5. mu.L, made up to 25. mu.L with EDPC water.
The invention is a one-tube multiplex amplification reaction, so that enhanced DNAsaq polymerase is needed, and the amplification between genes can be ensured not to influence. The reverse transcription and the amplification reaction are simultaneously carried out in one tube by one step, so that the whole amplification reaction system is carried out in a totally closed state, the subsequent amplification operation is simpler and more convenient, and the pollution possibility is avoided. The selection of PCR reaction solution is particularly important in a multiplex primer probe reaction system comprising 8 pairs of specific amplification reaction primers and 8 multichannel fluorescence labeling probes, and the PCR reaction solution ensures that the amplification efficiency of each gene primer probe in the system is similar to that of the corresponding gene primer probe during single amplification, and the primers or the primers and the probes in the system are not mutually inhibited and provide the amplification environment for each group of primer probes. In addition, the DNATaq polymerases of different brands and the mixture ratios of the DNATaq polymerases and other components need to be screened and verified, so that the optimal efficiency of the whole multiple amplification system can be ensured.
Preferably, in the kit, the test sample is mRNA extracted from a pathological tissue sample. The mRNA is preferably extracted by using a total RNA extraction kit (DP439) for paraffin-embedded tissue sections of Tiangen Biochemical technology (Beijing) Ltd, and the quality is monitored, preferably with OD260/280 of 1.8-2.0.
The invention also provides a use method of any one of the kits, which takes mRNA extracted from a pathological tissue sample as a template to carry out RT-PCR amplification, and the PCR reaction conditions are as follows: reverse transcription reaction at 55 deg.C for 10min, pre-denaturation at 95 deg.C for 1min, denaturation at 94 deg.C for 10s, annealing and extension at 60 deg.C for 30s, and 45 cycles. The amplification conditions are the optimal PCR amplification efficiency conditions obtained by screening.
Preferably, in the above method, the method for determining the detection result includes: and (3) taking a clinical sample with definite pathological information as a positive control, determining a positive judgment value of an ROC curve of a detection result, and determining breast cancer molecular typing according to the detected positive value.
Compared with the prior art, the invention has the following beneficial effects:
the primer and the probe provided by the invention relate to expression regions closely related to breast cancer molecule typing, specifically distinguish 4 molecular markers of breast cancer typing, have high detection sensitivity, and have better detection efficiency on a template with low copy level. Particularly, the probe sequence and the target position are specific exon-spanning fragments, and compared with a method for detecting only by using a PCR specific primer, the sensitivity and the accuracy of detection can be obviously improved.
The selected internal reference genes are closely related genes of the breast cancer, and are closer to reality as a contrast, the expression levels of ER, PR, HER2 and KI-67mRNA can be relatively and quantitatively calculated, the breast cancer molecular typing of the patient is judged according to the expression levels of the four target genes, and clinical basis is provided for the subsequent treatment scheme.
The four genes of ER, PR, HER2 and KI-67 and the four genes of CALM2, RPL37A, OAZ1 and B2M can be placed in two reaction holes for amplification reaction, and the provided primers are not mutually interfered in the same tube, so that the method is suitable for detecting the gene expression quantity of multiple multi-channel fluorescent polygenes. The PCR is carried out in two tubes, on one hand, the amplification between the target gene and the reference gene cannot interfere with each other, and on the other hand, the comprehensive expression quantity of the reference gene can be calculated in one tube of reaction to eliminate the interference between the reactions, so that the relative expression quantities of the ER, PR, HER2 and KI-67 genes are more accurate. The invention has social popularization, provides a more standard interpretation means in breast cancer molecular typing, has small influence of subjective factors, is suitable for being stably developed in different laboratories, and has high accuracy.
The related detection samples are relatively easy to obtain, the detection method is simple to operate, the interpretation is objective and visual, the result is obtained within 8 hours, the universal fluorescent quantitative PCR instrument can meet the detection requirement, and the whole set of experiment process adopts a one-stop totally-closed form, so that the possibility of cross contamination is avoided.
The method has the advantages of high detection sensitivity, better detection efficiency on the template with low copy level and the like. The kit has high sensitivity and high accuracy in detection, and is suitable for detection of breast cancer molecular typing.
Drawings
FIG. 1 shows PCR amplification curves for four target genes;
FIG. 2 is a PCR amplification curve of four reference genes;
FIG. 3 is a ROC area curve for the ER gene;
FIG. 4 is a ROC area curve of PR gene;
FIG. 5 is a ROC area curve for the HER2 gene;
FIG. 6 is a KI-67 gene ROC area curve.
Detailed Description
The technical solutions of the present invention are clearly and specifically explained and illustrated below in connection with specific embodiments so that those skilled in the art can better understand the present invention and implement it.
The reagents and experimental procedures used in the examples are, unless otherwise specified, commercially available reagents and procedures commonly used in the art.
Related genes: 4 target genes ER, PR, HER2 and KI-67; 4 reference genes CALM2, RPL37A, OAZ1 and B2M.
Example 1 detection of a detection assay for breast cancer molecular typing detection kit based on RT-PCR
Because mRNA is detected, RNA is extremely unstable, the pretreatment of a sample and the storage process of the RNA need to be considered, the RNA extraction quality is ensured, and the stability of a system and the accuracy of a detection result need to be ensured in the subsequent detection process. Therefore, the detection kit needs to have two requirements: the stability of sample pretreatment and the accuracy of a detection system.
The kit components are as follows:
Figure BDA0002829262640000061
Figure BDA0002829262640000071
456 breast cancer samples (from shanfu one in zhong) with known positive pathological information results (expression levels of ER, PR, HER2, Ki-67) were selected: 353 cases were identified as breast invasive ductal carcinoma, 27 breast invasive lobular carcinoma, 5 breast invasive medullary carcinoma, 46 breast invasive papillary carcinoma, and 25 cases were identified as breast invasive mucinous carcinoma.
1. Extracting RNA from the 456 different types of breast cancer samples;
2. performing RNA extraction on the sample by adopting a paraffin-embedded tissue section total RNA extraction kit (Tiangen Biochemical (Beijing) science and technology limited company, measuring the extraction quality and ensuring the stability of sample pretreatment;
3. preparing PCR reaction liquid and primer probe mixed liquid;
4. the PCR reaction conditions are as follows: reverse transcription reaction at 55 deg.C for 10min, pre-denaturation at 95 deg.C for 1min, denaturation at 94 deg.C for 10s, annealing and extension at 60 deg.C for 30s, and 45 cycles.
5. Preparation of amplification reaction System
PCR reaction solution (25. mu.L/person)
Components One part addition amount (mu L)
Reverse transcriptase (1U/. mu.L) 0.5
DNA polymerase (1U/. mu.L) 0.5
dNTPs(25mM) 3
Mg2+(1.5mM) 4
10 XDNA polymerase buffer 5
DEPC water Make up to 25. mu.L
A primer probe mixed liquid system:
Figure BDA0002829262640000081
PCR amplification System:
Figure BDA0002829262640000082
the PCR amplification procedure was as follows:
a first amplification stage: reverse transcription reaction at 55 deg.c for 10 min;
a second amplification stage: pre-denaturation at 95 ℃ for 1 min;
a third amplification stage: denaturation at 94 ℃ for 10s, annealing and extension at 60 ℃ for 30s, and 45 cycles.
6. Analysis of detection results
456 breast cancer samples (from Zhongshan monkshood) were tested in total using the above kit reaction system, which included 353 cases identified as breast invasive ductal carcinoma, 27 cases of breast invasive lobular carcinoma, 5 cases of breast invasive medullary carcinoma, 46 cases of breast invasive papillary carcinoma, and 25 cases identified as breast invasive mucinous carcinoma.
Based on the pathological results of ER, PR, HER2 and KI-67 as controls, the cut-off values of the four molecular markers are calculated by taking ER as a limit of 1% pathology, PR as a limit of 1% pathology, HER2 as a limit of 0/1 and 3+ negative and positive, and KI-67 as a limit of 14%. Particularly, HER2 has a 2+ result in pathological results, and for uncertainty, HER2 is finally given as a negative and positive result by performing verification and comparison through Fluorescence In Situ Hybridization (FISH).
The calculation formulas of the four genes including ER, PR, HER2 and KI-67 are as follows:
Ctmean internal referenceThe average value of Ct values of four reference genes (CALM2, RPL37A, OAZ1 and B2M)/4, namely CALM2, RPL37A, OAZ1 and B2M.
ΔΔCtER═ sample CtERSample Ctmean internal reference) - (Positive quality control Ct)ERPositive quality control Ctmean internal reference)
ΔΔCtPR═ sample CtPRSample Ctmean internal reference) - (Positive quality control Ct)PRPositive quality control Ctmean internal reference)
ΔΔCtHER2═ sample CtHER2Sample Ctmean internal reference) - (Positive quality control Ct)HER2Positive quality control Ctmean internal reference)
ΔΔCtKI-67═ sample CtKI-67Sample Ctmean internal reference) - (Positive quality control Ct)KI-67Positive quality control Ctmean internal reference)
In the above formula, the positive quality control is a Ct value obtained by detecting a positive quality control product (a cell line RNA fragment containing a target gene and an internal reference gene).
And obtaining delta Ct values of the four molecular markers including the ER, the PR, the HER2 and the KI-67 in 456 samples to be detected according to the calculation formula, obtaining corresponding ROC curve areas by referring to corresponding immunohistochemical results, and determining cut-off values of the four molecular markers including the ER, the PR, the HER2 and the KI-67. The corresponding ROC curve area is shown in the attached figures 1-6, and the following tables show the comparison condition of the coincidence rate of the ER, PR and KI-67 immunohistochemical results and the molecular detection results, and the comparison condition of the coincidence rate of the HER2 immunohistochemical results and the FISH results and the molecular detection results.
Figure BDA0002829262640000091
Figure BDA0002829262640000092
As can be seen from the above table, the results of the detection of the ER, PR, HER2 and KI-67 molecules are consistent with the results of the immunohistochemical detection by 93.1%, 94.3%, 88.8% and 98.3%, respectively. The molecular detection result in HER 22 + is 96% consistent with the FISH result. Therefore, compared with the conventional clinical means, the invention has the advantages that the consistency meets the clinical requirement, and compared with the methodology, the immunohistochemistry or FISH is less influenced by the factors of laboratories, subjective human beings and the like, is easier to be established into the standardized operation, and is used for wide popularization in different regions.
The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.
SEQUENCE LISTING
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<120> composition and kit for breast cancer molecular typing detection based on RT-PCR
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<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 10
ttcttgccat cattgtcag 19
<210> 11
<211> 17
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 11
tgtggttcct gcatgaa 17
<210> 12
<211> 17
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 12
gacagcggaa gtggtat 17
<210> 13
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 13
ctcctaagcc tgcacagc 18
<210> 14
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 14
cccggacccg ggtta 15
<210> 15
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 15
ctgtgctcgc gctactct 18
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 16
ctttccattc tctgctggat 20
<210> 17
<211> 17
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 17
attctggagt gtacaca 17
<210> 18
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 18
atactaaatg aacagcggat g 21
<210> 19
<211> 17
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 19
acgagtgtgt gggcgag 17
<210> 20
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 20
atcatcaagg aacagcct 18
<210> 21
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 21
gatgctgatg gtaatggc 18
<210> 22
<211> 13
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 22
tggctggcgg tgc 13
<210> 23
<211> 15
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 23
agtgagagtt ccagg 15
<210> 24
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> Based on Homo sapiens
<400> 24
ctatccagcg tactccaa 18

Claims (8)

1. A composition for breast cancer molecular typing detection based on RT-PCR comprises a target gene detection primer group and an internal reference gene detection primer group, and is characterized in that,
the nucleotide sequence of the target gene detection primer group is as follows:
ER Gene RT-PCR Forward primer (ER-F1): 1 of the amino acid sequence shown in SEQ ID NO,
ER Gene RT-PCR reverse primer (ER-R1): 2, SEQ ID NO;
PR Gene RT-PCR Forward primer (PR-F1): 3 of the amino acid sequence shown in SEQ ID NO,
PR Gene RT-PCR reverse primer (PR-R1): 4, SEQ ID NO;
HER2 gene RT-PCR forward primer (HER 2-F1): 5 of the amino acid sequence shown in SEQ ID NO,
HER2 gene RT-PCR reverse primer (HER 2-R1): 6, SEQ ID NO;
KI-67 Gene RT-PCR Forward primer (KI-67-F1): 7 of the amino acid sequence shown in SEQ ID NO,
KI-67 gene RT-PCR reverse primer (KI-67-R1): 8 in SEQ ID NO;
the nucleotide sequence of the reference gene detection primer group is as follows:
CALM2 gene RT-PCR forward primer (CALM 2-F1): 9 of the amino acid sequence shown in SEQ ID NO:9,
CALM2 gene RT-PCR reverse primer (CALM 2-R1): 10 in SEQ ID NO;
RT-PCR forward primer of RPL37A gene (RPL 37A-F1): 11 of the amino acid sequence shown in SEQ ID NO:11,
RT-PCR reverse primer for RPL37A gene (RPL 37A-R1): 12 is SEQ ID NO;
OAZ1 Gene RT-PCR Forward primer (OAZ 1-F1): 13 of the amino acid sequence shown in SEQ ID NO:13,
OAZ1 Gene RT-PCR reverse primer (OAZ 1-R1): 14, SEQ ID NO;
B2M Gene RT-PCR Forward primer (B2M-F1): 15 of the amino acid sequence shown in SEQ ID NO:15,
B2M gene RT-PCR reverse primer (B2M-R1): 16 in SEQ ID NO.
2. The composition of claim 1, further comprising MGB probes corresponding to the target gene and the reference gene,
the MGB probe of the target gene has the following specific nucleotide sequence:
ER Gene RT-PCR Probe (ER-FP 1): 17 in SEQ ID NO;
PR Gene RT-PCR Probe (PR-FP 1): 18 in SEQ ID NO;
HER2 gene RT-PCR probe (HER2-FP 1): 19 in SEQ ID NO;
KI-67 Gene RT-PCR Probe (KI-67-FP 1): 20 in SEQ ID NO;
the MGB probe of the internal reference gene has the following specific nucleotide sequence:
CALM2 gene RT-PCR probe (CALM2-FP 1): 21, SEQ ID NO;
RT-PCR probe for RPL37A gene (RPL37A-FP 1): 22 is SEQ ID NO;
OAZ1 Gene RT-PCR Probe (OAZ1-FP 1): 23, SEQ ID NO;
B2M Gene RT-PCR Probe (B2M-FP 1): 24 is SEQ ID NO;
carrying out different fluorescence channel marking modification among different target genes; different fluorescence channel marking modification is carried out among different reference genes.
3. A kit for molecular typing detection of breast cancer, comprising the composition of claim 1 or 2.
4. The kit according to claim 3, wherein the target gene detection primer set and the MGB probe of the target gene are placed in one tube, and the internal reference gene detection primer set and the MGB probe of the internal reference gene are placed in one tube.
5. The kit according to claim 4, further comprising PCR reaction solutions each comprising 0.5. mu.L of 1U/. mu.L reverse transcriptase, 0.5. mu.L of 1U/. mu.L Taq DNA polymerase, 3. mu.L of 25mM dNTPs, and 1.5mM Mg2+mu.L of 10 XDNA polymerase buffer 5. mu.L, made up to 25. mu.L with EDPC water.
6. The kit according to claim 4, wherein the test sample is mRNA extracted from a pathological tissue sample.
7. The method of using the kit according to any one of claims 3 to 6, wherein the mRNA extracted from the pathological tissue sample is used as a template for RT-PCR amplification under the following conditions: reverse transcription reaction at 55 deg.C for 10min, pre-denaturation at 95 deg.C for 1min, denaturation at 94 deg.C for 10s, annealing and extension at 60 deg.C for 30s, and 45 cycles.
8. The method according to claim 7, wherein the interpretation method of the detection result is: and (3) taking a clinical sample with definite pathological information as a positive control, determining a positive judgment value of an ROC curve of a detection result, and determining breast cancer molecular typing according to the detected positive value.
CN202011438465.7A 2020-12-10 2020-12-10 Composition and kit for breast cancer molecular typing detection based on RT-PCR Withdrawn CN112322744A (en)

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