CN109609633B - Serum miRNA marker related to breast cancer auxiliary diagnosis and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开一种与乳腺癌辅助诊断相关的血清miRNA标志物及其应用，该标志物为let‑7b‑5p,miR‑106a‑5p,miR‑16‑5p,miR‑19a‑3p,miR‑19b‑3p,miR‑20a‑5p,miR‑223‑3p,miR‑25‑3p,miR‑425‑5p,miR‑451a,miR‑92a‑3p和miR‑93‑5p中的一种或多种。血清miRNA作为新型的生物标志物，具有稳定性好、微创易获取，灵敏性和特异性高的特点。这类分子标志物的开发利用将为包括肿瘤在内的各种疾病的诊断以及进一步治疗提供新的方向。本研究将更有针对性的得出具有临床诊断潜能的乳腺癌血清miRNA标记物。研究证实了这组miRNA作为诊断乳腺癌的无创标记物的可靠性以及可重复性。

The invention discloses a serum miRNA marker related to auxiliary diagnosis of breast cancer and its application. The marker is let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR- One or more of 19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5p . Serum miRNA, as a new type of biomarker, has the characteristics of good stability, minimally invasive and easy access, and high sensitivity and specificity. The development and utilization of such molecular markers will provide new directions for the diagnosis and further treatment of various diseases including tumors. This study will be more targeted to obtain breast cancer serum miRNA markers with clinical diagnostic potential. The study confirmed the reliability and reproducibility of this group of miRNAs as non-invasive markers for the diagnosis of breast cancer.

Description

Serum miRNA marker related to breast cancer auxiliary diagnosis and application thereof

Technical Field

The invention belongs to the fields of genetic engineering and oncology, and relates to a serum miRNA marker related to breast cancer auxiliary diagnosis and application thereof.

Background

Breast Cancer (BC) is the most common malignancy in women, and accounts for 25.2% of all worldwide malignancies according to the latest World Cancer Report 2014. In China, the incidence of breast cancer is on the rising trend, and in 2015, the incidence of breast cancer of Chinese women is the first of malignant tumors and the fatality rate is the sixth. The existing examination means such as breast ultrasonic examination, molybdenum target examination, nuclear magnetic resonance examination and hollow needle aspiration biopsy all need a certain volume of tumor tissue to detect, and have certain limitation in early diagnosis of breast cancer; the tumor markers commonly used in clinic, such as carbohydrate antigen 15-3(CA15-3), are not highly sensitive or specific. With the development of biotechnology such as genomics, proteomics, and metabolomics, more and more biomarkers have been discovered or studied. Therefore, the discovery of a new marker capable of early diagnosis of breast cancer is indicated as "Nissan", thereby facilitating early intervention and treatment of breast cancer and prolonging the life of patients.

Micro RNA (miRNAs) is a small non-coding RNA molecule with evolution conservation and about 18-25 nucleotides in length, and is widely involved in a plurality of physiological and pathological processes of organisms including tumor occurrence, invasion, metastasis and the like through regulation and control after transcription. The expression of mirnas is both time-specific and tissue-specific, and some mirnas can be involved in specific physiopathologies as well as in specific disease processes. Researches show that the expression of miRNA has different degrees of up-regulation and down-regulation in tumors, and lays a foundation for the miRNA to be used as a new tumor marker. In 2008, Mitchell detected free mirnas in peripheral blood, which were found to be able to stably exist in peripheral blood and could be used as a non-invasive marker for diagnosing tumors. This finding has opened the way that numerous researchers around the world have begun exploring circulating mirnas as noninvasive markers. The existing research proves the potential diagnosis value of circulating miRNA in breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer and other tumors. However, the results of the study are not completely consistent due to differences in the study methods and the included population. Therefore, the research aims to search serum miRNA with potential diagnosis value for breast cancer through the research on the breast cancer serum with large samples based on the relative quantification method of qRT-PCR. And the expression of the miRNA in breast cancer tissues and peripheral exosomes is verified so as to further define the relation between the miRNA and the breast cancer. If a diagnostic kit aiming at breast cancer is designed according to the miRNA, the diagnosis and treatment level of breast cancer in China can be promoted, and a thought is provided for further research on breast cancer in the future.

Disclosure of Invention

The invention aims to provide a serum miRNA marker related to breast cancer auxiliary diagnosis.

The invention also aims to provide the application of the serum miRNA marker and the primer thereof in the preparation of an auxiliary breast cancer diagnosis kit and the preparation of a medicament for treating breast cancer.

The invention also aims to provide a kit and a medicament for auxiliary diagnosis and treatment of breast cancer.

The purpose of the invention can be realized by the following technical scheme:

a serum miRNA marker related to breast cancer aided diagnosis is provided, wherein the marker is one of let-7b-5p (ugagguagugguguguu), miR-106a-5p (aaagugcuacuagugcaguguaggug), miR-16-5p (uagaauggg), miR-19a-3p (ugcaaaaugugaggugcg), miR-19b-3p (ugcaaaaugcaaaacaauga), miR-19b-3p (ugcaaaccuccuagugaggacaaguguaaguaaguagguuga), miR-20a-5p (uaagcuuuagugacagguagguagguagguuag), miR-223-3p (ugagucuuucuuucuaaccgcca), miR-25-3p (cauuca) and miR-425-5p (uguaagucaagucaguca), miR-5 p (ugucaguca-uca), miR-25-3p (guucaaguca) and miR-93 (miR-agucaaguca) or miR-5 p (ugucaagucaagucaagucaaguca) and miR-3 p (ugucaagucaagucaaguca). The serum miRNA marker is preferably a combination of two or more of let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5p, and is further preferably a combination of two or more of let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p and miR-425-5p, a combination of twelve miRNAs of miR-451a, miR-92a-3p and miR-93-5 p.

The application of the serum miRNA marker in auxiliary diagnosis of breast cancer.

The serum miRNA marker is applied to the preparation of an auxiliary breast cancer diagnosis kit or a medicine for treating breast cancer.

A primer of a serum miRNA marker related to breast cancer aided diagnosis, which comprises a primer of one or more miRNAs in let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5 p; preferably primers comprising two or more miRNAs in serum let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5 p; further preferred are primers comprising twelve miRNAs of let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5p in serum miRNA.

The primer is applied to auxiliary diagnosis of breast cancer or preparation of an auxiliary diagnosis kit for breast cancer.

A breast cancer auxiliary diagnosis kit contains primers of one or more miRNAs in let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5p in serum miRNA; preferably primers containing two or more miRNAs in serum let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5 p; further preferably, the primers contain twelve miRNAs of let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5p in serum miRNA.

The kit may also include reagents commonly used in PCR reactions, such as reverse transcriptase, buffers, dNTPs, MgCl₂DEPC water and Taq enzyme, etc.; standards and/or controls may also be included.

The sequence of each miRNA in the serum miRNA markers let-7b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5p related to the breast cancer diagnosis is disclosed, but the combination of the miRNA markers as the breast cancer auxiliary diagnosis marker requires creative labor of technicians in the field. The amplification primers of the miRNA markers can be obtained by market purchase, and the primers of the serum miRNA markers used in the embodiment of the invention are specific miRNA stem-loop RT-PCR primers synthesized and produced by Sharpbo, Guangzhou.

Specifically, the technical solution of the present invention to solve the problem includes: (1) establishing a unified specimen library and a database: standard procedures (SOP) were used to collect blood samples meeting the standards and the system collected complete demographic and clinical data. (2) Differential expression profiling of serum mirnas: differentially expressed serum miRNAs in breast cancer and normal control populations were analyzed and further large sample multi-stage validation of differentially expressed miRNAs was performed. (3) The ability of these mirnas to diagnose breast cancer is clear through multi-stage validation. (4) Development of a serum miRNA diagnosis kit: miRNAs diagnostic kit is developed according to the differential expression miRNA in the serum of the breast cancer and normal population, so as to realize noninvasive auxiliary diagnosis of breast cancer patients. (4) The expression conditions of the miRNAs in breast cancer tissues, venous serum and exosomes are analyzed, the relationship between the miRNAs and breast cancer is disclosed, and a basis is provided for developing medicaments possibly related to the miRNAs for treating breast cancer in the future.

The inventor collects blood samples meeting the standard by a Standard Operation Program (SOP), systematically collects complete demographic data and clinical data, and adopts an Exiqon miRNA qPCR panel chip, a qRT-PCR method and the like.

The experimental method of research mainly includes the following parts:

1. study sample selection: patients who were initially treated, were not operated and were intervened with chemoradiotherapy and then pathologically confirmed as breast cancer. The normal control is a normal population for physical examination in a hospital.

2, initial screening of an Exiqon miRNA qPCR panel chip: and (3) carrying out RNA extraction on the serum mixed sample by using a TRIZOL-LS reagent, and carrying out qRT-PCR operation to obtain a primary screening result.

3. Training set, validation set, and additional validation set: RNA extraction is carried out on each serum sample by using an AM1556 kit (ABI company), a cDNA sample is obtained through reverse transcription reaction, and a PCR primer and SYBR Green fluorescent dye are added for PCR reaction. And (5) comparing the Ct values of the standard substance to obtain the miRNA content in the sample.

4. The RNA in breast cancer and tissues beside the cancer is extracted by using a TRIZOL-LS reagent, the RNA in exosomes is extracted by using an ExoQuick kit (SBI company) and an AM1556 kit (ABI company), and the expression difference of miRNA in tissues, venous serum and exosomes is detected by a qRT-PCR method.

5. Statistical analysis: exercise chi²Tests, paired t tests, and non-parametric rank-sum tests compare the differences in miRNA expression levels among different study groups. And (4) confirming the diagnostic value of the serum miRNA by calculating a risk value and analyzing a ROC curve.

By carrying out systematic expression analysis on miRNA in peripheral serum of a breast cancer patient, the research group of the invention has found a group of 12 breast cancer serum microRNA markers (let-7 b-5p, miR-106a-5p, miR-16-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5 p) with clinical diagnosis potential.

The invention has the beneficial effects that:

1. compared with the traditional tumor marker, the serum miRNA is used as a novel biomarker, and has the characteristics of good stability, minimally invasive and easy acquisition, and high sensitivity and specificity. The development and utilization of the molecular markers can provide a new direction for the diagnosis and further treatment of various diseases including tumors.

2. Researchers carry out rigorous and multistage verification and evaluation on differential expression miRNA in the serum of breast cancer and normal control population through an Exiqon miRNA qPCR panel chip and a qRT-PCR-based relative quantification method. The reliability and repeatability of this group of mirnas as noninvasive markers for diagnosing breast cancer were confirmed.

3. Researchers find that miR-16-5p, miR-25-3p, miR-425-5p, miR-93-5p and miR-106a-5p are expressed in breast cancer tissues higher than in paracancer tissues and consistent with the expression in serum; while let-7b-5p is under-expressed in breast cancer tissues. Meanwhile, the expression of other 10 miRNAs except miR-20a-5p and miR-223-3p in serum exosomes is higher than that of a normal control. These results will provide new ideas for future studies on the mechanism of these mirnas for breast cancer and for the treatment of breast cancer with these mirnas.

Drawings

FIG. 1: flow chart of experiment

FIG. 2: 12 miRNAs highly expressed in breast cancer serum

FIG. 3: ROC curve analysis was performed on the obtained mirnas.

A: a collection of training and validation sets; b: training a set; c: a verification set; d: an external verification set.

FIG. 4: expression of 12 miRNAs in breast cancer tissue

FIG. 5: expression of 12 miRNAs in breast cancer serum exosomes

Detailed Description

The inventor collects a large amount of venous serum samples of breast cancer patients and normal physical examination people from the first subsidiary hospital of Nanjing medical university in 2012 to 2014, and selects 216 samples of breast cancer and 214 samples of normal control from the samples through sorting sample data as experimental samples of an Exiqon miRNA qPCR panel chip primary screen and a subsequent series of qRT-PCR verification. At the same time, 32 pairs of breast cancer and tissues beside the cancer are reserved. Selected patient serum samples were all from patients who were initially treated, were not operated, and had undergone chemo-and chemo-radiation intervention, and were subsequently pathologically confirmed as breast cancer. And the system collects the demographic data and clinical data of the samples.

Referring to the flow chart (fig. 1), 40 breast cancer samples (including 10 HER2+ samples, 10 HR + samples, 10 triple negative breast cancer samples and 10 pooled samples) and 10 normal controls were randomly selected from the breast cancer and normal control serum samples and mixed into 4 breast cancer serum pooled samples and 1 normal pooled sample (one pooled sample was pooled from 10 200ul serum samples to form a 2ml sample), respectively. The 4 mixed samples were subjected to preliminary screening and analysis of the Exiqon miRNA qPCR panel chip, and the specific steps refer to the instructions of the Exiqon miRNAqPCR panel chip:

1. serum extraction

Serum samples were removed and centrifuged at 3000x g for 5min after thawing to remove some debris and some insoluble components. The supernatant was transferred to a new 1.5ml tube and after adding 750ul TRIZOL-LS, shaken vigorously for 5 s.

2. Two-phase separation

After homogenization the sample is incubated for 5 minutes at 15 to 30 ℃. 0.2ml of chloroform was added to 1ml of the sample homogenized with TRIZOL-LS reagent, and the cap was closed. After manually shaking the tube vigorously for 15 seconds, the tube is incubated at 15 to 30 ℃ for 2 to 3 minutes. Centrifuge at 13,000g for 15 minutes at 4 ℃.

RNA precipitation

The aqueous phase was transferred to a fresh centrifuge tube. The aqueous phase was mixed with isopropanol to precipitate the RNA therein, the amount of isopropanol added was: each sample was homogenized by adding 1ml of TRIZOL-LS reagent together with 0.5ml of isopropanol and 5ul of glycogen. Standing at 4 ℃ for half an hour to separate out RNA as much as possible. Centrifuge at 13,000g for 15 minutes at 4 ℃.

RNA washing

The supernatant was removed and at least 1ml of 75% (v/v) ethanol was added to each 1ml of the TRIZOL-LS reagent homogenate sample to wash the RNA pellet. The mixture was allowed to stand for 10 minutes and then centrifuged at 10000g at 4 ℃ for 5 minutes.

5. Re-solubilization of RNA pellets

The ethanol solution was removed, the RNA pellet was air-dried for 5-10 minutes, repeatedly blown several times with a gun by adding RNase-free water, and then incubated at 55 to 60 ℃ for 10 minutes.

6. And (3) measuring the concentration:

typically, 5. mu.g RNA/50ml serum is obtained.

cDNA Synthesis

(1) Diluting template RNA: 20-25 ng template RNA was diluted to 14ul (final concentration 1.492-1.786 ng/. mu.l) using DEPC water.

(2) Preparing a reaction solution: the 5 × Reaction Buffer and DEPC water were dissolved on ice and shaken well. The Enzyme mix was placed in an ice box at-20 ℃ and gently mixed before use and then placed on ice. All reagents were used after centrifugation.

(3) Preparing a reaction solution: the reaction solution in the following table was prepared

(4) Mix and centrifuge reagents: and shaking or pumping the reaction solution uniformly and then centrifuging to ensure that all the solutions are thoroughly and uniformly mixed.

(5) Reverse transcription and heat inactivation: after incubating the reaction solution at 42 ℃ for 60 minutes, the reverse transcriptase was inactivated by incubating at 95 ℃ for 5 minutes.

8.Real-Time PCR

Reagent:

Nuclease free water(Exiqon)

SYBR^TMGreen master mix(Exiqon)

cDNA template

ROX(Invitrogen)

miRNA PCR ARRAY(Exiqon)

The instrument comprises the following steps:

ABI PRISM7900system(Applied Biosystems)

(1) preparation of Real-time PCR reagents: the prepared cDNA template, DEPC water and SYBR^TMGreen master mix was dissolved on ice for 15-20 minutes.

(2) Diluting the cDNA template: the cDNA template obtained from the RT reaction was diluted 110-fold with nucleoease free water (for example, 2180. mu.l of nucleoease free water was added to 20. mu.l of the reaction solution).

(3) Mixing all reaction reagents:

A. after simple centrifugation of the PCR plate, the membrane was removed.

B. The 110-fold diluted cDNA template was mixed with 2 × SYBR Green master mix as described in 1: 1 and mixing.

C. Inverting and mixing the reaction solution and centrifuging

D. Adding the mixed reaction solution to each well in the plate

E. Resealing the PCR plate

(4) Subjecting the PCR plate to simple low-temperature centrifugation

(5) Real-time PCR amplification: real-time PCR amplification and dissolution curve analysis were performed according to the reaction conditions in the following table.

Real-time PCR cycling conditions are as follows:

and (3) data analysis: using the Delta Ct method

Preliminary data analysis was performed using software attached to the PCR instrument to obtain the original Cq value (Cp or Ct, which may vary from instrument to instrument).

We propose to use GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis) for standard and in-depth data analysis.

a. The Δ Ct for each pathway-associated gene in each treatment group was calculated.

ΔCt(group 1)＝average Ct–average of HK genes’Ct for group 1array

ΔCt(group 2)＝average Ct–average of HK genes’Ct for group 2array

b. The Δ Δ Ct for each gene in 2 PCR arrays (or two groups) was calculated.

Δ Δ Ct ═ Δ Ct (group 2) - Δ Ct (group 1)

Remarking: typicallygroup 1 is the control and group 2 is the experimental group.

c. The expression difference of the corresponding genes of the group 2 and thegroup 1 is calculated by 2-delta Ct.

After the initial screening of the chip, 28 differentially expressed mirnas were obtained as shown in the following table, including 22 high-expression mirnas (the union of mirnas more than 1.5-fold different from the normal sample in 4 breast cancer serum mixed samples) and 6 low-expression mirnas (the union of mirnas less than 0.67-fold different from the normal sample in 4 breast cancer serum mixed samples). Then, the 28 miRNAs are verified one by using qRT-PCR (quantitative reverse transcription-polymerase chain reaction) on 24 breast cancer serum samples and 24 normal control samples, and 19 differentially expressed miRNAs (the difference multiple is more than 1.5 times, and the P value is less than 0.05) are obtained.

Verifying 19 differential expression miRNAs obtained by primary screening by a relative quantitative method based on qRT-PCR through a training set, a verification set and an additional verification set, and specifically comprising the following steps:

1. serum RNA extraction: serum RNA extraction kit (AM1556) of ABI company is selected, 200ul of RNA is extracted from each sample according to the kit instruction, and finally 100ul of DEPC water is used for dissolving.

Preparation of cDNA:

1) reverse transcription experiment was performed using a 50. mu.L reaction system

The above reaction system was mixed well and after instantaneous centrifugation, the reaction was carried out according to the following procedure:

2) the following reactants are added into the reaction system after the reaction

3.qPCR

1) Using a 5. mu.L reaction system, the following ratio was used for the test

The reaction system is mixed evenly, placed in a real-time quantitative PCR instrument after instantaneous centrifugation, and reacted according to the following procedures:

the dissolution profile was added after the reaction was complete.

And (3) data analysis: statistical analysis is carried out by using SPSS 22.0 software, a group of miRNA with the expression levels being consistent with 12 miRNA in breast cancer serum in a training set is obtained: let-7b-5P, miR-106a-5P, miR-16-5P, miR-19a-3P, miR-19b-3P, miR-20a-5P, miR-223-3P, miR-25-3P, miR-425-5P, miR-451a, miR-92a-3P and miR-93-5P (P value is less than 0.05 in both training set and validation set, FIG. 2). By using the 12 miRNAs, the ROC curve of each sample can be calculated, as shown in FIG. 3, and the molecular marker consisting of the 12 miRNAs can well distinguish breast cancer patients from normal people. The additional training set further confirms the reliability of the results.

The 12 miRNAs are further detected in the expression of exosomes in breast cancer tissues and serum after the research group, TRIZOL is used for extracting RNA from the breast cancer tissues, and the exosome extraction kit is an ExoQuick kit (SBI company). Exosome RNA extracted from 200ul serum was resuspended in 200ul DEPC water, and exosome RNA was extracted using AM1556 kit (ABI) in the same procedure as the serum RNA extraction.

By using nonparametric inspection and analysis, the miR-16-5p, miR-25-3p, miR-425-5p, miR-93-5p and miR-106a-5p show high expression in breast cancer tissues and are consistent with the expression in serum; whereas let-7b-5P is under-expressed in breast cancer tissues, as opposed to serum (P <0.05, fig. 4). Meanwhile, the expression of 10 miRNAs except miR-20a-5P and miR-223-3P in serum exosomes is higher than that of a normal control (P <0.05, figure 5).

The kit comprises a batch of serum miRNAqRT-PCR primers, and can also comprise common reagents required by corresponding PCR technologies, such as: reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, fluorescent probes, RNase inhibitors, Taq enzyme and the like can be selected according to the specific experimental method, the common reagents are well known to those skilled in the art, and in addition, standard substances and controls (such as quantitative standard normal human samples and the like) can be provided. The kit has the value that only serum is needed, other tissue samples are not needed, and the expression content of miRNA in the serum sample is detected through the simplest fluorescence method, so as to assist in diagnosing the possibility of suffering from breast cancer of a patient from which the sample is derived. The serum miRNA is stable, convenient to detect and accurate in quantification, and sensitivity and specificity of disease diagnosis are greatly improved, so that the kit can help to guide diagnosis and further individualized treatment when put into practice.

Claims (2)

Translated fromChinese

1.检测血清miRNA标志物的引物在制备乳腺癌辅助诊断试剂盒中的应用，其特征在于，该血清miRNA标志物由let-7b-5p，miR-106a-5p，miR-16-5p，miR-19a-3p，miR-19b-3p，miR-20a-5p，miR-223-3p，miR-25-3p，miR-425-5p，miR-451a，miR-92a-3p和miR-93-5p组成。1. The application of the primers for detecting serum miRNA markers in the preparation of breast cancer auxiliary diagnosis kits, wherein the serum miRNA markers are composed of let-7b-5p, miR-106a-5p, miR-16-5p, miR -19a-3p, miR-19b-3p, miR-20a-5p, miR-223-3p, miR-25-3p, miR-425-5p, miR-451a, miR-92a-3p and miR-93-5p composition.2.根据权利要求1所述的应用，其特征在于，所述的试剂盒中还包括PCR技术常用的试剂。2 . The application according to claim 1 , wherein the reagent kit also includes commonly used reagents for PCR technology. 3 .

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