CN113699243B - Primer probe set, kit and detection method for detecting BCL2-IGH chromosome translocation - Google Patents
Primer probe set, kit and detection method for detecting BCL2-IGH chromosome translocation Download PDFInfo
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Abstract
The invention discloses a primer probe set, a kit and a detection method for detecting BCL2-IGH chromosome translocation; wherein the primer probe group comprises a BCL2-IGH specific primer and a BCL2-IGH specific probe combination, the nucleotides of which are shown as SEQ ID NO.1 to SEQ ID NO. 32; the kit comprises a primer probe set, and the detection method is to detect 14 different BCL2-IGH chromosome translocation situations by adopting the kit. The detection method is realized based on TaqMan probe PCR technology, has very high specificity and detection sensitivity, and effectively avoids the interaction between primers, between primers and probes and between probes through reasonable primer and probe matching, improves the specificity of BCL2-IGH chromosome translocation detection, reduces false positive and reduces detection errors.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a primer probe set, a kit and a detection method for detecting BCL2-IGH chromosome translocation.
Background
BCL2-IGH is a t (14:18) (q32: q21) chromosomal translocation product that integrates the BCL2 protooncogene in chromosome 18, region q21, into IGH in chromosome 14, region 32, found in 85% -90% Follicular Lymphoma (FL) and 30% diffuse large B-cell lymphoma (DLBCL). And are rarely seen in Chronic Lymphocytic Leukemia (CLL) and Acute Lymphocytic Leukemia (ALL). BCL2 has a total length of 230kb, contains 3 exons, and has diversified break points, wherein 60-70% of translocation occurs in a 150bp untranslated region at the 3' end of the 3 rd exon in a BCL2 gene sequence and is a main break point determining region (MBR); 20% -30% of the DNA sequence occurs at 25kb 3' end of 3 rd exon of BCL2 gene sequence, and is called Minor breakpoint determinant (MCR); the remaining breakpoints are distributed in the other regions of the BCL2 gene. BCL2-IGH fusion leads BCL2 gene to be highly expressed in B cells at the center of follicular cells, inhibits B cell apoptosis, enables the B cells to continuously proliferate, and initiates tumors. BCL2-IGH negative patients have a significantly increased disease-free survival time (P <0.00001) compared to positive patients, who have a 48-fold higher risk of relapse than the former. In addition, BCL2-IGH is the most important molecular biological characteristic of FL, and the detection of the gene has important significance on the discovery, identification and prognosis of FL.
The currently clinically applied BCL2-IGH chromosome translocation detection method mainly comprises four methods: cytogenetic analysis, fluorescence in situ hybridization, Southern hybridization analysis and fluorescence TaqMan probe PCR method. Cytogenetic analysis is relatively labor-consuming and expensive, requires long detection time, is complex to operate, and has low sensitivity; the operation requirement of fluorescence in situ hybridization is high, the analysis cost is moderate, the sensitivity is not high, and the average turnover time is long; the Southern hybridization analysis has high sample requirement, complex and long flow, longest turnover time and harmful radioactive probes used in the experiment to human body; therefore, the clinical popularization of the three methods has certain limitations. The fusion gene multi-detection method which is most widely applied clinically at present is a fluorescence PCR method, and has the main advantages of (1) closed tube operation and closed state detection, and the possibility of template pollution and false positive is reduced; (2) post-treatment of PCR products is not needed, so that the operation is simpler, more convenient and faster; (3) the sensitivity is high, and single copy genes can be detected; (4) the specificity is high, and the fluorescence PCR can specifically detect the target gene by the specific combination of the Taqman probe and the target sequence.
The current method for detecting BCL2-IGH chromosome translocation cannot meet the requirements of high efficiency, accuracy, simplicity and the like, is difficult to solve for the detection of small (small) amount of samples, and cannot determine the approximate fracture position, so that a method which is rapid, effective, accurate and suitable for detecting BCL2-IGH chromosome translocation in small samples is urgently needed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention redesigns a primer probe set, a kit and a detection method for detecting BCL2-IGH chromosome translocation based on the latest scientific research dynamics and clinical application. The detection method is simple, convenient, rapid, accurate and effective, has simple and easily-distinguished result, and is still applicable to small samples.
The first technical scheme of the invention is as follows:
a primer probe set for detecting BCL2-IGH chromosome translocation, which comprises a primer probe set with nucleotides shown as SEQ ID NO.1 to SEQ ID NO.32, and the primer probe set comprises a BCL2-IGH specific primer and a BCL2-IGH specific probe.
Preferably, in the primer probe set, the primer probe set contains 14 different BCL2-IGH chromosome translocation genes and 1 tube family gene ABL 1.
Preferably, in the above primer probe set, the primer probe set shown in SEQ ID No.1 to SEQ ID No.32 includes the following combinations:
combination 1:
SEQ ID NO.1:5'-TGACCTTTAGAGAGTTGCTTTACG-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.2: FAM-CTGTTTCAACACAGACCCACCCAGAG-BHQ1；
and (3) combination 2:
SEQ ID NO.3: 5'-GGAGAATTGCTTGAACCCAAT-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.4: FAM-CACACCATTGTACTCC-MGB；
and (3) combination:
SEQ ID NO.5:5'-AATCTGACGTTCAGTCATTTCTGA-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.6: FAM-CCAGAGAGCTATTCAAT- MGB；
and (4) combination:
SEQ ID NO.7: 5'-AATGGAGCTGGAACAACTGG-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.8: FAM-ATGGATCAGAAATGAATGCCATCTCAAATC-BHQ1；
and (3) combination 5:
SEQ ID NO.9:5'-CCTGGCTTCCTTCCCTCTGT-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.10: FAM-TCTCTGGGGAGGAGTGGAAAGGAAGG-BHQ1；
and (4) combination 6:
SEQ ID NO.11: 5'-GCACCTGCTGGATACAACACTG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.12: FAM-ATGCATACTACCTTTTGCTCTCAAACCTTTCAAG-BHQ1；
and (3) combination 7:
SEQ ID NO.13:5'-CATGTAGGGTATTGAGTATCATGCC -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.14: FAM-TATGGTATTTCGATTCCAATGGATATCGTTTGAA -BHQ1；
and (4) combination 8:
SEQ ID NO.15: 5'-GCATCTTAACTGCTCTTTATGAATGAA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.16:FAM-CAGTCCTCTGTATGTACTCCTCTTTACACTGGCC -BHQ1；
combination 9:
SEQ ID NO.17:5'-AAGCAGATACTACCCAGCGGAG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.18: FAM-AATTAATCTTTCTGTCAGCACCAAGGAAACAAGTAA -BHQ1；
combination 10:
SEQ ID NO.19: 5'-AATCTCAGCCCTACACCATCCTA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.20: FAM-TCCTCCATTTGGACATATTTCACATAGCAACAT -BHQ1；
combination 11:
SEQ ID NO.21:5'-TCTTCCAGTTTAGAATCAGCCTTG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.22: FAM-CATTGATGGAATAACTCTGTGGCATTATTGCA -BHQ1；
combination 12:
SEQ ID NO.23: 5'-CCTTCTGAAAGAAACGAAAGCA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.24: FAM-CAGGAACACTCATTTGTGCCATTGTGTTT -BHQ1；
combination 13:
SEQ ID NO.25:5'-TAGACTCCCAATCGGCTGAAT -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.26: FAM-TCCTTGTGTCCTCAGTGGAGACATGCA -BHQ1；
combination 14:
SEQ ID NO.27: 5'-ATGACTGGCTGCTGCTGCT -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.28: FAM-TCCGACTTTCCTTCTGCCCTCCTTC -BHQ1；
and (3) combining 15:
SEQ ID NO.29: 5'-ACCTGAGGAGACGGTGACC -3'；
SEQ ID NO.30: 5'-CGGGAACCTCCTGGACTACC -3'；
SEQ ID NO.31: VIC-CAGCAGCACCACGGCGTTCACC -BHQ1。
preferably, in the primer probe set, the 5 'end of the probe in the primer probe set is modified by FAM or VIC, and the 3' end of the probe is modified by NFQ-MGB or BHQ 1.
The invention also provides a kit using the primer probe set.
Preferably, the kit comprises a positive control solution and a blank control solution; the positive control solution contains 15 plasmid DNAs; the blank control is TE buffer solution; adding a carrier RNA solution with a plasmid protection effect into the positive control solution, wherein the final concentration of the carrier RNA solution is 10-50 ng/mu L; and the 15 plasmid DNAs contain 14 different BCL2-IGH chromosome translocation genes and 1 tube family gene ABL1, respectively.
The invention also provides a method for detecting different BCL2-IGH chromosome translocation genes by using the kit, which comprises the following steps:
obtaining genome DNA of a sample to be detected;
performing fluorescence PCR amplification by using BCL2-IGH specific primers and BCL2-IGH specific probes of a primer probe set in the kit and using the genomic DNA as a template, and collecting fluorescence signals;
and after the fluorescence signal collection is finished, a data result is exported and analyzed.
Preferably, in the detection method, the reaction system in the fluorescent PCR amplification further comprises the following components: TaqMan FAST advance Master Mix and ultrapure water.
Preferably, in the detection method, the reaction conditions of the reaction system in the fluorescent PCR amplification at different stages are as follows:
pretreatment: pretreating for 2 minutes at 50 ℃;
pre-denaturation: pre-denaturation at 95 ℃ for 5 min;
wherein the pre-denaturation stage further requires 45 cycles of execution according to the following reaction conditions:
first, pre-denaturation at 95 ℃ for 15 seconds;
then, the mixture was pre-denatured at 60 ℃ for 60 seconds.
From the above description, the present invention has the following advantages compared with the prior art:
1. the detection method is realized based on TaqMan probe PCR technology, and has high specificity and detection sensitivity.
2. The detection method of the invention can effectively avoid the interaction between the primers, between the primers and the probes and between the probes through reasonable primer and probe matching, such as 15 combinations shown in SEQ ID NO.1 to SEQ ID NO.32, thereby improving the specificity of BCL2-IGH chromosome translocation detection, greatly reducing false positive and reducing detection errors.
3. The invention provides recombinant plasmid DNA samples as positive controls aiming at different BCL2-IGH chromosome translocation forms, so that the fluorescence analysis of BCL2-IGH chromosome translocation is more accurate.
Drawings
FIGS. 1a and 1b are graphs showing the results of the blank limit amplification curves of the BCL2-IGH chromosome translocation combination 1 detection system of the present invention confirmed by using 30 samples of genomic DNA of healthy persons in example 1;
FIG. 2 is a data summary of the detection of 14 translocations of BCL2-IGH chromosome using recombinant plasmid DNA to verify the reproducibility of the detection system of the present invention in example 3;
FIG. 3 is a graph showing the results of an amplification curve for verifying the reproducibility of the detection system of the present invention using BCL2-IGH chromosomal translocation combination 1 detected by recombinant plasmid DNA in example 3;
FIG. 4 is a summary of data showing the detection limits of the sensitivity of the detection system of the present invention in example 4 using recombinant plasmid DNA to detect BCL2-IGH chromosomal translocation;
FIGS. 5a, 5b, 5c are graphs showing the results of amplification curves for verifying the sensitivity of the detection system of the present invention by using recombinant plasmid DNA to detect BCL2-IGH chromosomal translocation combination 1 in example 4;
FIG. 6 is a graph showing the results of detecting BCL2-IGH chromosomal translocation using human clinical genomic DNA samples in example 5;
FIG. 7 is a graph showing the results of detecting 14 translocation forms of BCL2-IGH chromosome and the reference gene ABL1 using the positive recombinant plasmid DNA in example 6.
Detailed Description
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
The primer probe group for detecting BCL2-IGH chromosome translocation is composed of a BCL2-IGH specific primer and a BCL2-IGH specific probe, wherein the nucleotides of the primer probe group are shown as SEQ ID NO.1 to SEQ ID NO. 32. Due to the diversification of BCL2-IGH fusion forms, 15 groups can be combined, wherein 14 (namely, the combination 1-14) primer probe groups are used for detecting BCL2-IGH chromosome translocation, and the other group, namely, the combination 15 is used for detecting an internal reference gene ABL 1.
The Tm values of the BCL2-IGH specific primers designed by the invention are all around 58 ℃, and the primers in each combination can be integrated and matched, so that the generation of dimers among the primers is avoided as much as possible; meanwhile, Tm values of probes selected for BCL2-IGH chromosome translocation detection are all around 68 ℃, so that the combination between the probes and other non-specific probe sequences is avoided, and the formation of complex dimers between the probes and primers is also avoided, thereby ensuring the PCR amplification specificity and the amplification efficiency of DNA in BCL2-IGH chromosome translocation.
Preferably, in one embodiment, the 5 'end of the probe (i.e., the fluorescent quantitative PCR probe) in the primer probe set is modified by FAM or VIC, and the 3' end of the fluorescent quantitative PCR probe is modified by NFQ-MGB or BHQ 1.
Compared with the traditional PCR, the TaqMan PCR technology is one of real-time fluorescent PCR, and a probe with two ends respectively marked with a fluorescent reporter group and a quenching group is added in a reaction system. When the probe structure is complete, the energy of fluorescence emitted by the fluorescence reporter group is transferred to the quenching group, and the quenching effect is presented. If a target sequence exists in the amplification process, the probe molecules are gradually hydrolyzed and cut off in the amplification process, the fluorescent reporter group and the quenching group are mutually dissociated, the fluorescent resonance energy transfer effect between the fluorescent reporter group and the quenching group is blocked, and the fluorescent reporter group sends out a fluorescent signal. As amplification proceeds, the fluorescent signal exhibits a linear increase as the target fragment is amplified.
Aiming at the primer probe set, the invention also provides a kit for detecting BCL2-IGH chromosome translocation; the kit contains the primer probe group, and has the advantage of high detection sensitivity.
The kit also comprises a positive control solution and a blank control solution. The positive control solution contains 15 kinds of plasmid DNA, and a formula capable of ensuring the stability of fusion positive plasmids is provided; the carrier RNA solution with the effect of protecting plasmids is added into the positive control solution, the final concentration of the carrier RNA solution is 10-50 ng/mu L, and 15 kinds of plasmid DNA respectively contain 14 different BCL2-IGH chromosome translocation genes and 1 seed gene ABL1 and respectively correspond to the 15 groups of gene polymorphism combinations. The blank control is TE buffer solution; wherein, the final concentration of the carrier RNA solution is preferably 31 ng/. mu.L.
The invention also provides a method for detecting different BCL2-IGH chromosome translocation genes by using the kit, which comprises the following steps:
s1, obtaining genome DNA of a sample to be detected;
s2, performing fluorescent PCR amplification by using BCL2-IGH specific primers and BCL2-IGH specific probes of the primer probe sets in the kit and using the genomic DNA in the step S1 as a template, and collecting fluorescent signals;
after the fluorescence signal collection at step S3 and step S2, the data result is derived from Software 7500 Software v2.3 and analyzed.
In step S2, the fluorescence PCR amplification reaction system is 20 μ L total reaction system, which includes 10 μ L TaqMan FAST advance Master Mix, 600nM each of the upstream primer and the downstream primer, 400nM probe, 100 ng-1 μ g genomic DNA, and ddH for the rest2And (4) supplementing and finishing.
Wherein, in a reaction system in the fluorescent PCR amplification, the reaction conditions of different stages are as follows:
pretreatment: pretreating for 2 minutes at 50 ℃;
pre-denaturation: pre-denaturation at 95 ℃ for 5 minutes, and said pre-denaturation phase also requires 45 cycles of execution according to the following reaction conditions:
first, pre-denaturation at 95 ℃ for 15 seconds;
then, pre-denatured at 60 ℃ for 60 seconds for collecting fluorescence signals.
When the BCL2-IGH chromosome translocation gene is detected, in a fluorescence PCR amplification reaction system, a BCL2-IGH specific primer is used for amplifying a target gene region of a DNA sample, and a BCL2-IGH specific probe is combined with the target gene, is gradually hydrolyzed in the amplification process and then releases a fluorescence signal. The probe is used as a linear oligonucleotide, two ends of the probe are respectively marked with a fluorescence reporter group and a fluorescence quenching group, when the probe is complete, a fluorescence signal emitted by the reporter group is absorbed by the quenching group, a fluorescence monitoring system cannot detect the fluorescence signal, and in the PCR amplification process, the probe is cut off, so that the reporter fluorescent group and the quenching fluorescent group are separated, and the fluorescence monitoring system can detect the fluorescence signal.
In addition, the position of the probe is as close to the upstream primer of the corresponding target gene region as possible, in the PCR reaction process, the DNA template in the sample to be detected is denatured into single strands by using high temperature, the temperature is reduced for annealing, and the primer is combined with the DNA template of the target region; meanwhile, the probe can be combined with the DNA template of the target region, and finally, a complementary strand is synthesized along the DNA strand by the primer under the action of polymerase, so that the target region is amplified, an amplification product is continuously extended in the amplification process, and the polymerase hydrolyzes the probe combined with the DNA template to release a corresponding fluorescent signal.
The present invention is further illustrated by the following examples, which are intended to be illustrative, but not limiting, of the present invention.
Example 1: the detection system blank of the invention was confirmed using genomic DNA samples from 30 healthy persons.
In this example, 30 samples of genomic DNA from healthy persons were used to confirm the blank limit of the detection method of the present invention.
The specific detection method comprises the following steps:
by adopting the PCR reaction system and the reaction conditions which have been described in the specification, 30 healthy human genome samples are selected, the concentration is 40 ng/muL-100 ng/muL, the detection is repeated once in each batch and among batches, each translocation form detection system generates 120 reactions, the threshold line and the blank limit ct value of each detection system are confirmed, and the blank limit ct value calculation formula is as follows: detecting the average value of ct values and three times of standard deviation values by using a healthy person sample; for example: the BCL2-IGH chromosome translocation combination 1 detection system finally confirms that the threshold line is 0.5, the blank limit ct value is 37.7, and the amplification curve result detected by the BCL2-IGH chromosome translocation combination 1 is shown in FIGS. 1a and 1 b; wherein, the mark (i) represents a positive plasmid sample amplification curve, and the mark (ii) represents a healthy human sample amplification curve. The results of confirming the blank limit of 14 translocation detection systems of BCL2-IGH chromosome by using genome DNA samples of 30 healthy people are summarized as follows: the threshold lines of the detection systems of the combinations 1, 2, 3 and 8 are set to be 0.5, the blank limit ct values are respectively 37.7, 39.6, 42.24 and 41.44, the threshold lines of the other 10 combination detection systems are set to be 0.2, and the blank limit ct values are all larger than 45.
Therefore, 14 BCL2-IGH chromosome translocation form detection systems can specifically amplify corresponding recombinant plasmid DNA positive samples, and a threshold line and a blank limit ct value are set for each detection system, so that false positive is effectively avoided.
Example 2: the recombinant plasmid DNA is adopted to confirm 14 translocation forms of BCL2-IGH chromosome and a standard curve equation of an internal reference gene ABL 1.
In this example, a recombinant plasmid DNA sample was used as a template for RT-PCR fluorescence detection, and the plasmid copy number was obtained by digital PCR quantification.
The specific detection method comprises the following steps:
carrying out detection according to a qPCR system and an amplification step by using a carrier RNA solution with 31 ng/mu L as a dilution matrix and carrying out gradient dilution with 10 times of high-copy fusion gene positive plasmids to obtain the amplification efficiency of the corresponding fusion genes; selecting 10 times diluted DNA samples of the medium-low copy plasmid, quantifying by a digital PCR method to obtain the absolute copy number of the high-copy fusion gene positive plasmid, inputting the copy number into Software 7500 Software v2.3, and automatically obtaining 14 translocation forms of BCL2-IGH chromosomes and a standard curve equation of an internal reference gene ABL1 by an instrument, wherein the standard curve equation mainly comprises PCR amplification efficiency, intercept, slope and R2The values, finally calculated using the standard curve equation, are as follows:
copy number of fusion gene to be detected =10e；
Wherein e = (ct value-intercept of fusion gene to be detected)/slope, and the standard curve equation data are as follows: amplification efficiency of combination 1 was 97%, intercept was 37.627, slope was-3.4, R2A value of 0.999; amplification efficiency of combination 2 was 95%, intercept was 37.229, slope was-3.441, R2A value of 0.999; amplification efficiency of combination 3 was 98%, intercept was 37.18, slope was-3.364, R2A value of 0.999; amplification efficiency of combination 4 was 92%, intercept was 38.355, slope was-3.523, R2A value of 0.999; amplification efficiency of combination 5 was 97%, intercept was 35.698, slope was-3.398, R2A value of 0.999; amplification efficiency of combination 6 was 101%, intercept was 36.456, slope was-3.306, R2A value of 0.999; amplification efficiency of combination 7 was 98%, intercept was 37.894, slope was-3.365, R2A value of 0.998; amplification efficiency of combination 8 was 97%, intercept was 37.696, slope was-3.398, R2A value of 0.998; amplification efficiency of combination 9 was 94%, intercept was 38.371, slope was-3.478, R2A value of 0.999; combination 10 amplification efficiency was 100%, intercept was 37.295, slope was-3.315, R2A value of 0.998; amplification efficiency of combination 11 was 97%, intercept was 36.555, slope-3.396, R2A value of 0.999; combination 12 amplification efficiency 93%, intercept 36.671, slope-3.511, R2A value of 0.999; amplification efficiency of combination 13 was 97%, intercept was 35.981, slope was-3.399, R2A value of 1; combination 14 amplification efficiency was 97%, intercept was 36.289, slope-3.397, R2A value of 0.999; amplification efficiency of combination 15 was 100%, intercept was 36.854, slope was-3.31, R2The value was 0.999.
Example 3: the recombinant plasmid DNA is adopted to detect BCL2-IGH chromosome translocation to verify the repeatability of the detection system.
In this example, the precision of the detection system of the present invention was also verified by using recombinant plasmid DNA samples as templates.
The specific detection method comprises the following steps:
and constructing reference substances with high copy-positivity (10^6 copies/mL), medium copy (10^4 copies/mL) and low copy number (10^3 copies/mL) by using the synthesized fusion gene positive plasmid sample, wherein the plasmid copy number is obtained quantitatively by the constructed fluorescence PCR standard curve equation. Each sample was repeated 20 times, and the reaction was performed in 4 batches, and 60 reactions were detected for each fusion gene. And calculating the CV values among batches, wherein the results show that the CV values of the detection results of the fusion genes among the batches are all less than 3%, and the data summary of the repeatability confirmation of the BCL2-IGH chromosome translocation combination 1-14 detection system is shown in the attached figure 2. For example: the results of amplification curves of three concentration positive plasmid samples in the BCL2-IGH chromosome translocation combination 1 detection system are shown in the attached figure 3, wherein, the mark (a) represents a high copy positive (10^6 copies/mL) plasmid sample amplification curve, the mark (a) represents a medium copy positive (10^4 copies/mL) sample amplification curve, and the mark (b) represents a low copy positive (10^3 copies/mL) sample amplification curve.
Example 4: the detection limit of the sensitivity of the detection system is confirmed by detecting BCL2-IGH chromosome translocation by adopting recombinant plasmid DNA.
In this example, the sensitivity of the detection system of the present invention was also confirmed by using a recombinant plasmid DNA sample as a template.
The specific detection method comprises the following steps:
selecting a reference substance with low copy number (10^3 copies/mL) concentration in the precision experiment, and performing 3-fold gradient dilution twice to construct samples with three concentrations. For each sample, 20 replicates were run and 60 responses were tested for each fusion gene. The sample concentration with the fusion gene detection rate of more than or equal to 95% is selected as the lowest detection concentration (lowest detection limit) of the fusion detection system, the sensitivity confirmation data of the BCL2-IGH chromosome translocation combination 1-14 detection system is summarized as shown in figure 4, and the detection minimum data value of the sensitivity of the BCL2-IGH chromosome translocation combination 1-14 detection system is as follows: the lowest detection limit of the combination 9 detection system is 1000 copies/mL, and the lowest detection limit of the other 13 combinations is 500 copies/mL. For example: the amplification curve results of three concentration positive plasmid samples of BCL2-IGH chromosome translocation combination 1 detection system are shown in FIGS. 5a, 5b and 5c, wherein symbol (c) represents a low copy positive (10^3 copies/mL) plasmid sample amplification curve, symbol (b) represents a plasmid sample amplification curve diluted by 3 times, and symbol (c) represents a plasmid sample amplification curve diluted by 9 times.
Example 5: BCL2-IGH chromosomal translocations were detected using human clinical genomic DNA samples.
In this example, BCL2-IGH chromosome translocation assay was performed using human clinical genomic DNA samples as templates and the assay system of the present invention.
In this example, the PCR reaction system and the reaction conditions described in this specification are used, the specific operation method is the same as that in other examples of the present invention, but the template is a human clinical genomic DNA sample, and the result of BCL2-IGH chromosomal translocation detection is shown in fig. 6, where the marker i represents an amplification curve of the internal reference gene ABL1, the marker ii represents an amplification curve of BCL2-IGH chromosomal translocation combination 1, the positive copy of BCL2-IGH chromosomal translocation combination 1 is 535 copies/mL, the copy number of the internal reference gene ABL1 is 1559094 copies/mL, the translocation ratio is 0.034%, and the translocation ratio is the ratio of the positive copy number of the fusion gene to the copy number of the internal reference gene ABL 1.
Example 6: and detecting 14 translocation forms of the BCL2-IGH chromosome and an internal reference gene ABL1 by adopting positive recombinant plasmid DNA.
In this example, the positive control is used as a template, and 14 translocation forms of BCL2-IGH chromosome and reference gene ABL1 are detected according to the detection system of the present invention.
In this embodiment, the PCR reaction system and the reaction conditions described in this specification are used, the specific operation method is the same as that in other embodiments of the present invention, but the template is a constructed positive control sample, and the result of the BCL2-IGH chromosome translocation detection is shown in fig. 7, where the identifier iii indicates an amplification curve of the internal reference gene ABL1, and the remaining curves are BCL2-IGH chromosome translocation combinations 1 to 14 amplification curves.
The results show that the BCL2-IGH chromosome translocation fluorescence PCR detection system can accurately and reliably carry out rapid detection on 14 translocation forms of the BCL2-IGH chromosome. Compared with other detection methods, the fluorescent PCR quantitative detection system has higher sensitivity, specificity and accuracy, has simple detection procedures, greatly shortens the detection time, saves a large amount of manpower, material resources and energy, provides a more brand-new, rapid, simple and convenient technology for detecting BCL2-IGH chromosome translocation, and has wide clinical application value.
It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Sequence listing
<110> Shenzhen silvergrass Beiken precision medicine Limited
SHANGHAI TISSUEBANK MEDICAL INSPECTION Co.,Ltd.
SHANGHAI TISSUEBANK BIOTECHNOLOGY Co.,Ltd.
Shanghai Yushuo Beiken Gene Technology Co., Ltd.
<120> primer probe set, kit and detection method for detecting BCL2-IGH chromosome translocation
<141> 2021-10-21
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<170> SIPOSequenceListing 1.0
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<213> Artificaial Sequence
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tgacctttag agagttgctt tacg 24
<210> 2
<211> 26
<212> DNA
<213> Artificaial Sequence
<400> 2
ctgtttcaac acagacccac ccagag 26
<210> 3
<211> 21
<212> DNA
<213> Artificaial Sequence
<400> 3
ggagaattgc ttgaacccaa t 21
<210> 4
<211> 16
<212> DNA
<213> Artificaial Sequence
<400> 4
cacaccattg tactcc 16
<210> 5
<211> 24
<212> DNA
<213> Artificaial Sequence
<400> 5
aatctgacgt tcagtcattt ctga 24
<210> 6
<211> 17
<212> DNA
<213> Artificaial Sequence
<400> 6
ccagagagct attcaat 17
<210> 7
<211> 20
<212> DNA
<213> Artificaial Sequence
<400> 7
<210> 8
<211> 30
<212> DNA
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<400> 8
atggatcaga aatgaatgcc atctcaaatc 30
<210> 9
<211> 20
<212> DNA
<213> Artificaial Sequence
<400> 9
<210> 10
<211> 26
<212> DNA
<213> Artificaial Sequence
<400> 10
tctctgggga ggagtggaaa ggaagg 26
<210> 11
<211> 22
<212> DNA
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<400> 11
gcacctgctg gatacaacac tg 22
<210> 12
<211> 34
<212> DNA
<213> Artificaial Sequence
<400> 12
atgcatacta ccttttgctc tcaaaccttt caag 34
<210> 13
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<212> DNA
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<400> 13
catgtagggt attgagtatc atgcc 25
<210> 14
<211> 34
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<400> 14
tatggtattt cgattccaat ggatatcgtt tgaa 34
<210> 15
<211> 27
<212> DNA
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<400> 15
gcatcttaac tgctctttat gaatgaa 27
<210> 16
<211> 34
<212> DNA
<213> Artificaial Sequence
<400> 16
cagtcctctg tatgtactcc tctttacact ggcc 34
<210> 17
<211> 22
<212> DNA
<213> Artificaial Sequence
<400> 17
aagcagatac tacccagcgg ag 22
<210> 18
<211> 36
<212> DNA
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<400> 18
aattaatctt tctgtcagca ccaaggaaac aagtaa 36
<210> 19
<211> 23
<212> DNA
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<400> 19
aatctcagcc ctacaccatc cta 23
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<400> 20
tcctccattt ggacatattt cacatagcaa cat 33
<210> 21
<211> 24
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<400> 21
tcttccagtt tagaatcagc cttg 24
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<400> 22
cattgatgga ataactctgt ggcattattg ca 32
<210> 23
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<400> 23
ccttctgaaa gaaacgaaag ca 22
<210> 24
<211> 29
<212> DNA
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<400> 24
caggaacact catttgtgcc attgtgttt 29
<210> 25
<211> 21
<212> DNA
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<400> 25
tagactccca atcggctgaa t 21
<210> 26
<211> 27
<212> DNA
<213> Artificaial Sequence
<400> 26
tccttgtgtc ctcagtggag acatgca 27
<210> 27
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<212> DNA
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<400> 27
atgactggct gctgctgct 19
<210> 28
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<400> 28
tccgactttc cttctgccct ccttc 25
<210> 29
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<212> DNA
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<400> 29
acctgaggag acggtgacc 19
<210> 30
<211> 20
<212> DNA
<213> Artificaial Sequence
<400> 30
cgggaacctc ctggactacc 20
<210> 31
<211> 22
<212> DNA
<213> Artificaial Sequence
<400> 31
cagcagcacc acggcgttca cc 22
<210> 32
<211> 22
<212> DNA
<213> Artificaial Sequence
<400> 32
catggctgac gagatctgag tg 22
Claims (8)
1. A primer probe group for detecting BCL2-IGH chromosome translocation, which is characterized by comprising a primer probe combination with nucleotides shown as SEQ ID NO.1 to SEQ ID NO.32 and used for detecting 14 different BCL2-IGH chromosome translocation genes and 1 tube family gene ABL 1; wherein the primer probe sets shown in SEQ ID NO.1 to SEQ ID NO.32 comprise the following combinations:
combination 1:
SEQ ID NO.1:5'-TGACCTTTAGAGAGTTGCTTTACG-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.2: FAM-CTGTTTCAACACAGACCCACCCAGAG-BHQ1；
and (3) combination 2:
SEQ ID NO.3: 5'-GGAGAATTGCTTGAACCCAAT-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.4: FAM-CACACCATTGTACTCC-MGB；
and (3) combination:
SEQ ID NO.5:5'-AATCTGACGTTCAGTCATTTCTGA-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.6: FAM-CCAGAGAGCTATTCAAT- MGB；
and (4) combination:
SEQ ID NO.7: 5'-AATGGAGCTGGAACAACTGG-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.8: FAM-ATGGATCAGAAATGAATGCCATCTCAAATC-BHQ1；
and (3) combination 5:
SEQ ID NO.9:5'-CCTGGCTTCCTTCCCTCTGT-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.10: FAM-TCTCTGGGGAGGAGTGGAAAGGAAGG-BHQ1；
and (4) combination 6:
SEQ ID NO.11: 5'-GCACCTGCTGGATACAACACTG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.12: FAM-ATGCATACTACCTTTTGCTCTCAAACCTTTCAAG-BHQ1；
and (3) combination 7:
SEQ ID NO.13:5'-CATGTAGGGTATTGAGTATCATGCC -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.14: FAM-TATGGTATTTCGATTCCAATGGATATCGTTTGAA -BHQ1；
and (4) combination 8:
SEQ ID NO.15: 5'-GCATCTTAACTGCTCTTTATGAATGAA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.16:FAM-CAGTCCTCTGTATGTACTCCTCTTTACACTGGCC -BHQ1；
combination 9:
SEQ ID NO.17:5'-AAGCAGATACTACCCAGCGGAG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.18: FAM-AATTAATCTTTCTGTCAGCACCAAGGAAACAAGTAA -BHQ1；
combination 10:
SEQ ID NO.19: 5'-AATCTCAGCCCTACACCATCCTA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.20: FAM-TCCTCCATTTGGACATATTTCACATAGCAACAT -BHQ1；
combination 11:
SEQ ID NO.21:5'-TCTTCCAGTTTAGAATCAGCCTTG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.22: FAM-CATTGATGGAATAACTCTGTGGCATTATTGCA -BHQ1；
combination 12:
SEQ ID NO.23: 5'-CCTTCTGAAAGAAACGAAAGCA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.24: FAM-CAGGAACACTCATTTGTGCCATTGTGTTT -BHQ1；
combination 13:
SEQ ID NO.25:5'-TAGACTCCCAATCGGCTGAAT -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.26: FAM-TCCTTGTGTCCTCAGTGGAGACATGCA -BHQ1；
combination 14:
SEQ ID NO.27: 5'-ATGACTGGCTGCTGCTGCT -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.28: FAM-TCCGACTTTCCTTCTGCCCTCCTTC -BHQ1；
and (3) combining 15:
SEQ ID NO.29: 5'-ACCTGAGGAGACGGTGACC -3'；
SEQ ID NO.30: 5'-CGGGAACCTCCTGGACTACC -3'；
SEQ ID NO.31: VIC-CAGCAGCACCACGGCGTTCACC -BHQ1。
2. a kit for detecting BCL2-IGH chromosomal translocation, comprising the primer probe set of claim 1.
3. The kit of claim 2, wherein the kit comprises a positive control solution and a blank control solution; the positive control solution contains 15 plasmid DNAs; the blank control is TE buffer solution; the primer probe sets shown in SEQ ID NO.1 to SEQ ID NO.32 corresponding to the 15 kinds of plasmid DNAs comprise the following combinations:
combination 1:
SEQ ID NO.1:5'-TGACCTTTAGAGAGTTGCTTTACG-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.2: FAM-CTGTTTCAACACAGACCCACCCAGAG-BHQ1；
and (3) combination 2:
SEQ ID NO.3: 5'-GGAGAATTGCTTGAACCCAAT-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.4: FAM-CACACCATTGTACTCC-MGB；
and (3) combination:
SEQ ID NO.5:5'-AATCTGACGTTCAGTCATTTCTGA-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.6: FAM-CCAGAGAGCTATTCAAT- MGB；
and (4) combination:
SEQ ID NO.7: 5'-AATGGAGCTGGAACAACTGG-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.8: FAM-ATGGATCAGAAATGAATGCCATCTCAAATC-BHQ1；
and (3) combination 5:
SEQ ID NO.9:5'-CCTGGCTTCCTTCCCTCTGT-3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.10: FAM-TCTCTGGGGAGGAGTGGAAAGGAAGG-BHQ1；
and (4) combination 6:
SEQ ID NO.11: 5'-GCACCTGCTGGATACAACACTG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.12: FAM-ATGCATACTACCTTTTGCTCTCAAACCTTTCAAG-BHQ1；
and (3) combination 7:
SEQ ID NO.13:5'-CATGTAGGGTATTGAGTATCATGCC -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.14: FAM-TATGGTATTTCGATTCCAATGGATATCGTTTGAA -BHQ1；
and (4) combination 8:
SEQ ID NO.15: 5'-GCATCTTAACTGCTCTTTATGAATGAA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.16:FAM-CAGTCCTCTGTATGTACTCCTCTTTACACTGGCC -BHQ1；
combination 9:
SEQ ID NO.17:5'-AAGCAGATACTACCCAGCGGAG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.18: FAM-AATTAATCTTTCTGTCAGCACCAAGGAAACAAGTAA -BHQ1；
combination 10:
SEQ ID NO.19: 5'-AATCTCAGCCCTACACCATCCTA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.20: FAM-TCCTCCATTTGGACATATTTCACATAGCAACAT -BHQ1；
combination 11:
SEQ ID NO.21:5'-TCTTCCAGTTTAGAATCAGCCTTG -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.22: FAM-CATTGATGGAATAACTCTGTGGCATTATTGCA -BHQ1；
combination 12:
SEQ ID NO.23: 5'-CCTTCTGAAAGAAACGAAAGCA -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.24: FAM-CAGGAACACTCATTTGTGCCATTGTGTTT -BHQ1；
combination 13:
SEQ ID NO.25:5'-TAGACTCCCAATCGGCTGAAT -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.26: FAM-TCCTTGTGTCCTCAGTGGAGACATGCA -BHQ1；
combination 14:
SEQ ID NO.27: 5'-ATGACTGGCTGCTGCTGCT -3'；
SEQ ID NO.32: 5'-CATGGCTGACGAGATCTGAGTG -3'；
SEQ ID NO.28: FAM-TCCGACTTTCCTTCTGCCCTCCTTC -BHQ1；
and (3) combining 15:
SEQ ID NO.29: 5'-ACCTGAGGAGACGGTGACC -3'；
SEQ ID NO.30: 5'-CGGGAACCTCCTGGACTACC -3'；
SEQ ID NO.31: VIC-CAGCAGCACCACGGCGTTCACC -BHQ1。
4. the kit according to claim 3, wherein a carrier RNA solution with a plasmid protecting effect is added into the positive control solution, and the final concentration of the carrier RNA solution is 10-50 ng/μ L.
5. The kit of claim 3, wherein the 15 plasmid DNAs contain 14 different BCL2-IGH chromosomal translocation genes and 1 housekeeping gene ABL1, respectively.
6. A method for detecting a different BCL2-IGH chromosomal translocation gene for a non-diagnostic purpose, comprising the steps of: obtaining genome DNA of a sample to be detected; performing fluorescence PCR amplification by using the primer probe group in the kit according to claim 2 and taking the genomic DNA as a template, and collecting a fluorescence signal; and after the fluorescence signal collection is finished, a data result is exported and analyzed.
7. The detection method according to claim 6, wherein the reaction system in the fluorescence PCR amplification further comprises the following components: TaqMan FAST advance Master Mix and ultrapure water.
8. The detection method according to claim 6 or 7, wherein the reaction conditions of the reaction system in the fluorescent PCR amplification at different stages are as follows:
pretreatment: pretreating for 2 minutes at 50 ℃;
pre-denaturation: pre-denaturation at 95 ℃ for 5 min;
wherein the pre-denaturation stage further requires 45 cycles of execution according to the following reaction conditions:
first, pre-denaturation at 95 ℃ for 15 seconds;
followed by pre-denaturation at 60 ℃ for 60 seconds.
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