CN111676286B - Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application - Google Patents

Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application Download PDF

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CN111676286B
CN111676286B CN202010479463.6A CN202010479463A CN111676286B CN 111676286 B CN111676286 B CN 111676286B CN 202010479463 A CN202010479463 A CN 202010479463A CN 111676286 B CN111676286 B CN 111676286B
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dna
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artificial sequence
lung cancer
multiplex pcr
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CN111676286A (en
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易吉
解青青
李泽卿
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Wuhan Igenebook Biotechnology Co ltd
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
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Abstract

The invention discloses a multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, a detection method and application. The multiplex PCR system for detecting the methylation of the free DNA of the lung cancer plasma comprises a first round multiplex PCR primer group and/or a second round multiplex PCR primer, wherein the first round multiplex PCR primer group comprises 55 pairs of PCR primer pairs, the sequences of the PCR primer pairs are respectively shown as SEQ ID NO. 1-SEQ ID NO.110 in a sequence table, and the sequences of the second round multiplex PCR primer pairs are shown as SEQ ID NO. 111-SEQ ID NO. 112. The method for detecting the methylation of the free DNA of the lung cancer plasma provided by the invention can simultaneously detect 55 methylation sites related to the lung cancer at one time, the mutual interference among multiple PCR primers is small, the specificity is strong, the amplification efficiency is high, the sample loss is reduced, the detection cost is reduced, the detection efficiency is improved, the detection rate of early lung cancer can be obviously improved by matching with a high-throughput sequencing technology, and the method is suitable for popularization and application.

Description

Multiplex PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application
Technical Field
The invention relates to the field of molecular biological detection, in particular to a multiplex PCR primer system for detecting the methylation of free DNA in lung cancer plasma, a detection method and application.
Background
Lung cancer has been the highest incidence and mortality cancer worldwide. The early diagnosis of lung cancer is mainly based on high-risk factors in the medical history and combined with means such as imaging, endoscopy and molecular biology to carry out early screening, so as to achieve the purpose of early detection and early treatment. However, these methods for early diagnosis and examination of lung cancer also have obvious disadvantages: low dose helical CT (LDCT) presents a risk of radiation; endobronchial endoscopy is often invasive and often has false negatives; the sputum cast-off cell examination also has the problems of low sensitivity and more false negatives. And the method of puncture tissue biopsy and the like has large trauma, and can only be applied to the confirmed diagnosis of cancer.
Clinical molecular diagnosis technology has been the research direction of cancer diagnosis, and tumor molecular markers are the key research targets. Abnormal methylation of lung cancer-associated genes is considered an early event in the development of lung cancer, and as the number of genes that develop abnormal methylation increases, the risk of developing lung cancer also gradually increases. For example, belinsky et al detected abnormal methylation of the p16 gene in pre-cancerous lesions in a mouse lung cancer model, and the frequency of abnormal methylation increased as the lesions progressed. Palmisano et al also detected abnormal methylation of the MGMT gene and/or the p16 gene in all sputum from patients diagnosed with lung cancer three years later. In addition, abnormal methylation of the lung cancer related gene can be detected not only in lung cancer tissues and sputum, but also in bronchial lavage fluid, bronchial brush test samples, serum or plasma of lung cancer patients, and has the advantage of easy sample acquisition. Therefore, the abnormal methylation of the lung cancer related gene can be used as a tumor marker of the lung cancer and used for early diagnosis or risk assessment of the lung cancer.
Plasma free DNA (cfDNA), also known as circulating free DNA, is extracellular nucleic acid present in plasma, generally fragmented, and varies in length from 150bp to 21 kbp. It has been found that abnormal methylation of tumor-associated genes can be detected in plasma cfDNA at an early stage of tumorigenesis, and that abnormal methylation in cfDNA always occurs simultaneously with abnormal methylation in tumor cells, while abnormal methylation of genes in plasma cfDNA has high sensitivity and specificity. The published table paper of Nature in 2017 shows that free DNA methylation can be used for screening early lung cancer and early pancreatic cancer, the sensitivity is 0.975 and 0.914 respectively, and the accuracy of the free DNA methylation on other cancers (breast cancer, colorectal cancer, renal cancer, bladder cancer and acute myelogenous leukemia) is about 0.9 or more. Therefore, the free DNA methylation is a stable and reliable cancer marker, and the application of the free DNA methylation detection to lung cancer screening has the feasibility of project implementation and the characteristics of non-invasiveness, simple operation, easy sampling, high sensitivity and high specificity required by large population screening, and has extremely important significance in early diagnosis and risk assessment of lung cancer.
In the current research, DNA methylation as a tumor marker is applied to tumor screening and is proved by extensive academic research, and some markers are commercialized, such as septin9 which is considered as the gold standard for intestinal cancer screening. While lung cancer DNA methylation markers are not widely used in the fields of large health screening and companion diagnostics. The early cancer screening can not only improve the survival rate of the cancer, but also reduce the family economic burden and the social burden of the patient. Therefore, there is an urgent need to develop a method for early screening of lung cancer based on the detection of free DNA methylation in plasma of lung cancer.
Disclosure of Invention
In view of the above, the invention provides a multiple PCR primer system for detecting methylation of free DNA in plasma of lung cancer, a detection method and application thereof, wherein the multiple PCR primer system can simultaneously detect 55 methylation sites related to lung cancer, has small mutual interference among primers, strong specificity and good amplification effect, can improve detection efficiency and reduce detection cost, can perform high-throughput sequencing by matching with a high-throughput sequencing technology, and has auxiliary screening and prompting effects on high-risk groups of lung cancer.
The technical scheme of the invention is realized as follows:
in a first aspect, the present invention provides a multiplex PCR primer system for detecting methylation of free DNA in plasma of lung cancer, comprising a first-round multiplex PCR primer set, wherein the first-round multiplex PCR primer set comprises 55 pairs of PCR primer pairs, and the sequences of the 55 pairs of PCR primer pairs are as follows:
site 1
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACCCCATCCTATTAAACATACA (SEQ ID NO. 1)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTGGGTAGTATAAAGTATTGGAT (SEQ ID NO. 2)
Site 2
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCCCACATTATCCTACTCT (SEQ ID NO. 3)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGATTTGAATGATATGAAGGG (SEQ ID NO. 4)
Position 3
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGATTTGGAAAGGAATGAATAAT (SEQ ID NO. 5)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACATCTAAATCCTTCATCACC (SEQ ID NO. 6)
Position 4
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGTTGGGAAGTAGTTATTATAGAT (SEQ ID NO. 7)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCTCCTCCTCTTCAATAACAT (SEQ ID NO. 8)
Position 5
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGAGATGATTTAGAATTAATAGTG (SEQ ID NO. 9)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTCCCTACTACTCTCTTCTA (SEQ ID NO. 10)
Position 6
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTCCTTTTCAAATCATAAAACT (SEQ ID NO. 11)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTTTAGAGTTGGTATGGAGTAAT (SEQ ID NO. 12)
Position 7
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTTTAAATATGTATAGTAGAGAGT (SEQ ID NO. 13)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCACCCTTAACCTATCTCA (SEQ ID NO. 14)
Position 8
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGAGATTTTGAGATAGTGAGAATT (SEQ ID NO. 15)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCCATCTACATCACCAATA (SEQ ID NO. 16)
Position 9
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTGGTTGGTAAGGAAATATAGG (SEQ ID NO. 17)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCCCCTATATCCAAACATA (SEQ ID NO. 18)
Site 10
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACCAACCTATTTCACTCCAA (SEQ ID NO. 19)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTGATTTAGATTTAGAGGATTGTA (SEQ ID NO. 20)
Position 11
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGTTTGTAAGGAGTGTGAT (SEQ ID NO. 21)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTCCATCTATTTAACCTCCTA (SEQ ID NO. 22)
Position 12
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGTGTAGGATGAAGGTATGA (SEQ ID NO. 23)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAACTCCCCATTACCCTTCTA (SEQ ID NO. 24)
Position 13
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACCAACCAACTCCTCTTATA (SEQ ID NO. 25)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGAGATTGGAAAGAAAGTATTGTG (SEQ ID NO. 26)
Site 14
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCATCAATCTATTAATTACACTA (SEQ ID NO. 27)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAATAGGTATAATATTAGGGTTTTT (SEQ ID NO. 28)
Position 15
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATCAATTCTCATCATACAACTCA (SEQ ID NO. 29)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTGTGTGTTAGTTAGTTTATGT (SEQ ID NO. 30)
Position 16
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGTTTTGGTATAAGTGTTTGTAA (SEQ ID NO. 31)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTTCCCAATCCTCCAATTT (SEQ ID NO. 32)
Position 17
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTGTTTGGTATAGAGAGTTGA (SEQ ID NO. 33)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACATTCCATAATCCCATATCC (SEQ ID NO. 34)
Position 18
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAACCATTTCCTATACCTTTTAA (SEQ ID NO. 35)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGTGGGGAGTTGTAAAAA (SEQ ID NO. 36)
Position 19
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAATTTAACAAAAATAATCTTACC (SEQ ID NO. 37)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGGAAGTTTGATATAGGAAATAATA (SEQ ID NO. 38)
Position 20
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAGAGATGGTATTTTATAGGGGA (SEQ ID NO. 39)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTAACTTCACCACATCCTTT (SEQ ID NO. 40)
Position 21
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCTCTTCATATCCCTAAAC (SEQ ID NO. 41)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGGAATTTTATTGTGTTGATTTAA (SEQ ID NO. 42)
Site 22
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGAGTTTAGTATGAAGTAATGTG (SEQ ID NO. 43)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCATCCTTTTACCCAAATC (SEQ ID NO. 44)
Position 23
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTGAGGGGAATAGGTTGGTA (SEQ ID NO. 45)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATCCCCCAATTCATCTTCAC (SEQ ID NO. 46)
Position 24
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTTGGAGAAGGTTAGTGGA (SEQ ID NO. 47)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACAACACCATTAAATTAATCATCA (SEQ ID NO. 48)
Position 25
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAGGGGTGGTTTAGGATAAA (SEQ ID NO. 49)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACAAATCAAATATTTCCAACATA (SEQ ID NO. 50)
Position 26
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTTTGGGATTTTTGTTTTTGGT (SEQ ID NO. 51)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACCATCATTTCAACCAATAACC (SEQ ID NO. 52)
Position 27
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTTTGTAATTGGAGGAGTGA (SEQ ID NO. 53)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAAATTTTTCCTTACAACCACTC (SEQ ID NO. 54)
Position 28
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGAGGTAGAGTTTAGAGAAGTA (SEQ ID NO. 55)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTACTCTCCACACTCCTCA (SEQ ID NO. 56)
Position 29
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGTGAAAAGAAAAGGAGTTA (SEQ ID NO. 57)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCCTTTCCTTAACCATTCCT (SEQ ID NO. 58)
Position 30
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTAGAAAGGGGTGTTAGGAA (SEQ ID NO. 59)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCCACAACCATAAAATCATTA (SEQ ID NO. 60)
Position 31
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCTCCCAATTCCCATACCTA (SEQ ID NO. 61)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGTTTTGGAAGAATAGGGTA (SEQ ID NO. 62)
Position 32
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACCCTCCTCACTATACTCA (SEQ ID NO. 63)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAAAGGGGAGTTGAGTTATA (SEQ ID NO. 64)
Position 33
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTGGGGGATGGGTTATATTTA (SEQ ID NO. 65)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAACCTTCTTTACACAAACACT (SEQ ID NO. 66)
Position 34
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGGGTTATGTAAGGGGATG (SEQ ID NO. 67)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACCAAACAACTACTAATCAAACA (SEQ ID NO. 68)
Position 35
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGTTGGAAGAGGGAAATTA (SEQ ID NO. 69)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCAAAAATCCTTCTACTCACCT (SEQ ID NO. 70)
Position 36
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTAGAGAGTGGTGGAGGA (SEQ ID NO. 71)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCAACACTACTCTTTCTCCA (SEQ ID NO. 72)
Position 37
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGTTAATTAGTAGGTATAGGTTT (SEQ ID NO. 73)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTCCCTTACAACCTATCCT (SEQ ID NO. 74)
Position 38
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACACCTCACTTTTCAACCTA (SEQ ID NO. 75)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAGGTGTTATGGTTGTGAT (SEQ ID NO. 76)
Position 39
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCACTCCAACCATTTAACT (SEQ ID NO. 77)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGATAGGGTATAGGGTATGT (SEQ ID NO. 78)
Position 40
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTATAGGTGTGTTGGGTGA (SEQ ID NO. 79)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCCAACCTACTTACTAAACT (SEQ ID NO. 80)
Position 41
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACTCTTAACTTCTCACCAAT (SEQ ID NO. 81)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTATTTTGGTTTTATGTGATGA (SEQ ID NO. 82)
Position 42
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGTTGGGAGTTAATGAGTAT (SEQ ID NO. 83)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCCAAAACCAAATCACTAATT (SEQ ID NO. 84)
Position 43
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGAAAGTAGGTAATGGGAGG (SEQ ID NO. 85)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACCAACTCTCCCAACAATTA (SEQ ID NO. 86)
Position 44
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAACCATTATATAAAAATACTACCA (SEQ ID NO. 87)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAGAGATTTGAGTTGTAGGGT (SEQ ID NO. 88)
Position 45
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACCAACACCCACTCTATAA (SEQ ID NO. 89)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTTGGGGAGAATGAGAATTGT (SEQ ID NO. 90)
Position 46
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGTGAGGGTTTATGTTTGTATA (SEQ ID NO. 91)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTTCCCTCACCTAAAATACA (SEQ ID NO. 92)
Position 47
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTAAGTAGGGGTGTGGTAGA (SEQ ID NO. 93)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTACCCTATCAACAACACA (SEQ ID NO. 94)
Position 48
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCAAAACCCATTCCTATTCA (SEQ ID NO. 95)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATATGTAGAGGGTAGGTGG (SEQ ID NO. 96)
Position 49
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTGGAGTTGTTTAAAAGGTT (SEQ ID NO. 97)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCACCCTCCCAATCTTAC (SEQ ID NO. 98)
Position 50
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAAAACTTCAACAAACATTCCA (SEQ ID NO. 99)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGGGGAATTTTTGAATTATTT (SEQ ID NO. 100)
Position 51
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTAGTGTATTTAAGAAATGTTGTAT (SEQ ID NO. 101)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAAATCAACCCAACACCAAC (SEQ ID NO. 102)
Position 52
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACAAACACAATAAAACCTAAATT (SEQ ID NO. 103)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTTGTATTTGTAAGTTGGGAG (SEQ ID NO. 104)
Position 53
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCACATCACCCTCTATTACC (SEQ ID NO. 105)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGTGGATGTTTAAGGTTAAGGT (SEQ ID NO. 106)
Position 54
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGAGTTTATAGTGAGGGTGAAA (SEQ ID NO. 107)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATCACAACTTCCTCTTCCT (SEQ ID NO. 108)
Position 55
Forward primer GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACCCACATCCACCCTAAATA (SEQ ID NO. 109)
Reverse primer TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGGTATAGTTTAGAGTTTAGTGGTA (SEQ ID NO. 110)
On the basis of the above technical solution, preferably, the multiplex PCR primer system further comprises a second round of multiplex PCR primers, and the sequences of the second round of multiplex PCR primers are as follows:
forward primer CAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG (SEQ ID NO. 111)
Reverse primer AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC (SEQ ID NO. 112)
Further, preferably, the second round of multiplex PCR primers contains 6-10 variable bases in sequence for labeling the sample DNA.
Further, preferably, the number of variable bases in the sequence of the second round of multiplex PCR primers is 8, and the sequence of the second round of multiplex PCR primers is as follows:
forward primer CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG (SEQ ID NO. 113)
Reverse primer AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC (SEQ ID NO. 114)
In a second aspect, the invention provides an application of the multiplex PCR primer system for detecting methylation of free DNA in plasma of lung cancer in preparing a reagent for screening and detecting methylation level of free DNA in plasma of lung cancer.
In a third aspect, the present invention provides a kit for detecting methylation of free DNA in plasma of lung cancer, comprising the multiplex PCR primer system for detecting methylation of free DNA in plasma of lung cancer according to the first aspect.
On the basis of the above technical solution, preferably, the kit for detecting methylation of free DNA in plasma of lung cancer further comprises a DNA extraction reagent, a bisulfite conversion reagent, a PCR amplification reagent, and a DNA purification reagent.
Further, preferably, the DNA purification reagent is a magnetic bead purification reagent.
In a fourth aspect, the present invention also provides a method for detecting the methylation level of free DNA in plasma of lung cancer, comprising the following steps:
s1, extracting sample plasma free DNA;
s2, performing bisulfite conversion and purification on the free DNA extracted in the step S1;
s3, performing multiplex PCR amplification by using the multiplex PCR primer system for detecting the methylation of the free DNA of the lung cancer plasma in the first aspect of the invention and taking the DNA transformed and purified in the step S2 as a template;
and S4, performing high-throughput sequencing on the product obtained by the multiplex PCR amplification in the step S3, comparing the sequencing result with the converted human genome reference sequence, and analyzing and judging the methylation level of the sample plasma free DNA.
Based on the above technical solution, preferably, the human genome reference sequence is hg19 or hg38.
Compared with the prior art, the multiple PCR primer system, the detection method and the application for detecting the methylation of the free DNA in the plasma of the lung cancer have the following beneficial effects:
(1) 2mL of peripheral blood only contains 10-20ng of DNA, only can meet the requirement of 1-2 PCR reactions, after bisulfite treatment, the plasma DNA is converted into a single strand, the plasma free DNA can be further degraded, if independent detection on a plurality of sites is required, the detection cannot be realized, the multiple PCR primer system designed for the methylation sites of the plasma free DNA comprises two primers, the first round of multiple PCR primer set comprises 55 pairs of PCR primer pairs, the amplification of 55 methylation sites can be completed in one reaction tube at one time, the mutual interference among the primers is small, the specificity is strong, the amplification effect is good, the detection efficiency can be improved, the problem of small amount of the plasma free DNA is perfectly solved, the detection cost is reduced, and the sample loss is reduced;
(2) Because the common next-generation sequencing method at present needs to carry out a plurality of steps of end repair, end A addition, adapter connection, amplification, purification, secondary amplification, sequencing primer addition, secondary purification and the like, the two-round PCR amplification library building system provided by the invention has the advantages that a section of 20-40 base universal sequence is respectively arranged on the forward and reverse primer upstream of the first round of multiplex PCR primer and is used for combining with the downstream sequence of the forward and reverse primer of the second round of multiplex PCR primer, the forward and reverse primer upstream sequence of the second round of multiplex PCR primer is used for high-throughput sequencing, the processes of end repair, end A addition and adapter connection are not needed, the library building can be completed only by simple two-step amplification, and the product can be directly used for universal SE50/SE100/SE150/PE50/PE100/PE150 after being purified;
(3) At present, sequencing flux of second-generation sequencing is extremely large (50 Gb-4 Tb), sequencing quantity of amplification products of a single sample is less than 10M, single sequencing flux cannot be met, samples are collected in 1000-10000 samples in a single day due to crowd screening, and detection flux is severely limited if each sample is independently built. The upstream and downstream of the second round of multiplex PCR primers of the invention contain 6-10 variable bases (N) for labeling samples, the random combination of the region is in the second round of PCR process, only 1 fixed combination is added to each sample, thereby realizing one-time up to the second round of forward primer 2 8 X second round reverse primer 2 8 65536 samples are used for second-generation sequencing, so that the detection time and the detection cost can be saved, and the detection automation is easy to realize.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments of the present invention, belong to the protection scope of the present invention.
Example 1 multiplex PCR primer System for detecting methylation of free DNA in Lung cancer plasma
The target region targeted by the primer design in this example is a region of 120 bases upstream and downstream of the methylation site specific to lung cancer. Since the maximum length of plasma-free DNA is generally not more than about 200bp, it is necessary to limit the amplification length of the primer to 120 bases or less.
The outline of the multiplex PCR primer design for detecting methylation of free DNA in plasma of lung cancer designed in this example is shown in Table 1.
TABLE 1 multiplex PCR primer design overview for detection of free DNA methylation in lung cancer plasma
Figure BDA0002516825220000161
Example 2 detection of free DNA methylation level in Lung cancer plasma Using multiplex PCR primer System designed according to the present invention
S1, extracting free DNA of lung cancer plasma
Extraction of lung cancer plasma free DNA was performed using the QIAamp Circulating Nucleic Acid Kit (Qiagen 55114) following the Kit instructions.
S2, conversion and purification of plasma free DNA bisulfite
10ng of plasma free DNA was transformed using EZ DNA Methylation-Gold Kit (zymo Research D5005 s) according to the Kit instructions, and the specific implementation steps were as follows:
1) Preparing CT Conversion Reagent Conversion solution according to the instruction, and preparing the solution for use at the moment;
2) Taking 20 mu L of untransformed DNA sample (the sample volume is less than 20 mu L, adding water for complementing), adding 130 mu L of CT Conversion Reagent Conversion solution, shaking to fully mix the solution, and centrifuging for a short time to collect the reaction solution to the bottom of the tube;
3) Placing the PCR tube in a PCR instrument, and carrying out the following reactions:
98℃ 10min
64℃ 2.5h
keeping at 4 ℃;
4) Adding 600 mu L M-Binding Buffer into a collecting column, adding a PCR product into the collecting column with the M-Binding Buffer, covering a cover, reversing and uniformly mixing for several times, centrifuging for 30s at 10000g, and discarding a filtrate;
5) Adding 100 mu L M-Wash Buffer, centrifuging for 30s at 10000g, and discarding filtrate;
6) Adding 200 μ L M-Desulphosphorylation Buffer, standing at room temperature for 15-20min, centrifuging at 10000g for 30s, and discarding the filtrate;
7) Adding 200 mu L M-Wash Buffer, centrifuging for 30s at 10000g, discarding the filtrate, and repeating the step once again;
8) Place the collection tube into a new 1.5mL centrifuge tube and add 9. Mu.L ddH 2 And O,10000g of the DNA sample is centrifuged for 30s, and the converted DNA sample is stored at the temperature of minus 20 ℃.
S3, first round multiple PCR amplification of DNA after transformation
Mixing 55 pairs of primers of the first round of multiplex PCR according to a preset proportion, and carrying out the first round of multiplex PCR amplification by taking the converted plasma free DNA as a template. The amplification system is shown in Table 2.
TABLE 2 first round multiplex PCR amplification System
Reagent Volume (μ L)
2×Taq Master Mix 10
Primer mixture (10. Mu.M each) 3
Form panel 7
Total volume 20
The PCR amplification procedure is shown in Table 3.
TABLE 3 first round multiplex PCR amplification procedure
Figure BDA0002516825220000181
S4, purifying the first round of multiple PCR amplification products
And (3) purifying the first round of multiplex PCR amplification products by using AMPure XP (Beckman Agencourt) magnetic beads, wherein the specific implementation steps are as follows:
1) Taking out AMPure XP magnetic beads from 2-8 ℃ 30min in advance, and standing to balance the temperature to room temperature;
2) Adding 20 mu L (1.2 x) of AMPure XP magnetic beads into the first round PCR product, blowing and beating for 10 times by using a pipette gun, fully and uniformly mixing, and incubating for 5min at room temperature to enable DNA to be combined on the magnetic beads;
3) The centrifuge tube was held on a magnetic stand and after the solution cleared (about 5 min), the supernatant was carefully removed;
4) The tube was held in a magnetic rack, 200. Mu.L of freshly prepared 80% ethanol solution was added to rinse the beads, incubated at room temperature for 30s, and the supernatant carefully removed;
5) Repeating the step 4) once;
6) Keeping the centrifugal tube on a magnetic rack, and opening the cover to dry for about 5-10min at room temperature;
7) The centrifuge tube was removed from the magnetic stand and 10. Mu.L ddH was added 2 And O, blowing and beating for 10 times by using a pipette gun, fully and uniformly mixing, standing for 5min at room temperature, placing on a magnetic frame, after the solution is clarified (about 5 min), carefully sucking 9 mu L of supernatant into a new sterilized PCR tube, and storing at-20 ℃.
S5, second round of multiplex PCR amplification
And performing second round multiplex PCR amplification by using second round multiplex PCR primers with different labels and taking the purified products of the first round PCR as templates. The amplification system is shown in Table 4.
TABLE 4 second round multiplex PCR amplification System
Reagent Volume (mu L)
2×Taq Master Mix 10
Primer mixture (10. Mu.M each) 1
Form panel 9
Total volume 20
The PCR amplification procedure is shown in Table 5.
TABLE 5 second round multiplex PCR amplification procedure
Figure BDA0002516825220000191
S6, purifying products of second round multiple PCR amplification
And (3) purifying the second round of multiplex PCR amplification products by using AMPure XP (Beckman Agencourt) magnetic beads, wherein the specific implementation steps are as follows:
1) Taking out AMPure XP magnetic beads from 2-8 ℃ in advance for 30min, and standing to balance the temperature to room temperature;
2) Adding water to the second round PCR product to complement to a system of 50 μ L, mixing well, adding 42.5 μ L (0.85X) of AMPure XP magnetic beads, and blowing and beating for 10 times by using a pipette gun to mix well;
3) Incubating at room temperature for 5min;
4) Placing the centrifuge tube on a magnetic rack, after the solution is clarified (about 5 min), carefully transferring the supernatant to a new sterilized PCR tube, and discarding the magnetic beads;
5) Shaking and mixing AMPure XP magnetic beads uniformly, sucking 5 mu L (0.1 x) of AMPure XP magnetic beads into supernatant, gently blowing and beating by using a pipette gun for 10 times, and mixing uniformly, and incubating for 5min at room temperature;
6) The centrifuge tube was held on a magnetic stand and after the solution cleared (about 5 min), the supernatant was carefully removed;
7) The tube was held in a magnetic rack, 200. Mu.L of freshly prepared 80% ethanol solution was added to rinse the beads, incubated at room temperature for 30s, and the supernatant carefully removed;
8) Repeating the step 7) once;
9) Keeping the centrifuge tube on a magnetic rack, and drying for about 5-10min at room temperature under a cover;
10 The centrifuge tube was removed from the magnetic rack, and 12.5. Mu.L ddH was added 2 And O, lightly blowing and fully mixing by using a pipette gun, standing at room temperature for 5min, placing on a magnetic frame, carefully sucking 10 mu L of supernatant into a new sterilized PCR tube after the solution is clarified (about 5 min), and storing at the temperature of minus 20 ℃.
S7, quality detection and sequencing
1) Measuring the concentration of the library by using a Qubit fluorescence quantifier;
2) Quality control of the library was performed using an Agilent 2100 bioanalyzer;
3) The sample library was sequenced using a Nextseq 500 sequencer.
S8, experimental results and data analysis
And performing data quality control on the offline data by using fastp, removing sequencing data with low sequencing quality, and splitting the offline data according to indexA and indexB. The off-line data for each sample was then aligned with the transformed human genome sequence hg19 or hg38 in the self-created database using Bowtie 2. Site information in the fixed site sequence was used to assess methylation levels at the site, and finally integrated to generate assay results, as shown in table 6.
TABLE 6 results of methylation level measurements
Figure BDA0002516825220000201
Figure BDA0002516825220000211
As shown in Table 6, the 55-site methylation level and the methylation levels of normal and lung cancer patients are correlated and compared, and then the sample is comprehensively evaluated through a self-built model, so that the lung cancer risk of the sample is predicted.
In conclusion, the method for detecting the methylation of the free DNA in the blood plasma simultaneously detects 55 methylation sites of the free DNA in the blood plasma, has the advantages of strong specificity of multiple PCR primers, high amplification efficiency, low sample loss and convenient and quick operation, greatly saves the detection cost while improving the detection efficiency, and has auxiliary screening and prompting functions on early lung cancer diagnosis by matching with a high-throughput sequencing technology.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Sequence listing
<110> Wuhan Aiji Baike Biotech Co., ltd
<120> multiple PCR primer system for detecting free DNA methylation of lung cancer plasma, detection method and application
<130> 2020-5-15
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<210> 76
<211> 53
<212> DNA
<213> (Artificial sequence)
<400> 76
tcgtcggcag cgtcagatgt gtataagaga cagggaggtg ttatggttgt gat 53
<210> 77
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 77
gtctcgtggg ctcggagatg tgtataagag acagcccact ccaaccattt aact 54
<210> 78
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 78
tcgtcggcag cgtcagatgt gtataagaga caggggatag ggtatagggt atgt 54
<210> 79
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 79
gtctcgtggg ctcggagatg tgtataagag acaggttata ggtgtgttgg gtga 54
<210> 80
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 80
tcgtcggcag cgtcagatgt gtataagaga cagccccaac ctacttacta aact 54
<210> 81
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 81
gtctcgtggg ctcggagatg tgtataagag acagactctt aacttctcac caat 54
<210> 82
<211> 56
<212> DNA
<213> (Artificial sequence)
<400> 82
tcgtcggcag cgtcagatgt gtataagaga cagggtattt tggttttatg tgatga 56
<210> 83
<211> 55
<212> DNA
<213> (Artificial sequence)
<400> 83
gtctcgtggg ctcggagatg tgtataagag acaggggttg ggagttaatg agtat 55
<210> 84
<211> 55
<212> DNA
<213> (Artificial sequence)
<400> 84
tcgtcggcag cgtcagatgt gtataagaga cagccccaaa accaaatcac taatt 55
<210> 85
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 85
gtctcgtggg ctcggagatg tgtataagag acagggaaag taggtaatgg gagg 54
<210> 86
<211> 53
<212> DNA
<213> (Artificial sequence)
<400> 86
tcgtcggcag cgtcagatgt gtataagaga cagaccaact ctcccaacaa tta 53
<210> 87
<211> 59
<212> DNA
<213> (Artificial sequence)
<400> 87
gtctcgtggg ctcggagatg tgtataagag acagaaacca ttatataaaa atactacca 59
<210> 88
<211> 55
<212> DNA
<213> (Artificial sequence)
<400> 88
tcgtcggcag cgtcagatgt gtataagaga cagaaagaga tttgagttgt agggt 55
<210> 89
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 89
gtctcgtggg ctcggagatg tgtataagag acagcaccaa cacccactct ataa 54
<210> 90
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 90
tcgtcggcag cgtcagatgt gtataagaga cagtttgggg agaatgagaa ttgt 54
<210> 91
<211> 56
<212> DNA
<213> (Artificial sequence)
<400> 91
gtctcgtggg ctcggagatg tgtataagag acagagtgag ggtttatgtt tgtata 56
<210> 92
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 92
tcgtcggcag cgtcagatgt gtataagaga caggcttccc tcacctaaaa taca 54
<210> 93
<211> 55
<212> DNA
<213> (Artificial sequence)
<400> 93
gtctcgtggg ctcggagatg tgtataagag acagtttaag taggggtgtg gtaga 55
<210> 94
<211> 53
<212> DNA
<213> (Artificial sequence)
<400> 94
tcgtcggcag cgtcagatgt gtataagaga caggctaccc tatcaacaac aca 53
<210> 95
<211> 55
<212> DNA
<213> (Artificial sequence)
<400> 95
gtctcgtggg ctcggagatg tgtataagag acagtccaaa acccattcct attca 55
<210> 96
<211> 53
<212> DNA
<213> (Artificial sequence)
<400> 96
tcgtcggcag cgtcagatgt gtataagaga cagaatatgt agagggtagg tgg 53
<210> 97
<211> 55
<212> DNA
<213> (Artificial sequence)
<400> 97
gtctcgtggg ctcggagatg tgtataagag acagggtgga gttgtttaaa aggtt 55
<210> 98
<211> 51
<212> DNA
<213> (Artificial sequence)
<400> 98
tcgtcggcag cgtcagatgt gtataagaga cagccaccct cccaatctta c 51
<210> 99
<211> 56
<212> DNA
<213> (Artificial sequence)
<400> 99
gtctcgtggg ctcggagatg tgtataagag acagcaaaac ttcaacaaac attcca 56
<210> 100
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 100
tcgtcggcag cgtcagatgt gtataagaga caggggggaa tttttgaatt attt 54
<210> 101
<211> 58
<212> DNA
<213> (Artificial sequence)
<400> 101
gtctcgtggg ctcggagatg tgtataagag acagtagtgt atttaagaaa tgttgtat 58
<210> 102
<211> 53
<212> DNA
<213> (Artificial sequence)
<400> 102
tcgtcggcag cgtcagatgt gtataagaga cagaaaatca acccaacacc aac 53
<210> 103
<211> 57
<212> DNA
<213> (Artificial sequence)
<400> 103
gtctcgtggg ctcggagatg tgtataagag acagacaaac acaataaaac ctaaatt 57
<210> 104
<211> 55
<212> DNA
<213> (Artificial sequence)
<400> 104
tcgtcggcag cgtcagatgt gtataagaga cagggttgta tttgtaagtt gggag 55
<210> 105
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 105
gtctcgtggg ctcggagatg tgtataagag acagccacat caccctctat tacc 54
<210> 106
<211> 55
<212> DNA
<213> (Artificial sequence)
<400> 106
tcgtcggcag cgtcagatgt gtataagaga cagtgtggat gtttaaggtt aaggt 55
<210> 107
<211> 56
<212> DNA
<213> (Artificial sequence)
<400> 107
gtctcgtggg ctcggagatg tgtataagag acagtgagtt tatagtgagg gtgaaa 56
<210> 108
<211> 53
<212> DNA
<213> (Artificial sequence)
<400> 108
tcgtcggcag cgtcagatgt gtataagaga cagaatcaca acttcctctt cct 53
<210> 109
<211> 54
<212> DNA
<213> (Artificial sequence)
<400> 109
gtctcgtggg ctcggagatg tgtataagag acagacccac atccacccta aata 54
<210> 110
<211> 58
<212> DNA
<213> (Artificial sequence)
<400> 110
tcgtcggcag cgtcagatgt gtataagaga cagtggtata gtttagagtt tagtggta 58
<210> 111
<211> 39
<212> DNA
<213> (Artificial sequence)
<400> 111
caagcagaag acggcatacg agatgtctcg tgggctcgg 39
<210> 112
<211> 43
<212> DNA
<213> (Artificial sequence)
<400> 112
aatgatacgg cgaccaccga gatctacact cgtcggcagc gtc 43
<210> 113
<211> 47
<212> DNA
<213> (Artificial sequence)
<400> 113
caagcagaag acggcatacg agatnnnnnn nngtctcgtg ggctcgg 47
<210> 114
<211> 51
<212> DNA
<213> (Artificial sequence)
<400> 114
aatgatacgg cgaccaccga gatctacacn nnnnnnntcg tcggcagcgt c 51

Claims (4)

1. A multiplex PCR primer system, comprising: the primer set comprises a first round of multiplex PCR primer set, wherein the first round of multiplex PCR primer set comprises 55 pairs of PCR primer pairs, and the sequences of the 55 pairs of PCR primer pairs are respectively shown as SEQ ID NO. 1-SEQ ID NO.110 in a sequence table; the primer sequence of the second round of multiplex PCR is shown as SEQ ID NO. 113-SEQ ID NO.114 in the sequence table.
2. A kit, characterized in that: comprising the multiplex PCR primer system of claim 1.
3. The kit of claim 2, wherein: also comprises a DNA extraction reagent, a bisulfite conversion reagent, a PCR amplification reagent and a DNA purification reagent.
4. The kit of claim 3, wherein: the DNA purification reagent is a magnetic bead purification reagent.
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CN114540497B (en) * 2022-02-25 2024-02-27 博尔诚（北京）科技有限公司 Marker for bladder cancer screening, probe composition and application thereof
CN114736968B (en) * 2022-06-13 2022-09-27 南京世和医疗器械有限公司 Application of plasma free DNA methylation marker in lung cancer early screening and lung cancer early screening device

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Denomination of invention: A multiplex PCR primer system, detection method, and application for detecting plasma free DNA methylation in lung cancer

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