CN113637759A - Composition and kit for early screening of lung cancer and detection method of early ctDNA methylation of lung cancer - Google Patents
Composition and kit for early screening of lung cancer and detection method of early ctDNA methylation of lung cancer Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a composition and a kit for early screening of lung cancer and a detection method for early ctDNA methylation of lung cancer. The composition for early screening of lung cancer provided by the invention can realize early screening of lung cancer by evaluating the methylation variation condition of ctDNA, and has the characteristics of high sensitivity and strong specificity.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a composition and a kit for early screening of lung cancer and a detection method for early ctDNA methylation of lung cancer.
Background
The lung cancer is a disease good in occult, clinical symptoms are often shown only when the disease develops to the advanced stage, 70-80% of lung cancer patients are already at the middle and advanced stages when the lung cancer symptoms are diagnosed, cancer cells are diffused, the best curing time is missed, and the five-year survival rate is low. For early-stage lung cancer patients, the survival rate and the survival quality of the patients can be greatly improved by 5 years and more through timely treatment. Early diagnosis of lung cancer and effective screening are therefore of paramount importance. Primary bronchogenic carcinoma (lung cancer for short) is one of the most common malignant tumors in the world at present, and the 'third national resident death cause survey' published by 2008 in 4 months of the Ministry of health of China shows that the incidence rate of lung cancer of China is increased greatly in the last 30 years, and the lung cancer is replaced to become the leading cause of death of the malignant tumors.
In recent years, although medical technology is continuously developed, the 5-year survival rate of lung cancer patients is not obviously improved and only improved to 10-15%, and one important reason of low survival rate of lung cancer is that the health consciousness of individuals is insufficient, the abnormal phenomenon of the individuals is not paid enough attention to, and the patients do not seek medical advice in time, so that the delayed diagnosis of the lung cancer is caused. Therefore, health knowledge publicity is enhanced, screening of the individual on the lung cancer risk factors is vigorously carried out, and the method has extremely important significance for reducing the incidence rate of lung cancer, improving the early diagnosis rate and improving the prognosis of patients. Because lung cancer often lacks specific expression in early stage, even some patients get ill with non-respiratory symptoms, resulting in missed diagnosis and misdiagnosis. Therefore, most patients have advanced at the time of visit. Therefore, early diagnosis and early treatment are of great significance in improving the prognosis and reducing the mortality of lung cancer patients.
Currently, lung cancer is mainly detected by imaging, tissue biopsy, carcinoembryonic antigen (CEA), and the like. However, imaging is subject to operator experience and is equipment-dependent, expensive, difficult to ensure accuracy, and difficult to apply widely and routinely, especially in situations where medical resources are limited. Tissue biopsy is the current gold standard for clinically diagnosing lung cancer, but the tissue biopsy has great limitations, such as difficulty in surgical sampling, or inconvenience in puncturing certain cancer parts, and certain clinical risks are brought by puncturing itself, and great pain is brought to patients by repeated puncturing screening. Currently, the most widely used serological detection is the detection of carcinoembryonic antigen (CEA), but the sensitivity and specificity of CEA to early lung cancer are not high. Therefore, the search for new lung cancer markers, especially early warning monitoring and early diagnosis markers, has very important significance for improving the diagnosis rate of early lung cancer, realizing early intervention treatment and reducing the mortality rate of lung cancer.
Disclosure of Invention
The invention mainly aims to provide a composition and a kit mainly used for early screening of lung cancer and a detection method of early ctDNA methylation of lung cancer, and aims to provide a marker capable of carrying out early screening of lung cancer.
In order to achieve the above objects, the present invention provides a composition for early screening of lung cancer, comprising a first specific primer and probe, a second specific primer and probe, a third specific primer and probe, a fourth specific primer and probe, a fifth specific primer and probe, a sixth specific primer and probe, a seventh specific primer and probe, an eighth specific primer and probe, and an internal reference primer and probe; wherein the content of the first and second substances,
the sequences of the first specific primer and probe are as follows:
a forward primer: ATTGTGTCTGCCTAGGAAAAGGGTGTG the flow of the air in the air conditioner,
reverse primer: AAAAGTCATTCTCGGCGGAGTGTTC the flow of the air in the air conditioner,
and (3) probe: FAM-CTCTCCGCAAGTGCCGCTCCT-BHQ 1;
the sequences of the second specific primer and probe are as follows:
a forward primer: TCGGCCCTGCCATGCCTCACAT the flow of the air in the air conditioner,
reverse primer: AGTGCCAGGGCAGGAAAGCCACAG the flow of the air in the air conditioner,
and (3) probe: FAM-CCCCAGGATGCCGCGGTGGGAACT-BHQ 1;
the sequences of the third specific primer and the probe are as follows:
a forward primer: AGAGAATCTCACCACAAATGAAA the flow of the air in the air conditioner,
reverse primer: TATGCCCTGCGATAGAAACAC the flow of the air in the air conditioner,
and (3) probe: FAM-ACCTCTCCCCTTTCACGTAGT-BHQ 1;
the sequences of the fourth specific primer and probe are as follows:
a forward primer: GGCAGGAAGTGTGAGGTGTTGAGCAGCTA the flow of the air in the air conditioner,
reverse primer: TCCGACTTCCGCTCCTCCTCACCACAA the flow of the air in the air conditioner,
and (3) probe: FAM-CCTCCTGCTCCCCGCCGCCTCC-BHQ 1;
the sequences of the fifth specific primers and probes are as follows:
a forward primer: ACTTCTGCGAGCCCAACCC the flow of the air in the air conditioner,
reverse primer: ACAGCAGGTCGCAGCCGTC the flow of the air in the air conditioner,
and (3) probe: FAM-CTGCAACGTCAGCTCGCACGGCAT-BHQ 1;
the sequences of the sixth specific primer and probe are as follows:
a forward primer: TGGCATAAAGTCTACTTGAGGGA the flow of the air in the air conditioner,
reverse primer: CGTGCACTCTGTTTAGTGTCT the flow of the air in the air conditioner,
and (3) probe: FAM-CAACCCGGCACGGTTACGTT-BHQ 1;
the sequences of the seventh specific primer and probe are as follows:
a forward primer: TTTCTCGGTATTTCGTTGTCAAGGCCAC the flow of the air in the air conditioner,
reverse primer: GAGCGGCCGGGAGAAACCCA the flow of the air in the air conditioner,
and (3) probe: FAM-CACCCCGGGATCCGACGGCAAGG-BHQ 1;
the sequences of the eighth specific primer and probe are as follows:
a forward primer: AGGCGCTCCTCGCTAGGGCTG the flow of the air in the air conditioner,
reverse primer: ACGGCATTCCTACATGAGATGGGGGTC the flow of the air in the air conditioner,
and (3) probe: FAM-CCACCGCACAGGAAACGGGGCAGG-BHQ 1;
the sequences of the internal reference primers and the probes are as follows:
a forward primer: GGTGCCAGATTTTCTCCATGTCGTC the flow of the air in the air conditioner,
reverse primer: ACGAGGCCCAGAGCAAGAGA the flow of the air in the air conditioner,
and (3) probe: VIC-TACCCCATCGAGCACGGCATCGTC-BHQ 1.
Furthermore, the invention also provides a kit for early screening of lung cancer, which comprises a PCR reaction solution, a methylation sensitive endonuclease and the composition for early screening of lung cancer.
Alternatively, the methylation sensitive endonuclease comprises a HinP1I methylation sensitive endonuclease, an HpaII methylation sensitive endonuclease, an AciI methylation sensitive endonuclease, and an HpyCH4IV methylation sensitive endonuclease.
Furthermore, the invention also provides a method for detecting the methylation of ctDNA in the early stage of lung cancer, which comprises the following steps:
providing a kit for early screening of lung cancer as described above;
extracting free DNA from a test plasma sample;
digesting the free DNA by using the methylation sensitive endonuclease to obtain a digestion product;
performing multiple PCR (polymerase chain reaction) pre-amplification on the digestion product by using the specific primer and the internal reference primer to obtain a pre-amplification product;
carrying out qPCR amplification on the pre-amplification product by using the specific primer and probe, the internal reference primer and probe to obtain an amplification product;
and obtaining the difference value delta Ct of the Ct values of the internal reference primer and the specific primer in the amplification product, and judging whether the tested plasma sample has methylation variation or not according to the difference value delta Ct.
Optionally, the step of digesting the free DNA with the methylation sensitive endonuclease to obtain a digested product comprises:
and (3) mixing the methylation sensitive endonuclease with the free DNA, incubating for 16h at 37 ℃, and inactivating the enzyme for 20min at 80 ℃ to obtain a digestion product.
Optionally, the step of obtaining a difference Δ Ct between Ct values of the internal reference primer and the specific primer in the amplification product, and determining whether the subject plasma sample has methylation variation according to the difference Δ Ct includes:
and obtaining the difference value delta Ct of the Ct values of the internal reference primer and the specific primer in the amplification product, when the difference value delta Ct is more than 1, determining that the tested plasma sample has methylation variation, and when the difference value delta Ct is less than or equal to 1, determining that the tested plasma sample has no methylation variation.
In the technical scheme provided by the invention, the composition for early screening of lung cancer comprises 8 pairs of specific primers and probes and 1 pair of internal reference primers and probes, wherein the primers and probes are used for determining methylation characteristics of ctDNA (deoxyribonucleic acid) in the early stage of Chinese non-small cell lung cancer by deeply excavating CGAC (CGAC) projects and public database resources, screening to obtain 8 site sequences capable of distinguishing early lung cancer risks, and then designing the specific primers, the internal reference primers and the probe sequences according to the site sequences in the last time, so that early screening of the lung cancer can be realized by evaluating the methylation variation condition of the ctDNA, and the composition has the characteristics of high sensitivity and strong specificity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows 8 pairs of methylation specific primers screened according to the present invention;
FIG. 2 is a diagram showing the result of obtaining the difference value Δ Ct in the detection method for early ctDNA methylation of lung cancer provided by the present invention;
FIG. 3 is a graph showing the results of methylation detection limit tests performed by the detection method of the present invention;
FIG. 4 is a diagram showing the results of the sensitivity and specificity tests performed by the detection method of the present invention.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B" including either A or B or both A and B. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Currently, lung cancer is mainly detected by imaging, tissue biopsy, carcinoembryonic antigen (CEA), and the like. However, imaging is subject to operator experience and is equipment-dependent, expensive, difficult to ensure accuracy, and difficult to apply widely and routinely, especially in situations where medical resources are limited. Tissue biopsy is the current gold standard for clinically diagnosing lung cancer, but the tissue biopsy has great limitations, such as difficulty in surgical sampling, or inconvenience in puncturing certain cancer parts, and certain clinical risks are brought by puncturing itself, and great pain is brought to patients by repeated puncturing screening. Currently, the most widely used serological detection is the detection of carcinoembryonic antigen (CEA), but the sensitivity and specificity of CEA to early lung cancer are not high. Therefore, the search for new lung cancer markers, especially early warning monitoring and early diagnosis markers, has very important significance for improving the diagnosis rate of early lung cancer, realizing early intervention treatment and reducing the mortality rate of lung cancer.
In order to realize early screening and diagnosis of lung cancer, the invention provides a composition for early screening of lung cancer, which comprises 8 pairs of specific primers, 1 pair of internal reference primers and a probe, wherein the specific primers comprise FAM signals and the internal reference primers comprise VIC signals. Specifically, the composition comprises a first specific primer and probe, a second specific primer and probe, a third specific primer and probe, a fourth specific primer and probe, a fifth specific primer and probe, a sixth specific primer and probe, a seventh specific primer and probe, an eighth specific primer and probe, and an internal reference primer and probe; wherein the sequences of the first specific primer and the probe are as follows: the sequence of the forward primer is shown as SEQ ID NO: 1 is as follows: ATTGTGTCTGCCTAGGAAAAGGGTGTG, and the reverse primer sequence is shown in SEQ ID NO: 2 is as follows: AAAAGTCATTCTCGGCGGAGTGTTC, and the probe sequence is shown in SEQ ID NO: 3, is as follows: FAM-CTCTCCGCAAGTGCCGCTCCT-BHQ 1; the sequences of the second specific primer and probe are as follows: the sequence of the forward primer is shown as SEQ ID NO: 4, is as follows: TCGGCCCTGCCATGCCTCACAT, and the reverse primer sequence is shown in SEQ ID NO: 5 is as follows: AGTGCCAGGGCAGGAAAGCCACAG, and the probe sequence is shown in SEQ ID NO: 6 is as follows: FAM-CCCCAGGATGCCGCGGTGGGAACT-BHQ 1; the sequences of the third specific primer and the probe are as follows: the sequence of the forward primer is shown as SEQ ID NO: shown in 7, is: AGAGAATCTCACCACAAATGAAA, and the reverse primer sequence is shown in SEQ ID NO: shown in 8, is: TATGCCCTGCGATAGAAACAC, and the probe sequence is shown in SEQ ID NO: shown in 9, is: FAM-ACCTCTCCCCTTTCACGTAGT-BHQ 1; the sequences of the fourth specific primer and probe are as follows: the sequence of the forward primer is shown as SEQ ID NO: shown in 10, is: GGCAGGAAGTGTGAGGTGTTGAGCAGCTA, and the reverse primer sequence is shown in SEQ ID NO: 11, is: TCCGACTTCCGCTCCTCCTCACCACAA, and the probe sequence is shown in SEQ ID NO: 12, is: FAM-CCTCCTGCTCCCCGCCGCCTCC-BHQ 1; the sequences of the fifth specific primers and probes are as follows: the sequence of the forward primer is shown as SEQ ID NO: 13, is: ACTTCTGCGAGCCCAACCC, and the reverse primer sequence is shown in SEQ ID NO: shown at 14, is: ACAGCAGGTCGCAGCCGTC, and the probe sequence is shown in SEQ ID NO: 15, is: FAM-CTGCAACGTCAGCTCGCACGGCAT-BHQ 1; the sequences of the sixth specific primer and probe are as follows: the sequence of the forward primer is shown as SEQ ID NO: 16, is: TGGCATAAAGTCTACTTGAGGGA, and the reverse primer sequence is shown in SEQ ID NO: 17, is: CGTGCACTCTGTTTAGTGTCT, and the probe sequence is shown in SEQ ID NO: 18, is: FAM-CAACCCGGCACGGTTACGTT-BHQ 1; the sequences of the seventh specific primer and probe are as follows: the sequence of the forward primer is shown as SEQ ID NO: 19, is: TTTCTCGGTATTTCGTTGTCAAGGCCAC, and the reverse primer sequence is shown in SEQ ID NO: 20 shows that: GAGCGGCCGGGAGAAACCCA, and the probe sequence is shown in SEQ ID NO: 21, is: FAM-CACCCCGGGATCCGACGGCAAGG-BHQ 1; the sequences of the eighth specific primer and probe are as follows: the sequence of the forward primer is shown as SEQ ID NO: 22, is: AGGCGCTCCTCGCTAGGGCTG, and the reverse primer sequence is shown in SEQ ID NO: 23, is: ACGGCATTCCTACATGAGATGGGGGTC, and the probe sequence is shown in SEQ ID NO: shown at 24, is: FAM-CCACCGCACAGGAAACGGGGCAGG-BHQ 1; the sequences of the internal reference primers and the probes are as follows: the sequence of the forward primer is shown as SEQ ID NO: shown at 25, is: GGTGCCAGATTTTCTCCATGTCGTC, and the reverse primer sequence is shown in SEQ ID NO: 26, is: ACGAGGCCCAGAGCAAGAGA, and the probe sequence is shown in SEQ ID NO: 27, is: VIC-TACCCCATCGAGCACGGCATCGTC-BHQ 1.
In the technical scheme provided by the invention, the composition for early screening of lung cancer comprises 8 pairs of specific primers and probes and 1 pair of internal reference primers and probes, wherein the primers and probes are used for determining methylation characteristics of ctDNA (deoxyribonucleic acid) in early stage of Chinese non-small cell lung cancer by deeply excavating CGAC (CGAC) projects and public database resources, screening to obtain 8 site sequences capable of distinguishing early lung cancer risks, then further screening hypomethylation targets in plasma of healthy people and hypermethylation targets in plasma of cancer patients according to the specific primers, the internal reference primers and the probe sequences which are designed according to the site sequences, and finally screening to obtain 8 pairs of methylation specific primers shown in figure 1, wherein the specific sequences are as described above. The composition for early screening of lung cancer provided by the invention can realize early screening of lung cancer by evaluating the methylation variation condition of ctDNA, and has the characteristics of high sensitivity and strong specificity.
The composition for early screening of lung cancer provided by the invention is based on the principle of ctDNA methylation, and is detected by a PCR amplification method. Further, the invention also provides a kit for early screening of lung cancer, which comprises a PCR reaction solution, a methylation sensitive endonuclease and the like in addition to the composition for early screening of lung cancer provided by the invention. According to the kit for early screening of lung cancer, provided by the invention, the characteristic that methylation sensitive endonuclease can not cut methylation sites is utilized, the methylation sensitive endonuclease is utilized to degrade cfDNA from a non-tumor source, the ctDNA from a tumor source is specifically amplified, and the early screening of the lung cancer is effectively realized.
Specifically, in a specific embodiment of the kit for early screening of lung cancer provided by the present invention, the methylation sensitive endonuclease includes HinP1I methylation sensitive endonuclease, HpaII methylation sensitive endonuclease, AciI methylation sensitive endonuclease and HpyCH4IV methylation sensitive endonuclease. The four methylation sensitive endonucleases are utilized to degrade a tested sample, so that the early screening of the lung cancer can be effectively realized, the detection sensitivity and specificity can be greatly improved, and the method has the advantages of short detection period, low cost, simplicity in operation and easiness in popularization.
When the methylation sensitive endonuclease is used for degrading a test sample, the composition of an enzyme digestion reaction solution is as follows: 1 Xenzyme digestion buffer solution, 2-10U of various methylation sensitive endonucleases and 20ng cfDNA. In addition, the present invention is not limited to the specific composition of the PCR reaction solution, and any PCR reaction solution may be used as long as it can perform PCR amplification on a DNA sample. In an embodiment of the present invention, the PCR reaction solution includes a pre-amplification PCR reaction solution and a qPCR reaction solution, wherein the pre-amplification PCR reaction solution includes: 1 × PCR master mix, 20 μ L of DNA template after enzyme digestion, 30-300 nM forward and reverse specific primers and 5-30 nM forward and reverse internal reference primers, wherein the qPCR reaction solution comprises: 1 XTaqman qPCR master mix, 1 mu L pre-amplification product, 50-200 nM forward and reverse specific primers, 10-50 nM specific probes, 50-200 nM forward and reverse internal reference primers and 10-50 nM internal reference probes.
In addition, the kit for early screening of lung cancer can also comprise an extraction kit for extracting DNA of a test sample. Specifically, the use flow of the kit for screening lung cancer is as follows: firstly, extracting free DNA from a tested plasma sample, then digesting the extracted free DNA by using the four methylation sensitive endonucleases, carrying out PCR pre-amplification on a digestion product by using the specific primer, then carrying out qPCR amplification by using the specific primer, the internal reference primer and the probe by using a pre-amplified product as a template to obtain a difference value delta Ct of Ct values of amplified internal reference foreign matters and the specific primer, and finally judging whether the tested plasma sample has methylation variation or not according to whether the difference value delta Ct exceeds a threshold value or not so as to judge the risk of lung cancer of a subject.
Furthermore, the invention also provides a method for detecting the methylation of ctDNA in the early stage of lung cancer, which comprises the following steps:
step S10, providing a kit for early screening of lung cancer as described above;
step S20, extracting free DNA from the tested plasma sample;
step S30, digesting the free DNA by using the methylation sensitive endonuclease to obtain a digestion product;
step S40, performing multiple PCR pre-amplification on the digestion product by using the specific primer and the internal reference primer to obtain a pre-amplification product;
step S50, carrying out qPCR amplification on the pre-amplification product by using the specific primer and the probe, the internal reference primer and the probe to obtain an amplification product;
and S60, obtaining a difference value delta Ct of the Ct values of the internal reference primer and the specific primer in the amplification product, and judging whether the tested plasma sample has methylation variation or not according to the difference value delta Ct.
The detection method of the methylation of the ctDNA in the early lung cancer provided by the invention is based on the kit for early lung cancer screening provided by the invention, firstly, the kit provided by the embodiment is provided, the kit comprises the composition for early lung cancer screening, PCR reaction liquid, methylation sensitive endonuclease and the like, and during detection, 2mL of tested plasma is taken firstly, and the Qiagen plasma extraction kit is used for extracting cfDNA. Then, 20ng of cfDNA was incubated with the four methylation sensitive endonucleases HinP1I, HpaII, AciI and HpyCH4IV to inactivate the enzymes, thereby obtaining a digested product. Specifically, the co-incubation temperature is 37 ℃ and the time is 16h, the enzyme inactivation temperature is 80 ℃ and the time is 20min, namely, the co-incubated product is treated at 80 ℃ for 20min to inactivate four methylation sensitive endonucleases in the system; the composition of the enzyme digestion reaction solution is as follows: 1 Xenzyme digestion buffer solution, 2-10U of various methylation sensitive endonucleases and 20ng cfDNA.
Then, taking the digestion product as a template, and carrying out multiple PCR (polymerase chain reaction) by using the configuration system of 8 pairs of specific primers and 1 pair of internal reference primers for 8 cycles to finish pre-amplification; and then 1 mu L of the pre-amplified product is taken as a template, the 8 pairs of specific primers, the 1 pair of internal reference primers and the corresponding Tagman probe are added, and qPCR reaction is carried out in an ABI 7500 fluorescent PCR instrument to complete qPCR amplification. The amplification procedures of the PCR pre-amplification and the qPCR amplification are not limited, as long as the PCR amplification can be performed on the DNA sample. In an embodiment of the present invention, the specific steps of the PCR pre-amplification and the qPCR amplification are shown in table 1 below:
TABLE 1 PCR Pre-amplification and qPCR amplification procedure
FAM and VIC channel signals are collected before the end of each cycle.
And finally, evaluating whether methylation variation occurs in the tested plasma sample according to the difference value delta Ct of the Ct values of the 1 pair of the internal reference primers and the 8 pair of the specific primers, and further evaluating the risk of the lung cancer of the tested subject. Specifically, the evaluation and interpretation is shown in FIG. 2 as: when the difference delta Ct is larger than 1, judging that the tested plasma sample has methylation variation, namely judging that the tested subject is at high risk of suffering from lung cancer, and recommending regular ultrasonic detection; and when the difference delta Ct is less than or equal to 1, judging that the tested plasma sample has no methylation variation, namely judging that the tested plasma sample has low risk of lung cancer.
FIG. 3 shows the results of methylation detection limit tests using fully methylated and fully unmethylated standards to prepare methylated samples at different content ratios and using the detection method of the present invention. As can be seen from FIG. 2, the detection method provided by the present invention can detect elevated changes in methylation levels as low as 0.01%.
FIG. 4 shows the sensitivity and specificity of the detection method provided by the present invention for assessing lung cancer risk by selecting 98 healthy controls and 125 plasma samples of patients with stage I-III lung cancer. As shown by comparison in FIG. 4, the sensitivity of the detection method provided by the invention on the risk assessment of lung cancer reaches 90.40%, and the specificity reaches 93.88%.
According to the detection method for early-stage ctDNA methylation of lung cancer, provided by the invention, the characteristic that methylation sites cannot be cut by methylation sensitive endonuclease is utilized, cfDNA from a non-tumor source is degraded, ctDNA from a tumor source is specifically amplified, and through a mode of evaluating the methylation variation condition of ctDNA, compared with a mode of a traditional serological detection method based on serum protein markers (such as CEA and the like), the sensitivity and specificity of the method provided by the invention are both remarkably improved; compared with the existing method based on ctDNA methylation, which is based on bisulfite conversion, the method provided by the invention has the advantages that massive nucleic acid degradation is caused in the conversion process, bias is caused, and the detection sensitivity is reduced, the detection method provided by the invention does not use bisulfite conversion, the cfDNA in-vitro degradation is avoided, and the detection sensitivity is improved; compared with the existing detection method based on the second-generation sequencing, the detection method provided by the invention has the advantages of short detection period, low cost, simplicity in operation and easiness in popularization, and has the mode of long detection period and high cost which is not beneficial to popularization. In conclusion, the detection method for early ctDNA methylation of lung cancer provided by the invention greatly improves the sensitivity and specificity of detection, wherein the sensitivity reaches 90.40%, and the specificity reaches 93.88%, and the detection method has the advantages of short detection period, low cost, simplicity in operation and easiness in popularization.
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.
SEQUENCE LISTING
<110> Nanjing Zhen Gene science and technology Co., Ltd
<120> composition and kit for early screening of lung cancer and detection of methylation of ctDNA in early lung cancer
Measuring method
<130> 20210830
<160> 27
<170> PatentIn version 3.5
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attgtgtctg cctaggaaaa gggtgtg 27
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aaaagtcatt ctcggcggag tgttc 25
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ctctccgcaa gtgccgctcc t 21
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tcggccctgc catgcctcac at 22
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agtgccaggg caggaaagcc acag 24
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ccccaggatg ccgcggtggg aact 24
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acctctcccc tttcacgtag t 21
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ggcaggaagt gtgaggtgtt gagcagcta 29
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tccgacttcc gctcctcctc accacaa 27
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ctgcaacgtc agctcgcacg gcat 24
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ccaccgcaca ggaaacgggg cagg 24
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Claims (6)
1. A composition for early screening of lung cancer, comprising a first specific primer and probe, a second specific primer and probe, a third specific primer and probe, a fourth specific primer and probe, a fifth specific primer and probe, a sixth specific primer and probe, a seventh specific primer and probe, an eighth specific primer and probe, and an internal reference primer and probe; wherein the content of the first and second substances,
the sequences of the first specific primer and probe are as follows:
a forward primer: ATTGTGTCTGCCTAGGAAAAGGGTGTG the flow of the air in the air conditioner,
reverse primer: AAAAGTCATTCTCGGCGGAGTGTTC the flow of the air in the air conditioner,
and (3) probe: FAM-CTCTCCGCAAGTGCCGCTCCT-BHQ 1;
the sequences of the second specific primer and probe are as follows:
a forward primer: TCGGCCCTGCCATGCCTCACAT the flow of the air in the air conditioner,
reverse primer: AGTGCCAGGGCAGGAAAGCCACAG the flow of the air in the air conditioner,
and (3) probe: FAM-CCCCAGGATGCCGCGGTGGGAACT-BHQ 1;
the sequences of the third specific primer and the probe are as follows:
a forward primer: AGAGAATCTCACCACAAATGAAA the flow of the air in the air conditioner,
reverse primer: TATGCCCTGCGATAGAAACAC the flow of the air in the air conditioner,
and (3) probe: FAM-ACCTCTCCCCTTTCACGTAGT-BHQ 1;
the sequences of the fourth specific primer and probe are as follows:
a forward primer: GGCAGGAAGTGTGAGGTGTTGAGCAGCTA the flow of the air in the air conditioner,
reverse primer: TCCGACTTCCGCTCCTCCTCACCACAA the flow of the air in the air conditioner,
and (3) probe: FAM-CCTCCTGCTCCCCGCCGCCTCC-BHQ 1;
the sequences of the fifth specific primers and probes are as follows:
a forward primer: ACTTCTGCGAGCCCAACCC the flow of the air in the air conditioner,
reverse primer: ACAGCAGGTCGCAGCCGTC the flow of the air in the air conditioner,
and (3) probe: FAM-CTGCAACGTCAGCTCGCACGGCAT-BHQ 1;
the sequences of the sixth specific primer and probe are as follows:
a forward primer: TGGCATAAAGTCTACTTGAGGGA the flow of the air in the air conditioner,
reverse primer: CGTGCACTCTGTTTAGTGTCT the flow of the air in the air conditioner,
and (3) probe: FAM-CAACCCGGCACGGTTACGTT-BHQ 1;
the sequences of the seventh specific primer and probe are as follows:
a forward primer: TTTCTCGGTATTTCGTTGTCAAGGCCAC the flow of the air in the air conditioner,
reverse primer: GAGCGGCCGGGAGAAACCCA the flow of the air in the air conditioner,
and (3) probe: FAM-CACCCCGGGATCCGACGGCAAGG-BHQ 1;
the sequences of the eighth specific primer and probe are as follows:
a forward primer: AGGCGCTCCTCGCTAGGGCTG the flow of the air in the air conditioner,
reverse primer: ACGGCATTCCTACATGAGATGGGGGTC the flow of the air in the air conditioner,
and (3) probe: FAM-CCACCGCACAGGAAACGGGGCAGG-BHQ 1;
the sequences of the internal reference primers and the probes are as follows:
a forward primer: GGTGCCAGATTTTCTCCATGTCGTC the flow of the air in the air conditioner,
reverse primer: ACGAGGCCCAGAGCAAGAGA the flow of the air in the air conditioner,
and (3) probe: VIC-TACCCCATCGAGCACGGCATCGTC-BHQ 1.
2. A kit for early screening of lung cancer, which comprises a PCR reaction solution, a methylation sensitive endonuclease and the composition for early screening of lung cancer as claimed in claim 1.
3. The kit for early screening of lung cancer according to claim 2, wherein the methylation sensitive endonuclease comprises HinP1I methylation sensitive endonuclease, HpaII methylation sensitive endonuclease, AciI methylation sensitive endonuclease and HpyCH4IV methylation sensitive endonuclease.
4. A method for detecting methylation of ctDNA in early stage of lung cancer is characterized by comprising the following steps:
providing a kit for early screening of lung cancer according to claim 2 or 3;
extracting free DNA from a test plasma sample;
digesting the free DNA by using the methylation sensitive endonuclease to obtain a digestion product;
performing multiple PCR (polymerase chain reaction) pre-amplification on the digestion product by using the specific primer and the internal reference primer to obtain a pre-amplification product;
carrying out qPCR amplification on the pre-amplification product by using the specific primer and probe, the internal reference primer and probe to obtain an amplification product;
and obtaining the difference value delta Ct of the Ct values of the internal reference primer and the specific primer in the amplification product, and judging whether the tested plasma sample has methylation variation or not according to the difference value delta Ct.
5. The method for detecting methylation of ctDNA in early stage of lung cancer according to claim 4, wherein the step of digesting the free DNA with the methylation sensitive endonuclease to obtain a digested product comprises:
and (3) mixing the methylation sensitive endonuclease with the free DNA, incubating for 16h at 37 ℃, and inactivating the enzyme for 20min at 80 ℃ to obtain a digestion product.
6. The method for detecting methylation of ctDNA in early lung cancer according to claim 4, wherein the step of obtaining the difference Δ Ct between Ct values of the internal reference primer and the specific primer in the amplification product and determining whether the methylation variation occurs in the plasma sample under test according to the difference Δ Ct comprises:
and obtaining the difference value delta Ct of the Ct values of the internal reference primer and the specific primer in the amplification product, when the difference value delta Ct is more than 1, determining that the tested plasma sample has methylation variation, and when the difference value delta Ct is less than or equal to 1, determining that the tested plasma sample has no methylation variation.
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| US20180274039A1 (en) * | 2017-03-02 | 2018-09-27 | Youhealth Biotech, Limited | Methylation markers for diagnosing hepatocellular carcinoma and lung cancer |
| CN112195245A (en) * | 2020-10-16 | 2021-01-08 | 中国药科大学 | Lung cancer related methylation gene combination in plasma and application thereof |
| CN113249487A (en) * | 2021-06-29 | 2021-08-13 | 北京求臻医学检验实验室有限公司 | Biomarker combination, detection method and kit for early screening of liver cancer |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180274039A1 (en) * | 2017-03-02 | 2018-09-27 | Youhealth Biotech, Limited | Methylation markers for diagnosing hepatocellular carcinoma and lung cancer |
| CN110603329A (en) * | 2017-03-02 | 2019-12-20 | 优美佳肿瘤技术有限公司 | Methylation markers for diagnosis of hepatocellular carcinoma and lung cancer |
| CN112195245A (en) * | 2020-10-16 | 2021-01-08 | 中国药科大学 | Lung cancer related methylation gene combination in plasma and application thereof |
| CN113249487A (en) * | 2021-06-29 | 2021-08-13 | 北京求臻医学检验实验室有限公司 | Biomarker combination, detection method and kit for early screening of liver cancer |
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