CN113136432A - Reagent and method for detecting relative expression quantity of PRAME gene in AML - Google Patents

Reagent and method for detecting relative expression quantity of PRAME gene in AML Download PDF

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CN113136432A
CN113136432A CN202110490656.6A CN202110490656A CN113136432A CN 113136432 A CN113136432 A CN 113136432A CN 202110490656 A CN202110490656 A CN 202110490656A CN 113136432 A CN113136432 A CN 113136432A
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陈雪青
王淑一
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Adicon Clinical Laboratories Center Inc
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Abstract

The invention discloses a primer, a probe, a method and a kit for detecting the relative expression quantity of a PRAME gene, wherein the primer and the probe comprise: (1) a primer PRAME-F, PRAME-R and a Probe PRAME-Probe for detecting the PRAME gene expression quantity; (2) primers abl-F and abl-R and a Probe abl-Probe for detecting the internal reference gene abl. The primer and the probe have the advantages of good specificity, high sensitivity and simple and convenient operation. The invention can rapidly detect whether the PRAME gene exists in the sample and the expression quantity of the PRAME gene, can provide accurate decision information and prognosis information for human Acute Myeloid Leukemia (AML) so as to assist in making an individualized treatment scheme and disease prognosis judgment, and has important clinical application value.

Description

Reagent and method for detecting relative expression quantity of PRAME gene in AML
Technical Field
The invention belongs to the fields of bioscience and biotechnology, and particularly relates to a method, a primer, a probe and a kit for detecting a PRAME gene in a sample, which adopt a fluorescent probe real-time quantitative PCR technology.
Background
Acute Myeloid Leukemia (AML), the most common type of Acute Leukemia, is a malignant disease of hematopoietic stem cells characterized by rapid proliferation of hematopoietic stem cells, manifested by abnormal accumulation of immature cells and suppression of normal hematopoietic cells. AML prognosis is variable, ranging from a survival period of several weeks to a complete remission and cure period. Among adult AML patients, the 5-year disease-free survival rate is closely related to age, with the average 5-year survival rate of AML patients being 21.4%, while the 5-year survival rate of patients over the age of 75 years is only 1%. Due to the different molecular mechanisms of AML progression, the prognosis of AML patients remains difficult to estimate accurately. AML prognosis is influenced by a number of factors, including general prognostic factors (age of onset, body weight, number of WBCs in primary peripheral blood, secondary AML, response to treatment) and genetic prognostic factors (recurrent chromosomal abnormalities, genetic mutations), of which the PRAME gene is a research hotspot.
The PRAME gene is a tumor-associated antigen which is firstly separated from melanoma, is positioned on a chromosome 22q11, is not expressed in most normal tissues and cells, is in a high expression state in normal testis tissues and lung cancer, kidney cancer and sarcoma, and belongs to a tumor-testis antigen. The product coded by the PRAME gene is a membrane protein and can inhibit an RARa pathway; in the presence of retinoic acid, PRAME can bind to RARa and block the activation process of the receptor, so that the function of RARE for starting downstream transcription is blocked by chemotactic Polycomb protein, and the effect of apoptosis is finally blocked.
Researches indicate that the simultaneous expression of PRAME and WT1 genes of leukemia patients can well detect minimal residual diseases of the leukemia patients, so that the survival rate of the leukemia patients is obviously improved. It is suggested that prognosis of AML can be assessed by detecting the expression of PRAME gene, and that individualized treatment regimens can be tailored to patients. Therefore, the PRAME gene expression level of AML patients can be used as an effective index for clinically evaluating the occurrence and development of diseases and judging prognosis.
RQ-PCR is a Real-time Quantitative polymerase chain reaction (RQ-PCR) method in which a fluorescent probe or a fluorescent dye capable of combining with a PCR product is added in a common PCR reaction, so that a fluorescent signal of the RQ-PCR increases proportionally with the increase of an amplification product, an instrument detects the fluorescence intensity after each cycle is finished in Real time, and a Quantitative result is obtained by comparing the fluorescence intensity with a standard curve. The initial number of the target gene can be directly detected, thereby dynamically monitoring the level of the target gene. The operation from amplification to product analysis is closed tube operation, which avoids pollution to the utmost extent and has no PCR post treatment. The designed specific Taq Man probe greatly improves the sensitivity and specificity of the whole system,
disclosure of Invention
The invention designs a primer and a probe sequence for detecting an internal reference/target gene, adopts a fluorescent quantitative PCR technology and utilizes a double-standard curve method to respectively construct quantitative standard curves of an internal reference gene ABL and a target gene PRAME, and detects the expression level of the target gene PRAME relative to the internal reference gene ABL. By adjusting the concentration and proportion of the primer and the probe, the reaction system and reaction conditions of PCR are optimized, and the amplification efficiency and the amplification rate can reach the best, so that the detection of the PRAME gene can be met, and the PRAME gene can be used for evaluating the treatment effect and predicting prognosis.
The invention provides a reagent for the relative expression quantity of a PRAME gene, which comprises (1) an upstream primer PRAME-F, a downstream primer PRAME-R and a Probe PRAME-Probe for detecting the PRAME gene, wherein,
PRAME-F：AGTGCTGATGAAGGGACAACA；
PRAME-R：GTCCTGATGAGAGTTCTTCCGTA；
PRAME-Probe：FAM-CTGGAGACCTTCAAAGCTGTGCTTGA-TAMRA。
(2) detecting an upstream primer ABL-F, a downstream primer ABL-R and a Probe ABL-Probe of an internal reference gene ABL, wherein the upstream primer ABL-F: GATACGAAGGGAGGGTGTACCA, respectively;
ABL-R：CTCGGCCAGGGTGTTGAA；
ABL-Probe：FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
the invention also provides a method for detecting the PRAME gene in a sample, which comprises the following steps:
(1) extracting RNA in a sample;
(2) reverse transcribing the RNA into cDNA;
(3) adding the cDNA into a reaction tube, and detecting a fluorescent signal of the PRAME gene in a sample by using an upstream primer and a downstream primer aiming at the PRAME and a probe; detecting a fluorescent signal of the ABL reference gene in the sample by using an upstream primer, a downstream primer and a probe aiming at the ABL reference gene;
(4) and determining the relative expression amount of the PRAME gene in the sample according to the fluorescence signal of the PRAME gene and the fluorescence signal of the ABL reference gene.
The invention finally provides a kit for detecting the relative expression quantity of the PRAME gene, the kit comprises a detection system PCR reaction solution, and the detection system PCR reaction solution comprises:
(1) an upstream primer PRAME-F, a downstream primer PRAME-R and a Probe PRAME-Probe for detecting the PRAME gene, wherein,
PRAME-F：AGTGCTGATGAAGGGACAACA；
PRAME-R：GTCCTGATGAGAGTTCTTCCGTA；
PRAME-Probe：FAM-CTGGAGACCTTCAAAGCTGTGCTTGA-TAMRA。
(2) detecting an upstream primer ABL-F, a downstream primer ABL-R and a Probe ABL-Probe of an internal reference gene ABL, wherein the upstream primer ABL-F: GATACGAAGGGAGGGTGTACCA, respectively;
ABL-R：CTCGGCCAGGGTGTTGAA；
ABL-Probe：FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
further, the kit also comprises a positive control substance, a negative control substance and a blank control substance, wherein the positive control substance is a positive plasmid solution containing a PRAME cDNA sequence, the negative control substance is a plasmid solution without the PRAME cDNA sequence, and the blank control substance is physiological saline or no substance.
Further, the kit also comprises a sample RNA extraction reagent, wherein the sample RNA extraction reagent comprises TRIzol, chloroform, isopropanol, 75% ethanol and RNase-free water.
Further, the sample RNA extraction reagent also comprises erythrocyte lysate, and the erythrocyte lysate comprises 16 mu mol/L ammonium chloride, 1mmol/L potassium bicarbonate and 12.5 mu mol/L EDTA.
The "PRAME cDNA sequence" in the present invention refers to a cDNA sequence produced by reverse transcription of mRNA produced by transcription of the PRAME gene, or directly synthesized by chemical synthesis based on the cDNA sequence. The PRAME cDNA sequence, whether reverse transcribed or directly produced by chemical synthesis, is inserted into a suitable plasmid and used as a positive control.
The invention has the beneficial effects that: the invention combines the real-time fluorescence PCR technology with a Taqman probe, respectively constructs quantitative standard curves of reference genes ABL and PRAME target genes by using a double-standard curve method, and detects the expression of PRAME in a tested body. Compared with the conventional immunohistochemical method and 2-△△CTThe method has the advantages of high accuracy, convenient result interpretation and the like. In addition, the method reasonably matches and optimizes primers and probes required by a reaction system, so that the experimental conditions are optimal, a complicated condition exploration link is omitted, and the experimental efficiency is greatly improved. In some samples tested by the present invention, 83% of PRAME gene expression levels were concentrated in 0.1-1 in normal persons, and 75% of PRAME gene expression levels were concentrated in 0-0.004 in AML patients. The invention has good specificity, high sensitivity and simple and convenient operation, can provide accurate decision information and prognosis information for human Acute Myelogenous Leukemia (AML) patients, is beneficial to the formulation of individualized medical schemes and disease prognosis judgment, and has important clinical application value.
Drawings
FIG. 1 is a graph showing the amplification of pUC57-T/PRAME positive plasmid at different concentrations.
FIG. 2 is a graph of amplification curves for different concentrations of template.
FIG. 3 is a graph of the PRAME gene and ABL internal reference amplification in blood samples of 24 AML patients.
FIG. 4 is a graph showing the PRAME gene and ABL internal reference amplification curves of 24 normal human blood samples.
Detailed Description
The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the conventional conditions and methods not described in the examples are generally employed by those skilled in the art according to the routine procedures: such as OsOb and Kingston, fourth edition, or following the manufacturer's suggested procedures and conditions.
Example 1
A kit for detecting PRAME gene in a sample, comprising:
(1) erythrocyte lysate comprising 16. mu. mol/L ammonium chloride, 1mmol/L potassium bicarbonate and 12.5. mu. mol/L EDTA.
(2) RNA extraction reagent comprising TRIzol, chloroform, isopropanol, 75% ethanol and RNase-free water.
(3) RNA reverse transcription reagent, ReverTra Ace qPCR RT Kit (TOYOBO Co.).
(4) Detection System PCR reaction solution, THERNDERBIRD Probe qPCR Mix (2X) (TOYOBO Co.). The PCR reaction solution of the detection system comprises an upstream primer PRAME-F, a downstream primer PRAME-R and a Probe PRAME-Probe for detecting the PRAME gene, and an upstream primer ABL-F, a downstream primer ABL-R and a Probe ABL-Probe for detecting the internal reference gene ABL,
PRAME-F：AGTGCTGATGAAGGGACAACA；
PRAME-R：GTCCTGATGAGAGTTCTTCCGTA；
PRAME-Probe：FAM-CTGGAGACCTTCAAAGCTGTGCTTGA-TAMRA。
ABL-F：GATACGAAGGGAGGGTGTACCA；
ABL-R：CTCGGCCAGGGTGTTGAA；
ABL-Probe：FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
(5) the kit comprises a positive control, a negative control and a blank control, wherein the positive control is a positive plasmid solution containing a PRAME cDNA sequence, the negative control is a plasmid solution without the PRAME cDNA sequence, and the blank control is normal saline or no substance.
The base sequences of the primers and probes SEQ ID NO. 1-6 used in the present invention are shown in Table 1.
TABLE 1 sequences of primers and probes
Figure BDA0003051874410000041
Figure BDA0003051874410000051
Example 2
The method comprises the following operation flows:
(1) extraction of RNA in blood: adding 1ml of the erythrocyte lysate in the example 1 into a clean 1.5ml centrifuge tube, taking 0.5ml of anticoagulation blood, uniformly mixing, and standing for 10min at room temperature; centrifuging at 5000rpm for 5min, discarding supernatant, and collecting cells at bottom; adding 0.5ml of the erythrocyte lysate in the example 1 again, centrifuging for 5min at 5000rpm, discarding the supernatant, and collecting the cells at the bottom; adding 1ml of TRIzol into the cells, repeatedly blowing and beating until the precipitate is completely dissolved, and standing at room temperature for 5 min; adding 0.2ml of chloroform, and shaking uniformly; centrifuging at 14000rpm at 4 ℃ for 10min, sucking the supernatant layer and transferring to another new centrifuge tube (not sucking the white middle layer); adding isopropanol with the same volume, mixing thoroughly, standing at room temperature for 10 min; centrifuging at 14000rpm and 4 ℃ for 10min, removing the supernatant, adding 1ml of 75% ethanol, and slightly reversing the upper part and the lower part to wash the tube wall; centrifuging at 14000rpm and 4 ℃ for 5min, and removing ethanol; drying at room temperature for 10-15min, adding 20ul RNase-free water to dissolve precipitate.
(2) Preparation of cDNA: the RNA prepared in (1) was inverted into cDNA by referring to the ReverTra Ace qPCR RT Kit instruction of TOYOBO.
(3) Preparing a reagent: preparing X ul of PCR reaction solution of a detection system according to the number of detected persons, and subpackaging 23ul of each person as shown in table 2:
x ═ 23ul reaction solution X (8 parts of internal reference (standard curve) +8 parts of target gene (standard curve) + n parts of sample +1 part of positive control +1 part of negative control +1 part of blank control);
TABLE 2 PRAME reaction System
Figure BDA0003051874410000052
Wherein the Forward Primer, Reverse Primer and TagMan Probe are respectively selected from PRAME-F, PRAME-R and PRAME-Probe, or ABL-F, ABL-R and ABL-Probe.
(4) Sample adding: adding the PCR reaction solution of the detection system prepared in the step (3) and 2 mul of cDNA prepared in the step (2) into a hole or a reaction tube of a 96-well plate; directly adding 2 mul of positive control substance and negative control substance into the positive control substance and the negative control substance; blank control was supplemented with 2. mu.l of physiological saline or nothing. Each sample also required 2 replicates to ensure stable results.
(5) And (3) detection: the detection was performed on a real-time fluorescent PCR instrument, and available instruments include ABI7300, 7500 (Applied Biosystems, USA), and the like. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; 95 ℃ for 15s, 58 ℃ for 35sec for 40 cycles, and fluorescence signals were collected at 58 ℃ for 35 sec.
(6) And (5) judging a result: the threshold line is adjusted to be above the background signal and the negative amplification line, and the system automatically calculates the copy number according to the standard curve and the Ct value.
1) When the internal reference is positive, the detection result is considered to be effective;
2) positive judgment standard: ct <36, positive; ct is more than or equal to 36 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative.
Example 3 Positive plasmid detection and sensitivity detection
When preparing positive plasmids, PRAME cDNA was directly synthesized, and inserted into previously selected plasmids (the pUC57-T plasmid is exemplified herein) to prepare pUC57-T/PRAME positive plasmids.
The prepared pUC57-T/PRAME positive plasmid is subjected to gradient dilution to obtain the copy number of 108、107、106、105、104、103、102The results of the detection according to example 2 using copies/. mu.l of positive plasmids as templates are shown in FIG. 1, in which the concentration of positive plasmids corresponding to each amplification curve in FIG. 1 is 10 from left to right8、107、106、105、104、103、102. As can be seen from FIG. 1, PRAME appeared normal, and the primers and probes of the present invention were used to detect pUC57-T/PRAME positive plasmids. Subsequently, the log of positive plasmid at different concentrationsThe value is used as an abscissa, the Ct value obtained by detecting the positive plasmid with each concentration according to the example 2 is used as an ordinate, a standard curve is drawn, and the amplification efficiency is calculated to be 98%, so that the positive plasmid amplification efficiency can meet the requirement of the invention.
Extracting RNA from peripheral blood of 40 normal persons by a Trizol phenol-chloroform method, dissolving the RNA together to obtain total RNA, wherein the concentration of the total RNA needs to reach about 100ng/ul, inverting the total RNA into cDNA, using the cDNA as a standard substance, designing 4 dilutions on the basis, wherein the dilution multiple is 10, and making 4 concentration gradients from the highest concentration. PCR amplification is carried out by using the template as a positive template. As shown in FIG. 2, it is understood that the lower limit of the detection of the positive plasmid is 0.1ng/ul, and the corresponding Ct value is 38.
Example 4 testing of clinical blood samples
In 24 cases of each of the blood samples of normal human and leukemia patients to be tested, RNA was extracted from the samples, and reagents were prepared and tested as described in example 2. Each sample was added to 2ul of the PCR reaction solution in the detection system. And simultaneously making positive, negative and blank controls, and respectively copying standard curves of the reference gene/target gene. 2 replicates per sample to ensure stability of the results. By carrying out statistical analysis on the ratio of the expression level of the PRAME gene (PRAME expression level/ABL internal reference expression level), the following results can be obtained: the expression level of PRAME is concentrated in 0.1-1 in 83% of AML patients, while the expression level is extremely low in AML patients, and the expression level of PRAME gene is between 0-0.004. The results of the experiment are shown in table 3 and fig. 3 and 4.
TABLE 324 PRAME mRNA expression levels in blood samples of normal and leukemia patients
Figure BDA0003051874410000071
The research adopts a fluorescent quantitative PCR method to analyze the mRNA content of PRAME in blood, can become a potential non-invasive biological marker for screening and diagnosing AML, and can also make a more reasonable individualized treatment scheme by referring to information provided by PRAME gene expression quantity so as to continuously improve the prognosis of AML patients, thereby having important clinical research application value.
Sequence listing
<110> Hangzhou Aidikang medical inspection center Co., Ltd
<120> reagent and method for detecting relative expression level of PRAME gene in AML
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gtcctgatga gagttcttcc gta 23
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tgcttctgat ggcaagctct acgtctcct 29

Claims (4)

1. An agent for the relative expression level of the PRAME gene, comprising:
(1) an upstream primer PRAME-F, a downstream primer PRAME-R and a Probe PRAME-Probe for detecting the PRAME gene,
PRAME-F：AGTGCTGATGAAGGGACAACA；
PRAME-R：GTCCTGATGAGAGTTCTTCCGTA；
PRAME-Probe：FAM-CTGGAGACCTTCAAAGCTGTGCTTGA-TAMRA。
(2) an upstream primer ABL-F, a downstream primer ABL-R and a Probe ABL-Probe for detecting the internal reference gene ABL,
ABL-F：GATACGAAGGGAGGGTGTACCA；
ABL-R：CTCGGCCAGGGTGTTGAA；
ABL-Probe：FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA。
2. the reagent of claim 1, wherein PRAME-F, PRAME-R, PRAME-Probe is added at a ratio of 1:1: 1.
3. The reagent of claim 1, wherein the ABL-F, ABL-R, ABL-Probe is added in a ratio of 1:1: 1.
4. A method for detecting PRAME gene in a sample, comprising the steps of:
(1) extracting RNA from the sample and digesting DNA;
(2) reverse transcribing the RNA into cDNA;
(3) adding the cDNA into a reaction tube, and detecting a fluorescent signal of the PRAME gene in a sample by using an upstream primer and a downstream primer aiming at the PRAME and a probe; detecting a fluorescent signal of the ABL reference gene in the sample by using an upstream primer, a downstream primer and a probe aiming at the ABL reference gene;
(4) and determining the relative expression amount of the PRAME gene in the sample according to the fluorescence signal of the PRAME gene and the fluorescence signal of the ABL reference gene.
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