Preparation method and application of three-color probe for diagnosing myelodysplastic syndrome

Abstract

The invention relates to the field of molecular biology, in particular to a preparation method and application of a three-color probe for diagnosing myelodysplastic syndrome, the sample size can be reduced by using the three-color probe, meanwhile, a small amount of open-loop nucleotide (UNA) is doped in the three-color probe composition, the specificity of the probe is improved, and the application prospect is wide. The background noise is reduced, and the result analysis is simplified.

Description

Technical Field

[0001] This application relates to the field of molecular biology, and particularly relates to a three-color probe composition for diagnosing myelodysplastic syndrome and a preparation method thereof. Background Art

[0002] Myelodysplastic syndrome (MDS) is a heterogeneous myeloid clonal disease originating from hematopoietic stem cells, characterized by myeloid cell dysplasia, manifested as ineffective hematopoiesis, refractory cytopenia, and a high risk of transformation to acute myeloid leukemia. Gene mutations, epigenetic alterations, chromosomal abnormalities, or abnormal bone marrow hematopoietic microenvironment can be found in hematopoietic cells of some MDS patients, and these abnormal changes may be involved in the multi-factor, multi-step, continuous and dynamic occurrence and development process of MDS. The diagnosis of MDS requires excluding cytopenia caused by other diseases, and the results of bone marrow morphology determination are highly subjective with a high misdiagnosis rate. It is one of the common blood system diseases that are recognized as difficult to diagnose and treat at home and abroad.

[0003] Fluorescence in situ hybridization (FISH) is a technique that labels a nucleotide of a nucleic acid probe with a fluorophore. The target gene to be detected is homologous and complementary to the nucleic acid probe used. After denaturation, annealing, and renaturation, a hybrid of the target gene and the nucleic acid probe can be formed, and the target gene can be qualitatively, quantitatively, or relatively localized analyzed under a microscope through a fluorescence detection system. Applying a set of probes targeting common abnormalities in MDS for fluorescence in situ hybridization detection can improve the detection rate of cytogenetic abnormalities in some MDS patients. The Guidelines for the Diagnosis and Treatment of Myelodysplastic Syndrome with Excess Blasts (2022 Edition) states that for suspected MDS patients, when bone marrow dry tap, no metaphase cells, poor metaphase quality, or less than 20 analyzable metaphase cells are present, FISH detection should be performed. Usually, the probes should include: 5q, CEP7, 7q, CEP8, 20q, CEPY, and TP53.

[0004] Limitations of the FISH technique: There are many types of abnormalities in MDS patients. Currently, because of the large number of detection indicators in the conventional FISH technique, 8 sets of probes (CSF1R / D5S630, EGR1 / D5S630, D7S522 / CEP7, D7S486 / CEP7, 7q22 / CEP7, D20S108 / CEP8, CEPY / CEPX, p53 / CEP17) are required, and a large number of cell samples are needed. The detection samples are from patients' bone marrow samples, and a large amount of bone marrow needs to be drawn. However, bone marrow aspiration is difficult for some patients, and it is not easy to obtain specimens. The technical solution of this application can reduce the collection amount of patients' bone marrow specimens, reduce the workload of doctors during clinical detection, and reduce the detection cost of patients. Summary of the Invention

[0005] To solve at least one of the above technical problems, a preparation method of a three-color probe composition for diagnosing myelodysplastic syndrome is developed.

[0006] The first aspect of this application provides a preparation method of a three-color probe for diagnosing myelodysplastic syndrome, including the following steps: (1) Download the BAC clone gene sequences corresponding to the following loci from UCSC Genome Browser EGR1: RP11-166J22, RP11-461O14 CSF1R: RP11-100O5, RP11-432O16 D5S630: RP11-535C10, RP11-1044D21 7q22: RP11-745C6, RP11-343F22 7q31.2: RP11-1152C19, RP11-1113D14 p53: RP11-1081A10, RP11-107F4 D20S108: RP11-97M2, RP11-17F3; (2) Respectively use the perl plug-in program chunks.pl to split the BAC clone gene sequences corresponding to each locus obtained in step (1) into 1-kb-sized blocks and remove duplicate sequences; batch import the split blocks into OligoArray software for probe design and probe screening, then export the screened probes and SEQ No 1, SEQ No 2, SEQ No 3, and SEQ No 4 to an EXCEL table, and then add a 17-bp tag sequence to the 5' end and an 18-bp tag sequence to the 3' end of each probe respectively to obtain a series of probe sequences with tag sequences, where SEQ No 1 is the gene sequence corresponding to CEP7, SEQ No 2 is the gene sequence corresponding to CEP8, SEQ No 3 is the gene sequence corresponding to CEPY, and SEQ No 4 is the gene sequence corresponding to CEPX; (3) Use a DNA synthesizer to chemically synthesize the gene probe sequences with tag sequences obtained in step (2) respectively, mix the chemically synthesized probes at the same locus, and respectively prepare probe libraries for each locus; (4) Synthesize universal primers with a green fluorescent group at the 5'-end, universal primers with a red / orange-red fluorescent group at the 5'-end, and universal primers with a cyan fluorescent group at the 5'-end respectively. Use the universal primers of different colors to perform amplification labeling reactions on probes at different sites, and optionally add about 5% to about 10% of UNA triphosphate of the total amount of nucleotide raw materials in the amplification labeling reaction; (5) Purify and dilute the amplification labeling products of the probe library obtained in step (4) to obtain a fluorescently labeled probe library, and combine them as follows to obtain a three-color probe composition, where the fluorescent colors of different sites in the same combination are different; Combination 1: EGR1 / CSF1R / D5S630 Combination 2: 7q22 / 7q31.2 / CEP7 Combination 3: p53 / D20S108 / CEP8 Combination 4: CEPY / CEPX.

[0007] In a preferred embodiment of the present application, the UNA triphosphate is selected from UNA-A triphosphate, UNA-U triphosphate, UNA-G triphosphate, and UNA-C triphosphate.

[0008] In a preferred embodiment of the present application, the molar mass ratio of the UNA-A triphosphate, UNA-U triphosphate, UNA-G triphosphate, and UNA-C triphosphate is 1:1:1:1.

[0009] By adopting the above technical solution, the screening of probes in the probe preparation method mainly depends on the program, and the more stable PCR method is used to amplify and label the probes. The probe labeling rate is higher and more uniform. Since only the 5'-end of the probe is fluorescently labeled, it does not affect the hybridization pairing reaction of the probe. During the amplification labeling reaction, about 5% to about 10% of UNA triphosphate of the total amount of nucleotide raw materials is added to the substrate, so that the obtained probe sequence incorporates ring-opened nucleotides. The ring-opened nucleotides can change the conformation of the original sequence. The incorporation of a small amount of ring-opened nucleotides enhances the specificity while maintaining the affinity of the probe for the target, reduces the background noise, and at the same time enhances the stability of the probe.

[0010] In a preferred embodiment of the present application, the preparation method of the EGR1 probe includes the following steps: (1) Download the BAC clone gene sequences RP11-166J22 and RP11-461O14 corresponding to the locus EGR1 from the UCSC Genome Browser.

[0011] (2) The RP11-166J22 and RP11-461O14 sequences obtained in step (1) are respectively segmented into 1-kb blocks using the perl plug-in program chunks.pl and duplicate sequences are removed; the segmented blocks are batch-imported into the OligoArray software for probe design and probe screening, and then the screened probes are exported to an EXCEL table. Then, a 17-bp tag sequence is added to the 5' end and an 18-bp tag sequence is added to the 3' end of each probe to obtain a series of probe sequences with tag sequences. The probe design and probe screening methods are as follows: the probe length is 50-150 bp, the TM value is 85-99 °C, the GC ratio is 40-80%, it does not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes is 5 bp.

[0012] (3) Use a DNA synthesizer to chemically synthesize the gene probe sequences with tag sequences obtained in step (2), and mix the chemically synthesized probes at the same locus to prepare a probe library of EGR1. (4) Synthesize a universal primer with a green fluorescent group at the 5' end and use this universal primer to perform an amplification labeling reaction on the EGR1 probe library synthesized in step (3), wherein about 5% to about 10% of UNA triphosphate of the total amount of nucleotide raw materials is optionally added in the amplification labeling reaction; the universal primer sequences are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0013] (5) Purify and dilute the amplification labeling product of the probe library obtained in step (4) to obtain a fluorescently labeled EGR1 probe library.

[0014] Through the above technical solution, an EGR1 probe composition with a green fluorescent group is prepared. EGR1 belongs to the 5q region, and the 5q mutation is a common gene abnormality mutation in MDS. The EGR1 probe detection is one of the detection indicators of current MDS conventional FISH.

[0015] In the preferred embodiment of the present application, the preparation method of the CSF1R probe: the BAC clone gene sequences in step (1) are RP11-100O5 and RP11-432O16, and a universal primer with a red fluorescent group at the 5' end is synthesized in step (4). The other preparation processes are the same as those of the EGR1 probe preparation method.

[0016] Through the above technical solution, a CSF1R probe composition with a red fluorescent group is prepared. Both CSF1R and EGR1 belong to the 5q region, and the 5q mutation is a common gene abnormality mutation in MDS. Combining CSF1R and EGR1 can improve the detection accuracy and detection rate.

[0017] In a preferred embodiment of the present application, the method for preparing the probe of D5S630: In step (1), the BAC clone gene sequences are RP11-535C10 and RP11-1044D21. In step (4), a universal primer with a cyan fluorescent group at the 5' end is synthesized, and the other preparation processes are the same as those of the EGR1 probe preparation method.

[0018] Through the above technical solution, a D5S630 probe composition with a cyan fluorescent group is prepared. D5S630 is located in the region of 5q15.31. The D5S630 probe composition is used in combination with the CSF1R and EGR1 probe compositions as an internal reference probe for the three-color probe composition. By adding the internal reference probe, the influence brought by the probe itself during the FISH process can be excluded, and the accuracy of the detection result can be improved.

[0019] In a preferred embodiment of the present application, the method for preparing the 7q22 probe: In step (1), the BAC clone gene sequences are RP11-745C6 and RP11-343F22. In step (4), a universal primer with a green fluorescent group at the 5' end is synthesized, and the other preparation processes are the same as those of the EGR1 probe preparation method.

[0020] In a preferred embodiment of the present application, the method for preparing the 7q31.2 probe: In step (1), the BAC clone gene sequences are RP11-1152C19 and RP11-1113D14. In step (4), a universal primer with a red fluorescent group at the 5' end is synthesized, and the other preparation processes are the same as those of the EGR1 probe preparation method.

[0021] Through the above technical solution, a 7q22 probe composition with a green fluorescent group and a 7q31.2 probe composition with a red fluorescent group are prepared. In MDS, both 7q22 and 7q31.2 are common gene abnormalities on chromosome 7. The combined use of the two can improve the accuracy and detection rate of the detection.

[0022] In a preferred embodiment of the present application, the method for preparing the p53 probe: In step (1), the BAC clone gene sequences are RP11-1081A10 and RP11-107F4. In step (4), a universal primer with a green fluorescent group at the 5' end is synthesized, and the other preparation processes are the same as those of the EGR1 probe preparation method.

[0023] In a preferred embodiment of the present application, the method for preparing the D20S108 probe: In step (1), the BAC clone gene sequences are RP11-97M2 and RP11-17F3. In step (4), a universal primer with a red fluorescent group at the 5' end is synthesized, and the other preparation processes are the same as those of the EGR1 probe preparation method.

[0024] Through the above technical solutions, a p53 probe composition with a green fluorescent group and a D20S108 probe composition with a red fluorescent group are prepared. p53 is a common gene abnormality on chromosome 17 in MDS, and D20S108 is a common gene abnormality on chromosome 20.

[0025] In a preferred embodiment of the present application, the method for preparing the CEP7 probe comprises the following steps: (1) Export the sequence of SEQ No 1 to an EXCEL spreadsheet, and then add a 17bp tag sequence to the 5' end and an 18bp tag sequence to the 3' end of SEQ No 1 respectively to obtain the SEQ No 1 probe sequence with tag sequences; (2) Chemically synthesize the gene probe sequence with tag sequences obtained in step (1) using a DNA synthesizer to prepare a probe library of CEP7; (3) Synthesize a universal primer with a cyan fluorescent group at the 5' end and use this universal primer to perform an amplification labeling reaction on the CEP7 probe library synthesized in step (2), wherein about 5% to about 10% of UNA triphosphate of the total amount of nucleotide raw materials is optionally added in the amplification labeling reaction; the universal primer sequences are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0026] (4) Purify and dilute the amplification labeling product of the probe library obtained in step (3) to obtain a fluorescently labeled CEP7 probe library.

[0027] In a preferred embodiment of the present application, the method for preparing the CEP8 probe: Replace SEQ No 1 in step (1) with SEQ No 2, and the other preparation processes are the same as those of the CEP7 probe preparation method.

[0028] In a preferred embodiment of the present application, the method for preparing the CEPY probe: Replace SEQ No 1 in step (1) with SEQ No 3, and synthesize a universal primer with a green fluorescent group at the 5' end in step (3), and the other preparation processes are the same as those of the CEP7 probe preparation method.

[0029] In a preferred embodiment of the present application, the method for preparing the CEPX probe: Replace SEQ No 1 in step (1) with SEQ No 4, and synthesize a universal primer with a red fluorescent group at the 5' end in step (3), and the other preparation processes are the same as those of the CEP7 probe preparation method.

[0030] By adopting the above technical solution, probe compositions of CEP7, CEP8, CEPY and CEPX were prepared, which respectively correspond to the gene sequences of SEQ No 1, SEQ No 2, SEQ No 3 and SEQ No 4. The gene sequences of SEQ No 1, SEQ No 2, SEQ No 3 and SEQ No 4 are respectively the gene sequences of the centromeric regions on chromosomes 7, 8, Y and X. The above four gene sequences are highly specific, and the sequence similarity with other regions is less than 80%. Using the above specific sequences can enhance the specificity of probe recognition, reduce background noise, improve the accuracy of detection, and reduce the complexity of FISH result analysis.

[0031] In the preferred embodiment of the present application, the method for preparing the three-color probe of the above combination 1 is: mixing the EGR1 probe, CSF1R probe and D5S630 probe prepared by the above method at equal concentrations to obtain a three-color probe.

[0032] In the preferred embodiment of the present application, the method for preparing the three-color probe of the above combination 2 is: mixing the 7q22 probe, 7q31.2 probe and CEP7 probe prepared by the above method at equal concentrations to obtain a three-color probe.

[0033] In the preferred embodiment of the present application, the method for preparing the three-color probe of the above combination 3 is: mixing the p53 probe, D20S108 probe and CEP8 probe prepared by the above method at equal concentrations to obtain a three-color probe.

[0034] In the preferred embodiment of the present application, the method for preparing the two-color probe of the above combination 4 is: mixing the CEPY probe and CEPX probe prepared by the above method at equal concentrations to obtain a two-color probe.

[0035] Through the above technical solution, three groups of three-color probes and one group of two-color probes were prepared. Through the above four probe compositions, the detection of common gene abnormalities in MDS can be completed. Compared with using all two-color probes (such as CSF1R / D5S630, EGR1 / D5S630, D7S522 / CEP7, D7S486 / CEP7, 7q22 / CEP7, D20S108 / CEP8, CEPY / CEPX, p53 / CEP17) which require detecting 8 groups of probes, the sample size is reduced from 8 to 4. Since MDS patients often suffer from problems such as dry bone marrow aspiration and difficult bone marrow puncture, it is not easy to obtain samples, and insufficient sample size is exactly the technical problem existing in the fluorescence in situ hybridization detection process of MDS. The above technical solution can complete the detection by using four groups of probes, can solve the technical problem of insufficient sample size, and at the same time can reduce the detection cost and workload.

[0036] In a preferred embodiment of the present application, the concentration of the above-mentioned three-color probe is: 20-50 ng / μL for each probe composition.

[0037] In a preferred embodiment of the present application, the concentration of the above-mentioned three-color probe is: 20 ng / μL for each probe composition.

[0038] In a preferred embodiment of the present application, the concentration of the above-mentioned three-color probe is: 30 ng / μL for each probe composition.

[0039] In a preferred embodiment of the present application, the concentration of the above-mentioned three-color probe is: 40 ng / μL for each probe composition.

[0040] In a preferred embodiment of the present application, the concentration of the above-mentioned three-color probe is: 50 ng / μL for each probe composition.

[0041] In a preferred embodiment of the present application, the amplification-labeling reaction system is as follows: (1) Reaction buffer (2) Deoxynucleoside triphosphate substrates and 5%-10% UNA triphosphate substrates; (3) Prepared probe library template; (4) Synthesized fluorescently labeled universal primer; (5) DNA polymerase.

[0042] In a preferred embodiment of the present application, the polymerase used in the amplification-labeling reaction is selected from Therminator DNA polymerase.

[0043] Therminator DNA polymerase is a high-fidelity thermostable DNA polymerase suitable for PCR reactions. This enzyme has excellent amplification rates and high specificity. The substrates in the reaction system contain natural deoxynucleoside triphosphate substrates and 5%-10% UNA triphosphate substrates. Therminator DNA polymerase can simultaneously recognize natural deoxynucleoside triphosphate substrates and UNA triphosphate substrates and perform high-fidelity PCR amplification to obtain probe sequences incorporating a small amount of open-ring nucleotides.

[0044] In a preferred embodiment of the present application, the conditions for the amplification-labeling reaction are: 95°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 35 cycles; 72°C for 10 min.

[0045] The second aspect of the present application provides a probe composition prepared by the above method.

[0046] The third aspect of the present application provides the use of the above probe composition in the preparation of a diagnostic product for myelodysplastic syndrome.

[0047] In a preferred embodiment of the present application, the above-mentioned method for diagnosing myelodysplastic syndrome includes: (1) Sample processing: Place the bone marrow cell smear sample into a container containing 2×SSC, heat it in a microwave oven at high power for 3 min until the liquid boils, and then continue to heat it at medium-low power for 10 min. Immediately after the treatment, place the glass slide in gradient alcohol pre-cooled to -20°C for dehydration and air-drying. Divide the sample into 4 parts numbered 1-4 for standby; (2) Preparation of the triple-color probe hybridization mixture: Mix the probe compositions of combinations 1, 2, 3, and 4 labeled with fluorescence and the hybridization buffer in a volume ratio of 1:9 respectively; (3) Co-denaturation of the probe and sample: Hybridize and mix the sample No. 1 with 10 μL of the triple-color probe hybridization mixture of combination 1 in step (2), the sample No. 2 with 10 μL of the triple-color probe hybridization mixture of combination 2 in step (2), the sample No. 3 with 10 μL of the triple-color probe hybridization mixture of combination 3 in step (2), and the sample No. 4 with 10 μL of the dual-color probe hybridization mixture of combination 4 in step (2). Cover the slide with a 22×22 mm coverslip, seal the slide with rubber glue. After sealing, place the slide in a hybridization instrument for denaturation at 90°C for 1 min and hybridization at 37°C for 30-60 min; (4) Washing after hybridization: After hybridization is completed, remove the coverslip, and place the slide in a washing solution preheated to 60°C to wash away the unbound probes; (5) Counterstaining and microscopic examination: Drop 10 μL of an anti-quenching mounting medium on the air-dried slide for sealing, and then observe the hybridization result under a fluorescence microscope.

[0048] Through the above technical solution, the diagnosis and detection of myelodysplasia by triple-color probes are realized, the number of samples is reduced. At the same time, a small amount of open-loop nucleotides are incorporated into the probe sequence, enhancing the specificity of the probe, reducing background noise, reducing the difficulty of result analysis, and improving the accuracy and sensitivity of detection.

[0049] The fourth aspect of the present application provides a kit, which contains (1) A mixture of the probe composition and the hybridization buffer prepared in the present application; (3) DAPI counterstain.

[0050] In a preferred embodiment of the present application, the components of the above hybridization solution are 10 wt% deionized formamide, 20 wt% dextran sulfate, 0.6 M sodium chloride, and 10 mM citrate buffer.

[0051] In a preferred embodiment of the present application, the probe composition in the above kit contains 1, 2, 3, or 4 tubes of probe composition, and each tube of probe composition contains combinations 1, 2, 3, and 4 defined in the above technical solution respectively.

[0052] In a preferred embodiment of the present application, the probe composition of the above-mentioned kit comprises 1 tube of probe composition, and this tube of probe composition comprises the probe composition of Combination 1, Combination 2, Combination 3 or Combination 4. For example, it can be Combination 1, can be Combination 2, can be Combination 3 or is Combination 4.

[0053] In a preferred embodiment of the present application, the probe composition of the above-mentioned kit comprises 2 tubes of probe composition. The first tube of probe composition comprises any one group of the probe compositions of Combination 1, Combination 2, Combination 3 or Combination 4, and the second tube of probe composition comprises another group of probe composition other than the first tube of probe composition.

[0054] In a preferred embodiment of the present application, the first tube of probe composition comprises the Combination 1 probe composition, and the second tube of probe composition comprises the Combination 2 probe composition.

[0055] In a preferred embodiment of the present application, the first tube of probe composition comprises the Combination 1 probe composition, and the second tube of probe composition comprises the Combination 3 probe composition.

[0056] In a preferred embodiment of the present application, the first tube of probe composition comprises the Combination 1 probe composition, and the second tube of probe composition comprises the Combination 4 probe composition.

[0057] In a preferred embodiment of the present application, the first tube of probe composition comprises the Combination 2 probe composition, and the second tube of probe composition comprises the Combination 3 probe composition.

[0058] In a preferred embodiment of the present application, the first tube of probe composition comprises the Combination 2 probe composition, and the second tube of probe composition comprises the Combination 4 probe composition.

[0059] In a preferred embodiment of the present application, the first tube of probe composition comprises the Combination 3 probe composition, and the second tube of probe composition comprises the Combination 4 probe composition.

[0060] In a preferred embodiment of the present application, the probe composition of the above-mentioned kit comprises 3 tubes of probe composition. The first tube of probe composition comprises any one group of the probe compositions of Combination 1, Combination 2, Combination 3 or Combination 4. The second tube of probe composition comprises another group of probe composition other than the first tube of probe composition, and the third tube of probe composition comprises another group of probe composition other than the first tube of probe composition and the second tube of probe composition.

[0061] In a preferred embodiment of the present application, the first tube of probe composition comprises the Combination 1 probe composition, the second tube of probe composition comprises the Combination 2 probe composition, and the third tube of probe composition comprises the Combination 3 probe composition.

[0062] In a preferred embodiment of the present application, the first tube of probe composition contains the combination 1 probe composition, the second tube of probe composition contains the combination 2 probe composition, and the third tube of probe composition contains the combination 4 probe composition.

[0063] In a preferred embodiment of the present application, the first tube of probe composition contains the combination 1 probe composition, the second tube of probe composition contains the combination 3 probe composition, and the third tube of probe composition contains the combination 4 probe composition.

[0064] In a preferred embodiment of the present application, the first tube of probe composition contains the combination 2 probe composition, the second tube of probe composition contains the combination 3 probe composition, and the third tube of probe composition contains the combination 4 probe composition.

[0065] In a preferred embodiment of the present application, the probe composition of the kit contains 4 tubes of probe composition, and each tube of probe composition contains the combination 1, combination 2, combination 3, and combination 4 probe compositions respectively.

[0066] In a preferred embodiment of the present application, the first tube of probe composition contains the combination 1 probe composition, the second tube of probe composition contains the combination 2 probe composition, the third tube of probe composition contains the combination 3 probe composition, and the third tube of probe composition contains the combination 4 probe composition.

[0067] Through the above technical solution, the probe composition can be selected according to actual needs during the FISH detection process. The probe composition can be a set of three-color probes, two sets of three-color probes, three sets of three-color probes, or four sets of three-color probes. If it is necessary to focus on detecting gene abnormalities on chromosome 5, the three-color probes of combination one can be used alone. Using this technical solution can reduce the detection workload and lower the detection cost.

[0068] Definitions and Explanations Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered uncertain or unclear without a special definition, but should be understood according to the meaning understood by those of ordinary skill in the art. When a trade name appears in this article, it is intended to refer to the corresponding product or its active ingredient.

[0069] In the present invention, unless otherwise specified, the terms "comprising, including, and containing" or equivalents are open-ended expressions, meaning that in addition to the listed elements, components, or steps, other unspecified elements, components, or steps may also be covered.

[0070] Unless otherwise specified, the "multiple" in the terms of the present invention refers to an integer greater than or equal to 2, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, and 10, up to the maximum number of items in the options.

[0071] Unless otherwise specified, the term "optional" and / or "optionally" in this application means that it can be selected or not selected. When not selected, it means that the step, the limitation, or the component does not exist. When selected, it means that the relevant operations or limitations are carried out according to the following description.

[0072] Unless otherwise specified, the term "UNA" in this application represents a novel acyclic RNA derivative, which is similar to DNA or RNA. The general structural formula of UNA is where B represents the natural bases adenine, thymine, guanine, and cytosine.

[0073] Unless otherwise specified, the general structural formula of UNA triphosphate is

[0074] Unless otherwise specified, the structural formula of the term "UNA-A triphosphate" in this application is

[0075] Unless otherwise specified, the structural formula of the term "UNA-T triphosphate" in this application is

[0076] Unless otherwise specified, the structural formula of the term "UNA-G triphosphate" in this application is

[0077] Unless otherwise specified, the structural formula of the term "UNA-C triphosphate" in this application is

[0078] In summary, the present invention includes at least one of the following beneficial effects: 1. By using the three-color probe, the number of test samples is reduced from 8 to 4, which can solve the problems of few bone marrow samples and difficult sample acquisition for patients with myelodysplastic syndrome. At the same time, it can reduce the detection cost and simplify the detection process.

[0079] 2. Using the three-color probe makes the detection sites more comprehensive, improving the detection rate and accuracy.

[0080] 3. This application uses SEQ No 1, SEQ No 2, SEQ No 4, and SEQ No 4 probes with high specificity and low similarity to other sequences to detect the centromeric regions of CEP7, CEP8, CEPY, and CEPX respectively, which can improve the specificity of detection and reduce background noise.

[0081] 4. In the probe preparation method of this application, the screening of probes mainly depends on programs, and the more stable PCR method is used for amplification and labeling of probes. The probe labeling rate is higher and more uniform. Fluorescent labeling is only carried out at the 5' end of the probe, so it does not affect the hybridization pairing reaction of the probe, and the batch-to-batch stability is high.

[0082] 5. A small amount of unlocked nucleic acid (UNA) is contained in the probe of the present application. On the basis of a slight increase in fluorescence after probe binding, it can further improve the specificity and stability of the probe and reduce background noise. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 : Color development comparison between the EGR1 probe of Preparation Example 1 of the present application and the EGR1 probe without UNA in Comparative Example 1. (a) shows the color development result of the EGR1 probe in Comparative Example 1 for normal cells; (b) shows the color development result of the EGR1 probe in Preparation Example 1 for normal cells; Figure 2 : Color development comparison between the CSF1R probe of Preparation Example 2 of the present application and the CSF1R probe without UNA in Comparative Example 2. (a) shows the color development result of the CSF1R probe in Comparative Example 2 for normal cells; (b) shows the color development result of the CSF1R probe in Preparation Example 2 for normal cells; Figure 3 : Color development comparison between the D5S630 probe of Preparation Example 3 of the present application and the D5S630 probe without UNA in Comparative Example 3. (a) shows the color development result of the D5S630 probe in Comparative Example 3 for normal cells; (b) shows the color development result of the D5S630 probe in Preparation Example 3 for normal cells; Figure 4 : Result diagram of Test Example 2. (a) shows the color development result of the EGR1 / CSF1R / D5S630 three-color probe for normal cells; (b) shows the color development result of the EGR1 / CSF1R / D5S630 three-color probe for cells lacking CSF1R. DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention will be described in detail below by way of examples, but this does not mean any adverse limitation to the present invention. The present invention has been described in detail herein, and its specific embodiments have also been disclosed. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0085] Preparation of the EGR1 Probe in Preparation Example 1 Preparation method of the EGR1 probe: (1) Download the BAC clone gene sequences RP11-166J22 and RP11-461O14 corresponding to the locus EGR1 from the UCSC Genome Browser.

[0086] (2) The RP11-166J22 and RP11-461O14 sequences obtained in step (1) are respectively split into 1-kb blocks using the perl plug-in program chunks.pl and duplicate sequences are deleted; the split blocks are batch-imported into the OligoArray software for probe design and probe screening, and then the screened probes are exported to an EXCEL table. Then, a 17-bp tag sequence is added to the 5' end and an 18-bp tag sequence is added to the 3' end of each probe to obtain a series of probe sequences with tag sequences; the probe design and probe screening methods are as follows: the probe length is 50-150 bp, the TM value is 85-99 °C, the GC ratio is 40-80%, it does not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes is 5 bp.

[0087] (3) Use a DNA synthesizer to chemically synthesize the gene probe sequences with tag sequences obtained in step (2), and mix the chemically synthesized probes at the same locus to respectively prepare an EGR1 probe library. (4) Synthesize a universal primer with a green fluorescent group at the 5' end and use this universal primer to perform an amplification labeling reaction on the EGR1 probe library synthesized in step (3), wherein 5% of UNA triphosphate is added to the total molar amount of nucleotide raw materials in the amplification labeling reaction; the universal primer sequences are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0088] (5) Purify and dilute the amplification labeling product of the probe library obtained in step (4) to obtain a fluorescently labeled EGR1 probe library.

[0089] Preparation Example 2 Preparation of CSF1R Probe Preparation method of CSF1R probe: Replace the BAC clone sequence in step (1) with RP11-100O5 and RP11-432O16, replace the synthesis of the green fluorescent group in step (4) with a red fluorescent group, and the addition amount of UNA triphosphate is 8% of the total molar amount of raw materials. The other processes are the same as those in Preparation Example 1 to prepare a CSF1R probe.

[0090] Preparation Example 3 Preparation of D5S630 Probe Preparation method of D5S630 probe: Replace the BAC clone sequence in step (1) with RP11-535C10 and RP11-1044D21, replace the synthesis of the green fluorescent group in step (4) with a cyan fluorescent group, and the addition amount of UNA triphosphate is 10% of the total molar amount of raw materials. The other processes are the same as those in Preparation Example 1 to prepare a D5S630 probe.

[0091] Preparation Example 4: Preparation of 7q22 Probe Method for preparing 7q22 probe: Replace the BAC clone sequence in step (1) with RP11-745C6 and RP11-343F22, and use a green fluorescent group for labeling in step (4). The other procedures are the same as those in Preparation Example 2 to obtain the 7q22 probe.

[0092] Preparation Example 5: Preparation of 7q31.2 Probe Method for preparing 7q31.2 probe: Replace the BAC clone sequence in step (1) with RP11-1152C19 and RP11-1113D14, and use a red fluorescent group for labeling in step (4). The other procedures are the same as those in Preparation Example 2 to obtain the 7q31.2 probe.

[0093] Preparation Example 6: Preparation of p53 Probe Method for preparing p53 probe: Replace the BAC clone sequence in step (1) with RP11-1081A10 and RP11-107F4, and use a green fluorescent group for labeling in step (4). The other procedures are the same as those in Preparation Example 2 to obtain the p53 probe.

[0094] Preparation Example 7: Preparation of D20S108 Probe Method for preparing D20S108 probe: Replace the BAC clone sequence in step (1) with RP11-97M2 and RP11-17F3, and use a red fluorescent group for labeling in step (4). The other procedures are the same as those in Preparation Example 2 to obtain the D20S108 probe.

[0095] Preparation Example 8: Preparation of CEP7 Probe Method for preparing CEP 7 probe: (1) Export the sequence of SEQ No 1 to an EXCEL spreadsheet, and add a 17bp tag sequence to the 5' end and an 18bp tag sequence to the 3' end of the SEQ No 1 probe respectively to obtain the SEQ No 1 probe sequence with tag sequences. (2) Use a DNA synthesizer to chemically synthesize the gene probe sequence with tag sequences obtained in step (1) to prepare a probe library of CEP7. (3) Synthesize a universal primer with a cyan fluorescent group at the 5' end and use this universal primer to perform an amplification labeling reaction on the probe library of CEP7 synthesized in step (2), wherein 8% of UNA triphosphate is added to the total amount of nucleotide raw materials in the amplification labeling reaction; the universal primer sequences are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0096] (4) Purify and dilute the amplified labeled product of the probe library obtained in step (3) to obtain a fluorescence-labeled CEP7 probe library.

[0097] Preparation Example 9 Preparation of CEP8 Probe Preparation method of CEP8 probe: Replace SEQ No 1 in step (1) with SEQ No 2, and the other preparation processes are the same as those of the CEP7 probe preparation method.

[0098] Preparation Example 10 Preparation of CEPY Probe Preparation method of CEPY probe: Replace SEQ No 1 in step (1) with SEQ No 3, synthesize a universal primer with a green fluorescent group at the 5'-end in step (3), and the other preparation processes are the same as those of the CEP7 probe preparation method.

[0099] Preparation Example 11 Preparation of CEPX Probe Preparation method of CEPX probe: Replace SEQ No 1 in step (1) with SEQ No 4, synthesize a universal primer with a red fluorescent group at the 5'-end in step (3), and the other preparation processes are the same as those of the CEP7 probe preparation method.

[0100] Example 1 Preparation of EGR1 / CSF1R / D5S630 Three-Color Probe Preparation method of EGR1 / CSF1R / D5S630 three-color probe: Mix the probes prepared in Preparation Example 1, Preparation Example 2, and Preparation Example 3 at equal concentrations. The concentrations of the EGR1 probe, CSF1R probe, and D5S630 probe are all 3 ng / μL.

[0101] Example 2 Preparation of 7q22 / 7q31.2 / CEP7 Three-Color Probe Preparation method of 7q22 / 7q31.2 / CEP7 three-color probe: Mix the probes prepared in Preparation Example 4, Preparation Example 5, and Preparation Example 8 at equal concentrations. The concentrations of the 7q22 probe, 7q31.2 probe, and CEP7 probe are all 2 ng / μL.

[0102] Example 3 Preparation of p53 / D20S108 / CEP8 Three-Color Probe Preparation method of p53 / D20S108 / CEP8 three-color probe: Mix the probes prepared in Preparation Example 6, Preparation Example 7, and Preparation Example 9 at equal concentrations. The concentrations of the p53 probe, D20S108 probe, and CEP8 probe are all 5 ng / μL.

[0103] Example 4 Preparation of CEPY / CEPX Two-Color Probe Preparation method of CEPY / CEPX probe: Mix the probes prepared in Preparation Example 10 and Preparation Example 11 at the same concentration. The concentrations of both CEPY probe and CEPX probe are 4 ng / μL.

[0104] Preparation of EGR1 probe without UNA in Comparative Example 1 Preparation method of EGR1 probe without UNA: (1) Download the BAC clone gene sequences RP11-166J22 and RP11-461O14 corresponding to the locus EGR1 from UCSC Genome Browser.

[0105] (2) Use the perl plug-in program chunks.pl to split the RP11-166J22 and RP11-461O14 sequences obtained in step (1) into 1-kb blocks respectively and remove duplicate sequences; batch import the split blocks into OligoArray software for probe design and probe screening, then export the screened probes to an EXCEL table, and add a 17-bp tag sequence to the 5' end and an 18-bp tag sequence to the 3' end of each probe respectively to obtain a series of probe sequences with tag sequences; the probe design and probe screening methods are that the probe length is 50-150 bp, the TM value is 85-99 °C, the GC ratio is 40-80%, it does not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes is 5 bp.

[0106] (3) Use a DNA synthesizer to chemically synthesize the gene probe sequences with tag sequences obtained in step (2) respectively, mix the chemically synthesized probes at the same locus, and prepare an EGR1 probe library respectively; (4) Synthesize a universal primer with a green fluorescent group at the 5' end and use this universal primer to perform an amplification labeling reaction on the EGR1 probe library synthesized in step (3), wherein the substrate of the amplification labeling reaction is a natural deoxynucleoside triphosphate substrate, and the universal primer sequences are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

[0107] (5) Purify and dilute the amplification labeling product of the probe library obtained in step (4) to obtain a fluorescence-labeled EGR1 probe library without UNA.

[0108] Preparation of CSF1R probe without UNA in Comparative Example 2 Preparation method of CSF1R probe without UNA: Replace the BAC cloning sequence in step (1) of Comparative Example 1 with RP11-100O5 and RP11-432O16, and replace the synthesized green fluorescent group in step (4) with a red fluorescent group. The other processes are the same as those in Comparative Example 1 to prepare a CSF1R probe without UNA.

[0109] Preparation of D5S630 probe without UNA in Comparative Example 3 Preparation method of D5S630 probe without UNA: Replace the BAC cloning sequence in step (1) of Comparative Example 1 with RP11-535C10 and RP11-1044D21, and replace the synthesized green fluorescent group in step (4) with a cyan fluorescent group. The other processes are the same as those in Comparative Example 1 to prepare a D5S630 probe without UNA.

[0110] Test Example 1 Color development comparison between the UNA-containing probe and the UNA-free probe of the present application I. Test method: (1) Sample treatment: Place the normal bone marrow cell smear sample into a container containing 2×SSC, heat it in a microwave oven at high power for 3 min until the liquid boils, and then continue to heat it at medium-low power for 10 min. After the treatment is completed, immediately place the glass slide in gradient alcohol pre-cooled at -20°C for dehydration and air drying. Divide the sample into 6 parts numbered 1-6 for standby; (2) Preparation of probe hybridization mixture: Mix the fluorescently labeled EGR1, CSF1R, and D5S630 probe compositions prepared in Preparation Examples 1-3 and the hybridization buffer in a volume ratio of 1:9 respectively; (3) Preparation of control probe hybridization mixture: Mix the UNA-free fluorescently labeled EGR1, CSF1R, and D5S630 probe compositions prepared in Comparative Examples 1-3 and the hybridization buffer in a volume ratio of 1:9 respectively; (4) Probe-sample co-denaturation: Hybridize and mix the 1st sample with 10 μL of the EGR1 probe hybridization mixture in step (2), the 2nd sample with 10 μL of the CSF1R probe hybridization mixture in step (2), the 3rd sample with 10 μL of the D5S630 probe hybridization mixture in step (2), the 4th sample with 10 μL of the EGR1 probe hybridization mixture in step (3), the 5th sample with 10 μL of the CSF1R probe hybridization mixture in step (3), and the 6th sample with 10 μL of the D5S630 probe hybridization mixture in step (3). Cover each with a 22×22 mm coverslip, seal the slide with rubber cement. After sealing, place the slide in a hybridization instrument for denaturation at 90°C for 1 min and hybridization at 37°C for 30-60 min; (4) Post - hybridization washing: After hybridization is completed, remove the coverslip, and place the slide in a washing solution pre - heated to 60 °C to wash away the unbound probes. (5) Counter - staining and microscopy: Add 10 μL of anti - quenching mounting medium to the air - dried slide for mounting, and then observe the hybridization results under a fluorescence microscope respectively.

[0111] II. Experimental results are as shown Figures 1 - 3 below III. Experimental conclusions It can be seen from Figure 1 that Figure 1 (a) There is obvious background noise, while Figure 1 (b) the background noise is significantly reduced, and at the same time, the chromogenic fluorescence intensity for the EGR1 gene is strong and clear, and the chromogenic effect is good; It can be seen from Figure 2 that Figure 2 (a) There is obvious background noise, while Figure 2 (b) the background noise is significantly reduced, and at the same time, the chromogenic fluorescence intensity for the CSF1R gene is strong and clear, and the chromogenic effect is good; It can be seen from Figure 3 that Figure 3 (a) There is obvious background noise, while Figure 3 (b) the background noise is significantly reduced, and at the same time, the chromogenic fluorescence intensity for the D5S630 gene is strong and clear, and the chromogenic effect is good; In summary, the probe composition prepared in this application has strong binding ability, good chromogenic effect, strong probe specificity, and low background noise. Using the probe composition prepared by the preparation method of this application for the diagnosis of myelodysplastic syndrome can improve the accuracy of detection results and reduce the difficulty of FISH result analysis.

[0112] Test Example 2 Chromogenic reaction of the EGR1 / CSF1R / D5S630 three - color probe of this application on normal cells and CSF1R gene - abnormal cells I. Test method (1) Sample treatment: Respectively place the normal bone marrow cell smear sample and the CSF1R gene - abnormal cell smear sample into a container containing 2×SSC, heat - treat with high - power microwave for 3 min until the liquid boils, and then continue to heat - treat with medium - low power for 10 min. After the treatment is completed, immediately place the slide in gradient alcohol pre - cooled to - 20 °C for dehydration and air - drying for standby; (2) Preparation of probe hybridization mixture: Mix the EGR1 / CSF1R / D5S630 three - color probe composition prepared in Example 1 and hybridization buffer in a volume ratio of 1:9; (3) Co-denaturation of probe sample and normal cells: Take a normal cell sample and hybridize it with 10 μL of the probe hybridization mixture in step (2). Cover the sample with a 22×22 mm coverslip and seal the slide with rubber cement. After sealing, place the slide in a hybridization instrument and denature it at 90 °C for 1 min, then hybridize at 37 °C for 30 - 60 min; (4) Co-denaturation of probe sample and CSF1R gene abnormal cells: Take CSF1R gene abnormal cells and hybridize them with 10 μL of the probe hybridization mixture in step (2). Cover the sample with a 22×22 mm coverslip and seal the slide with rubber cement. After sealing, place the slide in a hybridization instrument and denature it at 90 °C for 1 min, then hybridize at 37 °C for 30 - 60 min; (5) Washing after hybridization: After hybridization is completed, remove the coverslip and place the slide in a pre-warmed washing solution at 60 °C to wash away the unbound probes; (6) Counterstaining and microscopy: Drop 10 μL of anti-quenching mounting medium on the dried slide to seal it, and then observe the hybridization results under a fluorescence microscope respectively.

[0113] II. Experimental results are as Figure 4 III. Experimental conclusions Using the three-color probe of the present application to detect normal cells and CSF1R gene abnormal cells respectively, the results are as shown in Figure 4 (a) and Figure 4 (b). The detection result of the three-color probe of the present application for normal cells is two green, two red, and two cyan signals. Among them, the green signal is the color development of the EGR1 gene, the red signal is the color development of the CSF1R gene, and the cyan signal is the color development of the D5S630 gene. The D5S630 probe is an internal reference probe, and the normal color development of the D5S630 gene can prove the effectiveness of the probe composition and exclude the influence brought by the probe itself. The color development of the three-color probe of the present application for normal cells can illustrate that the three-color probe composition prepared in the present application is effective, and at the same time, the detection result has low background noise, clear color development, and accurate results. For the detection of CSF1R gene abnormal cells, it shows two green, two cyan, and one red signal. The color development result clearly shows the absence of one red signal, indicating that the CSF1R gene is abnormal, which is consistent with the actual cells used. The results show that the three-color probe prepared in the present application can simultaneously perform color development of three colors, and multiple different gene detection results can be obtained from the same sample. Moreover, the color development result is clear, the background noise is low, and the result analysis is difficult. It can reduce the sample usage, solve the problem of few bone marrow samples and difficult sample acquisition for patients with myelodysplastic syndrome, and at the same time can reduce the detection cost and simplify the detection process.

[0114] The above are all preferred embodiments of the present application, and do not limit the protection scope of the present application. Therefore, all equivalent changes made according to the structure, shape, and principle of the present application shall be covered within the protection scope of the present application.

Claims

1. A preparation method of a three-color probe for diagnosing myelodysplastic syndrome, characterized in that It includes the following steps: (1) Download the BAC clone gene sequences corresponding to the following loci from the UCSC Genome Browser EGR1: RP11-166J22, RP11-461O14 CSF1R: RP11-100O5, RP11-432O16 D5S630: RP11-535C10, RP11-1044D21 7q22: RP11-745C6, RP11-343F22 7q31.2: RP11-1152C19, RP11-1113D14 p53: RP11-1081A10, RP11-107F4 D20S108: RP11-97M2, RP11-17F3 (2) Use the perl plug-in program chunks.pl to split the BAC clone gene sequences corresponding to each locus obtained in step (1) into 1-kb blocks and remove duplicate sequences; batch import the split blocks into the OligoArray software for probe design and probe screening, then export the screened probes and SEQ No 1, SEQ No 2, SEQ No 3, and SEQ No 4 to an EXCEL spreadsheet, and add a 17-bp tag sequence to the 5' end and an 18-bp tag sequence to the 3' end of each probe respectively to obtain a series of probe sequences with tag sequences, where SEQ No 1 is the gene sequence corresponding to CEP7, SEQ No 2 is the gene sequence corresponding to CEP8, SEQ No 3 is the gene sequence corresponding to CEPY, and SEQ No 4 is the gene sequence corresponding to CEPX; (3) Use a DNA synthesizer to chemically synthesize the gene probe sequences with tag sequences obtained in step (2) respectively, mix the chemically synthesized probes at the same locus, and prepare probe libraries for each locus respectively; (4) Synthesize a universal primer with a green fluorescent group at the 5' end, a universal primer with a red / orange fluorescent group at the 5' end, and a universal primer with a cyan fluorescent group at the 5' end respectively, and use the universal primers of different colors to perform amplification labeling reactions on the probes at different loci, and optionally add about 5% to about 10% of UNA triphosphate to the total amount of nucleotide raw materials in the amplification labeling reaction; (5) Purify and dilute the amplification labeling products of the probe libraries obtained in step (4) to obtain a fluorescently labeled probe library, and combine them according to the following combinations to obtain a three-color probe composition, where the fluorescent colors of different loci in the same combination are different, Combination 1: EGR1 / CSF1R / D5S630; Combination 2: 7q22 / 7q31.2 / CEP7; Combination 3: p53 / D20S108 / CEP8; Combination 4: CEPY / CEPX.

2. The preparation method according to claim 1, wherein the 17-bp tag sequence in step (2) is TGTAAAACGACGGCCAG, and the 18-bp tag sequence is GGTCATAGCTGTTTCCTG.

3. The preparation method according to claim 1, wherein the probe screening conditions in step (2) are as follows: the probe length is 50 - 150 bp, the TM value is 85 - 99 °C, the GC ratio is 40 - 80%, it does not contain TTTT / GGGG / AAAA / CCCC, and the minimum interval between probes is 5 bp.

4. The preparation method according to claim 1, wherein the universal primer sequences in step (4) are TGTAAAACGACGGCCAGT and CAGGAAACAGCTATGACC.

5. The preparation method according to claim 1, wherein the polymerase used in the amplification labeling reaction is Therminator DNA polymerase.

6. The preparation method according to claim 1, wherein the UNA triphosphate is selected from UNA - A triphosphate, UNA - T triphosphate, UNA - G triphosphate, and UNA - C triphosphate. Preferably, the molar mass ratio of UNA - A triphosphate, UNA - T triphosphate, UNA - G triphosphate, and UNA - C triphosphate is 1:1:1:

1.

7. The preparation method according to claim 1, wherein in the combination 1, EGR1 is labeled with a green fluorescent group, CSF1R is labeled with a red fluorescent group, and D5S630 is labeled with a cyan fluorescent group; in the combination 2, 7q22 is labeled with a green fluorescent group, 7q31.2 is labeled with a red fluorescent group, and CEP7 is labeled with a cyan fluorescent group; in the combination 3, p53 is labeled with a green fluorescent group, D20S108 is labeled with a red fluorescent group, and CEP8 is labeled with a cyan fluorescent group; in the combination 4, CEPY is labeled with a green fluorescent group and CEPX is labeled with a red fluorescent group.

8. A probe composition prepared by the preparation method according to any one of claims 1 - 7.

9. A kit, the kit contains (1) A mixture of the probe composition according to claim 8 and a hybridization buffer; (2) Denaturing solution; (3) DAPI counterstain.

10. The kit according to claim 9, wherein the probe composition contains 1, 2, 3, or 4 tubes of probe compositions, and each tube of probe composition contains the combination 1, combination 2, combination 3, and combination 4 described in claim 1 respectively.

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