CN115851921A

CN115851921A - Primer probe combination product, kit and application of primer probe combination product and kit in nasopharyngeal carcinoma methylation detection

Info

Publication number: CN115851921A
Application number: CN202111123748.7A
Authority: CN
Inventors: 高堂杰; 肖珂; 戴宏霜; 吴康; 刘佳; 戴立忠
Original assignee: Sansure Biotech Inc
Current assignee: Sansure Biotech Inc
Priority date: 2021-09-24
Filing date: 2021-09-24
Publication date: 2023-03-28
Anticipated expiration: 2041-09-24
Also published as: CN115851921B

Abstract

The invention relates to the field of molecular biology detection, in particular to a primer probe combination product, a kit and application thereof in nasopharyngeal carcinoma methylation detection. The combination product is suitable for early screening of patients with no symptoms of nasopharyngeal carcinoma, and can reduce the pain and detection cost of the patients.

Description

Primer probe combination product, kit and application of primer probe combination product and kit in nasopharyngeal carcinoma methylation detection

Technical Field

The invention relates to the field of molecular biology detection, in particular to a primer probe combination product, a kit and application thereof in nasopharyngeal carcinoma methylation detection.

Background

With the continuous development of biotechnology and the improvement of living standard, people pay more attention to their health, and the demand of screening and detecting technologies (products) for various diseases is increasing, wherein the early screening and early diagnosis of tumors are particularly urgent.

Epigenetic studies have shown that methylation of related genes has become increasingly clinically accepted as a marker for early tumor screening, with aberrant methylation of genes occurring early in tumorigenesis.

Taking colorectal cancer as an example, the traditional screening means comprises joint screening of fecal occult blood detection and enteroscopy detection, and the fecal occult blood detection sensitivity is only 30% -60%. The enteroscopy is the golden standard for colorectal cancer diagnosis, but because invasive examination of the enteroscopy is invasive, the operation is complex, patients often fear the psychology, and the like, the detection compliance is poor, and the popularization has great difficulty. With the registration permission of NMPA (negative matrix amplification) of in-vitro diagnostic reagents taking gene methylation as markers in recent years, clinical auxiliary diagnosis or screening for colorectal cancer is approved, and the problem of popularization and shortage of early tumor screening work is expected to be relieved. The wide popularization of early screening has important significance for improving the survival rate of colorectal cancer patients.

At present, a plurality of products for colorectal cancer auxiliary diagnosis exist in the market, but the products have the defects of single detection target, low clinical sensitivity and the like, particularly, the clinical sensitivity of Septin9 methylation detection based on plasma free DNA is only about 70%, and the clinical application and popularization of the products are greatly limited. TCGA and GEO literature data show that many gene methylation markers related to nasopharyngeal carcinoma are available, such as PCDH1020, TET1, ZNF38225, WIF1 and SFRP5; and research shows that the method adopting the multi-gene joint detection has better sensitivity.

Nasopharyngeal carcinoma (NPC) is a tumor sensitive to radiation, and the main treatment method is radiotherapy or chemotherapy at present. Despite the great advances in radiation physics and radiobiology, the overall 5-year survival rate of nasopharyngeal carcinoma remains less than ideal. Therefore, early screening plays a key role in improving the prognosis of patients with nasopharyngeal carcinoma. Currently, the definitive diagnosis of nasopharyngeal carcinoma relies on biopsy of the nasopharyngeal lesion to obtain a pathological diagnosis. Patients with typical clinical symptoms may have obvious lesions in the nasopharynx, so that the diagnosis is easier to be confirmed; but in fact most patients are already in the advanced stage of the disease at the time of diagnosis; obviously, the early diagnosis of nasopharyngeal carcinoma is very difficult in clinic, and because the position of the nasopharynx is hidden, most patients are asymptomatic in the early stage, and the diagnosis is easy to miss. Therefore, the method has extremely important significance for the diagnosis of asymptomatic early nasopharyngeal carcinoma patients.

The molecular basis of the pathogenesis of nasopharyngeal carcinoma is not well understood, but current research suggests that the pathogenesis involves a multistep process, including EBV (Epstein-Barr virus) viral infection, environmental factors, and genetic and epigenetic changes. The EBV related antibody serological examination can obviously improve the early diagnosis rate of the nasopharyngeal carcinoma, and is the earliest and most widely applied nasopharyngeal carcinoma early screening method at present. However, the early-adopted cytoserological technology (immunofluorescence, IFA) for nasopharyngeal carcinoma screening is laborious, time-consuming and poorly standardized, which makes it less suitable for screening of large populations. The anti-EBV antibody enzyme-linked immunosorbent assay (ELISA) kit developed later shows better performance in nasopharyngeal carcinoma detection. However, although about 90% of patients with nasopharyngeal carcinoma are infected with EBV, EBV virus carriers have low cancer rate, and EBV virus detection cannot well guide nasopharyngeal carcinoma screening. The most common clinical method for diagnosing tumors at the initial stage is serological marker detection, but the method has the defect of obvious insufficient sensitivity, and the detection performances of different markers are often greatly different. For nasopharyngeal carcinoma, the detection sensitivity based on a single tumor marker is not high, generally 20% -60%, and a large amount of false positives exist to cause over-treatment. And the multi-tumor marker combined diagnosis method can effectively avoid missed diagnosis of nasopharyngeal carcinoma patients caused by single-marker negativity, and greatly improve the diagnosis rate of the nasopharyngeal carcinoma. In conclusion, early screening or auxiliary diagnosis of nasopharyngeal carcinoma is of great clinical significance and social value, and the technology can be more in line with the health economics, so that the method has high-sensitivity detection capability and universal application prospect, and is a great challenge.

Compared with the pathological changes of the nasopharynx part observed under the eyes of the traditional indirect nasopharyngoscope, the diagnosis rate of the asymptomatic nasopharyngoma patient can be improved to a certain extent through high-definition intranasal microscopy, particularly narrow-band endoscopic NBI examination and enhanced MRI, but the clinical challenges exist in two aspects: 1. endoscopes have difficulty in diagnosing submucosal lesions, and MRI (magnetic resonance imaging) examinations have difficulty finding subtle lesions; even if an endoscopic examination or an MRI examination is performed, a positive finding is not necessarily found in a true asymptomatic patient; lack of specificity for diagnosis of benign proliferative lesions; 2. those asymptomatic need to undergo nasal endoscopy or MRI because more asymptomatic are not nasopharyngeal carcinoma and need to prevent over-diagnosis so as not to add unnecessary pain and expense; these methods are not well suited for early screening of nasopharyngeal carcinoma, either in terms of sensitivity, cost of treatment, or patient compliance.

Therefore, the construction of a multigene methylation detection system which can be used for the early screening of nasopharyngeal carcinoma is particularly urgent.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention adopts a unique primer design scheme, and the constructed multiple methylation PCR reaction system can realize high-sensitivity detection of multiple targets, is suitable for early screening of asymptomatic patients, can reduce the pain and detection cost of the patients, and can well solve the difficulties.

In order to achieve the above purpose of the present invention, the following technical solutions can be adopted:

the first aspect of the present invention relates to a primer probe combination product comprising a nucleotide sequence of SEQ ID NO:1 to 6 and SEQ ID NO:7 to 9.

A second aspect of the invention relates to a kit comprising a primer probe combination as described above.

The third aspect of the invention relates to the application of the primer probe combination product in the preparation of a diagnostic reagent or a kit for detecting nasopharyngeal carcinoma methylation.

The beneficial effects of the invention include:

the primer probe combination product adopted by the invention can realize high-sensitivity detection of multiple targets by the constructed multiple methylation PCR reaction body, is suitable for early screening of patients with no symptoms of nasopharyngeal carcinoma, and can reduce the pain and detection cost of the patients.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph of amplification curves for a standard methylated human genomic DNA sample in one embodiment of the present invention;

FIG. 2 is a graph showing amplification curves for wild-type unmethylated human genomic DNA in one embodiment of the present invention;

FIG. 3 is an amplification curve for a clinically positive sample in an embodiment of the invention;

FIG. 4 is a graph of amplification curves for a clinically negative sample in one embodiment of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In the present invention, the term "plural" means 2 or more in number. For example, "any one or more" means any one or any combination of at least 2 constituents.

One aspect of the invention relates to a primer probe combination product comprising a nucleotide sequence of SEQ ID NO:1 to 6 and SEQ ID NO:7 to 9.

The primer probe combination product can be used for detecting the methylation conditions of three gene promoters of WIF1, SFRP5 and DAPK1, after sample nucleic acid is treated by sulfite, unmethylated cytosine (C) is deaminated into uracil (U), and methylated cytosine (5 mC) is unchanged; then, the uracil (U) is converted into thymine (T) through fluorescent PCR amplification, and 5mC is restored into C again, so that the accumulation of a fluorescent signal in the whole PCR process can be monitored in real time, and the fluorescent signal can be compared with a standard curve for quantitative analysis.

SEQ ID NO: 1-2 and SEQ ID NO:7 is used for detecting the methylation condition of the WIF1 gene promoter;

WIF1 Pre-transformation sequence (Gene accession number NC-000012.12)

CTGAGTGCCCTTCTCCGGGTCCGCCAGCCCTACACGCCCACTTCGCGGGCGCTCCACTGGGCGCACCGCACTGTGAATGCAGCCTCGGGGGTCCCTCGCGGCCCCGCCCCCGGGGGGGCCCCACAGCGCCCCCAAGTGGCGGCCGCCCAGGCCTCGCGGGCCCCACTCCTCGCTCGCACCTCGCTCGCGCCAGCCCTTCCCGCTCTTCTGTTCTCGCTCTATTTGCCCCGCTGACTGCTGGCCTCGCCAGCTTTGCCAGTCTTACGTCTCTGCCGCCCCCACTCCCGCCCGCGCCCCATC(SEQ ID NO：13)

post-WIF 1 transformation sequence:

TTGAGTGTTTTTTTTCGGGTTCGTTAGTTTTATACGTTTATTTCGCGGGCGTTTTATTGGGCGTATCGTATTGTGAATGTAGTTTCGGGGGTTTTTCGCGGTTTCGTTTTCGGGGGGGTTTTATAGCGTTTTTAAGTGGCGGTCGTTTAGGTTTCGCGGGTTTTATTTTTCGTTCGTATTTCGTTCGCGTTAGTTTTTTTCGTTTTTTTGTTTTCGTTTTATTTGTTTCGTTGATTGTTGGTTTCGTTAGTTTTGTTAGTTTTACGTTTTTGTCGTTTTTATTTTCGTTCGCGTTTTATT(SEQ ID NO：16)

SEQ ID NO: 3-4 and SEQ ID NO:8, the probe is used for detecting the methylation condition of the SFR-HP3 gene promoter;

SFRP5 Pre-transformation sequence (Gene accession number: NC-000010.11)

GGCTGTGGCGGAGAGAGATAAGGAGGAAGGATGGAGAAGGCGAGGGTCGAGGAAAATGCCCAGGGAGGCAGGGAGCCCTGGGGAGAAACGCTGGGCGAGGCCAGGGCTGCGGCAGGGGAGCCGAGCCGGGAGAGGGGCGCAAGACCTGGCGCTGGGCGGGACGCTCGGGCAGGGTGCGGGAGGGGCTGAGGGCCGCCCCCACTGCCTCCCCGCCTTGGAGCTGGGGCCAGGCGGCCGGAGATTGGCTGGGGCGCACGGCGAGGCCCGGGCTGGAGCCCCGAGGTGGGAGGCGCCAGGATC(SEQ ID NO：14)

post-SFRP 5 transformation sequence:

GGTTGTGGCGGAGAGAGATAAGGAGGAAGGATGGAGAAGGCGAGGGTCGAGGAAAATGTTTAGGGAGGTAGGGAGTTTTGGGGAGAAACGTTGGGCGAGGTTAGGGTTGCGGTAGGGGAGTCGAGTCGGGAGAGGGGCGTAAGATTTGGCGTTGGGCGGGACGTTCGGGTAGGGTGCGGGAGGGGTTGAGGGTCGTTTTTATTGTTTTTTCGTTTTGGAGTTGGGGTTAGGCGGTCGGAGATTGGTTGGGGCGTACGGCGAGGTTCGGGTTGGAGTTTCGAGGTGGGAGGCGTTAGGATT(SEQ ID NO：17)

SEQ ID NO: 5-6 and SEQ ID NO:9, the probe is used for detecting the methylation condition of the DAPK1 gene promoter;

DAPK1 Pre-transformation sequence (Gene accession number: NC-000009.12):

TTATATTTTCTAGTGGCCACCTTTTAAAAAGTAAACAGGTGAGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCCAGGCGGGCGGATCACGAGGTCAAGAGATGGAGACCATCCTGGTCGACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGGCATGGTGACGCGCGACTGTAGTCCTAGCTACTGGGGAGGCCGAGGCAGGAGAATCACTTGAACCCTGGAGGTGGAGGTTGCCACGCTCCACTACACTCCAGCCTGGCGACAGAGTGAG(SEQ ID NO：15)

post-DAPK 1 transformation sequence:

TTATATTTTTTAGTGGTTATTTTTTAAAAAGTAAATAGGTGAGGTCGGGCGCGGTGGTTTACGTTTGTAATTTTAGTATTTTGGGAGGTTTAGGCGGGCGGATTACGAGGTTAAGAGATGGAGATTATTTTGGTCGATACGGTGAAATTTCGTTTTTATTAAAAATATAAAAATTAGTTGGGTATGGTGACGCGCGATTGTAGTTTTAGTTATTGGGGAGGTCGAGGTAGGAGAATTATTTGAATTTTGGAGGTGGAGGTTGTTACGTTTTATTATATTTTAGTTTGGCGATAGAGTGAG(SEQ ID NO：18)

according to the methylation specific amplification primer provided by the invention, the 3 'end base of the upstream primer F is located at the C base of the CpG site, the primer sequence contains 3-5 CpG sites, the CpG site is better close to the 3' end of the primer, and the length of an amplicon formed by the upstream primer F and the downstream primer R is not more than 160bp. Further, the GC content of the above primer is 55 to 60%, and the Tm value thereof is 60 to 66 ℃.

The combined product provided by the invention has high sensitivity and good specificity, and can be amplified in the same reaction system.

In some embodiments, the combination further comprises primers and probes for detecting an internal reference nucleic acid that does not contain CpG sites.

The reference gene can be selected from housekeeping genes known to those skilled in the art, such as Actin, tubulin, GAPDH; the internal reference gene exists in normal cells and cancer tissue cells, and sequence difference does not exist, so that a detection sample can be better monitored in real time, and the methylation ratio of a target gene in the sample can be reflected.

In some embodiments, the reference nucleic acid is a β -Actin gene, or fragment thereof.

In some embodiments, the nucleotide sequence of the primer that detects the reference nucleic acid is SEQ ID NO: 10-11, wherein the nucleotide sequence of the probe for detecting the reference nucleic acid is SEQ ID NO: shown at 12.

In addition, it is contemplated that, in one aspect, useful primers and probes include those that are identical to SEQ ID NO: 1-12 have a nucleotide sequence that is greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. Such primer and probe modifications are also contemplated and can be made according to standard techniques.

The term "% identity" in the context of two or more nucleotide or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example,% identity is relative to the entire length of the coding region of the sequences to be compared.

For sequence comparison, typically one sequence is used as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Percent identity can be determined using search algorithms such as BLAST and PSI-BLAST (Altschul et al, 1990, J Mol Biol 215, altschul et al, 1997, nucleic Acids Res25, 17, 3389-402).

The primer and probe modification can be carried out by a known method. Modified versions of these primer and/or probe sequences may include, by way of non-limiting example, adding one or more nucleotides to the 5 'end, adding one or more nucleotides to the 3' end, adding one or more nucleotides to the 5 'and 3' ends, adding a tail, shortening the sequence, lengthening the sequence, moving the sequence several bases upstream and downstream, or any combination thereof.

Base modifications such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2 ' -deoxyadenosine, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2 ' -deoxyadenosine-5 ' -triphosphate, 2,6-diaminopurine (2-amino-dA), inverted dT, inverted dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's), and including at least one mismatched base at one of the bases, or replacing at least one of the bases with an RNA base, to achieve, for example, an increase in nucleic acid interaction at the 3' end of the mutant-specific primer to increase Tm. The addition of a double-stranded stable base modification has a positive effect on PCR, enabling it to be performed at higher temperatures, in the range where Taq polymerase is known to show maximal activity. The modified probe should retain the ability to distinguish between the mutated site and the wild-type site to be detected.

In some embodiments, the probe is labeled with a detectable signal substance. In some embodiments, the signal species is a fluorophore, colorimetric label, colloidal gold, quantum dot, biotin, and other label molecules that can be used for detection (e.g., alkyne groups for raman diffraction imaging, cyclic olefins for click reactions, priming groups for polymer labeling), and can also be selected from polypeptide/protein molecules, LNA/PNA, unnatural amino acids and their analogs (e.g., peptidomimetics), unnatural nucleic acids and their analogs (nucleotide mimetics), and nanostructures (including inorganic nanoparticles, NV-centers, aggregation/assembly-induced emission molecules, rare earth ion ligand molecules, polyoxometalate, and the like).

In some embodiments, the probes are both self-quenching probes.

In some embodiments, the fluorescent emitting moiety of each probe is independently selected from any one or more of AMCA, pacific Blue, atto 425, BODIPY FL, FAM, alexa Fluor 488, TET, JOE, yakima Yellow, VIC, HEX, quasar 570, cy3, NED, TAMRA, ROX, aqua Phluor593, texas Red, atto 590, cy5, quasar 670, and Cy5.5. Examples of the fluorescent emitting group of each probe independently include, but are not limited to, those listed above.

In some embodiments, the signal substances labeled on each probe in the combination product are distinguishable; in some embodiments, the fluorescent signals carried by the fluorescent emitting groups of each probe in the combination product are distinguishable.

The invention can be carried out by adopting multiple channels at the same time, and only needs to adopt independent fluorescent primers in different channels. In some preferred embodiments, the nucleic acid sequence of SEQ ID NO: the fluorescence emission groups on the probes shown in 7-9 can be distinguished under the same reaction system; further, SEQ ID NO: the fluorescence emitting groups on the probes shown in 7-9 and 12 can be distinguished under the same reaction system.

In a specific embodiment, SEQ ID NO: the fluorescent emission groups on the probes shown in 7 to 9 are selected from three of Cy5, ROX, HEX, and FAM. In a specific embodiment, SEQ ID NO: the fluorescent emission groups on the probes shown in 7 to 9 and 12 are ROX, HEX, FAM and Cy5, respectively.

In some embodiments, examples of the quencher group of each probe independently include, but are not limited to, BHQ1, BHQ2, BHQ3, dabcyl, eclipse, and MGB.

In some embodiments, the quencher group of each probe is independently selected from any one or more of BHQ1, BHQ2, BHQ3, dabcyl, eclipse and MGB.

The invention also relates to a kit containing the primer probe combination product.

The term "kit" refers to any article of manufacture (e.g., a package or container) comprising at least one device, the kit may further comprise instructions for use, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.

In some embodiments, the kit further comprises an amplification buffer, dntps, mg ²⁺ At least one of a DNA polymerase, a positive control, a negative control, water, and a sulfite sequencing reagent. The positive control refers to a target spot containing methylation for monitoring the detection performance of the reagent, and the negative control refers to a target spot not containing methylation for monitoring whether the experiment is polluted or not.

In some embodiments, the DNA polymerase has 5' exonuclease activity, e.g., any one selected from the group consisting of Taq, bst, vent, phi29, pfu, tru, tth, tl1, tac, tne, tma, tih, tf1, pwo, kod, sac, sso, poc, pab, mth, pho, ES4 DNA polymerase, and Klenow fragment.

In some embodiments, the water is typically nucleic acid-free and/or nuclease-free water. The water may be distilled, deionized or reverse osmosis water.

In some embodiments, the positive sample contains the methylated target, preferably the proportion of the methylated target in the total DNA is 0.3-0.7% (mass percent), for example 0.5%.

In some embodiments, the negative sample is human genomic DNA free of methylation of the target gene.

In some embodiments, the sulfite sequencing reagent comprises sulfite.

The primer/primer set provided by the present invention needs to be used in combination with a sulfite sequencing method.

Sulfite sequencing is currently the most common methylation detection method, and the principle is that bisulfite treatment converts unmethylated C to U, which is converted to T by the PCR process, while methylated C is not changed by bisulfite treatment.

In some embodiments, the sulfite sequencing reagent further comprises exogenous DNA unrelated to the nucleic acid fragment to be detected; further, the exogenous DNA is lambda DNA without methylation modification.

The nucleic acid fragments to be detected are preferably treated with bisulfite together with foreign DNA, in particular lambda DNA without methylation modification, in order to convert unmethylated cytosines to uracil. Exogenous DNA is co-processed with the sample efficiently during bisulfite treatment, which protects trace DNA fragments and reduces the damage of bisulfite to trace DNA to the utmost extent.

The components are preferably realized in lyophilized form, for example in the form of one or more so-called lyophilized beads. Lyophilized beads are generally understood to mean lyophilizates which are compressed into spherical form after production (after which the substance is generally present as a powder). Thus, the components required for the PCR batch, in particular the DNA polymerase, the nucleic acid components and the reaction buffer components, may be provided, for example, in lyophilized form. In this way, the PCR process can be started directly in a very user-friendly manner by adding the sample to be quantified and optionally other desired components. In particular, the provision of a lyophilized form is very advantageous for automated applications.

The invention also relates to application of the primer probe combination product in preparing a reagent or a kit for detecting nasopharyngeal carcinoma methylation.

The invention also relates to a nasopharyngeal carcinoma methylation detection method, which comprises the following steps:

sulfite-converted DNA samples were amplified using the primer probe combination described above.

In some embodiments, the DNA sample is genomic DNA.

In some embodiments, the DNA sample is from blood, serum, plasma, cell culture supernatant, sputum, saliva, nasopharyngeal swab, tissue, or tissue lysate.

In some embodiments, the blood, serum, plasma is from peripheral blood or bone marrow blood.

As used herein, "tissue lysate" may also be used in common with "lysate", "lysed sample", "tissue or cell extract", and the like, and refers to a sample and/or biological sample material comprising lysed tissue or cells, i.e., wherein the structural integrity of the tissue or cells has been compromised. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemical agents to lyse, degrade, or disrupt the cell walls and membranes of such tissues or cells. The skilled artisan is well familiar with suitable methods for obtaining a lysate. This process is encompassed by the term "lysis".

In some embodiments, the primers and probes of the primer probe combination amplify the DNA sample in a single PCR reaction.

In some embodiments, the working concentration of the upstream and downstream primers for each primer is independently selected from 0.15. Mu.M to 0.45. Mu.M, preferably 0.2. Mu.M.

In some embodiments, the working concentration of each probe is selected from 0.05. Mu.M to 0.25. Mu.M, preferably 0.1. Mu.M.

In some embodiments, the probe concentration is 0.4 to 0.6 times the primer concentration.

In some embodiments, mg ²⁺ The working concentration of (B) is 5mM to 7.5mM, preferably 6.6mM.

In some embodiments, the working concentration of dNTPS is between 0.5mM and 0.8mM, preferably 0.66mM.

In some embodiments, the working concentration of DNA polymerase (e.g., taq enzyme) is 0.25. Mu.M to 0.5. Mu.M, preferably 0.33. Mu.M.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

According to the invention, data mining and clinical sample detection are carried out through a TCGA database to find that methylation of three gene promoters of WIF1, SFRP5 and DAPK1 is closely related to nasopharyngeal carcinoma, so that the methylation detection is carried out by designing specific primer probes for the three targets, and simultaneously, a sequence which does not contain CpG sites in beta-Actin (ACTB) genes is selected to carry out internal standard primer probe design, so that the whole system is monitored. We constructed PCR detection system to detect the above four genes, and the sequences of the primers are shown in Table 1.

TABLE 1 primer sequences

Positive samples: 1 ng/. Mu.L of DNA, wherein the proportion of methylated targets is 0.5%;

negative samples: sequencing verification shows that human genome DNA without target gene methylation is 2 ng/. Mu.L

Detection process

Nucleic acid transformation: 50ng of standard methylated human genome DNA and wild non-methylated human genome DNA are respectively taken, transformed and purified and recovered by a sulfite transformation kit (D5042) produced by ZYMO company, and the detection sample is prepared according to theoretical concentration for later use.

Preparing a PCR system: PCR reaction solutions were prepared according to the reagent formulations in Table 2.

TABLE 2 PCR System Components

Adding the sample into a PCR reaction tube according to 5 mu L/reaction, then sequentially adding 25 mu L of PCR reaction solution, covering a PCR tube cover, shaking and uniformly mixing, and performing instantaneous centrifugation for 5s.

Fluorescent PCR reaction and result analysis

And (3) placing the PCR reaction tube into a sample groove of an amplification instrument, and setting the names of the samples to be detected according to the corresponding sequence.

Fluorescence detection channel selection: selecting a CY5 channel (Reportere: CY5, quencher: none) to detect the WIF1; selecting a ROX channel (Reportere: ROX, quencher: none) to detect SFRP5; selecting a FAM channel (report: FAM, quencher: none) to detect DAPK1; selecting an HEX channel (Reporter: HEX, quencher: none) as an internal standard channel to detect a gene beta-Actin (ACTB), wherein the gene exists in a methylation mode in tumor tissues and normal leukocyte DNA;

3) The conditions for the fluorescent quantitative PCR reaction are shown in Table 3.

TABLE 3 fluorescent quantitative PCR reaction conditions

PCR amplification is divided into three stages, wherein the first stage is a Taq enzyme activation stage and the temperature is 94 ℃. The second stage cycle stage, DAN amplification and fluorescence signal collection stage, annealing temperature of 60 deg.C, total 40 cycles. The first stage is instrument cooling, 1 minute at 4 ℃.

4) Analysis of results

After the reaction is finished, the instrument automatically stores the result, and can automatically analyze the result by using the software of the instrument (or manually adjust the starting value, the ending value and the threshold line value of the baseline for analysis), and the intersection point of the amplification curve and the threshold line is called Ct (cycle threshold, which refers to the cycle value that the fluorescence signal in the PCR reaction tube passes through when reaching the set threshold). The reagent provided by the invention has the advantages that the detection performance and the sensitivity can reach 0.5 percent methylation ratio in 10 ng/reaction (figure 1), and meanwhile, the reagent has no non-specific amplification in 20 ng/reaction of non-methylated DNA (figure 2); the results of the system for detecting 155 clinical plasma free DNA samples are shown in Table 4, the sensitivity and specificity are 86.00% and 93.33% respectively, the schematic diagram of the clinical positive sample amplification curve is shown in FIG. 3, and the negative sample amplification is shown in FIG. 4.

TABLE 4 sample assay data

Note: other cancer species include, but are not limited to, liver cancer, lung cancer, pancreatic cancer, and the like.

Tissue sample detection sensitivity (%) = number of positive detections/total number of positive cases × 100% =100%;

plasma sample detection sensitivity (%) = number of positive detections/total number of positive cases × 100% =86.00%;

plasma sample detection specificity (%) = total number of negative detections/total number of negative samples × 100% =93.33%;

cancer species specificity (%) = number of negative detections for non-nasopharyngeal cancer patients/total number of non-nasopharyngeal cancer patients × 100% =96.00%;

wherein the negative samples are all non-nasopharyngeal carcinoma samples.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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<400> 12

cttgggagag gactgggcca ttctcc 26

<210> 13

<211> 300

<212> DNA

<213> artificial sequence

<400> 13

ctgagtgccc ttctccgggt ccgccagccc tacacgccca cttcgcgggc gctccactgg 60

gcgcaccgca ctgtgaatgc agcctcgggg gtccctcgcg gccccgcccc cgggggggcc 120

ccacagcgcc cccaagtggc ggccgcccag gcctcgcggg ccccactcct cgctcgcacc 180

tcgctcgcgc cagcccttcc cgctcttctg ttctcgctct atttgccccg ctgactgctg 240

gcctcgccag ctttgccagt cttacgtctc tgccgccccc actcccgccc gcgccccatc 300

<210> 14

<211> 300

<212> DNA

<213> artificial sequence

<400> 14

ggctgtggcg gagagagata aggaggaagg atggagaagg cgagggtcga ggaaaatgcc 60

cagggaggca gggagccctg gggagaaacg ctgggcgagg ccagggctgc ggcaggggag 120

ccgagccggg agaggggcgc aagacctggc gctgggcggg acgctcgggc agggtgcggg 180

aggggctgag ggccgccccc actgcctccc cgccttggag ctggggccag gcggccggag 240

attggctggg gcgcacggcg aggcccgggc tggagccccg aggtgggagg cgccaggatc 300

<210> 15

<211> 300

<212> DNA

<213> artificial sequence

<400> 15

ttatattttc tagtggccac cttttaaaaa gtaaacaggt gaggccgggc gcggtggctc 60

acgcctgtaa tcccagcact ttgggaggcc caggcgggcg gatcacgagg tcaagagatg 120

gagaccatcc tggtcgacac ggtgaaaccc cgtctctact aaaaatacaa aaattagctg 180

ggcatggtga cgcgcgactg tagtcctagc tactggggag gccgaggcag gagaatcact 240

tgaaccctgg aggtggaggt tgccacgctc cactacactc cagcctggcg acagagtgag 300

<210> 16

<211> 300

<212> DNA

<213> artificial sequence

<400> 16

ttgagtgttt tttttcgggt tcgttagttt tatacgttta tttcgcgggc gttttattgg 60

gcgtatcgta ttgtgaatgt agtttcgggg gtttttcgcg gtttcgtttt cgggggggtt 120

ttatagcgtt tttaagtggc ggtcgtttag gtttcgcggg ttttattttt cgttcgtatt 180

tcgttcgcgt tagttttttt cgtttttttg ttttcgtttt atttgtttcg ttgattgttg 240

gtttcgttag ttttgttagt tttacgtttt tgtcgttttt attttcgttc gcgttttatt 300

<210> 17

<211> 300

<212> DNA

<213> artificial sequence

<400> 17

ggttgtggcg gagagagata aggaggaagg atggagaagg cgagggtcga ggaaaatgtt 60

tagggaggta gggagttttg gggagaaacg ttgggcgagg ttagggttgc ggtaggggag 120

tcgagtcggg agaggggcgt aagatttggc gttgggcggg acgttcgggt agggtgcggg 180

aggggttgag ggtcgttttt attgtttttt cgttttggag ttggggttag gcggtcggag 240

attggttggg gcgtacggcg aggttcgggt tggagtttcg aggtgggagg cgttaggatt 300

<210> 18

<211> 300

<212> DNA

<213> artificial sequence

<400> 18

ttatattttt tagtggttat tttttaaaaa gtaaataggt gaggtcgggc gcggtggttt 60

acgtttgtaa ttttagtatt ttgggaggtt taggcgggcg gattacgagg ttaagagatg 120

gagattattt tggtcgatac ggtgaaattt cgtttttatt aaaaatataa aaattagttg 180

ggtatggtga cgcgcgattg tagttttagt tattggggag gtcgaggtag gagaattatt 240

tgaattttgg aggtggaggt tgttacgttt tattatattt tagtttggcg atagagtgag 300

Claims

1. A primer probe combination product comprising a nucleotide sequence of SEQ ID NO:1 to 6 and SEQ ID NO:7 to 9.

2. The combination product of claim 1, further comprising primers and probes for detecting an internal reference nucleic acid that does not contain a CpG site.

3. The combination product of claim 2, wherein the reference nucleic acid is a β -Actin gene or fragment thereof.

4. The combination product of claim 3, wherein the nucleotide sequence of the primer for detecting the internal reference nucleic acid is SEQ ID NO: 10-11, wherein the nucleotide sequence of the probe for detecting the reference nucleic acid is SEQ ID NO: shown at 12.

5. A combination according to any one of claims 1 to 4, wherein the probes are self-quenching probes.

6. The combination product of claim 5, the fluorescent emitting group of each probe is independently selected from any one or more of AMCA, pacific Blue, atto 425, BODIPY FL, FAM, alexa Fluor 488, TET, JOE, yakima Yellow, VIC, HEX, quasar 570, cy3, NED, TAMRA, ROX, aqua Phluor593, texas Red, atto 590, cy5, quasar 670, and Cy5.5.

7. A combination product according to claim 6, wherein the fluorescent signal carried by the fluorescent emitting group of each probe is distinguishable.

8. A combination product according to claim 5, the quencher group of each probe being independently selected from any one or more of BHQ1, BHQ2, BHQ3, dabcyl, eclipse and MGB.

9. A kit comprising the primer probe combination of any one of claims 1 to 8.

10. The kit of claim 9, further comprising amplification buffer, dntps, mg ²⁺ At least one of DNA polymerase, positive control, negative control, water, and sulfite sequencing reagent.

11. Use of the primer probe combination of any one of claims 1 to 8 in the preparation of a diagnostic reagent or kit for detecting nasopharyngeal carcinoma methylation.