CN112410329A

CN112410329A - Primer combination, kit and application of kit in early screening of ovarian cancer

Info

Publication number: CN112410329A
Application number: CN202011112543.4A
Authority: CN
Inventors: 陈建国; 陈川; 张瑜巨
Original assignee: Shenzhen Letu Biological Technology Co ltd
Current assignee: Shenzhen Letu Biological Technology Co ltd
Priority date: 2020-10-16
Filing date: 2020-10-16
Publication date: 2021-02-26

Abstract

A primer combination, a kit and an application thereof in early screening of ovarian cancer are disclosed, wherein each primer in the primer combination contains a specific sequence, and the specific sequence contains at least one of sequences shown as SEQ ID NO 1-SEQ ID NO 278 or complementary sequences thereof. Because the specific sequence can target the tumor driving gene, the amplification and enrichment of the mutation characteristics are realized; the designed specific primers are subjected to primer interaction algorithm simulation and screening test, the conditions of missed detection and data nonuniformity caused by primer interaction are effectively reduced, the first round of amplification primers are completed in the 1-tube amplification reaction, and the time, the reagent usage and the sample usage are effectively saved.

Description

Primer combination, kit and application of kit in early screening of ovarian cancer

Technical Field

The invention relates to the field of biomedicine, in particular to a primer combination, a kit and application thereof in early screening of ovarian cancer.

Background

Ovarian cancer is a gynecological tumor with high malignancy degree, and is the first cause of the mortality of gynecological cancer because the ovarian cancer is discovered to be a late stage, so the ovarian cancer is the genuine king of gynecological cancer. The relative survival rate data of 5 years of cancer in China shows that the ovarian cancer is 38.9 percent, the cervical cancer is 45.4 percent and the uterine cancer is 55.1 percent.

If ovarian cancer can be discovered early and treated in time, good treatment effect can be achieved. For example, 5-year survival rates after early ovarian cancer (stage I) treatment can reach 80% -90%, but rapidly decrease to 30% -40% or even lower once advanced stages (stage III, stage IV) are reached. Taking the ovarian cancer data in the united states as an example, distant metastasis has occurred in 60% of cases when diagnosed, with a 5-year relative survival rate of only 28.8%.

At present, the clinical diagnosis of ovarian cancer mainly comprises gynecological pelvic cavity examination, marker detection of serum CA125, human epididymis protein 4 and the like, imaging examination of ultrasound, CT, MRI and the like, and combined application of the means, but the detection means has certain limitations in early diagnosis and disease condition monitoring of ovarian cancer. For example, the most commonly used clinical CA125 is commonly used for diagnosis and treatment of ovarian cancer, evaluation of curative effect and monitoring of recurrence, but has low sensitivity to early ovarian cancer, only 50-60% of patients with stage I ovarian cancer are raised, and the sensitivity can be raised in female inflammatory diseases such as chocolate ovarian cyst, follicular cyst, pregnancy, menstruation and the like, so that accurate diagnosis of ovarian cancer is influenced, and the specificity and sensitivity are still low. Human epididymin 4(HE4) is highly expressed in different types of ovarian cancer, particularly serous and endometrial types in epithelial ovarian cancer. HE4 is currently widely used for diagnosis of epithelial ovarian cancer. While HE4 is the first FDA approved biomarker available for detecting patients with recurrent ovarian cancer following CA 125. Particularly when HE4 and CA125 are used in combination, this helps to identify patients with ovarian cancer from those with pelvic masses. In the process of diagnosing the malignant ovarian cancer, the sensitivity of serum HE4 is 82.35%, the specificity is 96.03%, and the pathological diagnosis coincidence rate is 80.29%. However, in healthy people, when HE4 serum level is 31.08pmol/L, sensitivity is 81.91%, specificity is 50.79%, and it is seen that specificity is low, which easily causes excessive medical intervention, causes loss to patients, and further needs comprehensive consideration when applied to large-scale screening.

Meanwhile, with the development of imaging technology, imaging examination plays an increasingly important role in the diagnosis, clinical staging, postoperative follow-up and diagnosis of recurrence and metastasis of ovarian cancer. Various imaging examination methods have advantages and disadvantages for diagnosis, staging and postoperative follow-up of ovarian cancer. But is limited by the dependence on professionals and equipment, cost and detection sensitivity, and is not suitable for screening common people, particularly middle-low risk people.

Disclosure of Invention

The invention provides a primer combination, a kit and application thereof in early screening of ovarian cancer.

According to a first aspect, in one embodiment, a primer combination is provided, wherein each primer of the primer combination comprises a specific sequence comprising at least one of the sequences shown as SEQ ID No. 1 to SEQ ID No. 278 or the complement thereof.

According to a second aspect, in one embodiment, there is provided a primer combination comprising a primer combination according to the first aspect for a first round of PCR amplification and a primer combination for a second round of PCR amplification.

According to a third aspect, in one embodiment, there is provided a kit comprising a primer combination according to the first or second aspect.

According to a fourth aspect, in one embodiment, there is provided the use of a primer combination according to the first aspect in multiplex nested PCR amplification.

According to a fifth aspect, in an embodiment, there is provided the use of a primer combination according to the first aspect in a single-tube PCR amplification.

According to a sixth aspect, in one embodiment, there is provided the use of a primer combination of the first or second aspects, or a kit of the third aspect, for early screening for cancer.

According to a seventh aspect, in an embodiment, there is provided a PCR amplification method comprising: obtaining a DNA extraction product of a sample, and performing a first round of PCR amplification on the sample by adopting the primer combination of the first aspect, and then performing a second round of PCR amplification to obtain an amplification product.

According to the primer combination of the embodiment, the specific sequence can target a tumor driving gene, so that the amplification and enrichment of mutation characteristics are realized; the designed specific primers are subjected to primer interaction algorithm simulation and screening test, the conditions of missed detection and data nonuniformity caused by primer interaction are effectively reduced, the first round of amplification primers are completed in the 1-tube amplification reaction, and the time, the reagent usage and the sample usage are effectively saved.

Drawings

FIG. 1 shows quality control diagrams of the library fragments in example 1;

FIG. 2 is a schematic diagram showing the structure of the first round amplification primer in example 1;

FIG. 3 is a graph showing the results of quality control in example 1.

FIG. 4 is a statistical chart showing the number of positive detection cases in Tao Brush and plasma samples in example 2.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning.

The term "primer" or "primer sequence" as used herein refers to a short linear oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., a DNA template to be amplified) to prime a nucleic acid synthesis reaction. The primer may be an RNA oligonucleotide, a DNA oligonucleotide, or a chimeric sequence. The primer may contain natural, synthetic or modified nucleotides. The upper and lower limits of primer length are empirically determined. The lower limit of the primer length is the minimum length required to form a stable duplex upon hybridization to a target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (typically less than 3-4 nucleotides in length) are not capable of forming thermodynamically stable duplexes with target nucleic acids under the hybridization conditions. The upper limit is generally determined by the probability of duplex formation in regions of the target nucleic acid other than the predetermined nucleic acid sequence. Suitable primer lengths typically range from about 4 to 40 nucleotides in length.

In one embodiment, the primer combination realizes targeted enrichment of a target gene sequence, has small primer interaction, can target a high-frequency driving gene with close ovarian cancer occurrence, can be used for detecting low-frequency mutation in a sample to be detected, and realizes early screening of ovarian cancer.

In one embodiment, the specific primers are subjected to primer interaction algorithm simulation and screening test, so that the conditions of missed detection and data nonuniformity caused by primer interaction are effectively reduced, 139 pairs of primers are completed in 1-tube amplification reaction, the operation flow is effectively simplified, the detection time is shortened, and the use amount of samples and reagents is reduced.

In one embodiment, the primer combination is a primer combination for targeted amplification of an ovarian oncogene.

In one embodiment, the primer combination specifically targets at least one of the following genes: PTEN, TP53, APC, PIK3CA, CTNNB1, KRAS, PIK3R1, NRAS, AKT1, FGFR2, pool, PPP2R1A, CDKN2A, FBXW7, RNF43, BRAF, MAPK1, EGFR.

In one embodiment, the primer combination is a primer combination for early detection of ovarian cancer.

In one embodiment, each primer in the primer combination further comprises a bridging sequence linked to the specific sequence.

In one embodiment, the bridging sequence is linked to the 5' end of the specific sequence.

In one embodiment, the bridging sequence is a universal bridging sequence.

In one embodiment, when the primer in the primer combination is the forward primer, the bridging sequence is 5'-CTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO:299) or its complement.

In one embodiment, when the primer in the primer combination is a downstream primer, the bridging sequence is 5'-CTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO:300) or its complement.

In one embodiment, when the primer in the primer combination is an upstream primer or a downstream primer, a molecular tag sequence is connected between the bridging sequence and the specific sequence. All the upstream primers may be connected with a molecular tag sequence, all the downstream primers may be connected with a molecular tag sequence, or a part of the upstream primers and a part of the downstream primers may be connected with a molecular tag sequence.

In one embodiment, the molecular tag sequence contains 6-16 random bases, including but not limited to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, preferably 14. The random base is used for distinguishing original DNA mutation from false mutation introduced in the process of library amplification and sequencing, if the number of the base is too small, the randomness is not strong, and if the number of the base is too large, the amplification efficiency of the primer is influenced.

In one embodiment, the 3' end of the specific primer is modified. The modified primer has stronger specificity, is not easy to be degraded by nuclease and has relatively uniform Tm value.

In one embodiment, the method of modifying the 3' end of the specific primer includes, but is not limited to, at least one of thio-modification, phosphorylation, LNA modification, and the like.

LNA (Locked Nucleic acid) is an oligonucleotide derivative, and the 2'-O and 4' -C positions of beta-D-ribofuranose in the structure form a rigid structure through the effect of shrinkage. LNA nucleotides include A, C, G, T, U, mC six bases, and Locked Nucleic Acid (LNA) is a modified RNA in which a portion of the 2 'and 4' carbons of the ribose sugar are linked together.

In one embodiment, the modification position is a phosphodiester bond between the penultimate and second nucleotides located at the 3' end of the specific primer.

In one embodiment, each primer in the primer combination has a Tm of 55-65 ℃ including, but not limited to, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, preferably 58 ℃.

The Tm (melting temperature) is the temperature at which 50% of the double strand is cleaved into single strands.

In one embodiment, the primer combination is a primer combination for multiplex PCR amplification.

In one embodiment, the primer combination for the second round of PCR amplification comprises at least one of the sequences shown in SEQ ID NO. 279 to SEQ ID NO. 298 or the complementary sequences thereof.

In one embodiment, the primer set forth in SEQ ID NO. 1 or its complement is combined with the sequence set forth in SEQ ID NO. 140 or its complement as a pair of upstream and downstream primers, the primer set forth in SEQ ID NO. 2 is combined with the sequence set forth in SEQ ID NO. 141 as a pair of upstream and downstream primers, and so on. The primer shown by SEQ ID NO. 1 or the complementary sequence thereof can be an upstream primer or a downstream primer, and the sequence shown by SEQ ID NO. 140 or the complementary sequence thereof can be an upstream primer or a downstream primer.

In one embodiment, the kit is a kit for early screening of ovarian cancer.

In one embodiment, the early stage ovarian cancer patient is a clinically asymptomatic or atypical ovarian cancer patient, with tumors generally in stage IA/B. The standard of cancer staging is referred to the surgical pathology staging of the international Federation of obstetrics and gynecology (FIGO).

In one embodiment, the kit further comprises other reagents required for PCR amplification, including but not limited to at least one of PCR amplification enzymes, dntps, deionized water, PCR buffer, and the like.

According to a fourth aspect, in one embodiment, there is provided the use of a primer combination of the first or second aspect, or a kit of the third aspect, in multiplex nested PCR amplification.

In one embodiment, the primer combination of the first aspect is used in a first round of PCR amplification.

According to a fifth aspect, in an embodiment, there is provided the use of a primer combination according to the first aspect in a single-tube multiplex PCR amplification. The single-tube PCR amplification can effectively save time, reagents and samples and improve the detection efficiency.

According to a sixth aspect, in one embodiment, there is provided the use of a primer combination of the first or second aspects, or a kit of the third aspect, for early screening for cancer. When the early cancer screening is carried out, amplification products for sequencing are obtained by adopting the primer combination or the kit, and the result obtained based on the amplification products is only an intermediate reference result, and the final disease diagnosis result cannot be directly obtained only according to the sequencing result, and the final diagnosis result can be obtained by combining the existing examination methods such as cytology, imaging, serology and the like, so the application does not belong to the diagnosis and treatment method of the disease.

In one embodiment, the cancer comprises ovarian cancer.

According to a seventh aspect, in an embodiment, there is provided a PCR amplification method comprising: obtaining a DNA extraction product of a sample, and performing a first round of PCR amplification on the sample by adopting the primer combination of the first aspect, and then performing a second round of PCR amplification to obtain an amplification product. The amplification product can be used for on-machine sequencing.

In one embodiment, in the first round of PCR amplification, the primers required for the first round of PCR amplification are added to the same reaction vessel for the amplification reaction.

In one embodiment, the first round PCR amplification product is subjected to a second round of universal primer amplification, such that different sample bands are provided with molecular tags (index barcodes) for distinguishing samples and sequencing primers are added, thereby obtaining sequencing primers for computer.

In one embodiment, the method further comprises purifying the amplification product after the first round of PCR amplification, and then performing a second round of PCR amplification on the purified amplification product. The purification is to remove the residual impurities such as primers, dNTPs, enzymes, etc.

In one embodiment, the first round amplification products are purified using magnetic beads. Magnetic beads are commercially available.

In one embodiment, the sample includes, but is not limited to, at least one of a plasma sample, a serum sample, a tumor tissue shed cell sample, and the like.

In one embodiment, the source of the sample includes, but is not limited to, at least one of cervix, ovary, endometrium, fallopian tube, pleural effusion, and the like.

In one embodiment, the main objective of the present invention is to establish a non-invasive early-stage auxiliary screening method for ovarian cancer, which can be combined with the existing cytology, imaging, serology and other examination methods, so as to improve the early-stage screening coverage and detection rate of the above cancers, and achieve early detection, early diagnosis and early treatment, so as to improve the survival time and life quality of patients.

In one embodiment, multiple PCR primers, a kit and a method for detecting early screening of ovarian cancer based on a high-throughput targeted sequencing technology are provided, and a plasma free DNA low-frequency mutation detection method based on multiple PCR amplification and high-throughput sequencing is established. By selecting high-frequency mutant genes closely related to ovarian cancer, a specific amplification primer is designed, and UMI (single molecule label) is designed and added at the upstream of the primer, so that background noise is effectively eliminated, and mutation caused by factors such as amplification, sequencing and the like is reduced. The established plasma low-frequency mutation detection technology is used for detecting mutation information in tumor free DNA in peripheral blood of high risk groups, so that early screening of ovarian cancer is realized.

In one embodiment, the kit can simultaneously detect a plurality of ovarian cancer high-frequency driving genes in a plurality of samples, and introduces a molecular tag sequence outside a specific PCR primer to eliminate false positive of the primer in the PCR and sequencing processes, so that the detection sensitivity and accuracy are improved.

In one embodiment, a high-frequency driving gene closely related to ovarian cancer occurrence is selected, a specific primer is designed to amplify and enrich a mutation hotspot, and the early screening of ovarian cancer is realized by detecting low-frequency tumor mutation contained in a sample to be detected.

In one embodiment, the designed specific primers are subjected to primer interaction algorithm simulation and screening test, so that the conditions of missed detection and data nonuniformity caused by primer interaction are effectively reduced, and the 139 pairs of primers are completed in 1-tube amplification reaction;

in one embodiment, the designed specific primer is added with a molecular tag sequence, so that false positive caused by library amplification and sequencing introduction can be effectively reduced, and the lower limit of detection sensitivity on low-frequency mutation can reach 0.05 percent and is higher than 0.1 percent of common tumor liquid biopsy.

In one embodiment, the primer combination and the kit thereof are suitable for detecting free DNA of plasma and also suitable for detecting a tumor tissue or an ovarian/cervical exfoliated cell sample, and can obviously improve the sensitivity and accuracy of screening early ovarian cancer under the condition of simultaneously detecting and collecting the free DNA of the plasma and the exfoliated ovarian cell sample of a detected person.

Example 1

This embodiment provides a method of screening for ovarian cancer, comprising: screening tumor driving genes related to ovarian carcinogenesis, and selecting 139 pairs of specific primers designed by 18 genes such as PTEN, TP53, APC, PIK3CA, CTNNB1, KRAS, PIK3R1, NRAS, AKT1, FGFR2, POLE, PPP2R1A, CDKN2A, FBXW7, RNF43, BRAF, MAPK1, EGFR and the like for first round PCR amplification, wherein the amplification target region of each pair of specific primers is 100-170bp in size; after the second round of amplification, the length of the ex-warehouse library is about 300bp after adding index barcode and a sequencing linker sequence. FIG. 1 shows a quality control diagram of library fragments, the abscissa represents the capillary electrophoresis time, the DNA ladder introduced by electrophoresis is converted into fragment length, and the length of the library fragment is about 300 bp; the ordinate is fluorescence signal intensity, and the concentration of the library (ng/muL) is converted according to the introduced DNA marker, so that the library-out concentration is proved to meet the sequencing requirement.

As shown in FIG. 2, the structure of the first round of amplification primers is schematically represented, and the sequence structure of the primers used in the first round of PCR amplification sequentially comprises a universal bridging sequence, a molecular tag sequence and a specific primer sequence from 5 'to 3'. The primer sequences of the different capture intervals are identical in the universal bridging sequence portion, and the sequences are identical or complementary with the ligation sequence with index barcode sequencing joint and are used for the amplification and elongation of the library in the second round of PCR amplification. The molecular tag sequence of this example is 14 bases and is used to distinguish the original DNA mutation from the spurious mutations introduced during library amplification and sequencing.

The principle of the molecular label for mutation signal identification and correction is as follows: when the specific primer captures the target fragment, the product carrying the most original mutation carries a different molecular tag sequence, and if the molecular tag sequences on the read sequences corresponding to the mutations are identical, the mutation is most likely to be a false negative introduced during the library amplification process. The technical principle of the molecular tag sequence is roughly as follows: adding a molecular label to the amplified product DNA molecules of the targeted enrichment, then carrying out two-round amplification to extend the library and carrying a sequencing joint, and then carrying out high-depth sequencing by a computer. In the sequencing analysis process, the amplicon is subjected to clustering analysis through a molecular tag sequence, errors generated by DNA polymerase amplification and base misjudgment in the sequencing process are randomly scattered and distributed in the high-depth sequencing process, and the randomly generated error sequences can be filtered after the consistency sequence is counted. Only the original mutated sequence with the various molecular tags is retained, thus obtaining the corrected precise sequence, which can be used for 'absolute quantification' of the mutation frequency in the whole sample. "

Two standard DNA products HD780 and HD786 (purchased from Shenzhen Jingjingjingji technology Limited) are used, 40ng is used as the initial input amount, the mutation is subjected to gradient dilution, the tumor mutation frequencies after dilution are respectively 5%, 1%, 0.5%, 0.2% and 0.1%, and then library construction and sequencing are respectively carried out to evaluate the lower detection limit and positive consistency of the positive mutation.

Performing first round of target region amplification enrichment by using a primer combination containing specific primers shown in SEQ ID NO. 1-SEQ ID NO. 278, wherein a PCR reaction system is as follows: 2 mu L of specific upstream primer and downstream primer mixture; 40ng of DNA template; 2xPCR MIX 12.5 μ L; nuclease free water made up the volume to 25. mu.L. Among them, 2 × PCR MIX was purchased from Beijing Quanjin Biotechnology Ltd.

The PCR amplification reaction program is as follows:

72 ℃ for 5 min: 1 cycle.

Purifying the amplification product of each sample by using the purified magnetic beads, and removing the residual impurities such as the primers, the dNTP, the enzyme and the like; the magnetic beads are Agencour AMPure XP magnetic beads purchased from BECKMAN COULTER (Beckmann Coulter, USA).

Second round amplification is performed on the purified first round amplification purification product using second round amplification primers, library length is extended and sequencing adaptor sequences are added.

The second round of PCR reaction system is: 2XPCR MIX 12.5 uL, first round amplification purification product 5 uL, universal second round amplification primer mixture 2 uL, the residual volume with no nuclease water make up to 25 uL. Wherein the second round amplification primers used for each sample contain different index barcode sequences to distinguish the samples during the in-flight process.

The amplification procedure for the second round of amplification was:

72 ℃ for 5 min: 1 cycle.

After obtaining the secondary amplification products of the above 10 samples, 5 μ L of each sample was mixed, and then purified using 1.2X magnetic beads, the concentration of the purified product was controlled using Qubit 3.0, and the fragment was controlled using Agilent 2100, with the results of quality control shown in fig. 3.

The sample numbers are in the horizontal coordinate of fig. 3, representing different sample libraries, and the fragment lengths (bp) are in the vertical coordinate. The results in FIG. 3 show that most of the libraries were successful and that the library fragments were single without non-specific amplification.

And (3) after the quality control is qualified, performing on-machine sequencing by using an Illumina sequencing platform, wherein the sequencing strategy is PE150, and each sample has 10G data.

The method of this example analyzed the sensitivity and specificity of detection in standard DNA at different mutation frequencies, as shown in table 3.

The sensitivity is the ratio (or percentage) of the number of mutations detected by sequencing the standard by the method of the invention in the example to the mutation data possessed by the standard itself in the case of a specific data amount.

Specificity is 100% minus the percentage of the number of mutant sites detected by sequencing according to the method of the invention that are not present in the standard, to the total number of detected sites.

Sensitivity, specificity can be characterized as a percentage or as a decimal.

In table 3, "whole" refers to the comprehensive statistical results of different standards and their technical repeats.

In table 3, the values in the columns of sample attributes are mutation frequency and average sequencing depth, respectively, and the mutation frequency is a simulated mutation frequency sample of standard DNA after gradient dilution.

TABLE 3

As can be seen from Table 3, the method of this example has high sensitivity and specificity when used for detecting samples with different mutation frequencies.

Example 2

134 subjects were selected, 83 positive subjects and 51 negative subjects were tested for mutations in exfoliated cells collected by plasma-free DNA and Tao Brush endometrial sampler, respectively, according to the clinical diagnosis results, and the test method was performed according to example 1, and mutations in single plasma-free DNA and mutations in exfoliated ovarian cells were evaluated.

FIG. 4 is a statistical graph showing the number of positive detection cases in Tao Brush and Plasma samples, where Tao Brush represents the exfoliated cell sample collected by the Tao Brush endometrial sampler and Plasma represents the Plasma sample. In FIG. 4, 25 is the number of samples in which a positive mutation was detected in the exfoliated cell sample alone, and 13 is the number of samples in which the same subject was detected positive in both the exfoliated cells and plasma; 23 is the number of samples in which tumor mutations were detected only in the plasma of the subject.

Two false positive results appear in the detected positive samples, both are from plasma samples, and false mutation may be introduced in amplification or sequencing links.

Taking the plasma free DNA sample type as an example, among 83 ovarian cancer positive subjects, tumor mutations were detected in the plasma samples of 36 patients in total, and the positive agreement of the plasma sample was 36 ÷ 83 ≈ 43% by dividing the 36 positive patients who were positively read using the plasma sample alone by the total of 83 positive subjects who were clinically confirmed. The consistency of the results of the detached cell samples was calculated in the same manner as 38 ÷ 83 ≈ 46%.

As can be seen from fig. 4, the consistency of the plasma sample is about 43%, and the consistency of the exfoliated cell sample is about 46%; the mutation results of the exfoliated cells collected by the free DNA of the plasma and the Tao Brush are integrated for interpretation, and the ovarian cancer auxiliary diagnosis is carried out, wherein the detection sensitivity of the method is about 73.5 percent, and the specificity (also called specificity) is about 98.5 percent.

Sensitivity 73.5% is the percentage of all ovarian cancer positive subjects (including plasma free DNA and exfoliated cell samples) detected, the number of cases in which tumor mutations were detected in both sample types and in one of the sample types, divided by the total positive subject cases, i.e., (25+13+23) ÷ 83 ≈ 73.5%; specificity was 1 minus the percentage of 2 false positive cases detected divided by the total subject cases, i.e., 1- (2 ÷ 134) ≈ 100% ≈ 98.5%.

The method is a target area capture sequencing technology based on a high-throughput sequencing platform, and through detection and evaluation of a standard substance and a batch sample, the library building power of the method developed in the embodiment is greater than 99.5%, the capture efficiency is greater than 40%, and the detection sensitivity is as low as 1% o under the condition that the specificity is greater than 90%.

The pooling power is the number of all samples of the standard and examples that were successfully pooled and qualified sequencing data obtained, divided by the total number of samples used for pooling.

The capture efficiency is effective data, namely the data volume of the gene interval to be detected is compared with the total sequencing data volume in the data analysis link.

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

SEQUENCE LISTING

<110> Shenzhen Letu Biotech Limited

<120> primer combination, kit and application thereof in early screening of ovarian cancer

<130> 20I30317

<160> 300

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence

<400> 1

acacccccag gattcttaca g 21

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

gatgtggctc gccaattaac 20

<210> 3

<211> 18

<212> DNA

<213> Artificial sequence

<400> 3

agccacaggc tcccagac 18

<210> 4

<211> 26

<212> DNA

<213> Artificial sequence

<400> 4

gctgcatatt tcagatattt ctttcc 26

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence

<400> 5

ggatttcctg cagaaagact tg 22

<210> 6

<211> 29

<212> DNA

<213> Artificial sequence

<400> 6

aatagttgtt ttagaagata tttgcaagc 29

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence

<400> 7

aagattcagg caatgtttgt tagtatt 27

<210> 8

<211> 29

<212> DNA

<213> Artificial sequence

<400> 8

cagtaagata cagtctatcg ggtttaagt 29

<210> 9

<211> 29

<212> DNA

<213> Artificial sequence

<400> 9

tttcttattc tgaggttatc tttttacca 29

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<400> 10

ccataaccca ccacagctag a 21

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence

<400> 11

gcaattcact gtaaagctgg aaa 23

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence

<400> 12

aaacccaaaa tctgttttcc aa 22

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence

<400> 13

tcaatttggc ttctcttttt tttc 24

<210> 14

<211> 28

<212> DNA

<213> Artificial sequence

<400> 14

gcgctatgtg tattattata gctacctg 28

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence

<400> 15

gctacctgtt aaagaatcat ctgga 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence

<400> 16

aggcatttcc tgtgaaataa tactg 25

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<400> 17

tgtggtctgc cagctaaagg 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<400> 18

ttgagttccc tcagccgtta 20

<210> 19

<211> 27

<212> DNA

<213> Artificial sequence

<400> 19

tttttttttt aggacaaaat gtttcac 27

<210> 20

<211> 26

<212> DNA

<213> Artificial sequence

<400> 20

tctatgtgat caagaaatcg atagca 26

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence

<400> 21

tttaacaaaa aatgatcttg acaaagc 27

<210> 22

<211> 26

<212> DNA

<213> Artificial sequence

<400> 22

tgggttttca ttttaaattt tctttc 26

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

tagaggagcc gtcaaatcca 20

<210> 24

<211> 27

<212> DNA

<213> Artificial sequence

<400> 24

ggtccatttt cagtttattc aagttta 27

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence

<400> 25

gcagccagaa atgttttggt a 21

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<400> 26

gcagagtatt tgggcgaatg 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<400> 27

gctgggcatc actgtaaacc 20

<210> 28

<211> 24

<212> DNA

<213> Artificial sequence

<400> 28

ggaaataaat gtgatttgcc ttct 24

<210> 29

<211> 23

<212> DNA

<213> Artificial sequence

<400> 29

ttctcccttc tcaggattcc tac 23

<210> 30

<211> 27

<212> DNA

<213> Artificial sequence

<400> 30

tttacctcta ttgttggatc atattcg 27

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence

<400> 31

gtctgtgtgg tgcccagttt 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence

<400> 32

cccatcccag gagcttactt 20

<210> 33

<211> 21

<212> DNA

<213> Artificial sequence

<400> 33

tccttgtagc caatgaaggt g 21

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence

<400> 34

gccacctgaa gtccaaaaag 20

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence

<400> 35

atgtcatctc tcctccctgc t 21

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence

<400> 36

gttccgagag ctgaatgagg 20

<210> 37

<211> 17

<212> DNA

<213> Artificial sequence

<400> 37

ccctggctcc ttcccag 17

<210> 38

<211> 22

<212> DNA

<213> Artificial sequence

<400> 38

aagaagaaaa cggcattttg ag 22

<210> 39

<211> 23

<212> DNA

<213> Artificial sequence

<400> 39

ttttatcacc tttccttgcc tct 23

<210> 40

<211> 19

<212> DNA

<213> Artificial sequence

<400> 40

cgtgtttgtg cctgtcctg 19

<210> 41

<211> 21

<212> DNA

<213> Artificial sequence

<400> 41

tgcctcttgc ttctcttttc c 21

<210> 42

<211> 22

<212> DNA

<213> Artificial sequence

<400> 42

tggctctgac tgtaccacca tc 22

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence

<400> 43

tgtgatgatg gtgaggatgg 20

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence

<400> 44

gtggaaggaa atttgcgtgt 20

<210> 45

<211> 18

<212> DNA

<213> Artificial sequence

<400> 45

gtccccaggc ctctgatt 18

<210> 46

<211> 19

<212> DNA

<213> Artificial sequence

<400> 46

gccatggcca tctacaagc 19

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence

<400> 47

ctccgtcatg tgctgtgact 20

<210> 48

<211> 22

<212> DNA

<213> Artificial sequence

<400> 48

gccctgactt tcaactctgt ct 22

<210> 49

<211> 21

<212> DNA

<213> Artificial sequence

<400> 49

gcattgaagt ctcatggaag c 21

<210> 50

<211> 19

<212> DNA

<213> Artificial sequence

<400> 50

ctgcaccagc agctcctac 19

<210> 51

<211> 19

<212> DNA

<213> Artificial sequence

<400> 51

agctcccaga atgccagag 19

<210> 52

<211> 21

<212> DNA

<213> Artificial sequence

<400> 52

gcaatggatg atttgatgct g 21

<210> 53

<211> 21

<212> DNA

<213> Artificial sequence

<400> 53

tgactgctct tttcacccat c 21

<210> 54

<211> 19

<212> DNA

<213> Artificial sequence

<400> 54

agccccctag cagagacct 19

<210> 55

<211> 21

<212> DNA

<213> Artificial sequence

<400> 55

ccttccaatg gatccactca c 21

<210> 56

<211> 20

<212> DNA

<213> Artificial sequence

<400> 56

cgacacccac agaggaaaag 20

<210> 57

<211> 20

<212> DNA

<213> Artificial sequence

<400> 57

aatgccagtg atgacgacaa 20

<210> 58

<211> 22

<212> DNA

<213> Artificial sequence

<400> 58

acatggggat gatctcactc tt 22

<210> 59

<211> 23

<212> DNA

<213> Artificial sequence

<400> 59

ttcccttctg agagtgtcag tgt 23

<210> 60

<211> 21

<212> DNA

<213> Artificial sequence

<400> 60

acattcaacc cacacaagag g 21

<210> 61

<211> 20

<212> DNA

<213> Artificial sequence

<400> 61

gccatggaac cagacagaaa 20

<210> 62

<211> 20

<212> DNA

<213> Artificial sequence

<400> 62

ccccctccat caacttcttc 20

<210> 63

<211> 21

<212> DNA

<213> Artificial sequence

<400> 63

ttattccaga cgcatttcca c 21

<210> 64

<211> 25

<212> DNA

<213> Artificial sequence

<400> 64

caatgaatta agggaaaatg acaaa 25

<210> 65

<211> 23

<212> DNA

<213> Artificial sequence

<400> 65

gcatgccaat ctcttcataa atc 23

<210> 66

<211> 23

<212> DNA

<213> Artificial sequence

<400> 66

tttgatgaca ttgcatacat tcg 23

<210> 67

<211> 23

<212> DNA

<213> Artificial sequence

<400> 67

ttgagacagg ccagtgttta cat 23

<210> 68

<211> 23

<212> DNA

<213> Artificial sequence

<400> 68

ttgtttttct gtttctccct ctg 23

<210> 69

<211> 21

<212> DNA

<213> Artificial sequence

<400> 69

gaagtcccaa ccatgacaag a 21

<210> 70

<211> 25

<212> DNA

<213> Artificial sequence

<400> 70

aaaccttaag atgagcattg ttttg 25

<210> 71

<211> 23

<212> DNA

<213> Artificial sequence

<400> 71

tcagggaaga agtgaatgaa aaa 23

<210> 72

<211> 23

<212> DNA

<213> Artificial sequence

<400> 72

gggttttggg ctgatattaa aac 23

<210> 73

<211> 24

<212> DNA

<213> Artificial sequence

<400> 73

tgtttccatg tcagctattt tgtt 24

<210> 74

<211> 25

<212> DNA

<213> Artificial sequence

<400> 74

tctaggatca agttgtcaaa gaaga 25

<210> 75

<211> 28

<212> DNA

<213> Artificial sequence

<400> 75

agataatatt gaagctgtag ggaaaaaa 28

<210> 76

<211> 27

<212> DNA

<213> Artificial sequence

<400> 76

tgcagtaaga gattgttcta tgaaagg 27

<210> 77

<211> 23

<212> DNA

<213> Artificial sequence

<400> 77

ccaaatgaaa aggacagcta ttg 23

<210> 78

<211> 21

<212> DNA

<213> Artificial sequence

<400> 78

tttgaagaac agtgccagac c 21

<210> 79

<211> 20

<212> DNA

<213> Artificial sequence

<400> 79

tttcttttgc ctgcaggatt 20

<210> 80

<211> 26

<212> DNA

<213> Artificial sequence

<400> 80

ttgacagtag aagaagattg gaagaa 26

<210> 81

<211> 22

<212> DNA

<213> Artificial sequence

<400> 81

cctgaattgt agcaatcacc aa 22

<210> 82

<211> 20

<212> DNA

<213> Artificial sequence

<400> 82

gatgaagatt tgccccatca 20

<210> 83

<211> 20

<212> DNA

<213> Artificial sequence

<400> 83

cccaaggcat ctcatcgtag 20

<210> 84

<211> 24

<212> DNA

<213> Artificial sequence

<400> 84

tgattatgtt tttgacacca atcg 24

<210> 85

<211> 22

<212> DNA

<213> Artificial sequence

<400> 85

caacatgact gtcctttcac ca 22

<210> 86

<211> 22

<212> DNA

<213> Artificial sequence

<400> 86

tgttacccag ctcctcttca tc 22

<210> 87

<211> 20

<212> DNA

<213> Artificial sequence

<400> 87

gagaacgcgg aattggtcta 20

<210> 88

<211> 20

<212> DNA

<213> Artificial sequence

<400> 88

ggcaactacc atccagcaac 20

<210> 89

<211> 21

<212> DNA

<213> Artificial sequence

<400> 89

gccaaagtca tggaagaagt g 21

<210> 90

<211> 27

<212> DNA

<213> Artificial sequence

<400> 90

gtattctaat ttggcataag gcataga 27

<210> 91

<211> 24

<212> DNA

<213> Artificial sequence

<400> 91

aagtcggaaa attcaaatag gaca 24

<210> 92

<211> 25

<212> DNA

<213> Artificial sequence

<400> 92

gttctatgcc ttatgccaaa ttaga 25

<210> 93

<211> 24

<212> DNA

<213> Artificial sequence

<400> 93

tgatggttat ggtaaaagag gtca 24

<210> 94

<211> 27

<212> DNA

<213> Artificial sequence

<400> 94

aagatgatga aagtaagttt tgcagtt 27

<210> 95

<211> 20

<212> DNA

<213> Artificial sequence

<400> 95

agccgaccta gcccataaaa 20

<210> 96

<211> 28

<212> DNA

<213> Artificial sequence

<400> 96

ggatgataat gatggagaac tagataca 28

<210> 97

<211> 23

<212> DNA

<213> Artificial sequence

<400> 97

agatgagcag ttgaactctg gaa 23

<210> 98

<211> 21

<212> DNA

<213> Artificial sequence

<400> 98

atgtggttgg aacttgaggt g 21

<210> 99

<211> 22

<212> DNA

<213> Artificial sequence

<400> 99

tcgatttgtt tctgaaccat tg 22

<210> 100

<211> 21

<212> DNA

<213> Artificial sequence

<400> 100

attttggaca gcaggaatgt g 21

<210> 101

<211> 22

<212> DNA

<213> Artificial sequence

<400> 101

ccaatggttc agaaacaaat cg 22

<210> 102

<211> 25

<212> DNA

<213> Artificial sequence

<400> 102

tttgttggtc tctcttcttc ttcat 25

<210> 103

<211> 22

<212> DNA

<213> Artificial sequence

<400> 103

tcaataggct gatccacatg ac 22

<210> 104

<211> 27

<212> DNA

<213> Artificial sequence

<400> 104

aagaagagag accaacaaat tatagca 27

<210> 105

<211> 21

<212> DNA

<213> Artificial sequence

<400> 105

cggttttact gctttgtcca g 21

<210> 106

<211> 23

<212> DNA

<213> Artificial sequence

<400> 106

ttccttcatc acagaaacag tca 23

<210> 107

<211> 22

<212> DNA

<213> Artificial sequence

<400> 107

ccgaacatat gtcttcaagc ag 22

<210> 108

<211> 29

<212> DNA

<213> Artificial sequence

<400> 108

gaaaaacata ttggagtatc ttctacaca 29

<210> 109

<211> 27

<212> DNA

<213> Artificial sequence

<400> 109

cttgcaaagt ttcttctatt aaccaag 27

<210> 110

<211> 29

<212> DNA

<213> Artificial sequence

<400> 110

gatgtagttc attatcatct ttgtcatca 29

<210> 111

<211> 20

<212> DNA

<213> Artificial sequence

<400> 111

gtgctgtgac actgctggaa 20

<210> 112

<211> 21

<212> DNA

<213> Artificial sequence

<400> 112

ggtcagctga agatcctgtg a 21

<210> 113

<211> 20

<212> DNA

<213> Artificial sequence

<400> 113

agaatcagcc aggcacaaag 20

<210> 114

<211> 20

<212> DNA

<213> Artificial sequence

<400> 114

ggtgctcaga cacccaaaag 20

<210> 115

<211> 20

<212> DNA

<213> Artificial sequence

<400> 115

caggagaccc cactcatgtt 20

<210> 116

<211> 21

<212> DNA

<213> Artificial sequence

<400> 116

gctccgttca gagtgaacca t 21

<210> 117

<211> 20

<212> DNA

<213> Artificial sequence

<400> 117

catgccacca agcagaagta 20

<210> 118

<211> 21

<212> DNA

<213> Artificial sequence

<400> 118

ctcaaacagc tcaaaccaag c 21

<210> 119

<211> 20

<212> DNA

<213> Artificial sequence

<400> 119

agcactcagg ctggatgaac 20

<210> 120

<211> 20

<212> DNA

<213> Artificial sequence

<400> 120

tttgccacgg aaagtactcc 20

<210> 121

<211> 19

<212> DNA

<213> Artificial sequence

<400> 121

gagcctcgat gagccattt 19

<210> 122

<211> 22

<212> DNA

<213> Artificial sequence

<400> 122

gggaatgaaa cagaatcaga gc 22

<210> 123

<211> 23

<212> DNA

<213> Artificial sequence

<400> 123

aaaaccaaga gaaagaggca gaa 23

<210> 124

<211> 27

<212> DNA

<213> Artificial sequence

<400> 124

aggacctatt agatgattca gatgatg 27

<210> 125

<211> 24

<212> DNA

<213> Artificial sequence

<400> 125

caacctgttt tgtgatggta gaag 24

<210> 126

<211> 19

<212> DNA

<213> Artificial sequence

<400> 126

tgtgccaggg accttacct 19

<210> 127

<211> 20

<212> DNA

<213> Artificial sequence

<400> 127

ctggatccca gaaggtgaga 20

<210> 128

<211> 20

<212> DNA

<213> Artificial sequence

<400> 128

cgatctgcac acaccagttg 20

<210> 129

<211> 20

<212> DNA

<213> Artificial sequence

<400> 129

tccctggtgt caggaaaatg 20

<210> 130

<211> 28

<212> DNA

<213> Artificial sequence

<400> 130

tgttttcctt tacttactac acctcaga 28

<210> 131

<211> 20

<212> DNA

<213> Artificial sequence

<400> 131

tgtgccacac atctttgacc 20

<210> 132

<211> 16

<212> DNA

<213> Artificial sequence

<400> 132

aggagctggg ccatcg 16

<210> 133

<211> 18

<212> DNA

<213> Artificial sequence

<400> 133

cttcctggac acgctggt 18

<210> 134

<211> 17

<212> DNA

<213> Artificial sequence

<400> 134

gaccccgcca ctctcac 17

<210> 135

<211> 16

<212> DNA

<213> Artificial sequence

<400> 135

ccgagtggcg gagctg 16

<210> 136

<211> 21

<212> DNA

<213> Artificial sequence

<400> 136

tggctctgac cattctgttc t 21

<210> 137

<211> 19

<212> DNA

<213> Artificial sequence

<400> 137

gggtcgggta gaggaggtg 19

<210> 138

<211> 17

<212> DNA

<213> Artificial sequence

<400> 138

agccttcggc tgactgg 17

<210> 139

<211> 16

<212> DNA

<213> Artificial sequence

<400> 139

ggggagagca ggcagc 16

<210> 140

<211> 20

<212> DNA

<213> Artificial sequence

<400> 140

cgcctgtcct catgtattgg 20

<210> 141

<211> 20

<212> DNA

<213> Artificial sequence

<400> 141

gattgtcagt gcgcttttcc 20

<210> 142

<211> 23

<212> DNA

<213> Artificial sequence

<400> 142

tgacagaaag gtaaagagga gca 23

<210> 143

<211> 27

<212> DNA

<213> Artificial sequence

<400> 143

catcatcaat attgttcctg tatacgc 27

<210> 144

<211> 27

<212> DNA

<213> Artificial sequence

<400> 144

tcttttctaa atgaaaacac aacatga 27

<210> 145

<211> 23

<212> DNA

<213> Artificial sequence

<400> 145

ttaatggtgg ctttttgttt gtt 23

<210> 146

<211> 20

<212> DNA

<213> Artificial sequence

<400> 146

gcaattaaat ttggcggtgt 20

<210> 147

<211> 28

<212> DNA

<213> Artificial sequence

<400> 147

ttttaaactt ttcttttagt tgtgctga 28

<210> 148

<211> 24

<212> DNA

<213> Artificial sequence

<400> 148

catgattgtc atcttcactt agcc 24

<210> 149

<211> 25

<212> DNA

<213> Artificial sequence

<400> 149

gcacatatca ttacaccagt tcgtc 25

<210> 150

<211> 22

<212> DNA

<213> Artificial sequence

<400> 150

tggtccttac ttccccatag aa 22

<210> 151

<211> 20

<212> DNA

<213> Artificial sequence

<400> 151

aggcacaaga ggccctagat 20

<210> 152

<211> 27

<212> DNA

<213> Artificial sequence

<400> 152

cactggtcta taatccagat gattctt 27

<210> 153

<211> 21

<212> DNA

<213> Artificial sequence

<400> 153

cgccactgaa cattggaata g 21

<210> 154

<211> 23

<212> DNA

<213> Artificial sequence

<400> 154

ggaaggatga gaatttcaag cac 23

<210> 155

<211> 20

<212> DNA

<213> Artificial sequence

<400> 155

tgaacttgtc ttcccgtcgt 20

<210> 156

<211> 27

<212> DNA

<213> Artificial sequence

<400> 156

gttctgtttg tggaagaact ctacttt 27

<210> 157

<211> 24

<212> DNA

<213> Artificial sequence

<400> 157

tttggatatt tctcccaatg aaag 24

<210> 158

<211> 22

<212> DNA

<213> Artificial sequence

<400> 158

tgcacgctct atactgcaaa tg 22

<210> 159

<211> 23

<212> DNA

<213> Artificial sequence

<400> 159

aagtatcggt tggctttgtc ttt 23

<210> 160

<211> 20

<212> DNA

<213> Artificial sequence

<400> 160

acaagtcaac aacccccaca 20

<210> 161

<211> 25

<212> DNA

<213> Artificial sequence

<400> 161

aggttcattg tcactaacat ctggt 25

<210> 162

<211> 25

<212> DNA

<213> Artificial sequence

<400> 162

ctgatcttca tcaaaaggtt cattc 25

<210> 163

<211> 22

<212> DNA

<213> Artificial sequence

<400> 163

ctgacaccac tgactctgat cc 22

<210> 164

<211> 21

<212> DNA

<213> Artificial sequence

<400> 164

actgccatcg acttacattg g 21

<210> 165

<211> 22

<212> DNA

<213> Artificial sequence

<400> 165

tctggtgtca gagatggaga tg 22

<210> 166

<211> 20

<212> DNA

<213> Artificial sequence

<400> 166

tcttccctct ctccaccaga 20

<210> 167

<211> 28

<212> DNA

<213> Artificial sequence

<400> 167

tttcagtgtt acttacctgt cttgtctt 28

<210> 168

<211> 21

<212> DNA

<213> Artificial sequence

<400> 168

tgtactggtc cctcattgca c 21

<210> 169

<211> 28

<212> DNA

<213> Artificial sequence

<400> 169

tgactgaata taaacttgtg gtagttgg 28

<210> 170

<211> 20

<212> DNA

<213> Artificial sequence

<400> 170

gactgcccac aggaaggtaa 20

<210> 171

<211> 20

<212> DNA

<213> Artificial sequence

<400> 171

tggcatttga cattgagacg 20

<210> 172

<211> 21

<212> DNA

<213> Artificial sequence

<400> 172

agggtctgac gggtagagtg t 21

<210> 173

<211> 19

<212> DNA

<213> Artificial sequence

<400> 173

gaggctgtca gtggggaac 19

<210> 174

<211> 21

<212> DNA

<213> Artificial sequence

<400> 174

agtctgagtc aggcccttct g 21

<210> 175

<211> 20

<212> DNA

<213> Artificial sequence

<400> 175

taggaaggca ggggagtagg 20

<210> 176

<211> 20

<212> DNA

<213> Artificial sequence

<400> 176

cttctccccc tcctctgttg 20

<210> 177

<211> 20

<212> DNA

<213> Artificial sequence

<400> 177

ccagccaaag aagaaaccac 20

<210> 178

<211> 25

<212> DNA

<213> Artificial sequence

<400> 178

caagacttag tacctgaagg gtgaa 25

<210> 179

<211> 20

<212> DNA

<213> Artificial sequence

<400> 179

gcttcttgtc ctgcttgctt 20

<210> 180

<211> 21

<212> DNA

<213> Artificial sequence

<400> 180

gcggagattc tcttcctctg t 21

<210> 181

<211> 18

<212> DNA

<213> Artificial sequence

<400> 181

gtggcaagtg gctcctga 18

<210> 182

<211> 21

<212> DNA

<213> Artificial sequence

<400> 182

tcatcttggg cctgtgttat c 21

<210> 183

<211> 20

<212> DNA

<213> Artificial sequence

<400> 183

cttaacccct cctcccagag 20

<210> 184

<211> 22

<212> DNA

<213> Artificial sequence

<400> 184

cgaaaagtgt ttctgtcatc ca 22

<210> 185

<211> 19

<212> DNA

<213> Artificial sequence

<400> 185

accagccctg tcgtctctc 19

<210> 186

<211> 22

<212> DNA

<213> Artificial sequence

<400> 186

caacaagatg ttttgccaac tg 22

<210> 187

<211> 18

<212> DNA

<213> Artificial sequence

<400> 187

gggggtgtgg aatcaacc 18

<210> 188

<211> 20

<212> DNA

<213> Artificial sequence

<400> 188

cccttcccag aaaacctacc 20

<210> 189

<211> 20

<212> DNA

<213> Artificial sequence

<400> 189

cagaatgcaa gaagcccaga 20

<210> 190

<211> 20

<212> DNA

<213> Artificial sequence

<400> 190

tgggaaggga cagaagatga 20

<210> 191

<211> 18

<212> DNA

<213> Artificial sequence

<400> 191

cggtgtagga gctgctgg 18

<210> 192

<211> 20

<212> DNA

<213> Artificial sequence

<400> 192

tcatctggac ctgggtcttc 20

<210> 193

<211> 19

<212> DNA

<213> Artificial sequence

<400> 193

cagcccaacc cttgtcctt 19

<210> 194

<211> 18

<212> DNA

<213> Artificial sequence

<400> 194

actgccttcc gggtcact 18

<210> 195

<211> 20

<212> DNA

<213> Artificial sequence

<400> 195

ggaccaagga tatgccacac 20

<210> 196

<211> 20

<212> DNA

<213> Artificial sequence

<400> 196

tgtgtagggc gaagtgtgag 20

<210> 197

<211> 20

<212> DNA

<213> Artificial sequence

<400> 197

tacttccgga acctgtgctc 20

<210> 198

<211> 18

<212> DNA

<213> Artificial sequence

<400> 198

tggtgatgcc cactctgc 18

<210> 199

<211> 21

<212> DNA

<213> Artificial sequence

<400> 199

atctatgtcc ctgaagcagc a 21

<210> 200

<211> 20

<212> DNA

<213> Artificial sequence

<400> 200

cctcaggatt gcctttacca 20

<210> 201

<211> 20

<212> DNA

<213> Artificial sequence

<400> 201

gaaaaagccg aaggtcacaa 20

<210> 202

<211> 22

<212> DNA

<213> Artificial sequence

<400> 202

acattcacgt aggttgcaca aa 22

<210> 203

<211> 25

<212> DNA

<213> Artificial sequence

<400> 203

ctccatttta gcacttacct gtgac 25

<210> 204

<211> 20

<212> DNA

<213> Artificial sequence

<400> 204

tccaaagcct cttgctcagt 20

<210> 205

<211> 25

<212> DNA

<213> Artificial sequence

<400> 205

gatccaatcc atttttgttg tccag 25

<210> 206

<211> 20

<212> DNA

<213> Artificial sequence

<400> 206

cagtctctgg atcccacacc 20

<210> 207

<211> 20

<212> DNA

<213> Artificial sequence

<400> 207

ctgcaacatg acccatcaaa 20

<210> 208

<211> 20

<212> DNA

<213> Artificial sequence

<400> 208

cggacactca aagtgtggaa 20

<210> 209

<211> 25

<212> DNA

<213> Artificial sequence

<400> 209

cattttagta gacgcatctc gtacc 25

<210> 210

<211> 23

<212> DNA

<213> Artificial sequence

<400> 210

cagctatatt ccctggctta cct 23

<210> 211

<211> 22

<212> DNA

<213> Artificial sequence

<400> 211

ccatatttcc catctcgatg aa 22

<210> 212

<211> 25

<212> DNA

<213> Artificial sequence

<400> 212

tgtaattttt tccctacagc ttcaa 25

<210> 213

<211> 20

<212> DNA

<213> Artificial sequence

<400> 213

ctgggatgtg cgggtatatt 20

<210> 214

<211> 20

<212> DNA

<213> Artificial sequence

<400> 214

aaaactcacc tgggatgtgc 20

<210> 215

<211> 20

<212> DNA

<213> Artificial sequence

<400> 215

gggtctggca ctgttcttca 20

<210> 216

<211> 21

<212> DNA

<213> Artificial sequence

<400> 216

ttctttctca ttgccttcac g 21

<210> 217

<211> 21

<212> DNA

<213> Artificial sequence

<400> 217

agcacaagaa caagggaaac a 21

<210> 218

<211> 21

<212> DNA

<213> Artificial sequence

<400> 218

tactcagctg cctgcttctt c 21

<210> 219

<211> 21

<212> DNA

<213> Artificial sequence

<400> 219

tggtctctcg tctttctcag c 21

<210> 220

<211> 24

<212> DNA

<213> Artificial sequence

<400> 220

acgtatgaac agcattaaac caga 24

<210> 221

<211> 20

<212> DNA

<213> Artificial sequence

<400> 221

tctcccggac aagaaaagtg 20

<210> 222

<211> 23

<212> DNA

<213> Artificial sequence

<400> 222

aggacagtca tgttgccagt att 23

<210> 223

<211> 22

<212> DNA

<213> Artificial sequence

<400> 223

gaagaggagc tgggtaacac tg 22

<210> 224

<211> 20

<212> DNA

<213> Artificial sequence

<400> 224

tagaccaatt ccgcgttctc 20

<210> 225

<211> 23

<212> DNA

<213> Artificial sequence

<400> 225

aagttcctgg attttctgtt gct 23

<210> 226

<211> 20

<212> DNA

<213> Artificial sequence

<400> 226

ccatgacttt ggcaatctgg 20

<210> 227

<211> 25

<212> DNA

<213> Artificial sequence

<400> 227

cttctgtctt cctgagaggt atgaa 25

<210> 228

<211> 20

<212> DNA

<213> Artificial sequence

<400> 228

gtgtatgggc agcagagctt 20

<210> 229

<211> 23

<212> DNA

<213> Artificial sequence

<400> 229

tgtgtgacag atgagagaaa tgc 23

<210> 230

<211> 24

<212> DNA

<213> Artificial sequence

<400> 230

tgacctcttt taccataacc atca 24

<210> 231

<211> 20

<212> DNA

<213> Artificial sequence

<400> 231

ggattcaatc gagggtttca 20

<210> 232

<211> 26

<212> DNA

<213> Artificial sequence

<400> 232

tgcactatgt attttatggg ctaggt 26

<210> 233

<211> 28

<212> DNA

<213> Artificial sequence

<400> 233

tggtgtatct agttctccat cattatca 28

<210> 234

<211> 20

<212> DNA

<213> Artificial sequence

<400> 234

tgaaggactt tgccttccag 20

<210> 235

<211> 19

<212> DNA

<213> Artificial sequence

<400> 235

gtttgggtct tgcccatct 19

<210> 236

<211> 23

<212> DNA

<213> Artificial sequence

<400> 236

ccttgattgt ctttgctcac ttt 23

<210> 237

<211> 26

<212> DNA

<213> Artificial sequence

<400> 237

gacccaaaca cataatagaa gatgaa 26

<210> 238

<211> 26

<212> DNA

<213> Artificial sequence

<400> 238

cctgtttata ctgagagcac tgatga 26

<210> 239

<211> 24

<212> DNA

<213> Artificial sequence

<400> 239

ttcttgacac aaagactggc ttac 24

<210> 240

<211> 28

<212> DNA

<213> Artificial sequence

<400> 240

tcttcagagt aacgttcact ataattgg 28

<210> 241

<211> 24

<212> DNA

<213> Artificial sequence

<400> 241

tcaaaatgta agccagtctt tgtg 24

<210> 242

<211> 27

<212> DNA

<213> Artificial sequence

<400> 242

tgataagcct accaattata gtgaacg 27

<210> 243

<211> 25

<212> DNA

<213> Artificial sequence

<400> 243

aaaatgactg tttctgtgat gaagg 25

<210> 244

<211> 21

<212> DNA

<213> Artificial sequence

<400> 244

cgtcatgtgg atcagcctat t 21

<210> 245

<211> 21

<212> DNA

<213> Artificial sequence

<400> 245

tgattctgcc tcttggcatt a 21

<210> 246

<211> 20

<212> DNA

<213> Artificial sequence

<400> 246

agccttttga ggctgaccac 20

<210> 247

<211> 22

<212> DNA

<213> Artificial sequence

<400> 247

ccatccaagt tctgcacaga gt 22

<210> 248

<211> 26

<212> DNA

<213> Artificial sequence

<400> 248

tgattacatc ctatttcatc ttcagc 26

<210> 249

<211> 25

<212> DNA

<213> Artificial sequence

<400> 249

gctgacctag ttccaatctt ttctt 25

<210> 250

<211> 20

<212> DNA

<213> Artificial sequence

<400> 250

cagacgacac aggaagcaga 20

<210> 251

<211> 21

<212> DNA

<213> Artificial sequence

<400> 251

tcgctcctga agaaaattca a 21

<210> 252

<211> 20

<212> DNA

<213> Artificial sequence

<400> 252

aacatgagtg gggtctcctg 20

<210> 253

<211> 20

<212> DNA

<213> Artificial sequence

<400> 253

ctggcaatcg aacgactctc 20

<210> 254

<211> 21

<212> DNA

<213> Artificial sequence

<400> 254

atgccactta ccattccact g 21

<210> 255

<211> 20

<212> DNA

<213> Artificial sequence

<400> 255

aggaggtggt ggaggtgttt 20

<210> 256

<211> 21

<212> DNA

<213> Artificial sequence

<400> 256

gcagcttgct taggtccact c 21

<210> 257

<211> 20

<212> DNA

<213> Artificial sequence

<400> 257

agcatctgga agaacctgga 20

<210> 258

<211> 22

<212> DNA

<213> Artificial sequence

<400> 258

ggacctaagc aagctgcagt aa 22

<210> 259

<211> 23

<212> DNA

<213> Artificial sequence

<400> 259

cccattgtca ttttcctgaa ctg 23

<210> 260

<211> 24

<212> DNA

<213> Artificial sequence

<400> 260

tggttttcat ttgattcttt aggc 24

<210> 261

<211> 28

<212> DNA

<213> Artificial sequence

<400> 261

tcaatatcat catcatctga atcatcta 28

<210> 262

<211> 22

<212> DNA

<213> Artificial sequence

<400> 262

ttggcatggc agaaataata ca 22

<210> 263

<211> 22

<212> DNA

<213> Artificial sequence

<400> 263

ggtggaggta attttgaagc ag 22

<210> 264

<211> 20

<212> DNA

<213> Artificial sequence

<400> 264

ccatgccaac aaagtcatca 20

<210> 265

<211> 19

<212> DNA

<213> Artificial sequence

<400> 265

ggagaagctc ccaaccaag 19

<210> 266

<211> 19

<212> DNA

<213> Artificial sequence

<400> 266

cccacacagc aaagcagaa 19

<210> 267

<211> 19

<212> DNA

<213> Artificial sequence

<400> 267

gcatctgcct cacctccac 19

<210> 268

<211> 22

<212> DNA

<213> Artificial sequence

<400> 268

gcagcatgtc aagatcacag at 22

<210> 269

<211> 22

<212> DNA

<213> Artificial sequence

<400> 269

aactgttcaa actgatggga cc 22

<210> 270

<211> 21

<212> DNA

<213> Artificial sequence

<400> 270

ctgtaggacc ttcggtgact g 21

<210> 271

<211> 25

<212> DNA

<213> Artificial sequence

<400> 271

acaaattctc agatcatcag tcctc 25

<210> 272

<211> 18

<212> DNA

<213> Artificial sequence

<400> 272

gcaggtaccg tgcgacat 18

<210> 273

<211> 18

<212> DNA

<213> Artificial sequence

<400> 273

gctcctcagc caggtcca 18

<210> 274

<211> 19

<212> DNA

<213> Artificial sequence

<400> 274

caccagcgtg tccaggaag 19

<210> 275

<211> 17

<212> DNA

<213> Artificial sequence

<400> 275

gggtcgggtg agagtgg 17

<210> 276

<211> 17

<212> DNA

<213> Artificial sequence

<400> 276

ctcccgctgc agaccct 17

<210> 277

<211> 20

<212> DNA

<213> Artificial sequence

<400> 277

ggcctccgac cgtaactatt 20

<210> 278

<211> 19

<212> DNA

<213> Artificial sequence

<400> 278

cacctcctct acccgaccc 19

<210> 279

<211> 53

<212> DNA

<213> Artificial sequence

<400> 279

caagcagaag acggcatacg agattgcatg acgtgactgg agttcagacg tgt 53

<210> 280

<211> 53

<212> DNA

<213> Artificial sequence

<400> 280

caagcagaag acggcatacg agattgctat cggtgactgg agttcagacg tgt 53

<210> 281

<211> 53

<212> DNA

<213> Artificial sequence

<400> 281

caagcagaag acggcatacg agatcacaag cagtgactgg agttcagacg tgt 53

<210> 282

<211> 53

<212> DNA

<213> Artificial sequence

<400> 282

caagcagaag acggcatacg agattcgctg atgtgactgg agttcagacg tgt 53

<210> 283

<211> 53

<212> DNA

<213> Artificial sequence

<400> 283

caagcagaag acggcatacg agatcgcttt gtgtgactgg agttcagacg tgt 53

<210> 284

<211> 53

<212> DNA

<213> Artificial sequence

<400> 284

caagcagaag acggcatacg agatccgatg aagtgactgg agttcagacg tgt 53

<210> 285

<211> 53

<212> DNA

<213> Artificial sequence

<400> 285

caagcagaag acggcatacg agattgacca cagtgactgg agttcagacg tgt 53

<210> 286

<211> 53

<212> DNA

<213> Artificial sequence

<400> 286

caagcagaag acggcatacg agatgaagcc atgtgactgg agttcagacg tgt 53

<210> 287

<211> 53

<212> DNA

<213> Artificial sequence

<400> 287

caagcagaag acggcatacg agatttctgg tggtgactgg agttcagacg tgt 53

<210> 288

<211> 53

<212> DNA

<213> Artificial sequence

<400> 288

caagcagaag acggcatacg agatccgaaa acgtgactgg agttcagacg tgt 53

<210> 289

<211> 53

<212> DNA

<213> Artificial sequence

<400> 289

caagcagaag acggcatacg agatcgaaaa gggtgactgg agttcagacg tgt 53

<210> 290

<211> 53

<212> DNA

<213> Artificial sequence

<400> 290

caagcagaag acggcatacg agataaccag ctgtgactgg agttcagacg tgt 53

<210> 291

<211> 57

<212> DNA

<213> Artificial sequence

<400> 291

aatgatacgg cgaccaccga gatctacact atagcctaca ctctttccct acacgac 57

<210> 292

<211> 57

<212> DNA

<213> Artificial sequence

<400> 292

aatgatacgg cgaccaccga gatctacaca tagaggcaca ctctttccct acacgac 57

<210> 293

<211> 57

<212> DNA

<213> Artificial sequence

<400> 293

aatgatacgg cgaccaccga gatctacacc ctatcctaca ctctttccct acacgac 57

<210> 294

<211> 57

<212> DNA

<213> Artificial sequence

<400> 294

aatgatacgg cgaccaccga gatctacacg gctctgaaca ctctttccct acacgac 57

<210> 295

<211> 57

<212> DNA

<213> Artificial sequence

<400> 295

aatgatacgg cgaccaccga gatctacaca ggcgaagaca ctctttccct acacgac 57

<210> 296

<211> 57

<212> DNA

<213> Artificial sequence

<400> 296

aatgatacgg cgaccaccga gatctacact aatcttaaca ctctttccct acacgac 57

<210> 297

<211> 57

<212> DNA

<213> Artificial sequence

<400> 297

aatgatacgg cgaccaccga gatctacacc aggacgtaca ctctttccct acacgac 57

<210> 298

<211> 57

<212> DNA

<213> Artificial sequence

<400> 298

aatgatacgg cgaccaccga gatctacacg tactgacaca ctctttccct acacgac 57

<210> 299

<211> 30

<212> DNA

<213> Artificial sequence

<400> 299

ctctttccct acacgacgct cttccgatct 30

<210> 300

<211> 30

<212> DNA

<213> Artificial sequence

<400> 300

ctggagttca gacgtgtgct cttccgatct 30

Claims

1. A primer combination, wherein each primer in the primer combination contains a specific sequence, and the specific sequence contains at least one of sequences shown as SEQ ID NO. 1 to SEQ ID NO. 278 or complementary sequences thereof.

2. The primer combination of claim 1, wherein the primer combination is a primer combination for targeted amplification of an ovarian oncogene;

optionally, the primer combination specifically targets at least one of the following genes: PTEN, TP53, APC, PIK3CA, CTNNB1, KRAS, PIK3R1, NRAS, AKT1, FGFR2, pool, PPP2R1A, CDKN2A, FBXW7, RNF43, BRAF, MAPK1, EGFR.

3. The primer combination of claim 2, wherein each primer in the primer combination further comprises a bridging sequence that is linked to the specific sequence;

optionally, when the primer in the primer combination is an upstream primer, the bridging sequence is 5'-CTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO:299) or a complementary sequence thereof; when the primer in the primer combination is a downstream primer, the bridging sequence is 5'-CTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO:300) or a complementary sequence thereof;

optionally, the bridging sequence is linked to the 5' end of the specific sequence;

optionally, when the primer in the primer combination is an upstream primer or a downstream primer, a molecular tag sequence is connected between the bridging sequence and the specific sequence;

optionally, the molecular tag sequence contains 6-16 random bases.

4. The primer combination according to claim 1, wherein the primer shown in SEQ ID NO. 1 or the complementary sequence thereof and the sequence shown in SEQ ID NO. 140 or the complementary sequence thereof are combined into a pair of upstream and downstream primers, the primer shown in SEQ ID NO. 2 or the complementary sequence thereof and the sequence shown in SEQ ID NO. 141 or the complementary sequence thereof are combined into a pair of upstream and downstream primers, and so on;

optionally, the 3' end of the specific sequence is modified;

optionally, the method of modifying the 3' end of the specific primer is selected from at least one of thio-modification, phosphorylation, LNA modification;

optionally, the modification position is a phosphodiester bond between the penultimate and second nucleotides located at the 3' end of the specific primer;

optionally, the Tm value of each primer in the primer combination is 55-65 ℃.

5. A primer combination comprising the primer combination of any one of claims 1-4 for a first round of PCR amplification and a primer combination for a second round of PCR amplification;

optionally, the primer combination for the second round of PCR amplification contains at least one of the sequences shown in SEQ ID NO. 279 to SEQ ID NO. 298 or the complementary sequences thereof.

6. A kit comprising the primer combination of any one of claims 1 to 4 or the primer combination of claim 5;

optionally, the kit is a kit for cancer mutation type detection;

optionally, the kit is a kit for early screening for ovarian cancer;

optionally, the kit further comprises at least one of a PCR amplification enzyme, dntps, water, PCR buffer.

7. Use of the primer combination according to any one of claims 1 to 4, or the primer combination according to claim 5, or the kit according to claim 6 for multiplex nested PCR amplification, for a first round of PCR amplification.

8. Use of the primer combination of any one of claims 1-4 in single-tube multiplex PCR amplification.

9. Use of the primer combination of any one of claims 1 to 4, or the primer combination of claim 5, or the kit of claim 6 for early cancer screening;

optionally, the cancer comprises ovarian cancer.

10. A method of PCR amplification comprising: obtaining a DNA extraction product of a sample, carrying out first round PCR amplification on the sample by adopting the primer combination of any one of claims 1-4, and then carrying out second round PCR amplification to obtain an amplification product;

optionally, in the first round of PCR amplification, primers required for the first round of PCR amplification are added into the same reaction vessel for amplification reaction;

optionally, further comprising purifying the amplification product after the first round of PCR amplification, and then performing a second round of PCR amplification on the purified amplification product;

optionally, purifying the first round amplification product by using magnetic beads;

optionally, the sample is selected from at least one of a plasma sample, a serum sample, a sample of tumor tissue shed cells;

optionally, the sample is derived from at least one selected from the group consisting of cervix, ovary, endometrium, fallopian tube, and pleural effusion.