CN118064590A

CN118064590A - Nucleic acid combination product for detecting bladder cancer and application thereof

Info

Publication number: CN118064590A
Application number: CN202410322564.0A
Authority: CN
Inventors: 韩达; 刘振彬; 马倩
Original assignee: Zhenzhida Biotechnology Shanghai Co ltd
Current assignee: Zhenzhida Biotechnology Shanghai Co ltd
Priority date: 2024-03-20
Filing date: 2024-03-20
Publication date: 2024-05-24

Abstract

The application relates to the technical field of biomedicine, in particular to a nucleic acid combination product for detecting bladder cancer and application thereof. The nucleic acid combination product of the application adds the primer pair and the probe for detecting at least one of the NID2 and PENK auxiliary genes on the basis of the primer pair and the probe for detecting the single TWIST1 gene, improves the positive detection rate and the accuracy of bladder cancer, and provides more possibility for early detection and treatment of tumor diseases.

Description

Nucleic acid combination product for detecting bladder cancer and application thereof

Technical Field

The application relates to the technical field of biomedicine, in particular to a nucleic acid combination product for detecting bladder cancer and application thereof.

Background

Global Bladder Cancer (BC) is the 10 th most common cancer, with approximately 570,000 new cases and over 210,000 deaths each year. Clinically, routine examination of bladder cancer includes routine urine, B-ultrasound, cytological examination of urine shedding, and FISH examination of bladder endoscopy. Cystoscopy may lead to risk of urinary tract infections, urinary tract injuries, bladder injuries, etc. Ordinary cystoscopy leads to poor patient compliance, whereas painless cystoscopy has anesthesia risk, high tariffs, and unavoidable urinary tract infection risk. The sensitivity or specificity of the B ultrasonic and urine abscission cytology examination method is not high, and especially early cancer patients can not be accurately identified.

The traditional early screening method has a plurality of limitations, and if the early diagnosis rate of bladder cancer is to be effectively improved, a new technology is clinically needed for breaking ice. Liquid biopsy is a cancer prognosis technique that has been expected in recent years, and a genetic testing method for diagnosing tumors by collecting body fluid DNA on the human body using a non-invasive method, the main biomarkers including Circulating Tumor Cells (CTCs), ctDNA, and exosomes (exosomes). Such as the detection of the methylation status of the urine shed tumor cells, and the auxiliary evaluation of the occurrence, recurrence, drug efficacy and the like of bladder cancer.

A number of factors are associated with recurrence, progression of bladder cancer, and affect patient survival. There are a number of molecular markers currently available for the examination of bladder cancer, but their value is very limited. Thus, there is a need for new molecular markers for detection of bladder cancer patients (particularly those at high risk of progression and recurrence). Recently epigenetic markers have been used as an estimate of the high risk of developing cancer, providing new opportunities for treatment and prevention. DNA methylation has received increasing attention in recent years as a biomarker, as aberrant DNA methylation is a major feature of bladder cancer, playing a key role in the development and progression of bladder cancer.

Genetics is the study of genetic information on a genetic basis, whereas epigenetic is the study of reversible changes in heritable gene function without any DNA changes and other cellular phenotypes. DNA methylation occurs throughout the genome, including the addition of a methyl group on the cytosine loop of a CpG dinucleotide by a methyltransferase. DNA methylation is a key regulator of gene transcription and genome stability, in human cancers, abnormal changes of DNA methylation patterns activate high expression of oncogenes, and silencing of cancer suppressor genes does not express, so that cancerogenesis and cancer suppression are out of balance, and finally cancer is caused. Many cancers exhibit genome-wide hypomethylation, and regions of certain gene (proven to be cancer suppressor) promoters are highly methylated, which makes methylation possible as a marker for cancer. In addition, each tumor type may have its own unique methylation pattern, which allows the development of various tumor-specific methylation markers. Alterations in epigenetic events occur at the cancerous stage or early stages of cancer, and thus, gene methylation as a molecular diagnosis may be helpful in detecting cancer before symptoms or apparent imaging appear.

Significant progress has been made in the epigenetic study of bladder cancer. Since cancer suppressor gene promoter hypermethylation is common in bladder cancer, potential DNA methylation markers have been identified in serum, bladder wash, urine samples, and cancer tissues. Thus, DNA methylation can serve as a biological marker for early bladder discovery, effective treatment, and accurate prognosis.

There was a single-site study to diagnose bladder cancer by MS-qPCR detection of 4 methylation sites (VIM, RASSF1A, GDF and TMEFF 2) with a sensitivity of 82% and a specificity of 53%. The previous research results show that DNA methylation in urine is an effective marker for diagnosing bladder cancer, but the sensitivity and the specificity of the DNA methylation in urine still need to be improved.

Recently, huang Jian/Lin day and Kupffer team reported in the journal of Journal of Clinical Investigation under Chen Xu, titled Urine DNA methylation assay enables early detection and recurrence monitoring for bladder cancer, that the study identified 26 bladder cancer specific methylation sites by performing an integrated analysis on 3-queue bladder cancer sequencing data, and modeled and verified 2 methylation sites by single-center 313 and prospective multi-center 175 queues, with AUCs of the training and verification groups of 0.919 and 0.903, respectively, with an overall accuracy of 86.7%, a sensitivity of 90.0% and a specificity of 83.1% for bladder cancer patients.

In the whole, the existing noninvasive urine biomarker detection method for bladder cancer is not ideal in sensitivity or specificity, has high missing diagnosis rate and misdiagnosis rate, is difficult to meet clinical requirements, and has low clinical practical application rate.

In view of this, the present application has been made.

Disclosure of Invention

One or more embodiments of the present application provide a nucleic acid combination product for detecting bladder cancer, which can be used for bladder cancer diagnosis, and has good specificity and high sensitivity, and applications thereof.

One or more embodiments of the present application provide a nucleic acid combination product comprising a primer pair and a probe for detecting a biomarker;

The primer pair and the probe for detecting the biomarker comprise: primer pairs and probes for detecting TWIST1 genes, and primer pairs and probes for detecting one or more of PENK genes and NID2 genes;

The primer pair and the probe for detecting the TWIST1 gene comprise: a forward primer shown in any one of SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10 and SEQ ID NO.13, a reverse primer shown in any one of SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11 and SEQ ID NO.14, and a probe shown in any one of SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 and SEQ ID NO. 15;

The primer pair and the probe for detecting the PENK gene comprise: a forward primer shown in any one of SEQ ID No.16, SEQ ID No.19, SEQ ID No.22, SEQ ID No.25 and SEQ ID No.28, a reverse primer shown in any one of SEQ ID No.17, SEQ ID No.20, SEQ ID No.23, SEQ ID No.26 and SEQ ID No.29, a probe shown in any one of SEQ ID No.18, SEQ ID No.21, SEQ ID No.24, SEQ ID No.27 and SEQ ID No. 30;

the primer pair and the probe for detecting the NID2 gene comprise: a forward primer shown in any one sequence of SEQ ID No.31 and SEQ ID No.34, a reverse primer shown in any one sequence of SEQ ID No.32 and SEQ ID No.35, and a probe shown in any one sequence of SEQ ID No.33 and SEQ ID No. 36.

In some embodiments of the application, the primer pair and probe for detecting the TWIST1 gene comprises:

A forward primer shown as SEQ ID NO.1, a reverse primer shown as SEQ ID NO.2, and a probe shown as SEQ ID NO. 3; or alternatively

A forward primer shown as SEQ ID NO.4, a reverse primer shown as SEQ ID NO.5, and a probe shown as SEQ ID NO. 6; or alternatively

A forward primer shown as SEQ ID NO.7, a reverse primer shown as SEQ ID NO.8, and a probe shown as SEQ ID NO. 9; or alternatively

A forward primer shown as SEQ ID NO.10, a reverse primer shown as SEQ ID NO.11, and a probe shown as SEQ ID NO. 12; or alternatively

A forward primer shown as SEQ ID NO.13, a reverse primer shown as SEQ ID NO.14, and a probe shown as SEQ ID NO. 15.

In some embodiments of the application, the primer pair and probe for detecting PENK gene comprises:

A forward primer shown as SEQ ID NO.16, a reverse primer shown as SEQ ID NO.17, and a probe shown as SEQ ID NO. 18; or alternatively

A forward primer shown in SEQ ID NO.19, a reverse primer shown in SEQ ID NO.20, and a probe shown in SEQ ID NO. 21; or alternatively

A forward primer shown in SEQ ID NO.22, a reverse primer shown in SEQ ID NO.23, and a probe shown in SEQ ID NO. 24; or alternatively

A forward primer shown in SEQ ID NO.25, a reverse primer shown in SEQ ID NO.26, and a probe shown in SEQ ID NO. 27; or alternatively

A forward primer shown in SEQ ID No.28, a reverse primer shown in SEQ ID No.29, and a probe shown in SEQ ID No. 30.

In some embodiments of the application, the primer pair and probe for detecting NID2 gene comprises:

A forward primer shown as SEQ ID NO.31, a reverse primer shown as SEQ ID NO.32, and a probe shown as SEQ ID NO. 33; or alternatively

A forward primer shown as SEQ ID NO.34, a reverse primer shown as SEQ ID NO.35, and a probe shown as SEQ ID NO. 36.

In some embodiments of the application, the primer pair and probe for detecting a biomarker consists of the primer pair and probe for detecting a TWIST1 gene and the primer pair and probe for detecting a PENK gene, or consists of the primer pair and probe for detecting a TWIST1 gene and the primer pair and probe for detecting a NID2 gene.

In some embodiments of the application, in the primer pair and probe for detecting a biomarker, the 5 'end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; alternatively, the excitation light waves of the fluorophores of the probes for detecting different biomarkers are different.

In some embodiments of the application, the nucleic acid combination further comprises a primer pair and a probe for detecting a reference gene;

Optionally, the primer pair and the probe for detecting the reference gene comprise a forward primer shown in SEQ ID NO.37, a reverse primer shown in SEQ ID NO.38 and a probe shown in SEQ ID NO. 39;

Further alternatively, in the primer pair and the probe for detecting the reference gene, a fluorescent group is marked at the 5 'end of the probe, and a quenching group is marked at the 3' end of the probe;

further, the primer pair for detecting the reference gene and the probe in the probe and the primer pair for detecting the biomarker and the probe in the probe have fluorescent groups with different excitation light waves.

In yet another or more embodiments of the present application, there is provided a use of the nucleic acid combination product in the preparation of a bladder cancer diagnostic kit.

Still another or more embodiments of the present application provide a kit comprising the nucleic acid combination product.

In some embodiments of the application, the kit further comprises one or more of a DNA amplification reagent, a reagent that converts unmethylated cytosine bases to uracil, a DNA extraction reagent, and a DNA purification reagent.

Additional features, objects, and advantages of the application will be apparent from the description and claims, and from one or more embodiments of the application will be set forth in the description which follows.

Drawings

In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art.

FIG. 1 is a ROC curve of TWIST1 gene and PENK gene combined detection;

FIG. 2 is a ROC curve of the joint detection of TWIST1 gene and NID2 gene;

FIG. 3 shows the detection results corresponding to nucleic acid combination 1;

FIG. 4 shows the detection results corresponding to nucleic acid combination 2;

FIG. 5 shows the detection results corresponding to nucleic acid set 3;

FIG. 6 shows the detection results corresponding to nucleic acid set 4;

FIG. 7 shows the detection results corresponding to acid combination 5;

FIG. 8 shows the detection results corresponding to nucleic acid combination 6;

FIG. 9 shows the detection results corresponding to nucleic acid set 7;

FIG. 10 shows the detection results corresponding to nucleic acid set 8;

FIG. 11 shows the detection results corresponding to nucleic acid set 9;

FIG. 12 shows the detection results corresponding to nucleic acid set 10;

FIG. 13 shows the detection results corresponding to nucleic acid set 11;

FIG. 14 shows the detection results corresponding to the nucleic acid assembly 12;

FIG. 15 shows the results of the combined detection of nucleic acid combination 5 and nucleic acid combination 10;

FIG. 16 shows the results of a combined TWIST1 and NID2 test.

Detailed Description

The present application will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the application, it being understood that the application may be practiced without one or more of these details.

Unless defined otherwise, all 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. The terminology used herein in the description of the application is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the application.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).

The terms "plurality", "plural", "multiple", and the like in the present application refer to, unless otherwise specified, an index of 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present application can be implemented, the technical problem of the present application is solved, and the technical effect expected by the present application is achieved.

Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the application.

In the present application, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the application.

In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the present application, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Unless otherwise indicated, when a numerical range merely refers to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, herein, each integer is recited directly, such as t is an integer selected from 1-10, and t is any integer selected from the group of integers consisting of 1,2, 3, 4,5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

In the present application,% (w/w) and wt% each represent weight percent,% (v/v) represents volume percent, and% (w/v) represents mass volume percent.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical scheme of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.

In the reported literature, the sensitivity of single-gene methylation detection to bladder cancer is 65% -100%, the clinical sensitivity of the same gene in different researches is far away, and the considered factors are ① group-entering crowd differences and are influenced by the disease spectrum of each research institution; ② The same gene detected by each study, but the methylation sites of the promoter regions are different; ③ The differences in study types, retrospective and prospective, give bias to test results, (KIM AND KIM 2016). Genes traditionally reported for bladder cancer detection, for example: TWIST1, DAPK1, TERT, BCL2, p16 and RASSF1A, COL A2 have poor detection effect.

Early detection of bladder cancer (BCa) provides beneficial results to the patient and avoids the need for a cystectomy. The development of accurate and sensitive noninvasive BCa diagnostic test agents is imperative. DNA methylation is an early epigenetic event in the BCa development process. Certain specific aberrant methylation may serve as useful biomarkers. The object of the present application is to determine methylation biomarkers for early detection BCa. CpG methylation microarray analysis was performed on primary tumors and paired non-tumor tissues from different stages (T1-T4) of 9 BCa patients. Bisulphite pyrophosphate sequencing was performed to confirm the methylation status of candidate genes in tissue and urine sediment (n=51). Among them, PENK was selected as potential candidate and a set of independent 169 urine sediments (55 bca,25 benign urinary system diseases, 8 other urinary system cancers and 81 healthy controls) was used with quantitative methylation specific real-time PCR (mePENK-qMSP). All statistical analyses were performed using MedCalc software version 9.3.2.0. CpG methylation microarray analysis and stepwise verification of tissue and urine sediment by bisulfite pyrosequencing support abnormal methylation sites of the PENK gene as potential biomarkers for early detection BCa. Clinical validation of the mePENK-qMSP test using urinary sediment DNA showed a sensitivity of 86.5% (95% CI: 71.2-95.5%), a specificity of 92.5% (95% CI: 85.7-96.7%), and an area lower than that of ROC of 0.920 (95% CI: 0.863-0.959) when Ta advanced and advanced tumor stage (T1-T4) of BCa patients were examined. The sensitivities of the low-grade tantalum, high-grade tantalum, T1 and T2-T4 were 55.6, 83.3, 88.5 and 100%, respectively. The methylation status of PENK is independent of sex, age or stage and is related to the tumor grade of BCa. In this study, the present application analyzed the comprehensive pattern of DNA methylation, and found that PENK methylation has high potential as a biomarker for urine-based BCa early detection. Verification of PENK methylation demonstrates that it can significantly improve the sensitivity and specificity of the non-invasive detection of BCa. The kit provided by the application can detect the methylation states of the TWIST1, NID2 and PENK genes related to bladder cancer in urine abscission cells by adopting a methylation specific real-time fluorescence PCR method, and is an auxiliary diagnosis method for primarily diagnosing bladder cancer.

The term "diagnosis" includes auxiliary diagnosis, recurrence risk assessment, assessment of risk and extent of cancerous lesions, prognosis, and the like.

The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and typically more than ten. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotides may be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine and guanine.

The term "methylation" is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. DNA methylation refers to covalent binding of a methyl group at the 5 th carbon position of cytosine of a genomic CpG dinucleotide under the action of a DNA methyltransferase. DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression.

The term "methylation level" refers to whether or not cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/proportion/percentage of methylation, representing both qualitative and quantitative concepts.

In practical application, different detection indexes can be adopted to compare the DNA methylation level according to practical conditions. As in some cases, the comparison may be made based on Ct values detected by the sample; in some cases, the ratio of gene methylation in the sample, i.e., number of methylated molecules/(number of methylated molecules+number of unmethylated molecules). Times.100, can be calculated and then compared; in some cases, statistical analysis and integration of each index is also required to obtain a final decision index.

The term "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction, PCR) to amplify a sequence of interest based on a polynucleotide sequence corresponding to a gene of interest or a portion thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used. For example, for diagnostic and prognostic applications, the oligonucleotide primers will typically contain at least 10, 15, 20, 25 or more nucleotides, but may also contain fewer nucleotides, depending on the complexity of the target sequence. In the present disclosure, the term "primer" or "primer pair" refers to a pair of primers that hybridize to the double strand of a target DNA molecule or to regions of the target DNA molecule that flank the nucleotide sequence to be amplified.

The term "methylation-specific PCR" is one of the most sensitive experimental techniques currently studied for methylation, and a minimum of about 50pg of DNA methylation can be found. After the single-stranded DNA is subjected to bisulfite conversion, all unmethylated cytosines are deaminated to uracil, and methylated cytosines in CpG sites are kept unchanged, so that two pairs of primers aiming at methylated and unmethylated sequences are respectively designed, and the methylated and unmethylated DNA sequences can be distinguished through PCR amplification.

The term "methylation specific fluorescent quantitative PCR (QMSP)" is an experimental technique combining fluorescent quantitative PCR technology and methylation specific PCR technology. In the technology, proper primer pairs are designed based on sequence differences of DNA in different methylation states after bisulfite conversion, so that methylated sequences and unmethylated sequences are distinguished, but the final detection index of the q-MSP is a fluorescent signal, so that a fluorescent probe or a fluorescent dye is required to be added in addition to a methylation detection primer in a q-MSP reaction system. Compared with the traditional methylation specific PCR technology, the q-MSP detection DNA methylation level has higher sensitivity and specificity, is more suitable for detecting trace amounts of DNA fragments with abnormal methylation mixed in the DNA of patients in early cancer, does not need gel electrophoresis detection, and is simpler and more convenient to operate.

The term "probe" refers to a stretch of oligonucleotide sequences comprising a 5 'fluorescent group and a 3' quenching group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce because of the presence of a quenching group near the fluorescent group. During amplification, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of the DNA polymerase (e.g., taq enzyme) digests the probe and the fluorescent group is far from the quenching group, its energy is not absorbed, i.e., a fluorescent signal is generated. The fluorescence signal is also identical to the target fragment with a synchronous exponential increase per PCR cycle.

The term "bladder cancer" in the present application has the same meaning and means cancer of the stomach or stomach cells. These cancers may occur.

"Biomarker" in the present application refers to a substance such as a gene, a variable measurement associated with a disease, and can be used as an indicator or predictor of that disease. The presence or risk of the disease can be inferred from this parameter of the biomarker without the need to determine the disease itself.

In a first aspect of embodiments of the application, there is provided a nucleic acid combination product comprising a primer pair and a probe for detecting a biomarker;

In some examples, the primer pair and probe for detecting the TWIST1 gene comprises: a forward primer shown as SEQ ID NO.1, a reverse primer shown as SEQ ID NO.2, and a probe shown as SEQ ID NO. 3; or a forward primer shown as SEQ ID NO.4, a reverse primer shown as SEQ ID NO.5, and a probe shown as SEQ ID NO. 6; or a forward primer shown as SEQ ID NO.7, a reverse primer shown as SEQ ID NO.8, and a probe shown as SEQ ID NO. 9; or a forward primer shown in SEQ ID NO.10, a reverse primer shown in SEQ ID NO.11, and a probe shown in SEQ ID NO. 12; or a forward primer shown in SEQ ID NO.13, a reverse primer shown in SEQ ID NO.14, and a probe shown in SEQ ID NO. 15.

In some examples, the primer pair and probe for detecting the PENK gene comprises: a forward primer shown as SEQ ID NO.16, a reverse primer shown as SEQ ID NO.17, and a probe shown as SEQ ID NO. 18; or a forward primer shown as SEQ ID NO.19, a reverse primer shown as SEQ ID NO.20, and a probe shown as SEQ ID NO. 21; or a forward primer shown as SEQ ID NO.22, a reverse primer shown as SEQ ID NO.23, and a probe shown as SEQ ID NO. 24; or a forward primer shown in SEQ ID NO.25, a reverse primer shown in SEQ ID NO.26, and a probe shown in SEQ ID NO. 27; or a forward primer shown in SEQ ID No.28, a reverse primer shown in SEQ ID No.29, and a probe shown in SEQ ID No. 30.

In some examples, the primer pair and probe for detecting NID2 gene comprises: a forward primer shown as SEQ ID NO.31, a reverse primer shown as SEQ ID NO.32, and a probe shown as SEQ ID NO. 33; or a forward primer shown in SEQ ID NO.34, a reverse primer shown in SEQ ID NO.35, and a probe shown in SEQ ID NO. 36.

In some examples, the primer pair and probe for detecting a biomarker consists of the primer pair and probe for detecting a TWIST1 gene and the primer pair and probe for detecting a PENK gene. Alternatively, the primer pair and probe for detecting a biomarker includes nucleic acid combination 5 and nucleic acid combination 10, as described in example 1.

In some examples, the primer pair and probe for detecting a biomarker consists of the primer pair and probe for detecting a TWIST1 gene and the primer pair and probe for detecting a NID2 gene. Alternatively, the primer pair and probe for detecting a biomarker includes nucleic acid combination 5 and nucleic acid combination 12, as described in example 2.

The method of detection using the nucleic acid combination product of the present application is not particularly limited, and detection of the methylation level of the target region is achieved, for example, by one or more of the following methods: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome bisulfite sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution melting curve, methylation-sensitive restriction endonuclease, and methylation-specific fluorescent quantitative PCR.

In some examples, in the primer pair and probe of the detection biomarker, the 5 'end of the probe is labeled with a fluorescent group and the 3' end is labeled with a quenching group; alternatively, the excitation light waves of the fluorophores of the probes for detecting different biomarkers are different.

In some examples, the nucleic acid combination further comprises a primer pair and a probe that detect a reference gene;

In a second aspect of the embodiment of the application, the application of the nucleic acid combination product in preparing a bladder cancer diagnosis kit is provided.

In a third aspect of embodiments of the application, a kit is provided comprising the nucleic acid combination product.

In some examples, the kit further comprises one or more of a DNA amplification reagent, a reagent that converts an unmethylated cytosine base to uracil, a DNA extraction reagent, and a DNA purification reagent.

The nucleic acid combination and the kit provided by the application are suitable for clinical suspected bladder cancer such as haematuria, frequent urination, urgent urination, painful urination and the like, and clinical diagnosis suggestions for carrying out auxiliary diagnosis of a cystoscopy patient, and can be used as supplement and assistance of the existing diagnosis method for reference of clinicians.

The nucleic acid combination and the kit provided by the application can be used for detecting urine samples which are taken as detection objects. In the present application, "subject" or "patient" or "subject" includes human patients and other mammals, as well as any individual suffering from or suffering from bladder cancer, or who is desired to be analyzed or treated using the methods of the present application. Suitable mammals that fall within the scope of the application include, but are not limited to: primates, domestic animals (e.g., sheep, cattle, horses, monkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), pets (e.g., cats, dogs), and wild animals (e.g., foxes, deer, wild dogs) in containment. Preferably, the patient is a human patient.

The following are examples of nucleic acid combinations, kits, and methods of detecting urine samples using the nucleic acid combinations of the present application. It will be appreciated that a variety of other embodiments may be implemented in view of the general description provided above.

Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present application, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

TABLE 1 primer pairs and probes for detecting TWIST1 genes

TABLE 2 primer pairs and probes for detecting PENK Gene

TABLE 3 primer pairs and probes for detecting NID2 Gene

TABLE 4 primer pairs and probes for detecting GAPDH genes

In tables 1 to 4, the primer sequences are all 5 'to 3' terminal.

Example 1

1. Sample selection

360 Urine samples were taken, of which 94 urine samples for patients with bladder cancer, 114 urine samples for patients with interference, and 152 urine samples for healthy people.

Grouping criteria for samples:

the following conditions were selected for bladder cancer entry: the histological classification of bladder tumors recommended the classification standard of the classification of tumor of the WHO urinary system and male genital organs (4 th edition) in 2016, which was partially changed from 2004 edition. Bladder cancer staging criteria were based on the TNM staging system using the AJCC 2017 eighth edition of criteria. The final diagnosis is performed by histopathological examination as a gold standard, while relying on the expertise of the senior clinician.

Interference samples are grouped: the disease such as prostatic hyperplasia, vesical calculus, urothelial infection and the like is diagnosed, and the pathological result is negative bladder cancer is eliminated.

Three conditions (1) are selected for the healthy group, and subjects with common chronic diseases (hypertension, diabetes and coronary heart disease), tumor treatment history and major operation history are excluded according to a questionnaire; (2) no obvious clinical symptoms from the description; (3) The physical examination result is in the normal value range, and no obvious abnormality is seen; the examination result is out of the normal value range or abnormal, but has no clinical meaning by the judgment of doctors. (physical examination items include LDCT, abdomen color Doppler ultrasound, tumor marker 4 items, blood pressure, etc., as shown in the figure).

Exclusion criteria: less than 18 years old; pregnancy; the sample is repeated, undiagnosed, and unqualified.

2. DNA extraction

Cell DNA/cell-free DNA was easily purified from up to 40mL of urine using Quick-DNA ^TM Urine Kit Catalog No. D3061 (ZYMO RESEARCH). DNA yield-the DNA binding capacity of the column was 5. Mu.g.

Note that DNA yield may vary from urine to urine. Female urine generally produces more DNA than male urine. Urine DNA amounts for healthy female individuals range on average from 6ng/mL to 1000ng/mL. The average range of DNA for healthy male individuals is 2-20ng/mL.

3. Methylation detection

The DNA/cell-free DNA obtained by extraction and purification is subjected to bisulphite treatment and purification recovery, and a ZYMO methylation kit D5005 is adopted, and specific steps are referred to the instruction book of the kit, so that 20 mu L of Bis-DNA is finally obtained for each urine sample.

PCR reaction systems of TWIST1, PENK and reference gene GAPDH were prepared with reference to Table 5 using Bis-DNA as a template, and the primer pairs and probes used were the nucleic acid combinations 5 (TWIST 1-F5: SEQ ID NO.13; TWIST1-R5: SEQ ID NO.14; TWIST1-P5: SEQ ID NO. 15) as shown in Table 1, the nucleic acid combinations 10 (PENK-F4: SEQ ID NO.28; PENK-R4: SEQ ID NO.29; PENK-P4: SEQ ID NO. 30) as shown in Table 4, and the nucleic acid combinations 13 as shown in Table 4.

TABLE 5 reaction System

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TABLE 6 reaction conditions and instrumentation

Standard reference: the positive reference substance is bladder cancer tissue DNA; the negative control is leukocyte DNA. The reaction system and reaction conditions for the corresponding controls were as described in tables 5 and 6.

4. Basis of judgment

Ct values of the reference gene, TWIST1 gene and PENK gene are respectively represented by Ct0, ct1 and Ct2, deltaCt1= |Ct 1-Ct 0| and DeltaCt2= |Ct2-Ct 0|, and the Ct0 value is larger than 35 and is regarded as unqualified detection. The result was determined under the condition that Ct0 value was not more than 35.

Table 7, basis for determination of TWIST1 Gene and PENK Gene combination detection

Under the condition of single PENK gene detection, the following judgment basis is adopted: ct2 is less than or equal to 35, and delta Ct2 is less than 8, and the positive is judged; otherwise, the result is negative.

Table 8, results of experiments for combination detection of TWIST1 gene and PENK gene

Sensitivity is the proportion of positive methylation detection results in samples with positive pathological results; the specificity is the proportion of methylation detection results negative in samples with negative pathological results; the accuracy is the proportion of samples of the detection results in the samples. The ROC curve is shown in fig. 1.

Example 2

Use cases of TWIST1 gene and NID2 gene in combination.

1. Sample selection was the same as in example 1.

2. DNA extraction was the same as in example 1.

3. Methylation detection was performed substantially as in example 1, with the variation from example 1 in that nucleic acid combination 5 in Table 1, nucleic acid combination 13 in Table 4 were used in combination with nucleic acid combination 12 (NID 2-F2: SEQ ID NO.34, NID2-R2: SEQ ID NO.35, NID2-P2: SEQ ID NO. 36) in Table 3.

4. Basis of judgment

Ct values of the reference gene, TWIST1 gene and NID2 gene are respectively represented by Ct0, ct1 and Ct3, delta Ct 1= |Ct 1-Ct 0| and delta Ct 3= |Ct 3-Ct 0|, and the Ct0 value is larger than 35 and is regarded as unqualified detection. The result was determined under the condition that Ct0 value was not more than 35.

(1) The corresponding judgment basis of single TWIST 1: ct1 is less than or equal to 35, and the result is positive; otherwise, the result is negative.

(2) The corresponding judgment basis of single NID 2: ct3 is less than or equal to 35, and the result is positive; otherwise, the result is negative.

(3) The basis for the determination of TWIST1 and NID2 combination detection is as follows:

1) Ct1 is less than or equal to 35, and positive can be determined without determining NID 2;

2) Ct1 is more than 35 and less than or equal to 40, ct3 is more than or equal to 35, and delta Ct3 is less than 8, and the positive result can be judged;

1) And 2) other than that, it is judged as negative.

5. Results

Table 9, results of experiments for combination detection of TWIST1 gene and NID2 gene

The results in the table show: the sensitivity corresponding to the detection of the single TWIST1 is 70.21%, the specificity is 98.496%, and the accuracy is 91.11%; the detection of single NID2 has the corresponding sensitivity of 51.06%, the specificity of 97.74% and the accuracy of 85.56%; the sensitivity of TWIST1 and NID2 combined detection is 89.36%, the specificity is 93.98%, and the accuracy is 92.78%. The ROC curve is shown in fig. 2.

Example 3

In this embodiment, a total of 220 urine samples are blind-tested, the pathological condition of the urine samples is unknown (the hospital side knows the pathological condition), the detected result is submitted to the hospital, and the hospital side feeds back the coincidence rate of the experimental result.

An experiment was performed in the same manner as in example 1, and the final determination results are shown in Table 10.

Of the 220 samples: of the 78 positive samples, 74 samples were detected as positive, and 4 as negative; 4 samples among the 70 interference samples are detected as positive; the detection of 2 samples out of 72 healthy human samples was positive.

Table 10

The positive control (bladder cancer tissue DNA) was detected using the nucleic acid composition of table 1 with reference to the reaction system described in table 5, the reaction conditions and the apparatus described in example 1, and the results are shown in fig. 3 (corresponding to nucleic acid combination 1 of table 1), fig. 4 (corresponding to nucleic acid combination 2 of table 1), fig. 5 (corresponding to nucleic acid combination 3 of table 1), fig. 6 (corresponding to nucleic acid combination 4 of table 1), and fig. 7 (corresponding to nucleic acid combination 5 of table 1). From the detection results, it was found that the effect was optimal by using the nucleic acid combination 5 shown in Table 1.

The positive control (bladder cancer tissue DNA) was detected using the nucleic acid composition of table 2 with reference to the reaction system described in table 5, the reaction conditions and the apparatus described in example 1, and the results are shown in fig. 8 (corresponding to nucleic acid combination 6 of table 2), fig. 9 (corresponding to nucleic acid combination 7 of table 2), fig. 10 (corresponding to nucleic acid combination 8 of table 2), fig. 11 (corresponding to nucleic acid combination 9 of table 2), and fig. 12 (corresponding to nucleic acid combination 10 of table 2). From the results of the detection, the effect was optimal by using the nucleic acid combination 10 shown in Table 2.

The positive control (bladder cancer tissue DNA) was detected using the nucleic acid composition shown in table 3 with reference to the reaction system shown in table 5, the reaction conditions and the apparatus shown in table 6 of example 1, and the results are shown in fig. 13 (corresponding to nucleic acid combination 11 shown in table 3) and fig. 14 (corresponding to nucleic acid combination 12 shown in table 3). From the results of the detection, the effect was optimal by using the nucleic acid combination 12 shown in Table 3.

Referring to the reaction system shown in Table 5, the reaction conditions and the apparatus shown in Table 6 in example 1, positive controls (bladder cancer tissue DNA) were examined using nucleic acid set 5 and nucleic acid set 10, and the results are shown in FIG. 15, in which pink is the reference gene, blue is the PENK gene test result, and green is the TWIST1 gene test result, which is the result of the combined TWIST1 and PENK test, on the same graph.

Referring to the reaction system shown in Table 5, the reaction conditions and the apparatus shown in Table 6 in example 1, the positive control (bladder cancer tissue DNA) was detected using the nucleic acid set 5 and the nucleic acid set 12, and the results are shown in FIG. 16, in which pink is the reference gene, blue is the NID2 gene, and green is the TWIST1 gene, which is the result of the combined detection of TWIST1 and NID 2.

In general, the embodiments of the present application provide a nucleic acid combination product and a kit for detecting a TWIST1 gene and for jointly detecting at least one of a NID2 gene and a PENK gene, so that efficient, sensitive and highly specific diagnosis and detection of cancer can be achieved in a non-invasive manner.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely illustrate a few embodiments of the present application, which are convenient for a specific and detailed understanding of the technical solutions of the present application, but should not be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. A nucleic acid combination product comprising a primer pair and a probe for detecting a biomarker;

2. The nucleic acid combination product according to claim 1, wherein the primer pair and probe for detecting the TWIST1 gene comprises:

3. The nucleic acid combination of claim 1, wherein the primer pair and probe for detecting PENK gene comprises:

4. The nucleic acid combination of claim 1, wherein the primer pair and probe for detecting NID2 gene comprises:

5. The nucleic acid combination product according to any one of claims 1 to 4, wherein the primer pair and probe for detecting a biomarker consists of the primer pair and probe for detecting a TWIST1 gene and the primer pair and probe for detecting a PENK gene, or consists of the primer pair and probe for detecting a TWIST1 gene and the primer pair and probe for detecting a NID2 gene.

6. The nucleic acid combination according to any one of claims 1 to 4, wherein in the primer pair and the probe for detecting the biomarker, the 5 'end of the probe is labeled with a fluorescent group and the 3' end is labeled with a quenching group.

7. The nucleic acid combination of claim 6, wherein the excitation light waves of the fluorophores of the probes for detecting different biomarkers are different.

8. The nucleic acid combination according to any one of claims 1 to 4 and 7, further comprising a primer pair and a probe for detecting an internal reference gene.

9. The nucleic acid combination according to claim 8, wherein the primer pair and probe for detecting the reference gene comprises a forward primer shown in SEQ ID NO.37, a reverse primer shown in SEQ ID NO.38, and a probe shown in SEQ ID NO. 39.

10. The nucleic acid combination according to claim 9, wherein in the primer pair and the probe for detecting the reference gene, a fluorescent group is labeled at the 5 'end of the probe and a quenching group is labeled at the 3' end of the probe.

11. The nucleic acid combination of claim 10, wherein the primer pair and the probe of the detection reference gene and the primer pair and the probe of the detection biomarker have fluorescent groups of different excitation light waves.

12. Use of the nucleic acid combination of any one of claims 1 to 11 for the preparation of a bladder cancer diagnostic kit.

13. A kit comprising the nucleic acid combination of any one of claims 1 to 11.

14. The kit of claim 13, further comprising one or more of a DNA amplification reagent, a reagent that converts unmethylated cytosine bases to uracil, a DNA extraction reagent, and a DNA purification reagent.