CN101701253B - A dual fluorescent quantitative PCR kit for detecting gene expression - Google Patents

Abstract

A double fluorescent quantitative PCR kit for detecting gene expression, the kit is composed of PCR reaction liquid, DNA polymerase, standard, primer and fluorescent probe for detecting target gene, and primer and fluorescent probe for internal reference gene, characterized in that the sequence of upstream primer for detecting the gene by the internal reference gene abl is 5'-GGG CGG CCT GAA TGA AG-3', the sequence of downstream primer is 5'-GCG CTG AAC AAGTTG GTC TTT-3', the sequence of fluorescent probe is 5'-TGA GCG CCT TCT CCC CAA AGA-3'; the 5 'end of the fluorescent probe is marked with a fluorescent group, and the 3' end of the fluorescent probe is marked with a quenching group. The kit can achieve the detection accuracy and sensitivity of the existing single fluorescent quantitative PCR method, but the required sample amount and reagent amount are saved by nearly one time.

Description

A dual fluorescent quantitative PCR kit for detecting gene expression

technical field

The invention relates to the field of biochemistry, in particular to a quantitative detection method for gene expression.

Background technique

Real-time fluorescence quantitative PCR technology was launched by Applied Biosystems in 1996. Because this technology not only realized the leap from qualitative to quantitative PCR, but also compared with conventional PCR, it has stronger specificity, higher degree of automation, and effective solution. It has been widely used in the fields of medicine and food testing due to its characteristics such as PCR contamination.

The reaction system of fluorescent quantitative PCR is composed of a pair of primers (upstream primer P1 and downstream primer P2) and a probe T (as shown in Figure 1). The probe is marked with a fluorescent group R and a quencher group Q. Before the reaction, the fluorescent signal on the probe does not emit light due to the existence of the quenching group. When the reaction starts, the Taq enzyme hydrolyzes the probe and the quenching group falls off from the probe, and a fluorescent signal appears when irradiated by laser light. , the strength of the detected fluorescent signal is proportional to the number of PCR reactions.

The "primer + probe" mode reduces the occurrence of errors and can improve sensitivity and specificity at the same time. It has become the gold standard for PCR detection and can be applied to many aspects such as tumor detection, virus detection, and transgenic animal screening. The European Anti-Cancer Association believes that RQ-PCR technology is the gold standard for clinical detection of minimal residual leukemia with specific fusion genes.

Fluorescent quantitative PCR has been widely used to detect changes in gene expression. However, to detect changes in gene expression, it is necessary to detect the expression of the target gene and the internal reference gene at the same time, which requires a relatively large number of samples. For example, the internationally accepted bcr-abl _P210 detection actually includes the following three parts: ① Absolute quantification of the copy number of the bcr-abl _P210 gene in the sample, ② Absolute quantification of the copy number of the abl gene, ③ Absolute quantification of the copy number of the bcr-abl _P210 and abl gene ratio. Because ② can be used as a comprehensive quality control index for each link of RT-RQ-PCR to eliminate the errors in each sample processing process and experimental batches, therefore, ③ can more reliably reflect the changes in the content of fusion gene-positive cells in patients than ①, and also facilitates inter-laboratory comparisons. However, the existing RQ-PCR detection fusion gene (need a pair of primers and a corresponding probe) and internal reference gene (need another pair of primers and another probe) are carried out in different reaction tubes, namely Single-plex RQ-PCR; at the same time, in order to improve the accuracy of the experiment, multiple wells need to be set for each batch of experiments—calculated by setting 3 multiple wells. Therefore, a single detection of each specimen requires 2 genes to be tested × 3 Multiple wells = 6 tubes of specimens; and for accurate comparison, it is often necessary to repeat the detection of serial specimens at different time points, and more specimens are used. In order to ensure that enough samples are obtained, more bone marrow and blood samples from patients need to be drawn. Less sampling, high-throughput, and intensive detection are the inevitable direction of the development of modern laboratory diagnostic technology.

Double fluorescent quantitative PCR detection refers to two PCR reactions in the same reaction tube (as shown in Figure 2), including 2 sets of primers (P1, P2; P1', P2') and probes (T and T'), each group Containing probes with different fluorescent signals, the upstream primer P1 and the downstream primer P2 guide the site of the No. 1 reaction, and the fluorescence on the probe T indicates the progress of the No. 1 reaction; similarly, the No. 2 reaction is also carried out separately. There is no interaction between each other, and the fluorescent signals will not interfere with each other when the results are detected. Two signals can be detected with different wavelengths at the same time, and the reaction results can be compared with their individual reactions. Using double fluorescent quantitative PCR to detect gene expression changes can reduce the amount of reagents and samples by about half compared with single-weight. However, the requirements for the reaction system of dual fluorescent quantitative PCR are higher than those of single weight. Not only should the concentration of the six sequences (two pairs of primers and two probes) and the enzymes and dNTPs of the PCR system be optimally configured, but also should be as far as possible. Reduce interference between two pairs of primers and two probes. At present, using dual fluorescent quantitative PCR to detect gene expression changes usually requires re-selecting internal reference genes and designing primers and probes for internal reference genes every time a gene is detected, which is very troublesome.

Contents of the invention

The technical problem to be solved by the present invention is to provide a dual fluorescent quantitative PCR kit with a universal internal reference gene and its probe and primer.

The technical scheme that the present invention solves the above problems is:

A dual fluorescent quantitative PCR kit for detecting gene expression. The kit includes: PCR reaction solution, DNA polymerase, standard, primers and fluorescent probes for detecting target genes and primers and fluorescent probes for internal reference genes, characterized in that,

The internal reference gene is the abl gene;

The sequence of the upstream primer of the detection internal reference gene is 5'-GGG CGG CCT GAA TGA AG-3' (SEQ NO.1), and the sequence of the downstream primer is 5'-GCG CTG AAC AAG TTG GTC TTT-3' (SEQ NO.2), the sequence of the fluorescent probe is 5'-TGA GCG CCT TCT CCC CAAAGA-3' (SEQ NO.3); the 5' end of the fluorescent probe is labeled with a fluorescent group, such as HEX, 3 The ' end is labeled with a quenching group such as Elipse.

The target gene described in the kit of the present invention is the gene to be detected, which may be a mammalian gene, and corresponding primers should be matched for detecting different genes.

The using method of kit of the present invention is:

1. Preparation of Standard Curve

Set up 5 content gradients of standard substances, namely 1×10 ⁷ copies, 1×10 ⁶ copies, 1×10 ⁵ copies, 1×10 ⁴ copies and 1×10 ³ copies, and set 3 parallel tubes for each gradient. 15 standard reaction tubes;

According to the above setting, add target gene standard product and internal reference gene abl standard product, fluorescent quantitative PCR reaction solution, DNA polymerase, primers and fluorescent probes for detecting the target gene and internal reference gene abl primers and internal reference gene abl in each standard product reaction tube. Fluorescent probe, then add water to make up 50 μL. Finally, the reaction was performed according to the following procedure: 50°C for 3 minutes, 95°C for 10 minutes, then 94°C for 15 seconds, 60°C for 1 minute, and a total of 50 cycles of reaction. After the reaction, the Ct values of the target gene and the internal reference abl gene were read out through the machine's own software, and a standard curve was drawn with Ct as the dependent variable and the copy number of the standard as the independent variable.

2. Detection of samples

(1) Preparation of template cDNA: extract the RNA of the sample to be tested and reverse transcribe it into cDNA; set up 3 parallel tubes for each sample.

(2) PCR amplification: add cDNA template, fluorescent quantitative PCR reaction solution, DNA polymerase, primers and fluorescent probes for detecting the target gene, and primers and fluorescent probes for the internal reference gene abl in the sample reaction tube, and then add water to make up 50 μL . Finally, the reaction was performed according to the following procedure: 50°C for 3 minutes, 95°C for 10 minutes, then 94°C for 15 seconds, 60°C for 1 minute, and a total of 50 cycles of reaction. After the reaction, the Ct values of the target gene and the internal reference abl gene were read out through the machine's own software, and substituted into the obtained standard curve to obtain the copy numbers of the target gene and the internal reference abl gene, and the ratio of the two was calculated.

The kit of the present invention realizes the PCR detection of the target gene and the internal reference gene in the same reaction tube at the same time. It can be seen from the method of use that only 15 standard reaction tubes and 3 sample reaction tubes are needed to detect a sample, which is different from the existing one. Compared with existing technologies, it saves half the amount of reagents and samples.

The amplified DNA fragment of the primer for detecting the internal reference gene abl in the kit of the present invention is compared and retrieved in genebank (http://www.ncbi.nlm.nih.gov/), and it is found that the fragment is similar to that of apes, monkeys, dogs Most of the matching rates of the abl genes of animals such as horses, mice, etc. are above 97.1%, and the lowest is 87.9%, while there is almost no complete matching region with other genes. It can be seen that the kit of the present invention has a wide range of applications and can be used to detect the expression levels of various genes in mammals, especially pathogenic genes, such as tumor fusion genes BCR-ABL, PML-RARa, AML1-ETO, SIL-TAL1, CBFB -MYH11, E2A-PBX1, MLL-AF4, TEL-AML1, etc. Therefore, when using the kit of the present invention to detect gene expression, only the primers and probes of the target gene need to be designed, and there is no need to redesign the primers and probes of the internal reference gene.

Description of drawings

Figure 1 is the reaction principle of fluorescent quantitative PCR, wherein P1 and P2 are primers, T is a probe, R is a fluorescent generating group, and Q is a fluorescent quenching group.

Figure 2 is a schematic diagram of the reaction of dual fluorescent quantitative PCR, wherein P1, P2, P1' and P2' are primers, T and T' are probes, R is a fluorescent group, and Q is a fluorescent quencher group.

Figure 3 is the amplification curve of bcr-abl _p210 fusion gene and abl gene when the standard plasmid concentration is 1×10 ⁵ copies/2μL, in which 1 to 3 are respectively the amplification of bcr-abl _p210 fusion gene in 4 parallel tubes Curves 4 to 6 are respectively the abl gene amplification curves of three parallel tubes.

Figure 4 is a logarithmic graph of the amplification curves of the standard plasmid Bcr-ab _lP210 TA at different concentrations, where 1 to 7 are the concentrations of 1×10 ⁷ copies/2 μL, 1×10 ⁶ copies/2 μL, and 1×10 ⁵ , respectively. Copies/2μL, 1×10 ⁴ copies/2μL, 1×10 ³ copies/2μL, 1×10 ² copies/2μL and 1×10 ¹ copies/2μL standard plasmid amplification curve, 8 is the negative control, dummy The horizontal line is the threshold (threshold).

Fig. 5 is a standard curve diagram of the standard plasmid Bcr-ab _1P210T -A.

Detailed ways

example 1

One, the composition of kit of the present invention

1. 2×TaqMan universal PCR reaction solution (2×TaqMan universal PCR Mastermix, purchased from ABI Company in the United States): Contains polymerase and other components necessary for PCR reactions, except templates, primers and probes;

2. The standard product is the Bcr-ablP210 T-A vector, which is a control plasmid vector carrying bcr-ablP210 and the fragment of the internal reference gene abl at the same time. Its preparation method is as follows:

Prepare the following DNA solutions in a microcentrifuge tube, the total amount is 5 μl (Table 1);

Table 1

2) Add 5 μl (equal volume) of Solution I;

3) React at 16°C for 30 minutes.

Note: ①The ligation reaction can be carried out normally at room temperature (25°C), but the reaction efficiency is slightly reduced.

②The ligation reaction can be carried out normally even after 5 minutes, but the reaction efficiency is slightly reduced.

4) Add the full amount (10 μl) to 100 μl JM109 competent cells, and place in ice for 30 minutes;

5) After heating at 42°C for 45 seconds, place in ice for 1 minute;

6) Add 890 μl of SOC medium, shake and incubate at 37°C for 60 minutes;

7) Cultivate on an L-agar plate medium containing X-Gal, IPTG, and Amp to form a single colony. Count white and blue colonies;

8) Pick white colonies, and use the PCR method to confirm the length of the insert in the vector.

3. The primers and fluorescent probes of the internal reference gene abl are respectively:

The sequence of the upstream primer is SEQ NO.1, 5'-GGG CGG CCT GAA TGA AG-3'

The sequence of the downstream primer is SEQ NO.2, 5'-GCG CTG AAC AAG TTG GTC TTT-3'

The sequence of the fluorescent probe is SEQ NO.3, 5'-TGA GCG CCT TCT CCC CAA AGA-3', the 5' end of the fluorescent probe is labeled with a fluorescent group HEX, and the 3' end is labeled with a quenching reporter group Elipse.

4. The primers and fluorescent probes for detecting the bcr-ablP210 fusion gene, the sequences of which are respectively:

The primers and fluorescent probes of the bcr-ablP210 fusion gene can adopt the sequences recommended by the European Anti-Cancer Association (EAC), which are respectively:

The upstream primer is SEQ NO.4: 5'-TCC GCT GAC CAT CAA TAA GGA-3'

The downstream primer is SEQ NO.5: 5'-CAC TCA GAC CCT GAG GCT CAA-3'

The sequence of the fluorescent probe is SEQ NO.6: 5'-CCC TTC AGC GGC CAG TAG CAT CTG A-3', the 5' end of the fluorescent probe is labeled with the fluorescent group FAM, and the 3' end is labeled with a quenching reporter Group, TAMRA.

The above primers and probes were synthesized in Shanghai Yingjun Biotechnology Company and Dalian Baobio Company.

2. Samples to be tested

From May 2004 to December 2006, 46 patients with CML in chronic phase (CP) were outpatient or hospitalized in Nanfang Hospital, including 23 males and 23 females, aged 16 to 65 years, with a median age of 38.1 years. Peripheral blood samples were collected from the patients. Or bone marrow.

3. Sample processing

1. Isolate RNA: Take the bone marrow or peripheral blood of leukemia patients and separate them with Ficoll lymphocyte separation medium to obtain mononuclear cells, add 1mL Trizol Reagent at a concentration of 5×106 to 1×107 cells/tube, use immediately or store at -80°C , to isolate total RNA in one step. The A260/A280 value of RNA was measured by ultraviolet spectrophotometer, and the concentration and purity of RNA were determined.

2. Synthesize template cDNA: in 25μl system, containing 1.8μg total RNA, 0.5μg random primer, 40U RNase inhibitor, 1mmol/LdNTPs, 200U M-MLV reverse transcriptase, 50mM Tris-HCl (pH8.3), 75mM KCl, 3mM MgCl2, 10mM DTT. All of the above are Promega products. Mix total RNA with random primers, place at 65°C for 5 minutes, then add RNase inhibitors, dNTPs, reverse transcriptase and Buffer, and place at 37°C for 45 minutes.

4. Sample detection

1. detect with kit of the present invention

Instrument: Fluorescence quantitative PCR amplification instrument and analysis system of Biorad Company

(1) Preparation of standard curve:

Take the Bcr-ablP210 T-A carrier and dilute it with water to the following concentrations: 1×107 copies/2 μL, 1×106 copies/2 μL, 1×105 copies/2 μL, 1×104 copies/2 μL, 1×103 copies/2 μL, 1×102 copies/2 μL and 1 ×101copies/2μL;

Add 2 μL of Bcr-ablP210 T-A carrier and 25 μL of 2×TaqMan PCR universal reaction solution to each standard product reaction tube according to the above settings, and add primers for detection of bcr-abl fusion gene and primers for detection of internal reference gene abl to the concentration of each primer. 0.3 μmoL/L, the probe for detecting the bcr-abl fusion gene and the probe for detecting the internal reference gene abl until the concentration of each probe is 0.2 μmoL/L, add and then add water to make up 50 μL. Finally, the reaction was performed according to the following procedure: 50°C for 3 minutes, 95°C for 10 minutes, then 94°C for 15 seconds, 60°C for 1 minute for a total of 50 cycles. After the reaction, the Ct values of the bcr-abl gene and the internal reference abl gene were read out through the machine's own software, and a standard curve was drawn with Ct as the dependent variable and the copy number of the standard as the independent variable; each Bcr-ablP210 T-A vector Concentration set up 3 parallel tubes, a total of 15 standard reaction tubes.

(2) Detection of samples

Add 2 μL of template cDNA, bcr-ablP210 gene and abl internal reference gene primers (ie SEQ NO.4, SEQ NO.5, SEQNO.1 and SEQNO.2) to 0.3 μmoL/L each in the sample detection tube, bcr-ablP210 gene probe (SEQNO.6) and abl gene probe (SEQNO.3) to 0.2μmoL/L each, 25μL of 2×TaqMan PCR universal reaction solution, the total reaction system of each tube is 50μL, and the insufficient part is sterile double-distilled Water replenishment. Reaction program: 50°C×3min+95°C×10min, then 94°C×15s, 60°C×1min, a total of 50 cycles. After the reaction, the copy numbers of the bcr-ablP210 target gene and the abl internal reference were read out through the machine's built-in software, and the ratio of the two was calculated. Three parallel tubes were made for each specimen to ensure the accuracy of the results.

2. Comparative experiment 1 - detection by fluorescence in situ hybridization (FISH)

Perform operations such as specimen processing, denaturation, hybridization, and post-hybridization elution according to the interphase fluorescence in situ hybridization instructions provided by the kit.

3. Comparative experiment 2 - detection by single-plex fluorescent quantitative PCR

(1) Reagents: The standard plasmid is the plasmid Bcr-ablP210 plasmid containing the bcr-ablP210 gene and the T-A vector containing the abl internal reference gene, and the rest of the reagents are the same as the kit of the present invention.

(2) method:

① Drawing of Bcr-ablP210 gene standard curve

Take the Bcr-ablP210 plasmid vector and carry out 10-fold gradient dilution to contain 1×106copies, 1×105copies, 1×104copies, 1×103copies, 1×102copies, 1×101copies plasmids in each 2μL, and make 3 parallel tubes for each gradient , to ensure the accuracy of the results. Reaction system: add 2 μ L of positive templates to each tube, the upstream and downstream primers (ie, SEQ NO.4 and SEQ NO.5) of the bcr-ablP210 gene to 0.3 μmol/L each, and the FAM-labeled TaqMan probe of the bcr-ablP210 gene ( (SEQ NO.6) to 0.2μmoL/L; 2×TaqMan PCR universal common component (2×TaqMan universal PCR Mastermix, American ABI Company) 25μL, the total reaction system of each tube is 50μL, and the insufficient part is sterile double distilled water Replenish.

② Drawing of abl gene standard curve

Take the T-A vector with the fragment of the internal reference gene abl to be tested and make a 10-fold gradient dilution to contain 1×106copies, 1×105copies, 1×104copies, 1×103copies, 1×102copies, 1×101copies of plasmids in each 2μL, each gradient Three parallel tubes were made to ensure the accuracy of the results. Reaction system: Add 2 μL positive template to each tube, the upstream and downstream primers (i.e. SEQ NO.1 and SEQ NO.2) of the abl internal reference gene to 0.3 μmoL/L each, the HEX-labeled TaqMan probe of the abl gene (i.e. SEQ NO. .3) to 0.2 μmoL/L; 25 μL of 2×TaqMan universal PCR Mastermix (2×TaqMan universal PCR Mastermix, American ABI Company), the total reaction system of each tube is 50 μL, and the insufficient part is supplemented with sterile double distilled water.

Reaction program: 50°C×3min+95°C×10min, then 94°C×15s, 60°C×1min, a total of 50 cycles. After the reaction, read out the copy numbers of the bcr-ablP210 target gene and the abl internal reference through the machine's own software, calculate the ratio of the two, and draw their respective standard curves.

5. Results

1. The drawing of kit standard curve of the present invention

Fig. 3 shows that when the initial copy number of bcr-abl fusion gene and abl gene is the same, the Ct value of using respective primers to amplify is the same, illustrating that the standard curve of the standard plasmid of the present invention is used to replace bcr-abl fusion gene and A standard curve for the abl gene is available. Figure 4 is the concentration of 1 × 107copies/2μL, 1 × 106copies/2μL, 1 × 105copies/2μL, 1 × 104copies/2μL and 1 × 103copies/2μL of the standard plasmid and the amplification curve of the negative control. The standard curve drawn according to the results in Fig. 4 is shown in Fig. 5, and the standard curve equation is y=-3.381X+16.40, r=0.995. 1 * 10 to 10 copy number 7 specimen CV values of gradients (n=5) 4.89%, 3.88%, 4.34%, 1.90%, 3.93%, 4.26% and 3.45% respectively, indicating that the stability of the method of the present invention is good.

2. Test results of each group of samples

As shown in Table 2, the detection result of the kit of the present invention is basically the same as that of the single-fold fluorescence quantitative PCR detection result, indicating that the accuracy of the kit of the present invention is comparable to that of the single-fold fluorescence quantitative PCR, but the consumption of samples and reagents has saved one part. times.

Table 2

group Number of samples (example) Test result of the test kit of the present invention FISH test results   Detection results of single-plex fluorescent quantitative PCR 1 8 0.492±0.263 63.9%±16.1% 0.579±0.271 2 6 0.023±0.033 3.8%±1.8% 0.031±0.013 3 9 (4.33±3.84)×10 ^-4 Negative (3.99±1.32)×10 ^-4

6. Examples of typical cases:

The positive rate of bcr-abl gene detected by FISH was 95% in the newly treated patients.

After 3 months of Gleevec treatment, the positive rate of bcr-abl gene detected by FISH was 5%.

In patients who underwent allogeneic hematopoietic stem cell transplantation, the positive rate of bcr-abl gene detected by FISH was negative.

The above results show that the detection result of FISH is consistent with the result of clinical diagnosis, which is consistent with the detection result of the kit of the present invention.

Example 2

One, the composition of kit of the present invention:

2×TaqMan universal PCR reaction solution (2×TaqMan universal PCR Mastermix, purchased from ABI Company in the United States): Contains polymerase and other components necessary for PCR reactions, except templates, primers and probes;

The standard product is the Bcr-abl _P190 TA vector, which is a control plasmid vector carrying bcr-abl _P190 and the internal reference gene abl at the same time, and its preparation method is as follows:

1) Prepare the following DNA solutions in a microcentrifuge tube according to Table 2, the total volume is 5 μl.

table 3

2) Add 5 μl (equal volume) of Solution I.

3) React at 16°C for 30 minutes.

Note) ①The ligation reaction can be performed normally even at room temperature (25°C), but the reaction efficiency is slightly lower.

②The ligation reaction can be carried out normally even after 5 minutes, but the reaction efficiency is slightly reduced.

4) Add the whole amount (10μl) to 100μl JM109 competent cells, and place in ice for 30 minutes.

5) After heating at 42°C for 45 seconds, place in ice for 1 minute.

6) Add 890 μl of SOC medium, shake and incubate at 37°C for 60 minutes.

7) Cultivate on an L-agar plate medium containing X-Gal, IPTG, and Amp to form a single colony. Count white and blue colonies.

8) Pick white colonies, and use the PCR method to confirm the length of the insert in the vector.

The primers and fluorescent probes of the internal reference gene abl are

The sequence of the upstream primer is SEQ NO.1: 5'-GGG CGG CCT GAA TGA AG-3'

The sequence of the downstream primer is SEQ NO.2: 5'-GCG CTG AAC AAG TTG GTC TTT-3'

The sequence of the fluorescent probe is SEQ NO.3: 5'-TGA GCG CCT TCT CCC CAA AGA-3', the 5' end of the fluorescent probe is labeled with a fluorescent group HEX, and the 3' end is labeled with a quenching reporter group Elipse.

The primers and fluorescent probes of the bcr-abl _P190 fusion gene can adopt the sequences recommended by the European Anti-Cancer Association (EAC), which are respectively

The upstream primer is SEQ NO.7: 5'-CTG GCC CAA CGA TGG CGA-3';

The downstream primer is SEQ NO.5: 5'-CAC TCA GAC CCT GAG GCT CAA-3';

The sequence of the fluorescent probe is SEQ NO.6: 5'-CCC TTC AGC GGC CAG TAG CAT CTG A-3', the 5' end of the fluorescent probe is labeled with the fluorescent group FAM, and the 3' end is labeled with a quenching reporter Group TAMRA.

The above primers and probes were synthesized in Shanghai Handsome Biotechnology Company and Dalian Bao Biological Company.

2. Sample processing

1. Isolate RNA: Take the bone marrow or peripheral blood of leukemia patients and separate them with Ficoll lymphocyte separation medium to obtain mononuclear cells, add 1 mL Trizol Reagent at a concentration of 5×10 ⁶ to 1×10 ⁷ cells/tube, and use immediately or -80 Store at ℃, and isolate total RNA in a one-step method. The A ₂₆₀ /A ₂₈₀ value of RNA was measured by ultraviolet spectrophotometer, and the concentration and purity of RNA were determined.

2. Synthesize template cDNA: in 25 μl system, containing 1.8 μg total RNA, 0.5 μg random primer, 40 U RNase inhibitor, 1 mmol/LdNTPs, 200 U M-MLV reverse transcriptase, 50 mM Tris-HCl (pH8.3), 75 mM KCl, 3 mM _MgCl2 , 10 mM DTT. All of the above are Promega products. Mix total RNA with random primers, place at 65°C for 5 minutes, then add RNase inhibitors, dNTPs, reverse transcriptase and Buffer, and place at 37°C for 45 minutes.

3. Sample detection

Instrument: Fluorescence quantitative PCR amplification instrument and analysis system of Biorad Company

(1) Preparation of standard curve:

Take the Bcr-abl _P190 TA carrier and dilute it with water to contain 1×10 ⁶ copies, 1×10 ⁵ copies, 1×10 ⁴ copies, 1×10 ³ copies, 1×10 ² copies of Bcr-abl _P190 TA carrier per microliter , 1×10 ¹ copies;

Add 2 μL of Bcr-ab _lP190 TA carrier and 25 μL of 2×TaqMan PCR universal reaction solution to each standard product reaction tube according to the above settings, and add primers for detection of bcr-abl fusion gene and primers for detection of internal reference gene abl to the concentration of each primer 0.3 μmoL/L, the probe for detecting bcr-abl fusion gene and the probe for detecting the internal reference gene abl until the concentration of each probe is 0.2 μmoL/L, add water to make up 50 μL, and finally react according to the following procedure: 50℃×3min +95℃×10min, then 94℃×15s, 60℃×1min, a total of 50 cycles. After the reaction, the Ct values of the bcr-abl gene and the internal reference abl gene were read out through the machine's own software, and a standard curve was drawn with the Ct value as the dependent variable and the copy number of the standard as the independent variable; each Bcr-ab _lP190 TA Carrier concentration set up 3 parallel tubes, a total of 15 standard reaction tubes.

(2) Detection of samples

Add 2 μL of template cDNA, bcr-abl _P190 gene and abl internal reference gene primers (ie SEQ NO.7, SEQ NO.5, SEQ NO.1 and SEQ NO.2) to 0.3 μmoL/L each in the sample detection tube, bcr -Abl _P190 gene probe (SEQ NO.6) and abl gene probe (SEQ NO.3) to 0.2 μmoL/L each, 25 μL of 2×TaqMan PCR general reaction solution, 50 μL of total reaction system in each tube, insufficient Partially supplemented with sterile double distilled water. Reaction program: 50°C×3min+95°C×10min, then 94°C×15s, 60°C×1min, a total of 50 cycles. After the reaction, read out the copy numbers of the bcr-abl _P190 target gene and the abl internal reference through the machine's own software, and calculate the ratio of the two. Three parallel tubes were made for each specimen to ensure the accuracy of the results.

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Claims (1)

1. A dual fluorescent quantitative PCR kit for detecting gene expression, the kit comprising: PCR reaction solution, DNA polymerase, standard substance, primers and fluorescent probes for detecting target genes and primers and fluorescent probes for internal reference genes , characterized in that, The PCR reaction solution and DNA polymerase are TaqMan universal PCR amplification premixed reagents, The internal reference gene is the abl gene; The sequence of the upstream primer of the detection internal reference gene is 5'-GGG CGG CCT GAA TGA AG-3', the sequence of the downstream primer is 5'-GCG CTGAAC AAG TTG GTC TTT-3', and the sequence of the fluorescent probe is 5' -TGA GCG CCT TCT CCC CAA AGA-3'; the 5' end of the fluorescent probe is labeled with a fluorescent group HEX, and the 3' end is labeled with a quenching reporter group Elipse; The target gene is bcr-abl _p210 or bcr-abl _P190 , wherein, The target gene is the primer and fluorescent probe of bcr-abl _p210 respectively: the standard product is the TA vector carrying bcr-abl _p210 and internal reference gene abl; the sequence of the upstream primer for detection target gene bcr-abl _p210 The sequence of the downstream primer is 5'-TCC GCT GAC CAT CAA TAA GGA-3', the sequence of the downstream primer is 5'-CAC TCA GAC CCT GAG GCT CAA-3', and the sequence of the fluorescent probe is 5'-CCC TTC AGC GGCCAG TAG CAT CTG A-3'; the 5' end of the fluorescent probe is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching reporter group TAMRA; The target gene is the primer and fluorescent probe of bcr-abl _P190 respectively: the standard product is the TA vector carrying bcr-abl _P190 and the internal reference gene abl; the sequence of the upstream primer for detecting the target gene bcr-abl _P190 The sequence of the downstream primer is 5'-CTG GCC CAA CGA TGG CGA-3', the sequence of the downstream primer is 5'-CAC TCA GAC CCT GAG GCT CAA-3', and the sequence of the fluorescent probe is 5'-CCC TTC AGC GGC CAGTAG CAT CTG A -3'; the 5' end of the fluorescent probe is marked with a fluorescent group FAM, and the 3' end is marked with a quenching reporter group TAMRA.

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