Cell detection kit for abnormality of human chromosomes 3 and 10 and use thereof

Abstract

The present application relates to the technical field of in-vitro diagnosis, and in particular to a cell detection kit for the abnormality of human chromosomes 3 and 10 and a use thereof. A probe set comprised in the kit uses a specific chromosome locus or a centromere as a target, or uses a combination of a plurality of specific chromosome loci or centromeres as a target, so that the kit can rapidly and effectively detect a variety of cancer samples comprising lung cancer, breast cancer, intestinal cancer, esophageal cancer, bladder cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, cervical cancer or prostate cancer, and is suitable for cancer diagnosis and development and popularization of prognostic products.

Description

Human chromosome 3 and 10 abnormal cell detection kit and use thereof Technical Field

[0001] The present disclosure relates to the field of in vitro diagnostic technology, and in particular to a kit for detecting abnormal cells of human chromosomes 3 and 10 and a preparation method thereof. Background Art

[0002] In situ hybridization (ISH) refers to the process of using a specifically labeled nucleic acid of known sequence as a probe to hybridize with nucleic acids in cells or tissue sections, thereby accurately and quantitatively locating the specific nucleic acid sequence. Fluorescence in situ hybridization (FISH) is an in situ hybridization method that uses fluorescent labels instead of isotopic labels. This method has been widely used in clinical pathology and is an important auxiliary technology in clinical pathology diagnosis. It is mainly used for gene amplification, translocation and fusion, playing a role in helping to correctly diagnose diseases, guide clinical targeted treatments, and assess patient prognosis. It should be more widely used in cancer diagnosis, for example, in the detection of cancer-related targeted genes, early diagnosis, differentiation of benign and malignant pleural effusions, and prognosis.

[0003] Bacterial artificial chromosome library (BAC Library) is a commonly used genomic library. It is a recombinant DNA clone group containing random fragments of part or all genes of a certain organism constructed using BAC vectors. It is an information library that stores the entire genome sequence.

[0004] BAC vectors usually use Escherichia coli as hosts, are highly stable, and have no gene deletion, exchange, or mosaicism. The constructed genomic library is an important basic work for genomic research, especially for organisms with large and very large genomes. It plays an important role in physical mapping, gene map cloning, comparative genomics analysis, transgenic research, gene structure and regulation, random probe preparation, and other studies.

[0005] The use of BAC libraries to prepare in situ hybridization probes for cancer detection is currently one of the important ways to quickly obtain cancer diagnostic products. However, for the same chromosomal target (e.g., the same subband), the random probe sets prepared using BACs containing different target fragment regions for fluorescent in situ hybridization detection of cancer diagnosis have obvious differences in signal intensity and fluorescence background results. Therefore, it is necessary to optimize the target fragment regions contained in the BAC library to obtain a fluorescent in situ hybridization probe set that can effectively detect cancer.

[0006] Summary of the Invention

[0007] The purpose of the present disclosure is to provide a human chromosome abnormality detection kit and a preparation method thereof. The probe group contained in the kit targets a specific chromosome site or centromere, or a combination of multiple specific chromosome sites or centromeres, and can quickly and effectively detect a variety of cancer samples including lung cancer, breast cancer, intestinal cancer, esophageal cancer, bladder cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, cervical cancer and prostate cancer, and is suitable for the development and promotion of cancer diagnosis and prognosis products.

[0008] In order to solve the above technical problems and achieve the above objectives, the present disclosure provides the following technical solutions:

[0009] In a first aspect, the present disclosure provides a human chromosome abnormality detection kit, wherein the detected chromosome sites include at least one site among 3p, 3q, 10cen (10p11.1) or 10q, and the detection kit contains a fluorescent in situ hybridization probe set for amplifying the chromosome sites.

[0010] In an optional embodiment, the site targeted by the probe group includes at least one site among Chr3: 40.2-41.2 Mb, Chr3: 195.6-197.3 Mb, Chr10: 37.7-39.5 Mb or Chr10: 78.9-79.7 Mb.

[0011] In an optional embodiment, the chromosomal locus includes at least one of 3p22.1, 3q29, 10cen (10p11.1) or 10q22.3.

[0012] In an optional embodiment, the target fragment of the probe group for detecting the chromosome locus 3p22.1 includes Chr3:40,281,947~Chr3:41,223,565, the target fragment of the probe group for detecting the chromosome locus 3q29 includes Chr3:195,692,486~197,328,281, the target fragment of the probe group for detecting the chromosome locus 10cen (10p11.1) includes Chr10:37,709,476~39,201,629, and the target fragment of the probe group for detecting the chromosome locus 10q22.3 includes Chr10:78,908,277~Chr10:79,990,274.

[0013] Furthermore, the target fragment of the probe group for detecting chromosome locus 3p22.1 is selected from at least one of Chr3:40,281,947-40,476,764, Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255 or Chr3:40,382,551-40,613,653; the target fragment of the probe group for detecting chromosome locus 3q29 is Chr3:195,692,486-195,894,522; the target fragment of the probe group for detecting chromosome locus 10cen (10p11.1) ... The target fragment of the probe group is Chr10:37,709,476~38,214,862, preferably at least one of Chr10:37,709,476~37,861,144, Chr10:37,861,252~38,004,303 or Chr10:38,007,581~38,214,862; the target fragment of the probe group for detecting chromosome site 10q22.3 is selected from at least one of Chr10:79,491,820~79,670,444 or Chr10:79,499,153~79,703,867.

[0014] In an optional embodiment, the detection kit contains only any one of the following fluorescence in situ hybridization probe sets (a) to (d): (a) the fluorescence in situ hybridization probe set is used to amplify at least one of Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255 or Chr3:40,382,551-40,613,653; (b) the fluorescence in situ hybridization probe set is used to amplify Chr3:195,692,486-195,89 4,522; (c) the fluorescent in situ hybridization probe set is used to amplify a combination of Chr10:37,709,476~37,861,144, Chr10:37,861,252~38,004,303 and Chr10:38,007,581~38,214,862; (d) the fluorescent in situ hybridization probe set is used to amplify at least one of Chr10:79,491,820~79,670,444 or Chr10:79,499,153~79,703,867.

[0015] In another optional embodiment, the fluorescent in situ hybridization probe group of the detection kit is composed of the fluorescent in situ hybridization probe groups shown in the following (a) to (d): (a) the fluorescent in situ hybridization probe group is used to amplify at least one of Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255 or Chr3:40,382,551-40,613,653; (b) the fluorescent in situ hybridization probe group is used to amplify Chr3:195,692,486-1 95,894,522; (c) the fluorescent in situ hybridization probe set is used to amplify a combination of Chr10:37,709,476-37,861,144, Chr10:37,861,252-38,004,303 and Chr10:38,007,581-38,214,862; (d) the fluorescent in situ hybridization probe set is used to amplify at least one of Chr10:79,491,820-79,670,444 or Chr10:79,499,153-79,703,867.

[0016] In an optional embodiment, the detection kit further includes a sample collection device and consumables; the sample collected by the sample collection device is from blood, saliva, urine, pleural effusion or peritoneal effusion.

[0017] Furthermore, the sample from blood is a gradient separation sample of peripheral blood mononuclear cells.

[0018] In an optional embodiment, the detection kit further comprises an auxiliary detection reagent;

[0019] The auxiliary detection reagent includes at least one of a digestion solution, a cleaning solution, a blood sample preservation solution, a Ficoll separation solution or an organic reagent for FISH hybridization.

[0020] The second aspect of the present disclosure provides a method for preparing the detection kit described in the first aspect, the preparation method comprising preparing one or more BAC strains containing a target sequence at at least one site in human chromosome 3p, 3q, 10cen (10p11.1) or 10q, extracting DNA from the BAC strains, randomly fragmenting the extracted DNA using a transposase, and simultaneously adding transposase recognition sequences to both ends of the obtained random fragments, and then using the random fragments as amplification templates and sequences targeting the transposase recognition sequences as amplification primers, amplifying in an amplification system with a ratio of dUTP to dTTP+dUTP of 50% to 100%, to obtain a random probe composition targeting the target sequence; the transposase recognition sequence comprises a transposase; The invention relates to a random probe composition comprising an ME sequence and a linker sequence connected to the 5' end of the ME sequence of the transposase; the number of base A in the linker sequence is 4 to 20, and the number of dUTP bases inserted in the amplification step is adjusted by adjusting the content of base A in the linker sequence without affecting the efficiency of the transposase and the amplification efficiency; the dUTP in the random probe composition is also labeled, and the labeling method includes: adding the labeled dUTP to the amplification system, and labeling the dUTP in the random probe composition as the amplification reaction proceeds; or, labeling the dUTP in the obtained random probe composition during the amplification reaction or after the amplification is completed; the label is a fluorescent group label; and the fluorescent group is selected from fluorescein dyes, rhodamine dyes or cyanine dyes.

[0021] The third aspect of the present disclosure provides the use of the detection kit described in the aforementioned embodiment in the preparation of a cancer diagnosis and / or prognosis product. Wherein, the cancer is preferably lung cancer, breast cancer, intestinal cancer, esophageal cancer, bladder cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, cervical cancer or prostate cancer. Wherein, the diagnosis and / or prognosis method includes isolating and obtaining a cell sample from a sample to be diagnosed and / or a prognosis sample, hybridizing the obtained cell sample using the detection kit described in any one of the aforementioned embodiments or the probe set in the kit prepared by the preparation method described in the aforementioned embodiment X, detecting the hybridization signal and outputting the chromosome abnormality result of the cell sample.

[0022] A fourth aspect of the present disclosure provides a method for detecting cells with chromosomal abnormalities, comprising the following steps:

[0023] (1) Blood sample collection: Collect blood samples from subjects;

[0024] (2) Preparation of mononuclear cell suspension: isolating mononuclear cells from the blood sample collected in step (1) to obtain a mononuclear cell suspension;

[0025] (3) Probe hybridization: hybridizing the mononuclear cell suspension obtained in step (2) with the probe set in the aforementioned kit to obtain a hybridization sample;

[0026] (4) Image acquisition: performing imaging analysis on the hybridization sample obtained in step (3) to obtain an image of the hybridization sample;

[0027] (5) Image analysis: Analyze the image of the hybridization sample obtained in step (4), identify the signal distribution of cells with chromosomal abnormalities, and determine the chromosomal abnormal cells, wherein the signal distribution of cells with chromosomal abnormalities is manifested as an increase or loss of chromosomal sites.

[0028] The present invention constructs a BAC library targeting at least one site in human chromosomes 3p, 3q, 10cen (10p11.1) or 10q, and uses the constructed BAC library as raw material to prepare a random probe group, which can avoid defects such as chromosome positioning errors, non-specific site detection, high background noise, and weak specific signal intensity, and can quickly and accurately detect various cancer samples including lung cancer, breast cancer, intestinal cancer, esophageal cancer, bladder cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, cervical cancer or prostate cancer, and is suitable for the development and promotion of cancer diagnosis and prognosis products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is the Sp6 sequencing alignment result of the BAC targeting 3p22.1 in Example 1;

[0030] FIG2 is the T7 sequencing alignment result of the BAC targeting 3p22.1 in Example 1;

[0031] FIG3 is the Sp6 sequencing alignment result of the BAC targeting 3q29 in Example 1;

[0032] FIG4 shows the T7 sequencing alignment results of the BAC targeting 3q29 in Example 1, wherein the chr_alt chromosome, such as chr3_KI270779v1_alt, is an alternative sequence different from the reference genome and is currently available for some assemblies including danRer11, mm10, hg19, and hg38;

[0033] FIG5 is the Sp6 sequencing comparison result of the BAC targeting 10cen in Example 1;

[0034] FIG6 is the T7 sequencing alignment result of the BAC targeting 10cen in Example 1;

[0035] FIG7 is the Sp6 sequencing alignment results of the BAC targeting 10q22.3 in Example 1;

[0036] FIG8 is the T7 sequencing alignment result of the BAC targeting 10q22.3 in Example 1;

[0037] Figure 9 shows the hybridization results of a single target site of 3p BAC in Example 3;

[0038] Figure 10 is the specificity verification results of the four 3p BAC targets in Example 3;

[0039] Figure 11 is the hybridization result of 3q BAC single target in Example 4;

[0040] FIG12 is the validation result of hybridization specificity of different targets of 3q BAC in Example 4;

[0041] Figure 13 shows the hybridization results of single / combination targets of 10cen (10p11.1) in Example 5;

[0042] Figure 14 shows the specificity verification results after superposition of three BACs of 10cen (10p11.1) in Example 5;

[0043] FIG15 is a comparison of signal strength and coverage area after superposition of three BACs in Example 5;

[0044] FIG16 shows the hybridization results of 10q22.3-1 and 10q22.3-6 in Example 6;

[0045] FIG17 shows the specificity verification results of 10q22.3-1 and 10q22.3-6 in Example 6;

[0046] FIG18 shows the AUC values ​​obtained at different cell numbers in Example 9;

[0047] FIG19 is a Youden index curve under different threshold values ​​in Example 9;

[0048] FIG20 is the ROC curve when the cutoff value is greater than 0.000183 in Example 9. DETAILED DESCRIPTION

[0049] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present disclosure, and for ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0050] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0051] It will also be understood that in certain methods described herein that include more than one step or action, the order of the steps or actions of the method are not necessarily limited to the order in which the steps or actions of the method are listed unless the context dictates otherwise.

[0052] definition

[0053] As used herein, the terms "a," "an," "the," and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0054] As used herein, the terms "about," "substantially," and "similar to" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range may depend in part on how the value is measured or determined, or on limitations of the measurement system. As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0055] As used herein, the term "disease and / or condition" refers to a physical condition of the subject that is associated with the disease and / or condition described herein. As used herein, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, alleviate and / or relieve the disease or condition described herein.

[0056] As used herein, the term "cancer" refers to a disease or disorder caused by the proliferation of cells that are transformed by oncogenicity. "Cancer" should include any one or more of a wide range of benign or malignant tumors, including those that can, for example, invade and grow via the lymphatic system and / or bloodstream and metastasize through the human or animal body or part thereof. Although the present invention is particularly intended to diagnose or detect malignant tumors and solid cancers, as used herein, the term "tumor" includes benign and malignant tumors or solid masses. Cancer further includes (but is not limited to) carcinomas, lymphomas or sarcomas, such as ovarian cancer, colon cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, urethral cancer, uterine cancer, acute lymphocytic leukemia, Hodgkin's disease, small cell lung cancer, melanoma, neuroblastoma, glioma (such as glioblastoma), and soft tissue sarcomas, lymphomas, melanomas, sarcomas and adenocarcinomas, etc.

[0057] As used herein, the term "probe" refers to a single-stranded DNA or RNA fragment that can bind to a target sequence and be used to detect a complementary nucleic acid sequence. In some embodiments, the probe is a FISH probe. As used in the present invention, the term "fluorescence in situ hybridization (FISH)" is a technique that can be used to identify and analyze the presence of specific genes at precise locations. FISH detection is based on the principle of complementary nucleic acid base pairing. When the DNA molecule to be detected and the nucleic acid probe used are homologous and complementary, the two undergo denaturation-annealing-renaturation to form a hybrid of the target DNA and the nucleic acid probe. A reporter molecule, such as biotin or digoxigenin, is typically labeled on the nucleic acid probe. The immunochemical reaction between the reporter molecule and a specific avidin labeled with fluorescein is utilized to perform qualitative, quantitative, or relative positional analysis of the DNA to be detected through fluorescence microscopy. FISH technology can be used to detect gene amplification, deletion, and rearrangement, such as for the chromosomal localization of known genes or sequences, the study of uncloned genes or genetic markers, and chromosomal aberrations. "FISH probe" refers to a nucleic acid probe that is homologous and complementary to the DNA molecule to be detected and used in fluorescence in situ hybridization technology.

[0058] As used herein, the terms "negative reference slide" and "negative reference" are used interchangeably.

[0059] Specific technical solutions

[0060] In one aspect, the present disclosure provides a kit for detecting human chromosomal abnormalities, wherein the chromosomal loci detected include at least one of 3p, 3q, 10cen (10p11.1) or 10q, and the detection kit contains a fluorescent in situ hybridization probe set for amplifying the chromosomal loci.

[0061] In an optional embodiment, the site targeted by the probe group includes at least one site among Chr3: 40.2-41.2 Mb, Chr3: 195.6-197.3 Mb, Chr10: 37.7-39.5 Mb or Chr10: 78.9-79.7 Mb.

[0062] In an optional embodiment, the chromosomal locus includes at least one of 3p22.1, 3q29, 10cen (10p11.1) or 10q22.3.

[0063] In an optional embodiment, the sites targeted by the probe group include at least one of 40.2 to 41.2 Mb of chromosome site 3p22.1, 195.6 to 197.3 Mb of chromosome site 3q29, 37.7 to 39.5 Mb of chromosome site 10cen (10p11.1), or 78.9 to 79.7 Mb of chromosome site 10q22.3.

[0064] In an optional embodiment, the target fragment of the probe group for detecting chromosome locus 3p22.1 includes Chr3:40,281,947~Chr3:41,223,565, the target fragment of the probe group for detecting chromosome locus 3q29 includes Chr3:195,692,486~197,328,281, the target fragment of the probe group for detecting chromosome locus 10cen (10p11.1) includes Chr10:37,709,476~39,201,629, and the target fragment of the probe group for detecting chromosome 10q22.3 includes Chr10:78,908,277~Chr10:79,990,274.

[0065] Furthermore, the target fragment of the probe group for detecting chromosome locus 3p22.1 is selected from at least one of Chr3:40,281,947-40,476,764, Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255 or Chr3:40,382,551-40,613,653; the target fragment of the probe group for detecting chromosome locus 3q29 is Chr3:195,692,486-195,894,522; the target fragment of the probe group for detecting chromosome locus 10cen (10p11.1) ... The target fragment of the probe group is Chr10:37,709,476~38,214,862, preferably at least one of Chr10:37,709,476~37,861,144, Chr10:37,861,252~38,004,303 or Chr10:38,007,581~38,214,862; the target fragment of the probe group for detecting chromosome site 10q22.3 is selected from at least one of Chr10:79,491,820~79,670,444 or Chr10:79,499,153~79,703,867.

[0066] In an optional embodiment, the detection kit contains only any one of the following fluorescence in situ hybridization probe sets (a) to (d):

[0067] (a) The fluorescent in situ hybridization probe set is used to amplify at least one of Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255, or Chr3:40,382,551-40,613,653;

[0068] (b) The fluorescent in situ hybridization probe set is used to amplify Chr3:195,692,486-195,894,522;

[0069] (c) the fluorescent in situ hybridization probe set is used to amplify the combination of Chr10:37,709,476-37,861,144, Chr10:37,861,252-38,004,303 and Chr10:38,007,581-38,214,862;

[0070] (d) The fluorescent in situ hybridization probe set is used to amplify at least one of Chr10:79,491,820-79,670,444 or Chr10:79,499,153-79,703,867.

[0071] In another optional embodiment, the fluorescent in situ hybridization probe set of the detection kit consists of the fluorescent in situ hybridization probe sets shown in the following (a) to (d):

[0072] (a) The fluorescent in situ hybridization probe set is used to amplify at least one of Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255, or Chr3:40,382,551-40,613,653;

[0073] (b) The fluorescent in situ hybridization probe set is used to amplify Chr3:195,692,486-195,894,522;

[0074] (c) the fluorescent in situ hybridization probe set is used to amplify the combination of Chr10:37,709,476-37,861,144, Chr10:37,861,252-38,004,303 and Chr10:38,007,581-38,214,862;

[0075] (d) The fluorescent in situ hybridization probe set is used to amplify at least one of Chr10:79,491,820-79,670,444 or Chr10:79,499,153-79,703,867.

[0076] In an optional embodiment, the detection kit further includes a sample collection device and consumables; the sample collected by the sample collection device is from blood, saliva, urine, pleural effusion or peritoneal effusion.

[0077] Furthermore, the sample from blood is a gradient separation sample of peripheral blood mononuclear cells.

[0078] In an optional embodiment, the detection kit further comprises an auxiliary detection reagent;

[0079] The auxiliary detection reagent includes at least one of a digestion solution, a cleaning solution, a blood sample preservation solution, a Ficoll separation solution or an organic reagent for FISH hybridization.

[0080] The digestion solution includes digestive enzymes such as K proteinase, pepsin, trypsin, etc. The staining solution includes nucleic acid counterstaining solution such as DAPI (4',6-diamidino-2-phenylindole). The elution solution can be SSC eluent solution or other conventional eluents.

[0081] The second aspect of the present disclosure provides a method for preparing the detection kit described in the first aspect, the preparation method comprising preparing one or more BAC strains containing a target sequence at at least one site in human chromosome 3p, 3q, 10cen (10p11.1) or 10q, extracting DNA from the BAC strains, randomly fragmenting the extracted DNA using a transposase, and simultaneously adding transposase recognition sequences to both ends of the obtained random fragments, and then using the random fragments as amplification templates and sequences targeting the transposase recognition sequences as amplification primers, amplifying in an amplification system with a ratio of dUTP to dTTP+dUTP of 50% to 100%, to obtain a random probe composition targeting the target sequence; the transposase recognition sequence comprises a transposase; The invention relates to a random probe composition comprising an ME sequence and a linker sequence connected to the 5' end of the ME sequence of the transposase; the number of base A in the linker sequence is 4 to 20, and the number of dUTP bases inserted in the amplification step is adjusted by adjusting the content of base A in the linker sequence without affecting the efficiency of the transposase and the amplification efficiency; the dUTP in the random probe composition is also labeled, and the labeling method includes: adding the labeled dUTP to the amplification system, and labeling the dUTP in the random probe composition as the amplification reaction proceeds; or, labeling the dUTP in the obtained random probe composition during the amplification reaction or after the amplification is completed; the label is a fluorescent group label; and the fluorescent group is selected from fluorescein dyes, rhodamine dyes or cyanine dyes.

[0082] The third aspect of the present disclosure provides the use of the detection kit described in the aforementioned embodiment in the preparation of cancer diagnosis and / or prognosis products. Wherein, the cancer is preferably lung cancer, breast cancer, intestinal cancer, esophageal cancer, bladder cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, cervical cancer or prostate cancer. Wherein, the diagnosis and / or prognosis method includes isolating and obtaining a cell sample from the sample to be diagnosed and / or the prognosis sample, using the detection kit described in any one of the aforementioned embodiments or the probe group in the kit prepared by the preparation method described in the aforementioned embodiment to hybridize with the obtained cell sample, detecting the hybridization signal and outputting the chromosome abnormality result of the cell sample.

[0083] A fourth aspect of the present disclosure provides a method for detecting cells with chromosomal abnormalities, comprising the following steps:

[0084] (1) Blood sample collection: Collect blood samples from subjects;

[0085] (2) Preparation of mononuclear cell suspension: isolating mononuclear cells from the blood sample collected in step (1) to obtain a mononuclear cell suspension;

[0086] (3) Probe hybridization: hybridizing the mononuclear cell suspension obtained in step (2) with the probe set in the aforementioned kit to obtain a hybridization sample;

[0087] (4) Image acquisition: performing imaging analysis on the hybridization sample obtained in step (3) to obtain an image of the hybridization sample;

[0088] (5) Image analysis: Analyze the image of the hybridization sample obtained in step (4), identify the signal distribution of cells with chromosomal abnormalities, and determine the chromosomal abnormal cells, wherein the signal distribution of cells with chromosomal abnormalities is manifested as an increase or loss of chromosomal sites.

[0089] In an optional embodiment, in step (3), the mononuclear cell suspension obtained in step (2) is first dropped onto a glass slide, and then the probe group in the kit is added to the glass slide to allow the mononuclear cell suspension to hybridize with the probe group.

[0090] In an optional embodiment, step (5) image analysis also includes: obtaining the total number of scanned cells and the number of chromosomal abnormal cells, calculating the ratio of the number of chromosomal abnormal cells to the total number of scanned cells, and comparing the ratio with a preset threshold value; if the ratio is greater than the threshold value, the subject is considered positive; if the ratio is less than or equal to the threshold value, the subject is considered negative.

[0091] The embodiments of the present invention will be described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, if specific techniques or conditions are not specified, they are based on the techniques or conditions described in the literature in this area (e.g., with reference to "Molecular Cloning Experiment Guide" by J. Sambrook et al., translated by Huang Peitang et al., 3rd edition, Science Press) or according to product specifications. Reagents or instruments used that do not specify the manufacturer are all conventional products that can be purchased commercially.

[0092] Example 1: BAC library construction

[0093] The reference genome used in this example is the human reference genome GRCh38 / hg38.

[0094] 1.1 Construction of a BAC within the 40.2–41.2 Mb region of 3p22.1

[0095] Taking chromosome locus 3p22.1 as an example, the 40.2-41.2 Mb range of chromosome locus 3p22.1 was used as the target of the probe set to construct the corresponding BAC. The specific steps are as follows:

[0096] DNA isolated from a haploid cell line (CHM1htert) or healthy human leukocytes was used as starting material. The inserted DNA was digested with EcoRI restriction endonuclease and EcoRI methylase, and size fractionated by pulsed-field electrophoresis. DNA fragments of appropriate size were cloned into the pBACGK1 vector between two EcoRI sites. The ligated product was then transformed into competent E. coli cells using DH10B (T1-resistant) cells and electroporated. Successfully transfected clones were screened using antibiotic-containing medium. After culturing single clones, BAC DNA was extracted and identified by Sp6 and T7 paired-end sequencing. BACs with inserts within the 40.2-41.2 Mb range of chromosomal locus 3p22.1 (designated 3p22.1-8) were identified. Sequencing results for the selected clones are as follows:

[0097] The results of Sp6 sequencing are:

[0098] The results of T7 sequencing are:

[0099] The sequencing results were aligned using the UCSC BLAT tool. The SP6 sequence and T7 sequence alignment results had the highest matches with chr3:40382456-40383326 (as shown in Figure 1) and chr3:40612865-40613750 (as shown in Figure 2), respectively, indicating that the sequence range of the BAC insertion was between chr3:40382456 and 40613750.

[0100] 1.2 Construction of BACs within the 195.6–197.3 Mb range of 3q29

[0101] Taking chromosome locus 3q29 as an example, the probe set targeting the 195.6–197.3 Mb region of chromosome locus 3q29 was used to construct a corresponding BAC. The construction steps were the same as for 3p22.1-8. Sp6 and T7 paired-end sequencing was used to identify and screen BACs (designated 3q29-6) whose inserts fell within the 195.6–197.3 Mb region of chromosome locus 3q29. The sequencing results of the selected clones are as follows:

[0102] The results of Sp6 sequencing are:

[0103] The results of T7 sequencing are:

[0104] Sequencing results were aligned using the UCSC BLAT tool. The SP6 and T7 sequences aligned most closely to chr3:195893639–195894586 (Figure 3) and chr3:195692483–195693370 (Figure 4), respectively, indicating that the BAC insertion sequence ranged from Chr3:195692483 to 195894586. The chromosome location with the highest BAC paired-end sequencing match was consistent with expectations.

[0105] 1.3 Construction of 10cen BACs ranging from 37.7 to 39.5 Mb

[0106] Taking chromosome locus 10cen as an example, the probe set targeting the 37.7–39.5 Mb region of chromosome 10cen was used to construct a corresponding BAC, following the same construction steps as for 3p22.1-8. Sp6 and T7 paired-end sequencing was used to identify and screen for BACs (designated 10p11.1-1) with inserts within the 37.7–39.5 Mb region of chromosome 10cen. The sequencing results of the selected clones are as follows:

[0107] The results of Sp6 sequencing are:

[0108] The results of T7 sequencing are:

[0109] Sequence alignment using the UCSC BLAT tool revealed the highest matches between the SP6 and T7 sequences, respectively, at chr10:37860075–37861142 (Figure 5) and chr10:37709467–37710496 (Figure 6), indicating that the BAC insertion occurred within the sequence range of Chr10:37709467–37861142. The chromosome location of the BAC paired-end sequencing results with the highest match was consistent with expectations.

[0110] 1.4 Construction of a BAC within the 78.9–79.7 Mb range of 10q22.3 (designated 10q22.3-6)

[0111] Taking chromosome locus 10q22.3 as an example, the probe set targeting the 78.9–79.7 Mb region of chromosome locus 10q22.3 was used to construct a corresponding BAC. The construction steps were the same as for 3p22.1-8. Sp6 and T7 paired-end sequencing was used to identify and screen BACs (designated 10q22.3-6) whose inserts fell within the 78.9–79.7 Mb region of chromosome locus 10q22.3. The sequencing results of the selected clones are as follows:

[0112] The results of Sp6 sequencing are:

[0113] The results of T7 sequencing are:

[0114] Sequencing results were aligned using the UCSC BLAT tool. The SP6 and T7 sequences aligned with chr10:79841257-79842165 and chr10:79703126-79704034, respectively (as shown in Figure 7). The highest match was found in chr10:79499060-79500031 (as shown in Figure 8). Since the BAC insert is approximately 200 kb in length, this suggests that the BAC insert is located between Chr10:79499060-79704034. The chromosome location with the highest match from paired-end BAC sequencing was consistent with expectations.

[0115] Similarly, BAC libraries of other targets can be obtained similarly.

[0116] Example 2: Preparation of random probe sets

[0117] In this example, random probes targeting different BACs were prepared using a variety of different BAC libraries as raw materials, specifically including the following steps:

[0118] (1) Sequence acquisition

[0119] The BAC strain was cultured in LB medium containing chloramphenicol, and the BAC DNA was extracted using a BAC DNA purification kit or a plasmid purification kit (NucleoBond Xtra BAC, manufacturer: MACHEREY-NAGEL).

[0120] (2) Transposome (TTE Mix) Preparation

[0121] A 19 bps ME complementary sequence was designed based on the transposase Tn5. The sequence is as follows:

[0122] ME-A: 5'-AGATGTGTATAAGAGACAG-3' (SEQ ID NO.9)

[0123] ME-B: 5'-phos-CTGTCTCTTATACACATCT-NH2-3'(SEQ ID NO.10)

[0124] ME-A and ME-B were mixed at equal molar concentrations and annealed into double strands, and transposomes were prepared according to the instructions of transposase (TruePrep Tagment Enzyme, Novezan).

[0125] (3) DNA fragmentation and linker addition

[0126] Dilute BAC DNA to 50 ng / μL, prepare the reaction system (Table 1) according to the number of reaction tubes, and react at 55°C for 10 minutes:

[0127] Table 1 DNA fragmentation reaction system

[0128] After the reaction, the fragmented products were purified using magnetic beads or columns.

[0129] (4) PCR amplification

[0130] PCR amplification was performed using fragmented DNA as a template and a sequence that can complementarily bind to the ME sequence as a universal primer (the universal primer can be complementary to the entire ME sequence or partially complementary to it). During the amplification reaction, part or all of the dTTP was replaced with modified dUTP to conduct multiple groups of experiments. During amplification, dUTP was randomly inserted into the nucleic acid sequence, and the ratio of the dUTP to dTTP+dUTP was 50%, 67%, 75%, 80%, and 100%.

[0131] Prepare the reaction system according to the number of reaction tubes (Table 2):

[0132] Table 2 First PCR amplification reaction system

[0133] Run the following program (following the normal reaction program):

[0134] Purify the PCR product and measure the nucleic acid concentration using an ultramicro spectrophotometer. Take 10 ng of the product as a template for the next round of amplification and modification. The reaction system is as follows (Table 3):

[0135] Run the following program (following the normal reaction program):

[0136] Table 3 Second PCR amplification reaction system

[0137] The PCR products were purified to obtain random probes.

[0138] The specific BAC strain target regions and fluorescent labeling colors are shown in Table 4:

[0139] Table 4 BAC strain target regions and fluorescent labeling colors

[0140] Example 3: Screening of 3p BAC and its labeled probes

[0141] 3p BACs were amplified using a library construction method and labeled with Alexa Fluor 594 (red). Labeled probes were hybridized to negative reference slides to test labeling efficacy. Negative reference slides were prepared using mononuclear cells isolated from healthy human leukocytes. Labeled with Alexa Fluor 488 (green), the labeled probes were validated for specificity using metaphase slides (healthy human leukocytes treated with colchicine).

[0142] The BAC marker hybridization results showed that the number of signal points in a single cell of 3p22.1-3 hybridization was greater than 2 (as shown in Figure 9), indicating a specificity problem and excluding this BAC; the hybridization results of the three BACs mixed 3p22.1-1+2+3 (3p22.1-1, 3p22.1-2 and 3p22.1-3) showed that the number of signal points in a single cell was greater than 4, indicating that the positioning of the three BACs was inconsistent; the metaphase chromosome specificity of the BACs except 3p22.1-3 was then verified (co-hybridization with the OGT probe; if the signal points overlapped, it indicated that the positioning was consistent with the OGT probe).

[0143] The specificity results (shown in Figure 10) showed that 3p22.1-1 was not located on chromosome 3 (not on the same chromosome as the OGT probe). 3p22.1-2, 3p22.1-11, and 3p22.1-8 were located in the same region as the OGT probe. The specificity (shown in Figure 10) and hybridization signals of these four BACs met the requirements. 3p22.1-8 had the strongest hybridization signal. The hybridization FISH scanning signal parameters are shown in Table 5:

[0144] Table 5 FISH scanning signal parameters of 3p BAC labeled probe and OGT-3p probe hybridization

[0145] Example 4: Screening of 3q BAC and its labeled probes

[0146] 3q BAC was amplified using the library construction method and labeled with Alexa Fluor 488 dye (green). The labeled probe was hybridized with a negative reference slide to test the labeling effect. The labeled probe was labeled with Alexa Fluor 594 dye (red) and its specificity was verified using a metaphase slide.

[0147] BAC marker hybridization results showed strong hybridization signals for both 3q29-6 and 3q29-1. Specificity results (Figure 11) showed that 3q29-1 and 3q29-6 were located in the same region as the OGT-3q probe, but 3q29-1 had a weak nonspecific signal (Figure 12), suggesting that the sequence may have nonspecific binding to other chromosomal regions. 3q29-6 was located in the same region as the OGT-3q probe, so choosing 3q29-6 as the BAC for 3q29 was more effective. The FISH scanning signal parameters for the 3q BAC marker probe and the OGT-3q probe hybridization are shown in Table 6:

[0148] Table 6 FISH scanning signal parameters of 3q BAC labeled probe and OGT-3q probe hybridization

[0149] Example 5: Screening of 10cen (10p11.1) BAC and its labeled probe set

[0150] During the feasibility study, the labeling effect of the 10cen(10p11.1) BAC (10p11.1-6) was verified, but the signal was weak and did not meet the interpretation requirements. Therefore, five consecutive BACs (10p11.1-1 to 10p11.1-5) were selected for BAC stacking probe labeling testing. By stacking BACs, the probe coverage area was increased, thereby improving the signal strength.

[0151] The 10cen (10p11.1) BAC was amplified using the library construction method and labeled with iFluor 440 dye (blue). The labeled probe was hybridized to a negative reference slide to test labeling efficacy. The labeled probe was labeled with Atto 532 dye (gold) and its specificity was verified using a metaphase slide.

[0152] The BAC labeling results (shown in Figure 13) showed that the 10p11.1-4 and 10p11.1-5 marker probes had many nonspecific signal points, so only the 10p11.1-1, 10p11.1-2, and 10p11.1-3 were stacked for testing. Both the single BAC and the stacked BAC had two signal points, indicating that the three BACs were consistently positioned on the chromosome. Specificity verification results showed that the stacked probes were consistently positioned with the OGT-10cen (10p11.1) probe (shown in Figure 14), and the signal intensity of the three-BAC stacked probe was superior to that of the single BAC probe (shown in Figure 15).

[0153] Example 6: Screening of 10q BAC and its labeled probes

[0154] The 10q BAC was amplified using the library construction method and labeled with Atto 532 dye (gold). The labeled probe was hybridized to a negative reference slide to test labeling efficacy. The probe was labeled with Alexa Fluor 594 dye (red) and its specificity was verified using a metaphase slide.

[0155] The hybridization signals of the probes labeled 10q22.3-1 and 10q22.3-6 both met the interpretation requirements (Figure 16). The specificity verification results (Figure 17) showed that the two BACs were located consistently with the OGT-10q probe and there were no nonspecific signal spots. In addition, the 10q22.3-6 and OGT-10q probes were located consistently in the same sample. The hybridization FISH scanning signal parameters are shown in Table 7:

[0156] Table 7 FISH scanning signal parameters of 10q BAC labeled probe and OGT-10q probe hybridization

[0157] Example 7

[0158] According to the scanning results in Examples 3-6, the clinical performance of the four-color probe combination was set, and the four-color probes were a 3p probe for detecting the marker of the 3p22.1-8 target region, a 3q probe for detecting the marker of the 3q29-6 target region, a 10cen (10p11.1) probe for detecting the superimposed marker target region of 10p11.1-1, 10p11.1-2, and 10p11.1-3, and a 10q probe for detecting the marker of the 10q22.3-6 target region.

[0159] This example is based on a hybridization-on-analyzer detection process for blood samples. To improve hybridization imaging, this example optimizes the FISH probe hybridization process. The optimized sample used in the experiment was a negative reference slide. The fluorescence signal intensity and background intensity of each cell were obtained from imaging analysis using a pathology slide scanner (Duet System, BioView).

[0160] Hybridization of blood samples - The general hybridization process on the machine is as follows:

[0161] (1) Separating the sample to be tested using Ficoll separation solution to obtain a mononuclear cell suspension;

[0162] (2) taking the cell suspension and dropping it onto a glass slide to prepare a slide containing mononuclear cells;

[0163] (3) After the slides were immersed in the digestive solution, they were washed with 1× PBS and then immersed in 1% formaldehyde fixative and 70%, 85%, and 100% graded ethanol for dehydration at room temperature. The slides were allowed to stand until they were completely dry for later use.

[0164] (4) Pipette the probe mixture and slowly add it to the target area of ​​the slide. Immediately cover the target area of ​​the slide with a coverslip and seal the slide along the edge of the coverslip with sealing glue.

[0165] (5) placing the slide prepared in step (4) in a hybridizer and starting the hybridization process, which includes denaturation and hybridization;

[0166] (6) Immerse the hybridized slides in eluent-solution 1 for high-temperature elution (elution 1), then in eluent-solution 2 for elution at room temperature (elution 2), and then remove the slides from the light and dry them for later use;

[0167] (7) Add nucleic acid counterstaining solution to the center of the target area of ​​the slide obtained in step (6), cover with a coverslip and directly use it for imaging analysis using a pathology section scanner (Duet System, BioView).

[0168] When performing an optimization experiment, if the test sample is a negative reference plate slide, steps (1) and (2) in the above-mentioned general hybridization process of hybridization-on-machine can be omitted.

[0169] 7.1 Study on the appropriate working concentration and time of digestive fluid

[0170] Pepsin is used as the digestion solution. Pepsin is a proteolytic enzyme with an optimal pH of 2.0. Under this condition, it can digest proteins surrounding nucleic acids, increasing the chance of probe binding to nucleic acids and improving hybridization efficiency. The enzyme digestion conditions in the reaction system were tested and the optimal conditions were selected based on the quality of the fluorescence in situ hybridization signal.

[0171] (1) Preparation of digestive fluid

[0172] Refer to the instructions for the in situ hybridization protease (Yuezhu Xiebei No. 20190035, Zhuhai Shengmei Biodiagnostic Technology Co., Ltd.) to prepare the digestion solution. When preparing, first preheat the digestion buffer to 37±1°C, then use a pipette of appropriate volume to add 1mL of preheated digestion buffer to the digestion agent; cover the digestion agent lid, invert and mix thoroughly to dissolve, and transfer the solution in the bottle to the digestion buffer; repeat this step until all the digestion agent is completely dissolved and transferred to the digestion buffer. Prepare three digestion solutions with concentrations of 0.25mg / mL, 0.5mg / mL, and 1mg / mL. Place the prepared digestion working solutions of different concentrations at 37±1°C for activation for 15 minutes before use.

[0173] (2) Experimental setup

[0174] Orthogonal experiments were conducted using three digestion solutions of different concentrations at three different enzyme digestion times. The experimental groups are shown in Table 8:

[0175] Table 8 Digestion experiment grouping

[0176] Fluorescence in situ hybridization (FISH) was performed using three negative reference slides for each condition, which were then scanned using a pathology scanner. After scanning, the fluorescence signal intensity and background intensity of the samples under each condition were analyzed. The optimal digestion solution was selected as the working condition.

[0177] (3) Evaluation criteria:

[0178] Qualified: The intensity of the four fluorescent signals must reach level 2 or above, and the proportion of high background cells must be less than 10%.

[0179] Excellent: The intensity of all four fluorescent signals must reach level 3 and the proportion of high background cells must be less than 10%.

[0180] The signal strength levels include: level 1: weak signal; level 2: bright; level 3: very strong.

[0181] The digestion test results are shown in Table 9. When the digestion condition was pepsin 1 mg / mL-10 min, the experimental results were excellent with good repeatability and stability.

[0182] Table 9 Digestion test results

[0183] 7.2 Denaturation conditions

[0184] The DNA in the cell nucleus is denatured and then hybridized with the fluorescent probe to form a "chromosome-specific sequence probe" complex. At the appropriate denaturation temperature and time, the probe binds tightly to the nucleic acid, emitting a bright fluorescent signal. The denaturation conditions in the reaction system are studied to determine the optimal ones.

[0185] (1) Initial screening experiment setup for denaturation conditions:

[0186] Initial screening of denaturation conditions: set three different denaturation temperatures (70, 75, and 80°C) and three different denaturation times (1, 5, and 10 min) for orthogonal experiments. The experimental groups are shown in Table 10:

[0187] Table 10 Denaturation Conditions Initial Screening Experiment Grouping

[0188] Fluorescence in situ hybridization (FISH) was performed using three negative reference samples for each condition, followed by scanning using a pathology slide scanner. After scanning, the fluorescence signal intensity and background intensity of the samples under each condition were analyzed. The optimal condition was selected as the initial denaturation screening condition.

[0189] (2) Denaturation conditions rescreening:

[0190] The optimal denaturation temperature (±3°C) and denaturation time (±2 minutes) were increased and decreased from the initial screening condition. Three negative reference slides were used for fluorescence in situ hybridization (FISH) testing under each condition, followed by scanning using a pathology scanner. After scanning, the fluorescence signal intensity and background intensity of the samples under each condition were analyzed. The optimal denaturation condition was selected for rescreening.

[0191] (3) Confirmation of denaturation conditions:

[0192] Based on the optimal denaturation conditions identified in the rescreening, perform fluorescence in situ hybridization (FISH) using three negative reference samples. The samples are then scanned using a pathology scanner. After scanning, analyze the sample fluorescence signal intensity and background intensity. Finally, determine the final denaturation conditions.

[0193] (4) Evaluation criteria:

[0194] Qualified: The intensity of the four fluorescent signals must reach level 2 or above, and the proportion of high background cells must be less than 10%.

[0195] Excellent: The intensity of all four fluorescent signals must reach level 3 and the proportion of high background cells must be less than 10%.

[0196] The signal strength levels include: level 1: weak signal; level 2: bright; level 3: very strong.

[0197] (5) Denaturation conditions experimental results and analysis

[0198] The results of the initial denaturation screening experiment are shown in Table 11. Both 75°C for 5 minutes and 75°C for 10 minutes resulted in acceptable and excellent results, with no significant difference between the two conditions. Considering common FISH methods and time constraints, 75°C for 5 minutes was selected for further denaturation confirmation.

[0199] Table 11 Results of initial screening experiment on denaturation conditions

[0200] The results of the denaturation condition re-screening experiment are shown in Table 12. Among them, the experimental results under the condition of 78°C-5min were optimal, and this condition was selected as the subsequent denaturation confirmation condition.

[0201] Table 12 Denaturation condition rescreening test results

[0202] The results of the denaturation condition confirmation experiment are shown in Table 13. Among them, after the denaturation condition of 78°C-5min was confirmed three times, the experimental results were all excellent, with good repeatability and stability.

[0203] Table 13 Denaturation condition confirmation experiment results

[0204] In summary, the denaturation condition was selected as 78°C-5min.

[0205] 7.3 Hybridization Temperature and Time

[0206] Hybridization temperature is a key factor in hybridization success. At the appropriate temperature, the probe binds more tightly to the nucleic acid sequence. Excessively high or low temperatures can affect the hybridization efficiency between the probe and target DNA. Hybridization time also significantly influences the results. Too short a hybridization time can result in incomplete hybridization, while too long a time can increase nonspecific hybridization. Most DNA probe hybridization times are typically set between 16 and 20 hours.

[0207] In this example, the hybridization conditions in the reaction system were tested, and the optimal hybridization conditions were screened out based on the quality of the fluorescence in situ hybridization signal.

[0208] (1) Hybridization temperature and time conditions for initial screening experiment settings

[0209] Initial screening of hybridization temperature and time conditions: set three different hybridization temperatures and two different hybridization times for orthogonal experiments (Table 14):

[0210] Table 14 Hybridization temperature and time conditions initial screening experiment grouping

[0211] Fluorescence in situ hybridization (FISH) was performed using three negative reference samples for each condition, followed by scanning using a pathology slide scanner. After scanning, the fluorescence signal intensity and background intensity of the samples under each condition were analyzed. The optimal hybridization temperature and time conditions were selected for initial screening.

[0212] (2) Hybridization temperature and time conditions rescreening:

[0213] The optimal hybridization temperature for the initial screening was increased or decreased by 2°C (±2°C). One intermediate point was added within the optimal hybridization time range. Three negative reference samples were used for each condition for fluorescence in situ hybridization, and then scanned using a pathology slide scanner. After scanning, the fluorescence signal intensity and background intensity of the samples under each condition were analyzed. The optimal hybridization temperature and time conditions were selected for rescreening.

[0214] (3) Confirmation of hybridization temperature and time conditions:

[0215] Based on the optimal hybridization temperature and time conditions found in the rescreening, perform fluorescence in situ hybridization (FISH) using three negative reference samples. The samples are then scanned using a pathology slide scanner. After scanning, analyze the sample fluorescence signal intensity and background intensity. The final hybridization temperature and time conditions are determined.

[0216] (4) Evaluation criteria

[0217] Qualified: The intensity of the four fluorescent signals must reach level 2 or above, and the proportion of high background cells must be less than 10%.

[0218] Excellent: The intensity of all four fluorescent signals must reach level 3 and the proportion of high background cells must be less than 10%.

[0219] The signal strength levels include: level 1: weak signal; level 2: bright; level 3: very strong.

[0220] (5) Hybridization temperature and time experimental results and analysis

[0221] The results of the initial screening experiment on hybridization temperature and time are shown in Table 15. Among them, the test results of 34℃-24h, 39℃-16h, 39℃-24h, and 44℃-16h were all above the qualified level, and 16-24h at 39℃ was more stable overall. Therefore, 39℃ was selected as the optimal hybridization temperature, and 16-24h was selected as the hybridization time for the next re-screening.

[0222] Table 15 Hybridization temperature and time conditions preliminary screening test results

[0223] The results of the hybridization temperature and time rescreening experiments are shown in Table 16. The four-color fluorescence signal intensity of each sample in each of the nine experimental groups met the standard of level 2 or higher, and the high background ratio was less than 10%, meeting the expected standards. Based on the expected standards, the conditions with higher levels were preferentially selected as the optimal conditions. From the three experimental groups of hybridization temperatures mentioned above, 39°C / 16-24h, which showed higher and more stable four-color fluorescence signal intensity levels, was selected as the optimal hybridization condition for reconfirmation.

[0224] Table 16 Hybridization temperature and time conditions rescreening experimental results

[0225] The results of the hybridization temperature and time condition confirmation experiment are shown in Table 17. Among them, the results of the three confirmations all met the expected standards and were stably repeatable. Therefore, the optimal hybridization condition was 39°C / 16h~24h.

[0226] Table 17 Hybridization temperature and time conditions confirmation experiment results

[0227] In summary, the hybridization temperature and time conditions were selected as 39°C and 16h to 24h.

[0228] 7.4 Study on High-Temperature Elution Conditions

[0229] The main components of the eluent solution 1 in Elution 1 are 0.4×SSC / 0.3% NP-40. Under high temperature and low salt conditions, this increases the stringency of nucleic acid chains, enhancing repulsion between RNA and DNA, thereby reducing nonspecific hybridization signals. The Elution 1 conditions in this reaction system were studied and the optimal high-temperature Elution 1 condition was identified based on the quality of the fluorescence in situ hybridization signal.

[0230] (1) High temperature elution 1 condition initial screening experiment setup

[0231] High-Temperature Elution 1 Condition Initial Screening: Perform an orthogonal experiment using three different elution temperatures and three different elution times. See Table 18 for experimental groupings. Perform fluorescence in situ hybridization (FISH) using three negative reference samples for each condition. The samples are then scanned using a pathology slide scanner. After scanning, analyze the fluorescence signal intensity and background intensity of the samples under each condition. Select the optimal condition for the High-Temperature Elution 1 initial screening.

[0232] Table 18 High temperature elution 1 condition experimental grouping

[0233] (2) High temperature elution 1 condition rescreening:

[0234] The optimal high-temperature elution temperature for the initial screening was increased and decreased by 2°C (±2°C). Due to the shorter elution times, the rescreening was repeated at the three elution times (1 minute, 2 minutes, and 3 minutes) used in the initial screening. Fluorescence in situ hybridization was performed using three negative reference samples for each condition, and then scanned using a pathology slide scanner. After scanning, the fluorescence signal intensity and background intensity of the samples under each condition were analyzed. The optimal high-temperature elution condition was selected as the rescreening condition.

[0235] (3) Confirmation of high temperature elution conditions 1:

[0236] Based on the optimal high-temperature elution 1 conditions found during rescreening, perform fluorescence in situ hybridization (FISH) using three negative reference samples. The samples are then scanned using a pathology slide scanner. After scanning, analyze the sample fluorescence signal intensity and background intensity. Finally, determine the final high-temperature elution 1 conditions.

[0237] (4) Acceptable standards

[0238] Qualified: The intensity of the four fluorescent signals must reach level 2 or above, and the proportion of high background cells must be less than 10%.

[0239] Excellent: The intensity of all four fluorescent signals must reach level 3 and the proportion of high background cells must be less than 10%.

[0240] The signal strength levels include: level 1: weak signal; level 2: bright; level 3: very strong.

[0241] (5) Experimental results and analysis of high temperature elution condition 1

[0242] The results of the initial screening experiment under high-temperature elution 1 conditions are shown in Table 19. Among them, the blue signal intensity at 78°C-3min was unqualified at level 1, and the other 8 groups of experiments met the requirements. According to the requirement of selecting the best and more stable conditions as the screening criteria, 78°C and 73°C were excluded from the above 8 groups of experiments, and 68°C was preferentially selected as the most suitable elution 1 temperature for the next rescreening.

[0243] Table 19 High temperature elution 1 condition initial screening test results

[0244] The results of the high-temperature elution 1 rescreening experiment are shown in Table 20. The sample slide signals at various times under the 66°C condition failed to meet the requirements. Because the elution temperature and time within a certain range can meet the requirements of high signal intensity and low background, elution at 70°C for 2 minutes (optimal for a very stable blue signal) was selected within the validation conditions for experimental stability confirmation.

[0245] Table 20 High temperature elution 1 condition rescreening experimental results

[0246] The results of the high-temperature elution condition 1 confirmation experiment are shown in Table 21. Among them, after the elution condition 1 was confirmed three times at 70℃-2min, the experimental results were all qualified or excellent, with good repeatability and stability.

[0247] Table 21 High temperature elution 1 condition confirmation experimental results

[0248] In summary, the condition for high temperature elution 1 is elution at 70°C for 2 minutes.

[0249] 7.5 Study on elution conditions at room temperature

[0250] The main components of the eluent in Elution 2, Solution 2, are 2×SSC / 0.1% NP-40. Under the low-temperature, high-salt elution conditions, this solution removes most nonspecifically bound probes. The Elution 2 conditions in the reaction system were tested and the optimal Elution 2 conditions were selected based on the quality of the fluorescence in situ hybridization signal.

[0251] (1) Room temperature elution 2-condition initial screening experiment setup

[0252] Initial screening using room temperature elution conditions (2): Set three different elution times. See Table 22 for experimental settings. Since elution temperature 2 is room temperature and there is no clear value, the room temperature condition (1) was used as an orthogonal experiment. Fluorescence in situ hybridization (FISH) was performed using three negative reference samples for each condition, and then scanned using a pathology slide scanner. After scanning, analyze the fluorescence signal intensity and background intensity of the samples under each condition. Select the optimal condition as the initial screening condition.

[0253] Table 22 Room temperature elution 2 condition initial screening experiment grouping

[0254] (2)Condition confirmation:

[0255] Based on the optimal room temperature elution 2 conditions from the initial screening, perform fluorescence in situ hybridization (FISH) using three negative reference samples. The samples are then scanned using a pathology slide scanner. After scanning, analyze the sample fluorescence signal intensity and background intensity. The final room temperature elution 2 conditions are determined.

[0256] (3) Acceptable standards

[0257] Qualified: The intensity of the four fluorescent signals must reach level 2 or above, and the proportion of high background cells must be less than 10%.

[0258] Excellent: The intensity of all four fluorescent signals must reach level 3 and the proportion of high background cells must be less than 10%.

[0259] The signal strength levels include: level 1: weak signal; level 2: bright; level 3: very strong.

[0260] (4) Experimental results and analysis of room temperature elution conditions 2

[0261] The results of the preliminary screening experiment under the two conditions of room temperature elution are shown in Table 23. It can be seen that the effect of room temperature elution for 3 minutes is the best, so room temperature -3 minutes is preferred for repeated confirmation.

[0262] Table 23 Room temperature elution 2 condition initial screening test results

[0263] The results of the room temperature elution condition 2 confirmation experiment are shown in Table 24. It can be seen that after the elution condition of room temperature-3 min was confirmed three times, the experimental results were all qualified or excellent, and the repeatability and stability were good.

[0264] Table 24 Room temperature elution 2 condition confirmation experiment results

[0265] In summary, the elution condition was selected to be elution at room temperature for 3 minutes.

[0266] Example 8 Clinical Sample Experiment

[0267] 8.1 Sample Information

[0268] This example studied 130 samples stored by the applicant, all of which had clear diagnostic results, including non-surgical biopsy, surgical biopsy, or physical examination to determine whether they were benign. The demographic information of the samples is shown in Table 25:

[0269] Table 25 Demographic information of the sample

[0270] According to the 8th edition of the TNM staging system of the International Union Against Cancer and the American Joint Committee on Cancer, the pathology of 130 samples was classified. The classification results are shown in Table 26. When the maximum diameter of the primary tumor is less than or equal to 30 mm, it is stage IA.

[0271] Table 26 Pathological classification

[0272] 8.2 Experimental Procedure

[0273] The above samples were tested according to the following process:

[0274] (1) Collect blood samples from subjects and store them in cell preservation medium;

[0275] (2) Separate blood samples using the Ficoll method to obtain a mononuclear cell suspension;

[0276] (3) Take 20 μL of the cell suspension and drop it onto a glass slide to obtain a slide containing mononuclear cells;

[0277] (4) Immerse the slides in 1 mg / mL pepsin digestion solution for 10 minutes, wash with 1× PBS, immerse in 1% formaldehyde fixative for 5 minutes, wash, and then immerse in 70%, 85%, and 100% graded ethanol in sequence. Dehydrate at room temperature for 1 minute each, and let stand until completely dry for later use.

[0278] (5) Pipette 6 μL of the probe mixture (BAC-labeled probes prepared in Examples 3 to 6) and slowly add it to the target area of ​​the slide. Immediately cover the target area of ​​the slide with a coverslip and seal the slide along the edge of the coverslip with sealing glue.

[0279] (6) Place the slide from step 5 in the hybridizer and start the hybridization program: denaturation at 78 ± 1°C for 5 minutes and hybridization at 39 ± 1°C for 16 to 24 hours;

[0280] (7) Immerse the slide in 0.4×SSC / 0.3% NP-40 eluent-solution 1 preheated to 70±1°C for 2 minutes, remove the slide, use dust-free paper to absorb excess liquid at the bottom of the slide, and then immerse it in 2×SSC / 0.1% NP-40 eluent-solution 2 at room temperature for 3 minutes, then remove the slide and dry it in the dark for later use;

[0281] (8) Add 6 μL of nucleic acid counterstaining solution to the center of the target area of ​​the slide obtained in step (7), cover with a coverslip and directly use it for imaging analysis using a pathology section scanner (Duet System, BioView);

[0282] (9) Identify the first 500, 5000, 10000, 30000 and 50000 (including more than 50000) cells in the imaging results, classify the scanned cell signals, and record the number of cell scans and the number of abnormal cells in different samples.

[0283] (10) Identification criteria:

[0284] ① The signal distribution of cells with chromosomal abnormalities is manifested as an increase or loss of chromosomal sites.

[0285] ② Cells that were detected to have multiple chromosome gains (i.e., two probes for chromosome 3 had three or more signals or two probes for chromosome 10 had three or more signals) were defined as circulating abnormal cells (CAC).

[0286] ③ If there are one or more CACs, the sample is judged as an abnormal sample.

[0287] The first 500, 5,000, 10,000, 30,000, and 50,000 cells were scanned to detect circulating abnormal cells (CAC) at different cell counts. The AUC values ​​were calculated by comparing the FISH data from histopathological sections, as shown in Figure 18, where A to E represent scans of 500, 5,000, 10,000, 30,000, and 50,000 cells, respectively. It can be seen that good detection performance was achieved when scanning 500, 5,000, 10,000, 30,000, and 50,000 cells. The detection probe with 50,000 or more cells showed the best performance, achieving the best AUC.

[0288] 8.3 Calculation of the optimal positive judgment value

[0289] The number of cell scans was determined to be 50,000 or more, and the positive cell ratios of 130 applicants' own samples were counted. Combined with the FISH data from their pathological sections, the Youden Index was calculated for different cutoff values ​​of the positive cell ratio. The specific results are shown in Figure 19. The Youden Index was explored for cutoff values ​​of 0.00038051 to 0.000995096. Among them, the Youden Index was greater than 0.1 for the cutoff value of 0.000053632 to 0.000311579, and the Youden Index was greater than 0.5 for the cutoff value of 0.000177854 to 0.000274977, indicating that this protocol can effectively distinguish between negative and positive patients. When the cut-off value is greater than 0.000183 (for example, when the number of cell scans is 50,000, the number of positive cells is greater than 9), the Youden index is the highest, reaching 0.6282. At this cut-off value, the sensitivity of this scheme is 84.69%, and the specificity is 78.12%. The ROC curve is shown in Figure 20, and the AUC value is as high as 0.866, indicating that when the cut-off value is greater than 0.000183, the effect of distinguishing between positive and negative patients is best.

[0290] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention.

Claims

1. A human chromosome abnormality detection kit, wherein: The chromosomal loci to be detected include at least one of 3p, 3q, 10cen or 10q, and the detection kit contains a fluorescent in situ hybridization probe set for amplifying the chromosomal loci.

2. The detection kit according to claim 1, wherein The sites targeted by the probe group include at least one site among Chr3: 40.2-41.2 Mb, Chr3: 195.6-197.3 Mb, Chr10: 37.7-39.5 Mb or Chr10: 78.9-79.7 Mb.

3. The detection kit according to claim 1, wherein The chromosomal loci include at least one of 3p22.1, 3q29, 10cen or 10q22.

3.

4. The detection kit according to claim 3, wherein The target fragments of the probe group for detecting the chromosome locus 3p22.1 include Chr3:40,281,947~Chr3:41,223,565, the target fragments of the probe group for detecting the chromosome locus 3q29 include Chr3:195,692,486~197,328,281, the target fragments of the probe group for detecting the chromosome locus 10cen include Chr10:37,709,476~39,201,629, and the target fragments of the probe group for detecting the chromosome locus 10q22.3 include Chr10:78,908,277~Chr10:79,990,274.

5. The detection kit according to claim 4, wherein The target fragment of the probe set for detecting chromosome locus 3p22.1 is selected from at least one of Chr3:40,281,947-40,476,764, Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255 or Chr3:40,382,551-40,613,653; The target fragment of the probe set for detecting chromosome locus 3q29 is Chr3:195,692,486~195,894,522; The target fragment of the probe set for detecting chromosome locus 10cen is at least one of Chr10:37,709,476-37,861,144, Chr10:37,861,252-38,004,303 or Chr10:38,007,581-38,214,862; The target fragment of the probe group for detecting chromosome locus 10q22.3 is selected from at least one of Chr10:79,491,820-79,670,444 or Chr10:79,499,153-79,703,867.

6. The detection kit according to claim 1, wherein The detection kit contains only any one of the following fluorescence in situ hybridization probe sets (a) to (d): (a) The fluorescent in situ hybridization probe set is used to amplify at least one of Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255, or Chr3:40,382,551-40,613,653; (b) The fluorescent in situ hybridization probe set is used to amplify Chr3:195,692,486-195,894,522; (c) the fluorescent in situ hybridization probe set is used to amplify the combination of Chr10:37,709,476-37,861,144, Chr10:37,861,252-38,004,303 and Chr10:38,007,581-38,214,862; (d) The fluorescent in situ hybridization probe set is used to amplify at least one of Chr10:79,491,820-79,670,444 or Chr10:79,499,153-79,703,867.

7. The detection kit according to claim 1, wherein The fluorescent in situ hybridization probe set of the detection kit consists of the fluorescent in situ hybridization probe sets shown in the following (a) to (d): (a) The fluorescent in situ hybridization probe set is used to amplify at least one of Chr3:40,335,808-40,532,606, Chr3:40,331,951-40,518,255, or Chr3:40,382,551-40,613,653; (b) The fluorescent in situ hybridization probe set is used to amplify Chr3:195,692,486-195,894,522; (c) the fluorescent in situ hybridization probe set is used to amplify the combination of Chr10:37,709,476-37,861,144, Chr10:37,861,252-38,004,303 and Chr10:38,007,581-38,214,862; (d) The fluorescent in situ hybridization probe set is used to amplify at least one of Chr10:79,491,820-79,670,444 or Chr10:79,499,153-79,703,867.

8. The detection kit according to any one of claims 1 to 7, wherein The detection kit also includes a sample collection device and consumables; the sample collected by the sample collection device comes from blood, saliva, urine, pleural effusion or peritoneal effusion.

9. The detection kit according to claim 8, wherein The blood sample is a gradient separation sample of peripheral blood mononuclear cells.

10. The detection kit according to claim 8, wherein The detection kit further comprises at least one of a digestion solution, a cleaning solution, a blood sample preservation solution, a Ficoll separation solution or an organic reagent for FISH hybridization.

11. The method for preparing the detection kit according to any one of claims 1 to 10, wherein: The preparation method comprises preparing one or more BAC strains containing at least one target sequence at a locus in human chromosome 3p, 3q, 10cen or 10q, extracting DNA from the BAC strains, randomly fragmenting the extracted DNA using a transposase, and simultaneously adding transposase recognition sequences to both ends of the obtained random fragments. Then, using the random fragments as amplification templates and sequences targeting the transposase recognition sequences as amplification primers, amplification is performed in an amplification system with a ratio of dUTP to dTTP+dUTP of 50% to 100%, thereby obtaining a random probe composition targeting the target sequence; the transposase recognition sequence comprises an ME sequence of the transposase and a 5' end of the ME sequence connected to the transposase. The invention relates to a linker sequence having a plurality of bases A at the end; the number of base A in the linker sequence is 4 to 20, and the number of dUTP bases inserted in the amplification step is adjusted by adjusting the content of base A in the linker sequence without affecting the efficiency of the transposase and the amplification efficiency; the dUTP in the random probe composition is also labeled, and the labeling method includes: adding the labeled dUTP to the amplification system, and labeling the dUTP in the random probe composition as the amplification reaction proceeds; or, labeling the dUTP in the obtained random probe composition during or after the amplification reaction; the label is a fluorescent group label; and the fluorescent group is selected from fluorescein dyes, rhodamine dyes, or cyanine dyes.

12. Use of the detection kit according to any one of claims 1 to 10 or the kit prepared by the preparation method according to claim 11 in the preparation of cancer diagnosis and / or prognosis products; The method for diagnosis and / or prognosis comprises isolating a cell sample from a sample to be diagnosed and / or a sample to be prognosed, hybridizing the obtained cell sample with a probe set in the detection kit according to any one of claims 1 to 10 or a kit prepared by the preparation method according to claim 11, detecting the hybridization signal, and outputting a result of chromosomal abnormality of the cell sample.

13. The use according to claim 12, wherein The cancer includes lung cancer, breast cancer, intestinal cancer, esophageal cancer, bladder cancer, liver cancer, stomach cancer, pancreatic cancer, ovarian cancer, cervical cancer or prostate cancer.

14. A method for detecting cells with chromosomal abnormalities, comprising the following steps: (1) Blood sample collection: Collect blood samples from subjects; (2) Preparation of mononuclear cell suspension: isolating mononuclear cells from the blood sample collected in step (1) to obtain a mononuclear cell suspension; (3) Probe hybridization: hybridizing the mononuclear cell suspension obtained in step (2) with the probe set in the kit according to any one of claims 1 to 10 to obtain a hybridization sample; (4) Image acquisition: performing imaging analysis on the hybridization sample obtained in step (3) to obtain an image of the hybridization sample; (5) Image analysis: Analyze the image of the hybridization sample obtained in step (4), identify the signal distribution of cells with chromosomal abnormalities, and determine the chromosomal abnormal cells, wherein the signal distribution of cells with chromosomal abnormalities is manifested as an increase or loss of chromosomal sites.

15. The method according to claim 14, wherein In step (3), the mononuclear cell suspension obtained in step (2) is first dropped onto a glass slide, and then the probe set in the kit is added to the glass slide to allow the mononuclear cell suspension to hybridize with the probe set.

16. The method according to claim 14 or 15, wherein: Step (5) image analysis also includes: obtaining the total number of scanned cells and the number of chromosomal abnormal cells, calculating the ratio of the number of chromosomal abnormal cells to the total number of scanned cells, and comparing the ratio with a preset threshold value. If the ratio is greater than the threshold value, the subject is considered positive; if the ratio is less than or equal to the threshold value, the subject is considered negative.

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