阅读说明：本技术 ***的检测方法 (Method for detecting cervical cancer ) 是由 林东旭 魏国鹏 于 2018-07-13 设计创作，主要内容包括：本发明涉及一种宫颈癌的检测方法,其包括通过检测3号染色体上的3q扩增热点区域的相对拷贝数来确定受试者是否患有宫颈癌或处于发展宫颈癌的风险中。更具体地,本发明涉及通过实时定量PCR(qPCR)来检测3号染色体上的3q扩增热点区域的相对拷贝数。本发明还涉及用于检测宫颈癌的试剂盒。(The present invention relates to a method for detecting cervical cancer, which comprises determining whether a subject has or is at risk of developing cervical cancer by detecting the relative copy number of a 3q amplification hotspot region on chromosome 3. More specifically, the present invention relates to the detection of the relative copy number of the 3q amplification hotspot region on chromosome 3 by real-time quantitative pcr (qpcr). The invention also relates to a kit for detecting cervical cancer.)

1. Use of a reagent for detecting the relative copy number of a 3q amplification hotspot region on chromosome 3 in the preparation of a reagent or kit for use in a method of determining whether a subject has or is at risk of developing cervical cancer, the method comprising:

a. providing a sample from the subject,

preferably, the sample is a genomic sample from the subject,

b. detecting the copy number of the 3q amplification hotspot region on chromosome 3 in the sample,

c. detecting the copy number of the reference chromosomal region in said sample, and

d. normalizing the copy number of the 3q amplification hotspot region relative to the copy number of the reference chromosome region, thereby obtaining a relative copy number of the 3q amplification hotspot region,

wherein an increase in the relative copy number of the 3q amplification hotspot region in the subject's sample, as compared to the relative copy number of the 3q amplification hotspot region in the control sample, is indicative of the subject having, or being at risk of developing, cervical cancer.

2. The use of claim 1, wherein said 3q amplification hotspot region comprises a chromosomal region from 3q26.2 to 3q29,

for example, the 3q amplification hotspot region comprises the chromosomal region from 3q27 to 3q29,

for example, the 3q amplification hotspot region comprises a chromosomal region of the DLG1 gene.

3. The use of claim 1 or 2, wherein the reference chromosomal region comprises a chromosomal region of a single copy gene,

for example, the reference chromosomal region comprises a chromosomal region of the ALB gene.

4. The use of any one of claims 1-3, wherein the copy number of the 3q amplification hotspot region on chromosome 3 in the sample is detected by real-time quantitative PCR (qPCR) using a forward primer P1 and a reverse primer P2, wherein P1 and P2 hybridize to the target nucleic acid sequence on the 3q amplification hotspot region.

5. The use of any one of claims 1-4, wherein the copy number of a reference chromosomal region in the sample is detected by qPCR using a forward primer P3 and a reverse primer P4, wherein P3 and P4 hybridize to a target nucleic acid sequence on the reference chromosomal region.

6. A reagent or kit for determining whether a subject has, or is at risk for developing, cervical cancer comprising reagents for detecting the relative copy number of a 3q amplification hotspot region on chromosome 3 in a sample.

7. The reagent or kit of claim 6, wherein the reagents for detecting the relative copy number of the 3q amplification hotspot region on chromosome 3 comprise reagents for detecting the copy number of the 3q amplification hotspot region on chromosome 3 in the sample by real-time quantitative PCR (qPCR), and reagents for detecting the copy number of the reference chromosome region in the sample by real-time quantitative PCR (qPCR).

8. The reagent or kit of claim 6 or 7, wherein said 3q amplification hotspot region comprises a chromosomal region of 3q26.2 to 3q29,

for example, the 3q amplification hotspot region comprises the chromosomal region from 3q27 to 3q29,

for example, the 3q amplification hotspot region comprises a chromosomal region of the DLG1 gene.

9. The reagent or kit of claim 7 or 8, wherein the reference chromosomal region comprises a chromosomal region of a single copy gene,

for example, the reference chromosomal region comprises a chromosomal region of the ALB gene.

10. The reagent or kit of claim 9, wherein the reagents for detecting the copy number of the 3q amplification hotspot region on chromosome 3 in the sample by real-time quantitative pcr (qpcr) comprise a forward primer P1 and a reverse primer P2 that hybridize to the target nucleic acid sequence on the 3q amplification hotspot region.

11. The reagent or kit of claim 9, wherein the reagents for detecting the copy number of a reference chromosomal region in the sample by real-time quantitative pcr (qpcr) comprise a forward primer P3 and a reverse primer P4 that hybridize to a target nucleic acid sequence on the reference chromosomal region.

12. A method of characterizing copy number amplification of a 3q amplification hotspot region in a sample from a subject, comprising detecting the relative copy number of DLG1 gene in the sample, wherein an increase in the relative copy number of DLG1 gene in the sample of the subject compared to the relative copy number of DLG1 gene in a control sample indicates that the copy number of the 3q amplification hotspot region is amplified.

Technical Field

The present invention relates to a method for detecting cervical cancer, which comprises determining whether a subject has or is at risk of developing cervical cancer by detecting the relative copy number of a 3q amplification hotspot region on chromosome 3. More specifically, the present invention relates to the detection of the relative copy number of the 3q amplification hotspot region on chromosome 3 by real-time quantitative pcr (qpcr). The invention also relates to a kit for detecting cervical cancer.

Background

Cervical cancer is worldwide the most common disease in the 3 rd position and the 4th position of the fatality rate of women, and its age of onset has a marked tendency to become younger in recent years. Especially in developing countries, the incidence is 6 times higher than in developed countries due to the imperfect cervical cancer screening work, and the incidence rate is increasing at a rate of 2% -3% each year. There is a gradual evolutionary process in the development and progression of cervical cancer, which can range from years to decades, and it is generally accepted that the evolution is divided into several stages: mild (CINI), moderate (CINII) and severe intraepithelial neoplasia (CINIII), invasive carcinoma. Therefore, effective screening of CINIII can well reduce the prevalence rate of cervical cancer, thereby reducing the cancer mortality.

Etiological studies of cervical cancer have demonstrated that Human Papillomavirus (HPV) infection is predominant. There are many subtypes of HPV, about 40 involving genital tract infections, with persistent infections by high-risk types of HPV (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 69), especially HPV types 16, 18, which can trigger cervical cancer. However, HPV infection does not necessarily lead to cervical cancer, and furthermore, most of HPV infected people can eliminate HPV virus by the autoimmune system within 1 to 2 years, thereby avoiding cancer. However, the current early screening for cervical cancer still dominates cytological examination. TCT (TCT), namely liquid-based thin-layer cell detection, is a relatively advanced examination technology of cervical exfoliated cytology internationally at present, compared with the traditional cervical smear examination by scraping, the method obviously improves the satisfaction degree of a specimen and the detection rate of abnormal cervical cells, has data showing that the sensitivity and specificity of cytological screening of CINII and above grade lesions are respectively 51.5 percent and 73.4 percent, and the sensitivity and specificity of HPV detection of the same lesion are respectively 88.2 percent and 57.8 percent. The TCT method has high specificity and high HPV detection sensitivity. Both approaches have advantages and disadvantages for screening cervical cancer and precancerous lesions.

Although the HPV detection is widely applied, 5-10% of patients suffering from cervical cancer are found to be HPV negative, which indicates that the canceration mechanism of the patients is unrelated to HPV infection, and HPV early cancer screening of the population is ineffective. TCT assays are also problematic in that human factors are more influential and do not provide information at a deeper level, such as in the context of genes, due to the limitations of the methods themselves in observing only cytological morphology. The accuracy of colposcopy as a conventional clinical examination method depends greatly on whether the complete transformation region can be observed, but the colposcopy has the problems of insufficient diagnosis and over-diagnosis due to the differences of the age of the patient, the extent and severity of the affected cervix, the resolution of the colposcope, the operation and diagnosis level of the clinician, and the like. According to research, cervical biopsy pathology under a colposcope is compared with a final diagnosis pathological result, and the diagnosis deficiency rate of CINIII grade and in-situ cancer is 42%.

Thus, there remains a need for methods of using molecular markers to directly identify cervical cancer and monitor disease progression.

Summary of The Invention

The invention provides a Copy Number Variation (CNV) marker related to cervical cancer. Specifically, the present inventors have surprisingly found that copy number amplification of a specific region (referred to as a 3q amplification hot spot region in the present invention) in the long arm (3q) of chromosome three can be used as a marker for judging the occurrence of cervical cancer. In preferred embodiments, copy number amplification of 3q amplification hotspot regions is particularly suitable for detecting cervical cancer lesions of grade CINII and above.

In one aspect, the invention provides a method of determining whether a subject has, or is at risk of developing, cervical cancer, the method comprising:

a. providing a sample from said subject, and

b. detecting the relative copy number of the 3q amplification hotspot region on chromosome 3,

wherein an increase in the relative copy number of the 3q amplification hotspot region in the subject's sample, as compared to the relative copy number of the 3q amplification hotspot region in the control sample, is indicative of the subject having, or being at risk of developing, cervical cancer.

In a specific embodiment, the invention provides a method of determining whether a subject has, or is at risk for developing, cervical cancer, the method comprising:

a. providing a sample from the subject,

b. detecting the copy number of the 3q amplification hotspot region on chromosome 3 in the sample,

c. detecting the copy number of the reference chromosomal region in said sample, and

d. normalizing the copy number of the 3q amplification hotspot region relative to the copy number of the reference chromosome region, thereby obtaining a relative copy number of the 3q amplification hotspot region,

wherein an increase in the relative copy number of the 3q amplification hotspot region in the subject's sample, as compared to the relative copy number of the 3q amplification hotspot region in the control sample, is indicative of the subject having, or being at risk of developing, cervical cancer.

In a specific embodiment, the 3q amplification hotspot region comprises the chromosomal region 3q26.2 to 3q 29.

In a specific embodiment, the 3q amplification hotspot region comprises the chromosomal region 3q27 to 3q 29.

In a specific embodiment, the 3q amplification hotspot region comprises a chromosomal region of the DLG1 gene.

In particular embodiments, the reference chromosomal region comprises a chromosomal region of a single copy gene.

In a specific embodiment, the reference chromosomal region comprises a chromosomal region of an ALB gene.

In a specific embodiment, the copy number of the 3q amplification hotspot region on chromosome 3 in the sample is detected by real-time quantitative pcr (qpcr) using forward primer P1 and reverse primer P2, wherein P1 and P2 hybridize to the target nucleic acid sequence on the 3q amplification hotspot region.

In a specific embodiment, the copy number of the reference chromosomal region in the sample is detected by qPCR using a forward primer P3 and a reverse primer P4, wherein P3 and P4 hybridize to the target nucleic acid sequence on the reference chromosomal region.

In another aspect, the present invention provides the use of a reagent for detecting the relative copy number of a 3q amplification hotspot region on chromosome 3 in the preparation of a reagent or kit suitable for use in the methods of the invention as described above.

In another aspect, the present invention also provides a reagent or kit for determining whether a subject has or is at risk of developing cervical cancer, comprising a reagent for detecting the relative copy number of the 3q amplification hotspot region on chromosome 3 in a sample of the subject.

In specific embodiments, the reagents for detecting the relative copy number of the 3q amplification hotspot region on chromosome 3 comprise reagents for detecting the copy number of the 3q amplification hotspot region on chromosome 3 in the sample by real-time quantitative pcr (qpcr), and reagents for detecting the copy number of the reference chromosome region in the sample by real-time quantitative pcr (qpcr).

In a specific embodiment, the 3q amplification hotspot region comprises a chromosomal region from 3q26.2 to 3q 29.

In specific embodiments, the 3q amplification hotspot region comprises a chromosomal region from 3q27 to 3q 29.

In a specific embodiment, the 3q amplification hotspot region comprises a chromosomal region of the DLG1 gene.

In particular embodiments, the reference chromosomal region comprises a chromosomal region of a single copy gene.

In a specific embodiment, the reference chromosomal region comprises a chromosomal region of an ALB gene.

In specific embodiments, the reagents for detecting the copy number of the 3q amplification hotspot region on chromosome 3 in the sample by real-time quantitative pcr (qpcr) comprise a forward primer P1 and a reverse primer P2 that hybridize to the target nucleic acid sequence on the 3q amplification hotspot region.

In specific embodiments, the reagents for detecting the copy number of a reference chromosomal region in the sample by real-time quantitative pcr (qpcr) comprise a forward primer P3 and a reverse primer P4 that hybridize to a target nucleic acid sequence on the reference chromosomal region.

Drawings

FIG. 1 shows copy number amplification of the chromosome three region in different cervical cancer samples.

FIG. 2 shows the results of gel electrophoresis of target nucleic acids amplified by qPCR using designed primers.

Figure 3 shows the amplification curve for qPCR amplification using designed primers.

Detailed Description

The present invention has many specific embodiments, and technical details of the present invention depend on numerous patents, applications, and other references known to those skilled in the art. Thus, when a patent, application, or other reference is cited or repeated below, it is understood that the entire contents thereof are incorporated herein by reference.

The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. A detailed description of a suitable technique may be obtained by reference to the following examples. However, other equivalent conventional procedures may of course be used. Such conventional techniques and descriptions can be found in standard Laboratory manuals, such as genomic analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and molecular cloning: A Laboratory Manual (both from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Free, New York, Gas, "oligonucleotide Synthesis: A Practical apparatus" 1984, IRL Press, London and Colon (2000), Lehninger, Principles of Biochemistry 3^rdEd, W.H.Freeman pub, New York, N.Y. and Berg et al (2002) Biochemistry, 5^thEd, w.h.freeman pub., New York, n.y., all of which are incorporated herein by reference in their entirety for all purposes.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Various scientific dictionaries covering the terms included herein are well known and available to those skilled in the art.

Definition of

As used herein, the term "subject" refers to any individual or patient that performs the methods of the invention. Generally, the subject is a human, although the subject may be an animal, as recognized by one of skill in the art. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject of the invention.

As used herein, the term "sample" or "specimen" refers to any sample suitable for the methods provided herein. The cell sample used in the present methods can be obtained from a tissue sample or body fluid of the subject, or a tissue obtained by a biopsy procedure (e.g., needle biopsy) or a surgical procedure. Thus, exemplary samples include, but are not limited to, tissue samples, frozen tissue samples, biopsy samples, surgical samples, cell samples, exfoliated cell samples, cell lines, xenografts, tumors, fine needle aspirates, whole blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, serum, plasma, amniotic fluid, mucus, plasma, urine, chyle, stool, sputum, sweat, tears, semen, nipple aspirates, saliva, and any combination thereof.

In particular embodiments, the term "sample" or "specimen" includes a nucleic acid sample. As used herein, the term "nucleic acid sample" refers to a sample comprising nucleic acids. In particular embodiments, the nucleic acid sample can be a sample comprising a genome, e.g., a sample comprising all or a portion of the genome of a subject. In a preferred embodiment, the sample is a genomic sample from a subject.

As used herein, the term "control sample" or "control sample" refers to a sample known to have no copy number variation. For example, such a sample may be a sample from a healthy subject or a subject not having cervical cancer.

As used herein, the term "copy number" refers to the number of copies of a given chromosome or chromosomal region or gene that are present in a genome. In the context of the present invention, reference to a chromosome generally refers to a human chromosome.

As used herein, the term "3 q amplification hotspot region" refers to a region of the long arm of chromosome three (3q) that is associated with cervical cancer. The present inventors have found that copy number amplification of a 3q amplification hotspot region can be used as a marker for judging the occurrence of cervical cancer. In a specific embodiment, the 3q amplification hotspot region comprises or consists of the chromosomal region 3q26.2 to 3q29 on chromosome three. In a more specific embodiment, the 3q amplification hotspot region comprises or consists of a chromosomal region on chromosome three, 3q26.2, 3q26.31, 3q26.32, 3q26.33, 3q27.1, 3q27.2, 3q27.3, 3q28, or 3q29, or a chromosomal region between any of the foregoing loci. In a preferred embodiment, the 3q amplification hotspot region comprises or consists of the DLG1(Discs large homolog 1) gene located at 3q29 (from 197042560bp to 197299272bp) of the hotspot region.

As used herein, the term "copy number amplification" refers to an increase in the copy number of a given chromosome or chromosomal region or gene present in a genome as compared to a reference chromosome or chromosomal region or gene.

As used herein, "reference chromosome", "reference chromosomal region", or "reference gene" are used interchangeably to refer to a chromosome, chromosomal region, or gene that is different from the target chromosome, chromosomal region, or gene. It may be a single copy of a chromosome, chromosomal region or gene or a chromosome, chromosomal region or gene of known copy number. In some embodiments, the reference chromosome, chromosomal region, or gene is on the same or different nucleic acid molecule (e.g., chromosome) as the target chromosome, chromosomal region, or gene. In a preferred embodiment, a "reference chromosome", "reference chromosomal region" or "reference gene" is a single copy of a chromosome, chromosomal region or gene. Examples of single copy chromosomes, chromosomal regions or genes include, but are not limited to, genes encoding the following proteins and chromosomes or chromosomal regions containing such genes: CD3, CD4, 36B4 (which encodes acid ribosomal phosphoprotein P0(RPLP0)), ATP-synthase subunit 5B (A5B), tumor protein translation control protein 1(TPT1), signal recognition particle 14kDa (SRP14), TATA Binding Protein (TBP), eukaryotic elongation factor 1a1(EEF1a1), hypoxanthine phosphoribosyltransferase 1(HPRT1), polyubiquitin (Ubi), glyceraldehyde-3-phosphate dehydrogenase (G3PD), β -Actin (ACTB), and ALB. Suitable examples of other single copy genes are known to those skilled in the art. In a preferred embodiment, the reference chromosome or chromosomal region or gene is an ALB gene or a chromosomal region comprising an ALB gene. The present inventors found that the ALB gene located on chromosome 4 (4q13.2, from 73404255bp to 73421412bp) is a single copy gene with less occurrence of copy number variation, which is particularly suitable for use as a reference gene in the method of the present invention.

As used herein, "Polymerase Chain Reaction (PCR)" is a rapid nucleic acid amplification method well known in the art (see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188). PCR generally involves adding a polymerase, dntps, a buffer, primers (e.g., a pair of oligonucleotide primers) to a sample (template), and subjecting the PCR master mix to at least one cycle, including denaturation (melting), annealing (or hybridization), and extension (or extension) steps.

As used herein, the term "primer" refers to a polynucleotide that can serve as a point of initiation of nucleic acid synthesis in a suitable buffer and at an appropriate temperature, for example, in the presence of 4 different nucleoside triphosphates and a polymerase. Thus, a primer includes a targeting sequence that hybridizes to a target nucleic acid (template). The primer is typically an oligonucleotide and is single-stranded, however, the primer may refer to a polynucleotide having a double-stranded segment. The appropriate length of the targeting sequence for the primer depends on the intended use of the primer. Short primer molecules generally require lower temperatures to form sufficiently stable hybridization complexes with the template. The primer need not reflect the exact sequence of the nucleic acid template, but must be sufficiently complementary to hybridize with the nucleic acid template. In the methods described herein, the primers can be synthesized using the four naturally occurring deoxynucleotides dATP, dTTP, dCTP and dGTP. In some embodiments of the invention, the primer may also incorporate natural or synthetic deoxynucleotide analogs not normally present in DNA. Primers can be designed using standard primer design computer software techniques known to those skilled in the art. Variables considered during primer design can include primer length, GC pair content, melting temperature, and size of the target nucleic acid amplified by the primer or primer pair. In general, the primers should not form hairpin structures or self or heterologous primer pairs. In preferred embodiments, the primer may comprise a sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more bases complementary to a portion of the template.

As used herein, the term "complementary" or "substantially complementary" refers to hybridization or base pairing or duplex formation between nucleotides or nucleic acids. Two nucleic acids are considered to be complementary to each other at a given position if the nucleotides of the two nucleic acids are capable of forming hydrogen bonds with the nucleotides of the other nucleic acid at that position. Complementary nucleotides are typically A and T (or A and U) or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand are aligned to at least about 80%, usually at least about 90% to about 95%, and even about 98% to 100% of the other strand, with optimal alignment and comparison and appropriate nucleotide insertions or deletions.

As used herein, the term "hybridize" refers to the formation of a double-stranded structure of two single-stranded nucleic acids by base-complementary pairing. Hybridization can occur between fully complementary nucleic acid strands or between "substantially complementary" nucleic acid strands with a small amount of mismatched regions. Conditions under which hybridization can only occur between fully complementary nucleic acid strands are referred to as "stringent hybridization conditions" or "sequence-specific hybridization conditions". Stable duplexes of substantially complementary sequences may be obtained under non-stringent hybridization conditions. The degree of mismatch tolerated can be controlled by appropriate adjustment of the hybridization conditions. One skilled in the art can empirically determine the stability of a duplex by considering a number of variables. These variables include: length of oligonucleotide and concentration of base pairs, ionic strength, base pair mismatch ratio. Qualitative and quantitative considerations for establishing stringent and non-stringent hybridization conditions for designing oligonucleotides or probes of the invention can be found, for example, in Ausubel et al, Short Protocols in Molecular Biology (4th ed., John Wiley & Sons 1999); sambrook et al, Molecular Cloning: a Laboratory Manual (3d ed., Cold Spring harbor Laboratory Press 2001): nucleic Acid hybridization: a Practical Approach (B.D. Hames & S.J. Higgins eds., IRL Press 1985).

As used herein, the term "polymerase" refers to an enzyme that synthesizes a nucleic acid strand or polymer, including DNA polymerases and RNA polymerases. Preferably, the polymerase used herein is a DNA polymerase. One polymerase that may be used is Sequenase^TM(derived from phage 7 DNA polymerase, which is modified to improve its sequencing properties-see Tabor and Richarson, Proc. Nat. Acad. Sci. USA, 84:4767-4771(1987), available from, for example, United states Biochemical Corporation, Cleveland, Ohio). Can be used to replace Sequenase^TMIncluding but not limited to the Klenow fragment of DNA polymerase I, AMV reverse transcriptase and Taq polymerase. Further descriptions of polymerases that can be used in the methods described herein can also be found in WO05024010 and WO06120433, the entire contents of which are incorporated herein by reference.

The primer extension conditions generally used are polymerization conditions known in the art to be suitable for the polymerases described above. In Sequenase^TMIn the case of (a), the polymerization conditions include a temperature in the range of from about room temperature to about 45 ℃; a buffer of pH7 to 8, preferably pH 7.3 to 7.7; the enzyme concentration is about 0.01 units/microliter to about 1 unit/microliter, and the reaction time is about 1 to about 20 minutes, preferably 1 to 5 minutes. For sequenases^TMA typical buffer of (a) consists of: 0.040M Tris HCl (pH7.5), 0.050M sodium chloride, 0.010M magnesium chloride, 0.010M dithiothreitol. In the case of the Klenow fragment of DNA polymerase I, these typical conditions include a temperature in the range of about 10 ℃ to about 45 ℃, preferably about 15 ℃ to about 40 ℃; a buffer at pH 6.8 to 7.4, preferably pH 7.0 to 7.4; the enzyme concentration is about 0.01 units/microliter to about 1 unit/microliter, preferably about 0.02 to about 0.15 units/microliter, and the reaction time is about 1 to about 40 minutes. A typical buffer for the Klenow fragment of DNA polymerase I consists of: 0.05M Tris chloride, pH 7.50.05M magnesium chloride, 0.05M sodium chloride, 0.010M dithiothreitol.

It should be understood that these conditions are exemplary only. When other polymerases are used, the conditions most suitable for them should be used, since it is generally desirable to perform the polymerization reaction as quickly as possible. For this purpose, a temperature of 42 ℃ is generally used for the reverse transcriptase; 24 ℃ for Klenow polymerase; for Sequenase^TMAt 37 ℃; and 72 ℃ for Taq polymerase. Furthermore, in order to enhance the reaction, especially in case modified dntps are used, it may be advantageous to use a significant excess of dntps (over stoichiometry) or to modify other conditions such as salt concentration.

Preparation of genomic samples

Various methods of preparing genomic samples can be used in the methods of the invention. In the present invention, the preparation of the genome comprises digesting proteins and RNA in a sample, extracting and precipitating DNA, and resuspending the DNA (i.e., genome) pellet in a solution (e.g., water). Typically, a protease (e.g., proteinase K) is used to digest proteins contained in the sample, and an RNase (e.g., RNase A) is used to digest RNA. The remaining DNA is subsequently purified, for example by precipitation. Various methods of DNA purification are known in the art and may be used in the methods of the present invention.

Detection of relative copy number of 3q amplification hotspot region

Various techniques are known in the art for detecting changes in copy number of chromosomes and genes. Thus, the detection of the relative copy number of the 3q amplification hotspot region of the invention can be performed using any method known in the art to detect relative copy number. Such methods include, for example, Comparative Genomic Hybridization (CGH), multicolor fluorescence in situ hybridization (M-FISH), and real-time quantitative PCR (qPCR).

In a preferred embodiment of the present invention, the relative copy number of the 3q amplification hotspot region on chromosome 3 is detected by real-time quantitative pcr (qpcr).

As used herein, the terms "real-time PCR," "quantitative PCR," "real-time quantitative PCR," and "qPCR" are used interchangeably and are a PCR-based technique for amplifying and quantifying a target genetic material (e.g., a chromosome, a chromosomal region, or a gene). qPCR enables to sample DNACan be detected and quantified (relative or absolute levels of copy number can be quantified when normalizing to DNA addition or other reference genes). qPCR follows the general procedure of PCR and has the additional feature of quantifying the accumulated amplified DNA in real time after each cycle of qPCR. Such quantification can be accomplished by conventional methods of inserting a fluorescent substance into the accumulating DNA or a nucleic acid probe complementary to the amplified DNA sequence that produces a detectable signal upon binding to the accumulating DNA. qPCR methods are known to those skilled in the art and are described in the prior art, e.g., Sambrook and Russell, Molecular Cloning: a laboratory Manual. Volumes 1,2and 3(2001, 3)^rdEdition); "Guide to Forming ReglativeQuantitation of Gene Expression Using Real-Time Quantitative PCR," Part Number4371095, and Arya et al (2005) Expert Rev.mol. Diagn.5:209-19, each of which is incorporated herein by reference in its entirety.

Accordingly, in one embodiment, the present invention provides a method of determining whether a subject has or is at risk of developing cervical cancer by detecting the relative copy number of a 3q amplification hotspot region on chromosome 3 by real-time quantitative pcr (qpcr), the method comprising:

a. providing a sample from the subject,

b. detecting the copy number of the 3q amplification hotspot region on chromosome 3 in the sample,

c. detecting the copy number of the reference chromosomal region in said sample, and

d. normalizing the copy number of the 3q amplification hotspot region relative to the copy number of the reference chromosome region, thereby obtaining a relative copy number of the 3q amplification hotspot region,

wherein an increase in the relative copy number of the 3q amplification hotspot region in the subject's sample, as compared to the relative copy number of the 3q amplification hotspot region in the control sample, is indicative of the subject having, or being at risk of developing, cervical cancer.

In a specific embodiment, the 3q amplification hotspot region comprises a chromosomal region from 3q26.2 to 3q 29.

The present inventors also found that the DLG1 gene located at 3q29 (from 197042560bp to 197299272bp) of the hotspot region is particularly suitable for characterizing copy number amplification of the 3q amplification hotspot region, and thus is particularly suitable for diagnosing cervical cancer.

Thus, in one embodiment, the invention provides a method of characterizing copy number amplification of a 3q amplification hotspot region in a sample from the subject, comprising detecting the relative copy number of DLG1 gene in the sample, wherein an increase in the relative copy number of DLG1 gene in the sample of the subject compared to the relative copy number of DLG1 gene in a control sample indicates that the copy number of the 3q amplification hotspot region is amplified.

In another embodiment, the present invention provides a method of determining whether a subject has or is at risk of developing cervical cancer by detecting the relative copy number of the DLG1 gene by real-time quantitative pcr (qpcr), the method comprising:

a. providing a sample from the subject,

b. detecting the copy number of the DLG1 gene in the sample,

c. detecting the copy number of a reference chromosomal region or a reference gene in said sample, and

d. normalizing the copy number of the 3q amplification hotspot region relative to the copy number of the reference chromosome region, thereby obtaining a relative copy number of the 3q amplification hotspot region,

wherein an increase in the relative copy number of the DLG1 gene in the sample of the subject as compared to the relative copy number of the DLG1 gene in the control sample is indicative of the subject having or being at risk of developing cervical cancer.

Reagent and kit

The invention also relates to reagents or kits for detecting cervical cancer comprising reagents (e.g., primers, dyes or probes, polymerases, nucleotides, buffers, controls, instructions, etc.) necessary, sufficient, or useful for detecting relative copy number. For example, in some embodiments, the kit includes primers for amplification of a chromosomal region (or gene) of interest and a reference chromosomal region (or gene). In some embodiments, the kit includes analytical software (e.g., to analyze amplification data).

As previously described, the detection of the relative copy number of the 3q amplification hotspot region of the invention can be performed using any method known in the art to detect relative copy number. One skilled in the art can determine the reagents for detecting the relative copy number of the 3q amplification hotspot region on chromosome 3 in a sample from a subject according to the particular detection method. Such agents are also well known in the art.

For example, in embodiments where the relative copy number of the 3q amplification hotspot region on chromosome 3 is detected by qPCR, the reagents for detecting the relative copy number of the 3q amplification hotspot region on chromosome 3 comprise reagents for detecting the copy number of the 3q amplification hotspot region on chromosome 3 in the sample by real-time quantitative pcr (qPCR), and reagents for detecting the copy number of the reference chromosome region in the sample by real-time quantitative pcr (qPCR).

Reagents for qPCR may typically include, for example, polymerase, free nucleotides (e.g., dntps), buffers, fluorescent dyes (e.g., SYBR green), or fluorescent reporter probes (e.g., Taqman probes), as well as forward and reverse primers.

In particular embodiments, the kit can comprise forward primer P1 and reverse primer P2 that hybridize to the target nucleic acid sequence on the 3q amplification hotspot region, and forward primer P3 and reverse primer P4 that hybridize to the target nucleic acid sequence on the reference chromosomal region. In particular embodiments, the kit comprises, in addition to the primers described above, one or more of primers, a 2 × SYBRGreen qPCR Mix, deionized water, and a control sample.

In some embodiments, the kit includes one or more containers comprising primers, probes, polymerases, nucleotides, buffers, controls, and the like. In some embodiments, the components of the kit are packaged in separate containers. In some embodiments, the containers are packaged and/or shipped in the same kit or case for use together. In some embodiments, one or more components of the kit are shipped and/or packaged separately.

The invention has the beneficial technical effects

The present inventors have surprisingly found that amplification of the copy number of the 3q amplification hotspot region as described herein can be detected in all cervical cancer patients tested. Whereas copy number amplification of a chromosomal region on chromosome three outside the 3q amplification hotspot region as described herein could not be detected in all cervical cancer patients. Thus, the relative copy number of the 3q amplification hotspot region as described herein can be used to detect cervical cancer in a subject.

The inventor also finds that the cervical cancer can be detected by judging the amplification condition of a 3q amplification hot spot region by carrying out qPCR copy number detection on the DLG1 gene. The qPCR method not only can accurately detect the 3q amplification of the cervical cancer, but also reduces the experiment cost and shortens the detection period.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.