CN117448456B

CN117448456B - Molecular target, probe combination and kit for identifying benign and malignant salivary gland tumor and application of molecular target and probe combination

Info

Publication number: CN117448456B
Application number: CN202311798090.9A
Authority: CN
Inventors: 吴艳霞; 雷顺媚; 陈香华; 陈绍宇
Original assignee: Guangzhou Lbp Medicine Science And Technology Co ltd; Affiliated Hospital of Guangdong Medical University
Current assignee: Guangzhou Lbp Medicine Science And Technology Co ltd; Affiliated Hospital of Guangdong Medical University
Priority date: 2023-12-26
Filing date: 2023-12-26
Publication date: 2024-04-19
Anticipated expiration: 2043-12-26
Also published as: CN117448456A

Abstract

The application discloses a molecular target, a probe combination, a kit and application thereof for identifying benign and malignant salivary gland tumors. The application provides a new molecular target for identifying benign and malignant salivary gland tumor, and assists doctors to make diagnosis by detecting abnormal conditions such as aneuploidy of chromosome 3 and deletion of p16 gene in salivary gland tumor tissue, thereby being beneficial to defining the subsequent treatment scheme of patients and reducing excessive medical treatment of salivary gland tumor patients; the application also provides a probe combination and a kit for identifying benign and malignant salivary gland tumors based on the fluorescence in situ hybridization technology, and the kit provided by the application can improve the accuracy of identifying benign and malignant salivary gland tumors, and the result is easy to judge.

Description

Molecular target, probe combination and kit for identifying benign and malignant salivary gland tumor and application of molecular target and probe combination

Technical Field

The invention relates to the field of molecular diagnosis, in particular to a molecular target, a probe combination, a kit and application thereof for identifying benign and malignant salivary gland tumors.

Background

Salivary gland tumor is one of common tumors of head and neck, and accounts for about 2.3% of human tumor, is a tumor with high morbidity and organ specificity in histological structure, and has the rising trend year by year in recent years, and the relative survival rates of local, regional and distant lesions in 5 years are 95%, 69% and 44% respectively. The salivary gland tumors are in 43 major categories, and the pathological types are quite complex. Most large salivary gland tumors (70% -80%) are benign, such as polymorphic adenomas; malignant tumors account for about 16% and are most common as mucoepidermoid carcinoma. The normal salivary gland generation process and morphological structure are complex, so that the salivary gland tumor cell morphology, tissue structure and biological behaviors are diversified. Because salivary gland tumors are classified into complex and various forms, cross overlapping exists in morphology, for example, certain low-grade malignant tumors can also present benign manifestations in early stages, the disease course is longer, the disease course is easy to confuse with benign tumors, the repeatability of morphological classification of different pathologists is poor, and the benign malignancy of the tumors is difficult to accurately identify clinically, so that the improvement of the treatment effect and long-term survival rate of salivary gland tumor patients is limited.

Advanced diagnostic methods have been employed for many years for better differential diagnosis. Conventional high-frequency ultrasound is the first imaging method for detecting salivary gland lesions, and has more overlapping benign and malignant lesions and limited discrimination capability. Fine needle aspiration cytopathological diagnosis (FNAC) is an important tool for preoperative risk stratification. The accuracy of qualitative diagnosis of the fine needle aspiration cytology examination is higher, but the compliance rate of histological classification is about 80%, the compliance rate of specific diagnosis is 60% -75%, false negative diagnosis is easy to cause, the tumor cytology forms are obviously overlapped, and the benign and malignant of slight salivary gland lesions are difficult to distinguish, especially under the condition of limited biopsy materials, the experience of pathologists is relied on. The tissue classification of salivary gland tumors clearly has a decisive significance for the mode of treatment/the scope of surgery and reduces unnecessary medical treatment for the patient. Therefore, identification of benign and malignant salivary gland tumors, particularly identification of polymorphic adenomas in myxoepidermoid carcinoma, cannot rely on existing histological diagnosis and requires the assistance of molecular detection to identify benign and malignant salivary gland tumors.

Fluorescent in situ hybridization (fluorescence in situ hybridization, FISH) is a technology for directly observing specific genes in cells in vitro, can be used for researching chromosome and abnormal detection of genes, provides a new technical support for research of malignant tumors, and has the advantages of high sensitivity, good specificity, simple operation, accurate spatial positioning and direct and clear result observation, and is widely used for diagnosing malignant tumors such as urothelial cancer, blood system tumor, cervical cancer and the like.

The detection of chromosomal and genetic abnormalities in salivary gland tumor cells, which are responsible for the identification of benign and malignant tumors, is not currently seen as an effective molecular target, due to the large number of genetic alterations in salivary gland tumors. Chromosome 3 triploid is the most common genetic change, and in the case of triploid or polyploid in various cancers such as bladder cancer and B lymphomas, the P16 gene is located in human chromosome 9P21 and directly participates in the regulation of cell cycle, and is responsible for regulating cell proliferation and division, and the gene deletion is an early event of various cancers. Detection of abnormal chromosome 3 numbers and deletion of p16 tumor suppressor genes based on FISH technology has been applied to diagnosis and re-monitoring of bladder cancer. However, chromosome 3 and the P16 gene are not applied to diagnosis/benign and malignant identification of salivary gland tumors at present.

In summary, the development of a kit for accurately and sensitively identifying the benign and malignant tumors of the salivary glands has practical significance.

Disclosure of Invention

The invention aims to provide a molecular target for identifying benign and malignant salivary gland tumors.

It is another object of the present invention to provide a use of a molecular target.

It is another object of the present invention to provide a probe assembly.

Another object of the present invention is to provide a method for preparing a probe assembly.

It is another object of the present invention to provide a kit.

Another object of the present invention is to provide a method for identifying benign and malignant salivary gland tumors.

To solve the above technical problem, in a first aspect of the present invention, a molecular target for identifying benign and malignant salivary gland tumors is provided, wherein the molecular target comprises a first target and/or a second target;

Wherein the first target is located on chromosome 3 centromere; the second target is located at the P16 gene.

In some preferred embodiments, the starting position of the first target is chr3:90,489,294-90,493,199; the starting position of the second target is (chr 9:21,802,635-22,029,593).

In a second aspect of the present invention, there is provided a probe assembly comprising:

A first probe for detecting the first target, the first target being located on chromosome 3 centromere; and

And a second probe, wherein the first probe is used for detecting the second target, and the second target is positioned on the P16 gene.

In some preferred embodiments, the first probe is a polynucleotide capable of hybridizing to the first target.

In some preferred embodiments, the second probe is a polynucleotide capable of hybridizing to the second target.

In some preferred embodiments, the first probe has a first fluorescein labeled thereon, the second probe has a second fluorescein labeled thereon, and the first and second luciferins are different in color.

In some preferred embodiments, the first fluorescein is red or green in color.

In some preferred embodiments, the second fluorescein is red or green in color.

In some preferred embodiments, the first Fluorescein is TRITC (tetramethylrhodamine) or flurescein

(Fluorescein isothiocyanate).

In some preferred embodiments, the second Fluorescein is TRITC (tetramethylrhodamine) or flurescein

(Fluorescein isothiocyanate).

In some preferred embodiments, the method of preparing the first probe comprises the steps of: using human genome as a template, obtaining an amplification product through polymerase chain reaction, and marking the first fluorescein on the amplification product to obtain the fluorescent peptide;

The primers used in the polymerase chain reaction comprise a forward primer and a reverse primer, wherein the sequence of the forward primer is shown as SEQ ID NO. 1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.

In some preferred embodiments, the first fluorescein is labeled on dUTP of the first probe.

In some preferred embodiments, the method of preparing the second probe comprises the steps of: transforming a host cell by using a BAC vector containing a target gene fragment, culturing the host cell to obtain plasmid DNA, and marking the second fluorescein on the plasmid DNA to obtain the recombinant DNA;

Wherein, the BAC vector containing the target gene fragment is RP11-615P15 and RP11-478M20.

In some preferred embodiments, the second fluorescein is labeled at dUTP of the second probe.

In a third aspect of the invention, a kit is provided for identifying benign and malignant salivary gland tumors, the kit comprising a probe for detecting the molecular target of the first aspect of the invention.

In some preferred embodiments, the kit comprises a probe combination according to the second aspect of the invention.

In some preferred embodiments, hybridization buffers and human placental DNA are also included in the kit.

In some preferred embodiments, the kit further comprises an in situ hybridization staining solution.

In some preferred embodiments, the hybridization buffer comprises sodium citrate buffer (SSC), deionized formamide, and dextran sulfate (DSS).

In some preferred embodiments, the volume ratio of the deionized formamide in the hybridization buffer is 40-60%.

In some preferred embodiments, the mass concentration of dextran sulfate in the hybridization buffer is 0.1-0.2g/mL.

In some preferred embodiments, the in situ hybridization staining solution is an in situ hybridization blue staining solution, such as 4', 6-diamidino-2-phenylindole (DAPI).

In a fourth aspect of the invention, there is provided a method of preparing a probe combination according to the second aspect of the invention, the method comprising the steps of:

preparing a first probe comprising the steps of: using human genome as a template, obtaining an amplification product through polymerase chain reaction, and marking the first fluorescein on the amplification product to obtain the fluorescent peptide; the primers used in the polymerase chain reaction comprise a forward primer and a reverse primer, wherein the sequence of the forward primer is shown as SEQ ID NO. 1, and the sequence of the reverse primer is shown as SEQ ID NO. 2; and

Preparing a second probe comprising the steps of: transforming a host cell by using a BAC vector containing a target gene fragment, culturing the host cell to obtain plasmid DNA, and marking the second fluorescein on the plasmid DNA to obtain the recombinant DNA; wherein, the BAC vector containing the target gene fragment is RP11-615P15 and RP11-478M20.

In some preferred embodiments, the first fluorescein is labeled on the amplification product using a notch shift method.

In some preferred embodiments, the second fluorescein is labeled on the plasmid DNA using a nick translation method.

In a fifth aspect of the present invention, there is provided a method for identifying benign and malignant salivary gland tumors, said method comprising the steps of: the kit according to the third aspect of the present invention is used for detecting a sample to be detected.

In some preferred embodiments, the method comprises the steps of: s1, preprocessing a sample to be detected; s2, carrying out denaturation treatment and hybridization treatment on the mixed solution of the sample to be detected and the probe; s3, counterstaining the hybridized sample by using an in-situ hybridization staining solution; s4, observing the counterstained sample by using a fluorescence microscope, and calculating the abnormal cell percentage; s5, judging the type of the salivary gland tumor as benign or malignant according to the value of the abnormal cell percentage.

In a sixth aspect, the invention provides the use of the molecular target of the first aspect of the invention for the preparation of a reagent for identifying a benign and malignant salivary gland tumor kit, the molecular target comprising a first target and/or a second target, the first target being located on chromosome 3 centromere; the second target is located at (p 16 gene).

In some preferred embodiments, the abnormal cell is a chromosome 3 abnormal cell and/or a p16 gene abnormal cell.

The invention has at least the following advantages over the prior art:

(1) The invention provides a new molecular target for identifying benign and malignant salivary gland tumor, which assists doctors in making diagnosis by detecting abnormal conditions such as aneuploidy of chromosome 3 and deletion of p16 gene in salivary gland tumor tissue, and is beneficial to defining subsequent treatment schemes of patients and reducing excessive medical treatment of salivary gland tumor patients;

(2) The invention provides a probe combination and a kit for identifying benign and malignant salivary gland tumors based on a fluorescence in situ hybridization technology, which have high sensitivity and good specificity.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.

FIGS. 1a to 1c are graphs showing the results of detection of staining of cultured cells in human peripheral blood by using a FISH probe according to an embodiment of the present invention, 100-fold, scale 20. Mu.M;

FIGS. 2a-2b are representative graphs of normal cells of sample detection results, 100-fold scale 20. Mu.M, according to an embodiment of the present invention;

FIGS. 3a-3d are representative graphs of abnormal cells of the sample test results, 100-fold scale 20. Mu.M, according to an embodiment of the present invention;

FIG. 4 is a graph of ROC of chromosome 3 and p16 gene alone and in combination in a sample test of test example 2 according to an embodiment of the present invention.

Detailed Description

Through extensive and intensive research, the inventor discovers a novel molecular target for identifying benign and malignant salivary gland tumors, and develops a corresponding probe combination and a kit based on a fluorescence in situ hybridization technology, so that doctors can be assisted in identifying benign and malignant salivary gland tumors which cannot be clearly diagnosed, the accuracy of identifying benign and malignant salivary gland tumors is improved, excessive medical treatment of salivary gland tumor patients is reduced, and unnecessary time, labor and fund waste is reduced.

Molecular targets

The invention relates to a molecular target for identifying benign and malignant salivary gland tumors, which comprises at least one of a first target positioned in chromosome 3 centromere and a second target positioned in a P16 gene. Preferably, the molecular targets for identifying benign and malignant salivary gland tumors include a first target and a second target.

As used herein, the term "chromosome 3" refers to chromosome 3 of the human genome, which belongs to an autosome. Typically, the human autosomes contain two equatorial, separated dye monomers, which are joined to form a centromere. The central centromere type, the central centromere type only, and the proximal centromere type are classified according to the positions of centromeres. Chromosome 3 is the third largest human chromosome spanning about 1.98 hundred million base pairs (building blocks of DNA), accounting for about 6.5% of the total DNA of the cell. One skilled in the art can query the number of genes and sequence information currently known to be located on chromosome 3 via the NCBI database.

As used herein, the term "P16 gene" is used interchangeably with "MTS (multiple tumor suppressor 1) gene" at the 9P21 position of the human chromosome, and the complete P16 memory consists of two introns and three exons, which together encode an inhibitor of the known cyclin-dependent kinase CDK 4. Abbreviated as P16.

As used herein, the term "salivary gland tumor" refers to a tumor that occurs at the site of salivary glands, and can be divided into parotid gland tumor, submandibular gland tumor, sublingual gland tumor, small salivary gland tumor. Salivary gland tumors are classified as benign and malignant. Benign tumors mainly have symptoms of locally slowly-increased tumors such as benign tumors like polymorphic adenoma, warthin tumor and the like, malignant tumors tend to grow faster, and complications such as pain, facial paralysis, limited opening, eating difficulty and the like, such as mucous epidermoid carcinoma, adenoid cystic carcinoma and the like, can also occur. In a preferred embodiment of the invention, salivary gland tumors include benign tumors such as polymorphic adenoma, warthin tumors, malignant tumors such as mucous epidermoid carcinoma, adenoid cystic carcinoma, and the like.

The invention also relates to application of the molecular target, wherein the molecular target comprises at least one of a first target and a second target, and the molecular target is used for preparing a kit for identifying benign and malignant salivary gland tumors.

Probe combination

The invention also relates to a probe combination comprising a first probe for detecting a first target; and/or a second probe for detecting a second target.

As used herein, the term "probe" refers to a single-stranded DNA or RNA fragment that can bind to a target sequence and is used to detect a nucleic acid sequence complementary thereto. In some embodiments, the probe is a FISH probe. As used herein, the term "fluorescent in situ hybridization technique, FISH (Fluorescence In Situ Hybridization)" is a technique that can be used to identify and analyze the condition of a particular gene at an accurate site. According to the principle of nucleic acid base complementary pairing, when the DNA molecule to be detected is homologous complementary with the nucleic acid probe, the DNA molecule to be detected and the nucleic acid probe can form a hybrid of target DNA and the nucleic acid probe through denaturation, annealing and renaturation. Usually, a reporter molecule such as biotin, digoxin and the like is labeled on a nucleic acid probe, and qualitative, quantitative or relative positioning analysis is performed on the DNA to be detected by fluorescence microscopy by utilizing the immunochemical reaction between the reporter molecule and fluorescein-labeled specific avidin. FISH techniques can be used to detect gene amplifications, deletions, and rearrangements, such as for chromosomal location of known genes or sequences, investigation of unclonable genes or genetic markers, and chromosomal aberrations. "FISH probe" refers to a nucleic acid probe that is homologous and complementary to a DNA molecule to be detected as used in fluorescent in situ hybridization techniques.

In the present invention, a group which facilitates recognition may be labeled on the probe, and these groups may be a radioisotope such as 3H, 35S, 125I or 32P, or the like, or a non-radioactive substance such as fluorescein, biotin, digoxygenin, or the like. In some embodiments of the invention, the fluorescein molecule is labeled on a probe. In the present invention, the Fluorescein molecule labeled on the probe is a Fluorescein molecule commonly used for FISH probes, such as TRITC or flurescein. In a preferred embodiment of the invention, the first probe is labeled with a first fluorescein and the second probe is labeled with a second fluorescein, and the first and second luciferins are different in color. The first and second luciferin colors should meet the requirements that are distinguishable under a fluorescence microscope, such as red and green. In some more preferred embodiments of the invention, the first fluorescein is red in color and the second fluorescein is green in color; or the first fluorescein is green in color and the second fluorescein is red in color. In a most preferred embodiment of the invention, the first Fluorescein is TRITC (tetramethylrhodamine) and the second Fluorescein is flurescein (Fluorescein isothiocyanate); alternatively, the first Fluorescein is Fluorescein (Fluorescein isothiocyanate) and the second Fluorescein is TRITC (tetramethylrhodamine). Fluorescein is optionally labeled at any base of the probe sequence, or at each base. In a preferred embodiment of the invention, the fluorescein is labeled on dUTP of the probe. Methods for labeling fluorescein on probes are conventional in the art, nick translation (otherwise known as notch translation) or random priming, and in preferred embodiments of the invention fluorescein is labeled by notch translation.

Preferably, in the probe set of the present invention, the ratio of the amounts of the substances of the first luciferin labeled on the first probe and the second luciferin labeled on the second probe is (0.8 to 1.2): (0.8-1.2), more preferably 1:1.

Method for preparing probe combination

The probe combinations in the present invention can be obtained using the PCR method or the BAC cloning method.

In a preferred embodiment of the present invention, the method for preparing the first probe comprises the steps of: taking a human genome as a template, obtaining an amplification product through polymerase chain reaction, and marking a first fluorescein on the amplification product to obtain the PCR amplification product; the primers used in the polymerase chain reaction comprise a forward primer and a reverse primer, wherein the sequence of the forward primer is shown as SEQ ID NO. 1, and the sequence of the reverse primer is shown as SEQ ID NO. 2.

Sequence number	Sequence information
		SEQID NO:1	GTGGAATTTGCAAGTGGAGA
SEQID NO:2	CTTTTCCACCAATGGCCTCA

In a preferred embodiment of the present invention, the method for preparing the second probe comprises the steps of: transforming a host cell by using a BAC vector containing a target gene fragment, culturing the host cell (preferably an escherichia coli cell) to obtain plasmid DNA, and marking second fluorescein on the plasmid DNA to obtain the recombinant DNA; wherein BAC vectors containing target gene fragments are RP11-615P15 and RP11-478M20.

The method of labeling the above PCR amplification product or plasmid DNA with fluorescein is preferably a nick translation method. Specifically, the notch translation method comprises the steps of: the method comprises the steps of taking DNaseI, DNA polymerase I, DNA to be marked, dNTP, dUTP marked by fluorescein and buffer solution as raw materials, carrying out marking reaction at 25 ℃, inactivating enzyme after the reaction is finished to obtain a marked product, and purifying the obtained marked product.

Kit for detecting a substance in a sample

The invention also relates to a kit for identifying benign and malignant salivary gland tumors, which comprises a probe for detecting the molecular target. Preferably, the probe combinations of the invention are included in the kit of the invention.

To increase the stability of the system, hybridization buffers are included in the kits of the invention in addition to FISH probe combinations. As hybridization buffers, a combination of sodium citrate buffer (SSC), deionized formamide and dextran sulfate (DSS) is included. In a preferred embodiment, the volume ratio of the deionized formamide is 40 to 60%; the mass concentration of the dextran sulfate is 0.1-0.2g/mL; the balance of sodium citrate buffer solution. The formula of the hybridization buffer solution is favorable for maintaining the morphological structure of tissues, and the hybridization background and the detection effect are better.

Also included in the kit of the invention are in situ hybridization blue staining fluids (e.g., 4', 6-diamidino-2-phenylindole (DAPI)).

Human placental DNA, such as COT 1 Human DNA, is preferably also included in the kit of the invention, 50-300bp in length, rich in repetitive DNA sequences, for blocking nonspecific hybridization and reducing the background of nonspecific hybridization.

In the most preferred embodiment of the invention, the kit comprises the following components and proportions:

Component (A)	Each test (mu L)
		Hybridization buffer	7
Human placenta DNA (1 mg/mL)	1
		First probe	0.8
Second probe	0.8
		Purified water	Make up 10 mu L

Method for identifying benign and malignant salivary gland tumor

The invention also relates to a method for identifying benign and malignant salivary gland tumors, which comprises the step of detecting a sample to be detected by using the kit.

As used herein, a "sample to be tested" may be a tumor tissue slice, a cell (such as a puncture sample), a tissue, or the like. The sample to be tested is taken from a patient suffering from adenophoroma. Samples from patients need to be pre-processed to obtain sample pieces that facilitate subsequent hybridization operations. The pretreatment method of the sample to be tested from the patient is conventional in the art, and generally the pretreatment step comprises dewaxing by using a dewaxing agent, gradient rehydration by using ethanol solutions with different concentrations, sequential boiling, enzyme digestion treatment, and gradient dehydration and air drying by using ethanol solutions with different concentrations.

The sample pieces obtained after pretreatment may be subjected directly to denaturation and hybridization operations, the methods of which are well known to those skilled in the art. The operation method which can be referred to in the invention is as follows: the hybridization solution in the kit of the invention is fully mixed with the sample, and then denaturation and hybridization treatment are sequentially carried out. In an embodiment of the invention, the step of denaturing comprises denaturing at 85.+ -. 1 ℃ for 5 minutes; the hybridization treatment includes hybridization at 37+ -1deg.C for 10-18 hours.

After hybridization is completed, excess probe is washed away. The method of washing to remove the excess probe can be carried out in a conventional manner. The operation modes which can be referred to are as follows: washing in 0.1% NP-40/2 XSSC at 37.+ -. 1 ℃ for 5 minutes; soaking in 70% ethanol at room temperature for 3 min.

Washing to remove excessive probes and airing the hybridization sample, and taking in-situ hybridization staining solution in the kit for counterstaining to fully mix the sample with the in-situ hybridization staining solution. The counterstained samples were dried and directly observed under a fluorescent microscope and the results were interpreted. The benign and malignant of the salivary gland tumor is identified by counting and counting the fluorescent signals and calculating the ratio of abnormal cells to normal cells.

The kit is used for detecting a sample, a fluorescent microscope is used for observing cells, and the result interpretation method is referred as follows:

Observation result	Results	Chromosome variation
			Two red and two green	Normal cells
Green probe signal number >2	Abnormal cell of chromosome 3	Chromosome 3 amplification
			The number of red and green colors is more than or equal to 2	P16 abnormal cells	P16 heterozygous deletion
0 Red and green number is not less than 2	P16 abnormal cells	P16 homozygous deletion

An alternative method of positive and negative interpretation in the present invention is referenced below:

The same number of cells was counted per sample and the percentage value of abnormal cells of each type was calculated. Percentage value of abnormal cells of a certain type = number of abnormal cells of that type/total number of cells. For example, percent number 3 chromosome abnormal cells = number 3 chromosome abnormal cells/total number of cells. Positive is determined when the number 3 chromosome abnormal cell percentage is greater than its threshold value (10% of the threshold value in the present invention). Otherwise, the result is negative. In some embodiments of the invention, the sensitivity of the method is about 63.6% and the specificity is about 88.1%.

In the same manner, the P16 abnormal cell percentage=the number of P16 abnormal cells/total number of cells, and positive is determined when the P16 abnormal cell percentage is greater than its threshold value (in the present invention, the threshold value is 20%). Otherwise, the result is negative. In some embodiments of the invention, the sensitivity of the method is about 77.3% and the specificity is about 79.5%.

Another more preferred method of positive and negative interpretation in the present invention is referred to as follows:

Positive is determined when the number 3 abnormal cell percentage is greater than its threshold (10% of this threshold in the present invention) and the P16 abnormal cell percentage is greater than its threshold (20% of this threshold in the present invention). Otherwise, the result is negative. In some embodiments of the invention, the sensitivity of the method is about 90.9% and the specificity is around 83.3%.

The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, it is to be noted that the terms used herein are used merely to describe specific embodiments and are not intended to limit exemplary embodiments of the application.

The term "or" means and is used interchangeably with the term "and/or" unless otherwise indicated.

As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

Example 1 preparation of composition of FISH Probe for identification of benign and malignant salivary glands

In this example, a FISH probe combination for identifying benign and malignant salivary gland tumors is provided, which relates to detection probes for chromosome 3 and p16 genes. The chromosome 3 centromere detection probe and the p16 gene detection probe are labeled with fluorescein with different colors. And the mass ratio of the chromosome 3 centromere to the p16 gene is 1:1.

1. Chromosome centromere probe preparation:

(1) Primer design and synthesis: and searching and screening high-specificity regions of the chromosome 3 and chromosome 7 centromeres of the human beings to design primers, and finally obtaining a sequence with the optimal fragment marking effect.

The sequences and detection segments of the probe primers are shown in Table 1 below.

TABLE 1

Sequence number	Primer name	Sequence(s)
			SEQID NO:1	CSP 3-F	GTGGAATTTGCAAGTGGAGA
SEQID NO:2	CSP 3-R	CTTTTCCACCAATGGCCTCA
			SEQID NO:3	CSP 7-F	TGCAAGTGGAGATTTCAAGC
SEQID NO:4	CSP 7-R	CAAATATCCACTTGCAGACA

PCR amplification was performed using human genome as a template, and the PCR system formulation is shown in Table 2.

TABLE 2

Name of the name	Each test (mu L)
		10×PCR Buffer(Mg²⁺plus)	5
MgCL2(25mmol/L)	2
		dNTP Mixture(2.5mM each)	4
Human genome DNA (50 ng/. Mu.L)	1.6
		Probe primer F (100 pmol/. Mu.L)	0.2
Probe primer R (100 pmol/. Mu.L)	0.2
		TAKARA TAQ enzyme (5U/. Mu.L)	0.5
Purified water	Make up to 50 mu L

The total volume was 50uL. The cycle parameters were set using a PCR amplification apparatus as shown in Table 3.

TABLE 3 Table 3

Temperature (temperature)	Time of	Cycle number
			95℃	For 10 minutes	/
94℃	30 Seconds	40
			55℃	30 Seconds	40
72℃	35 Seconds	40
			72℃	10. Minute (min)	/

(3) Agarose gel electrophoresis: after completion of the PCR reaction, 1. Mu.L of the product was detected by 2% agarose gel electrophoresis, and the product had a bright band around 1000 bp.

(4) Labeling a probe: the amplified products were fluorescently labeled using notch shift, preferably Fluorescein, and the probe labeling reaction is shown in Table 4 below.

TABLE 4 Table 4

Component (A)	Each test (mu L)
		10 XNT buffer	5
DATP, dCTP and dGTP (1 mM)	1
		dTTP(1 mM)	0.6
DNaseI(0.001 U/μL)	3
		DNA polymerase I (5U/. Mu.L)	1.5
Fluorescein-labeled dUTP	2
		PCR products	300 ng
Purified water	Complement 50

After the system is prepared, the system is vibrated, evenly mixed and centrifuged, a PCR gene amplification instrument is used for setting up a program, the PCR gene amplification instrument is used for marking for 2 hours at 25 ℃, and the PCR gene amplification instrument is incubated for 10 minutes at 80 ℃ to inactivate enzymes.

(5) The labeled product was purified as follows:

a, according to 3M sodium acetate: absolute ethanol = 1:25, preparing a purified solution by mixing the components in proportion;

b, ethanol precipitation and concentration are carried out on the marked product. According to the product: purified solution=5:13 volume ratio, adding the marked product into a 1.5 mL centrifuge tube containing purified solution, mixing uniformly by vortex, then instantly separating, and standing the mixture in an ultralow temperature refrigerator at-80 ℃ for 30-60 minutes. The probe was pelleted by centrifugation at 13000 rpm for 2 minutes, the supernatant was discarded and dried in the dark.

C, adding 200 mu L of 70% ethanol, rinsing the precipitate gently, centrifuging briefly to remove ethanol thoroughly, and drying at 45 ℃ for 3 minutes.

And d, finally, dissolving the mixture in 2 mu L of purified water to obtain the marked CSP3 probe raw material, and storing the marked CSP3 probe raw material in a dark place.

The primer sequence was synthesized by Shanghai Bioengineering services Co.

2. The preparation method of the P16 gene probe comprises the following steps:

(1) BAC clone screening: clones containing the p16 gene sequence were screened and purchased from Invitrogen RP11 BAC clone library. BAC clones against the p16 gene are shown in Table 5 below.

TABLE 5

BAC	Insert start and stop position	Size (kb)
			RP11-615P15	chr9:21,764,403-21,981,385	217
RP11-478M20	chr9:21,947,304-22,110,179	163

Plasmid extraction: the BAC clones were individually extracted with plasmid DNA using commercially available plasmid extraction kits according to the kit instructions to obtain plasmid DNA, which was quantified by Nanodrop 2000.

Fluorescent labeling of plasmid DNA: the plasmid DNA is fluorescently labeled by using a notch translation method, and the fluorescein is preferably tetramethyl rhodamine (TAMRA), a PCR reaction system is prepared on ice under the condition of strict light shielding, and the probe labeling reaction system is shown in the following table 6.

TABLE 6

Component (A)	Each test (mu L)
		10×NT buffer	5
DATP, dCTP and dGTP (1 mM)	1
		dTTP(1 mM)	0.6
DNaseI(0.001 U/μL)	3
		DNA polymerase I (5U/. Mu.L)	1.5
TRITC-tagged dUTP	2
		Plasmid DNA	300 ng
Purified water	Make up 50 mu L

After the system is prepared, shaking, mixing and centrifuging are carried out, marking is carried out at 25 ℃ for 2 hours, and incubation is carried out at 80 ℃ for 10 minutes to inactivate enzymes.

The labeled product is then subjected to precipitation of nucleic acid using sodium acetate, high-speed centrifugation to obtain labeled probe, and the purification steps are as described above, and are not described in detail herein. And dissolving the probe by using purified water to obtain the labeled GSP 16 probe raw material.

To evaluate the sensitivity and specificity of the probe prepared in example 1, the probe in example 1 was used to test human peripheral blood cultured cells, respectively, 50 metaphase or interphase cells were analyzed, and fluorescent brightness, hybridization efficiency, hybridization position, etc. of the hybridization signal were analyzed, as a result, see FIGS. 1a-1c, wherein FIGS. 1a,1b, and 1c are fluorescent patterns of chromosome 3, chromosome 7, and chromosome P16, respectively, and it was found that the corresponding chromosome loci should be labeled with green and red fluorescent light, respectively, the signal was bright and there was no cross hybridization phenomenon between the chromosome loci, indicating that the hybridization efficiency of this example was 100% (50/50).

Example 2 kit for identifying benign and malignant salivary glands

In the embodiment, a kit for identifying benign and malignant salivary gland tumors is provided, so that signals can be directly observed in a tissue sample. The kit contains the hybridization solution of the FISH probe combination in example 1. Hybridization solutions included probes, hybridization buffer solutions, placental DNA, and in situ hybridization blue dye solutions (available from the company, inc. of pharmaceutical technologies, an Bi, guangzhou). The hybridization buffer solution contains sodium citrate buffer solution (SSC), deionized formamide and dextran sulfate (DSS), wherein the concentration of the deionized formamide is 40% -60%, the concentration of the DSS is 0.1-0.2g/mL, and the in situ hybridization blue dye solution is 4', 6-diamidino-2-phenylindole (DAPI).

More specifically, the composition and ratio of the kit hybridization solution are shown in Table 7 below.

TABLE 7

Component (A)	Each test (mu L)
		Hybridization buffer	7
COT human DNA(1 mg/mL)	1
		Probe-FITC labelling	0.8
Probe-TAMRA markers	0.8
		Sterilizing purified water	Make up 10 mu L

In the above examples, fluorescein-labeled dUTP was purchased from Roche (cat. No. 11373242910), TRITC-labeled dUTP was purchased from Roche (cat. No. 11534378910), COT human DNA was purchased from Roche (cat. No. 11581074001), DNaseI was purchased from NEB (cat. No. M0303), and DNA polymerase I was purchased from NEB (cat. No. M0209).

Example 3 FISH screening of probes for identification of benign and malignant salivary gland tumors

The kit described in example 2 is used for detecting tissue samples of patients suffering from salivary gland tumors, and the specific steps are as follows:

sample pretreatment:

placing the sample slice on a constant temperature heating table at 65 ℃ to bake the slice for 5-30 minutes; samples were dewaxed in room temperature environmental dewaxing agent (available from guangzhou An Bi plain pharmaceutical technologies, inc., docket No. 20220944) for 10 minutes; removing residual dewaxing agent in absolute ethyl alcohol at room temperature for 10 minutes; sequentially adding the mixture into room temperature absolute ethyl alcohol, 90% ethanol, 70% ethanol and purified water for 3 minutes for gradient rehydration; the sample piece is put into purified water with the temperature of 95-100 ℃ to be boiled for 25 minutes, 100-200 mu l of pepsin working solution with the temperature of 37+/-1 ℃ is dripped into the sample piece after the sample piece is air-dried, and the sample piece is digested for 3-15 minutes; washing in 2 XSSC at room temperature for 3 minutes; and then sequentially placing the materials into 70%, 90% and 100% gradient ethanol at room temperature to dehydrate for 2 minutes, and air-drying at room temperature.

Sample and probe were simultaneously denatured and hybridized (light protected): dripping 4-8 mu L of hybridization solution into the hybridization area, rapidly covering a cover glass, uniformly distributing the probes by light pressure, removing bubbles, and slowly expanding the sample by the probe mixed solution to cover the whole surface; the rubber glue is sealed along the edge of the cover glass, denatured for 5 minutes at 85+/-1 ℃ and hybridized for 10-18 hours at 37+/-1 ℃.

Post-hybridization washes (light-protected operation): gently tearing the rubber off the sample piece, carefully removing the cover glass, and placing the sample piece into 2 XSSC preheated at 37+ -1 ℃ for washing for 10 minutes; then put it into 0.1% NP-40/2 XSSC at 37+ -1deg.C for 5 minutes; soaking in 70% ethanol at room temperature for 3 min, and air drying the slide;

DAPI counterstain: after the dark place is dried, 5-7 mu L of in-situ hybridization blue staining solution (DAPI) is dripped into a hybridization area at room temperature, air bubbles are removed by light pressure after cover glass is covered, the air is dried at room temperature, and the result is observed and interpreted under a fluorescence microscope.

Analysis of detection results:

The relevant DAPI and fluorescence require the observation of the fluorescent signal using a suitable frit, wherein the CSP3 probe displays a green signal, the CSP7 probe displays a red signal, and the GCP P16 probe displays a red signal. Selecting 50 cells, selecting the parts with clear cell outline, uniform DAPI staining, no overlapping of cell nuclei and clear probe signals, counting and counting green and red fluorescent signals in the nuclei under a 100-time objective lens, and calculating the ratio of abnormal cells to normal cells.

Interpretation of counts and results:

(1) Normal cells: two red and two green;

(2) Typical abnormal cells:

a, green probe signal number >2, suggesting chromosome 3 amplification;

b, when 1R (one red) and the green number is more than or equal to 2, prompting P16 to generate heterozygote deletion; when the number of green colors is 0R (0 red) and is more than or equal to 2, prompting that P16 is subjected to homozygote deletion;

And judging whether the sample is negative or positive according to the ratio of typical abnormal signals, wherein at least one of the two probes is positive, namely judging that the sample is positive. The specific criteria are as follows:

Counting the same number of cells in each sample, counting the percentage of signal points with different abnormal conditions, if the percentage of signal points with abnormal conditions of any one probe of the chromosome 3 and the p16 gene of the sample is larger than a threshold value (the abnormal threshold value of the chromosome 3/7 is 10 percent, and the abnormal threshold value of the p16 gene is 20 percent), judging that the sample is a positive result, otherwise judging that the sample is a negative result, and if the sample is equal to the abnormal threshold value, increasing the number of the cells of the observation sample; the method for establishing the abnormal threshold comprises the following steps: the benign and malignant salivary gland tumors with definite histological results are detected by using the kit, the same number of cells are counted by each group of probes, the percentages of different types of abnormal cell numbers are counted, and the percentage for effectively distinguishing the benign and malignant salivary gland tumors is determined as a threshold value.

The results are shown in FIGS. 2a-2b and FIGS. 3a-3 d. Wherein, fig. 2a is a sample fluorescence image of detecting the normal of both chromosome 3 and P16 genes, fig. 2b is a sample fluorescence image of detecting the normal of chromosome 3 and chromosome 7, and it is seen that the percentage of abnormal cell number is lower than the abnormal threshold value by the 2 green fluorescence signals and the 2 red fluorescence signals of most cells in the visual field; FIG. 3a is a fluorescent picture of a sample with normal chromosome 3 and a p16 gene deleted, with normal green signals and less than 2 red signals in the field of view; FIG. 3b is a sample fluorescence image of chromosome 3 amplification and p16 gene deletion, FIG. 3c is a sample fluorescence image of both chromosome 3 and chromosome 7 abnormal amplification, and FIG. 3d is a sample fluorescence image of chromosome 3 amplification and p16 gene normal.

Test example 1:

Sample source: a total of 34 salivary gland tumor tissue samples with known clear pathological information results from the affiliated hospitals (Zhanjiang) of Guangdong medical university are selected, wherein the samples comprise 22 salivary gland malignant tumors (mucinous epidermoid carcinoma and adenoid cystic carcinoma), 12 benign tumors and 5 μm thick sample sections, and the pretreatment, denaturation hybridization and counterstaining of the samples are carried out according to the method. The results of the tests are shown in Table 8, "+" indicates positive for the probe test results and "-" indicates negative for the probe test results.

TABLE 8

Sample number	Cell number	FISH results	FISH results	FISH results	Pathological results
								CSP3	CSP7	GP3P P16
1	100	-	-	-	Mucinous epidermoid carcinoma
						2	100	+	+	+	Mucinous epidermoid carcinoma
3	100	+	-	+	Mucinous epidermoid carcinoma
						4	100	+	+	+	Mucinous epidermoid carcinoma
5	100	+	+	+	Mucinous epidermoid carcinoma
						6	100	+	-	-	Mucinous epidermoid carcinoma
7	100	-	-	+	Mucinous epidermoid carcinoma
						8	100	+	+	+	Mucinous epidermoid carcinoma
9	100	+	+	+	Mucinous epidermoid carcinoma
						10	100	+	-	+	Mucinous epidermoid carcinoma
11	100	-	-	+	Mucinous epidermoid carcinoma
						12	100	+	-	+	Mucinous epidermoid carcinoma
13	100	-	-	+	Mucinous epidermoid carcinoma
						14	100	+	-	+	Mucinous epidermoid carcinoma
15	100	+	+	+	Adenoid cystic carcinoma
						16	100	+	-	+	Mucinous epidermoid carcinoma
17	100	+	-	-	Mucinous epidermoid carcinoma
						18	100	+	-	-	Mucinous epidermoid carcinoma
19	100	+	-	+	Mucinous epidermoid carcinoma
						20	100	+	-	+	Mucinous epidermoid carcinoma
21	100	+	+	+	Mucinous epidermoid carcinoma
						22	100	-	-	-	Salivary duct cancer
23	100	-	-	-	Benign tumor of salivary gland
						24	100	-	-	-	Benign tumor of salivary gland
25	100	-	-	-	Benign tumor of salivary gland
						26	100	+	-	+	Benign tumor of salivary gland
27	100	-	-	-	Benign tumor of salivary gland
						28	100	-	-	-	Benign tumor of salivary gland
29	100	+	-	-	Benign tumor of salivary gland
						30	100	-	-	-	Benign tumor of salivary gland
31	100	-	-	-	Benign tumor of salivary gland
						32	100	-	+	-	Benign tumor of salivary gland
33	100	-	-	-	Benign tumor of salivary gland
						34	100	-	-	-	Benign tumor of salivary gland

The sensitivity and specificity test results are shown in Table 9.

TABLE 9

Probe name	Sensitivity of	Specificity (specificity)
			CSP3	77.3%(17/22)	88.1%(10/12)
CSP7	31.8% (7/22)	91.6%(11/12)
			GSP P16	77.3%(17/22)	83.3%(10/12)
CSP7+GSP P16	77.3%(17/22)	91.6%(11/12)
			CSP3+GSP P16	90.9%(20/22)	83.3%(10/12)
CSP3+CSP7	77.3%(17/22)	75.0%(9/12)
			CSP3+CSP7+GSP P16	90.9%(20/22)	75.0%(9/12)

According to the sample detection results, the specificity of the 3 probes for detecting salivary gland tissues is better and is more than 80%, but the sensitivity of the chromosome 7 probe is poor and is only 31.8%. The combination detection contrast of the 3 probes is that the CSP3+GSP16 combination detection has the best effect, the sensitivity is 90.9 percent, the specificity is 83.3 percent, and the detection effect is better than that of single probes or other probe combinations, and the detection effect on salivary gland malignant tumors is better.

Test example 2:

The sample size test was increased. Sample source: a total of 86 salivary gland tumor tissue samples with known clear pathological information results from the affiliated hospitals (Zhanjiang) of Guangdong medical university are selected, wherein the samples comprise 20 cases of mucinous epidermoid carcinoma, 13 cases of adenoid cystic carcinoma, 6 cases of squamous cell carcinoma, 5 cases of other types of malignant tumors, 19 cases of polymorphous adenoma, 11 cases of Warthin tumor, 9 cases of salivary gland tissue and 3 cases of other types of benign tumors, the thickness of a tissue sample slice is 5 mu m, and the sample pretreatment, denaturation hybridization and counterstaining are carried out according to a kit method.

The results of the pathological histology and the gold standard are shown in Table 10, and the sensitivity, the specificity and the AUC area of the detection of 86 samples are statistically analyzed.

Table 10

Probe name	Sensitivity of	Specificity (specificity)	AUC
				CSP3	63.6%(28/44)	88.1%(37/42)	0.736
GSP P16	77.3% (34/44)	79.5%(35/44)	0.842
				CSP3+GSP P16	90.9%(40/44)	83.3%(35/42)	0.858

For the 86 patients with clinical salivary gland tumors, the detection result of the kit provided by the invention is compared with the clinical practical pathological detection result, and at least one of the chromosome 3 centromere probe and the P16 gene probe is positive, namely, a positive sample is judged. The sensitivity of detecting salivary gland malignant tumor is 63.6% and 77.3% respectively, the specificity of detecting salivary gland benign tumor is 88.1% and 79.5% respectively, and the AUC is 0.736 and 0.842 respectively as shown in FIG. 4, chromosome 3 centromere probe and P16 gene probe; when any one of the two genes is detected positive, the sensitivity of the two genes for detecting the benign tumor of the salivary gland is 90.9%, the specificity for detecting the benign tumor of the salivary gland is 83.3%, and the ROC area is 0.858. The ROC graph is shown in fig. 4.

The result shows that in the detection performance comparison of the single probe, the sensitivity of the p16 gene is better, the specificity of the chromosome 3 probe is better, and when the combination detection of the chromosome 3 and the p16 gene has better detection effect, the detection sensitivity of the malignant tumor is better, but the specificity is reduced, and the detection rate of the malignant tumor can be improved by the combination detection of the chromosome 3 and the p16 gene. In a word, the novel molecular target selected by the invention and the specific section designed for the target can rapidly and accurately identify benign and malignant salivary gland tumors, and become a very important reference basis for clinicians to identify benign and malignant salivary gland tumors, and the method has high sensitivity and specificity and easy interpretation of results.

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims

1. Use of a FISH probe combination for preparing a kit for identifying benign and malignant salivary gland tumors by detecting the percentage of abnormal cells in a sample; chromosome 3 amplification and/or P16 gene deletion as abnormal cells;

the probe combination consists of a first probe and a second probe:

the first probe is used for detecting a first target, and the first target is positioned in chromosome 3 centromere; and

The second probe is used for detecting a second target, and the second target is positioned on the P16 gene;

The preparation method of the first probe comprises the following steps: taking a human genome as a template, obtaining an amplification product through polymerase chain reaction, and marking a first fluorescein on the amplification product to obtain the PCR amplification product; the primers used in the polymerase chain reaction comprise a forward primer and a reverse primer, wherein the sequence of the forward primer is shown as SEQ ID NO. 1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;

The preparation method of the second probe comprises the following steps: transforming a host cell by using a BAC vector containing a target gene fragment, culturing the host cell to obtain plasmid DNA, and marking second fluorescein on the plasmid DNA to obtain the recombinant DNA; wherein, the BAC vector containing the target gene fragment is RP11-615P15 and RP11-478M20.

2. The use according to claim 1, wherein the first probe has a first fluorescein labeled thereon, the second probe has a second fluorescein labeled thereon, and the first and second luciferins are different in color.

3. The use according to claim 1, wherein the first fluorescein is labeled on dUTP of the first probe;

and/or, the second fluorescein is labeled on dUTP of the second probe.

4. The use according to claim 1, wherein the first Fluorescein is TRITC or flurescein;

And/or, the second Fluorescein is TRITC or flurescein.

5. The kit is used for preparing a kit for identifying benign and malignant salivary gland tumors, and the kit is used for identifying the benign and malignant salivary gland tumors by detecting the abnormal cell percentage in a sample; chromosome 3 amplification and/or P16 gene deletion as abnormal cells;

The kit comprising the FISH probe combination of any one of claims 1-4, as well as hybridization buffer and human placental DNA.

6. The use according to claim 5, wherein the hybridization buffer comprises sodium citrate buffer (SSC), deionized formamide and dextran sulfate (DSS).

7. The use according to claim 6, characterized in that the volume ratio of the deionized formamide in the hybridization buffer is 40-60%.

8. The use according to claim 6, characterized in that the mass concentration of dextran sulphate in the hybridization buffer is 0.1-0.2g/mL.