CN111534600B

CN111534600B - Esophageal cancer gene methylation detection primer probe combination, kit and application thereof

Info

Publication number: CN111534600B
Application number: CN202010561492.7A
Authority: CN
Inventors: 邵琦; 张艳桥
Original assignee: Anhui Dajian Medical Technology Co ltd; GUANGZHOU TARGENE BIOTECH CO Ltd
Current assignee: Anhui Dajian Medical Technology Co ltd; GUANGZHOU TARGENE BIOTECH CO Ltd
Priority date: 2020-06-18
Filing date: 2020-06-18
Publication date: 2023-07-21
Anticipated expiration: 2040-06-18
Also published as: CN111534600A

Abstract

The invention provides an esophageal cancer gene methylation detection primer probe combination, a kit and application thereof. The esophageal cancer gene methylation detection kit provided by the invention takes cancer diagnosis, early screening and DNA methylation abnormality of a predicted important biological index as detection objects, can realize noninvasive detection by detecting the gene methylation states of esophageal cast-off cells and peripheral blood, can efficiently and sensitively complete PCR amplification of target nucleic acid fragments in a micro-reaction unit by adopting a digital PCR technology, acquires fluorescent signals for statistical analysis, thoroughly gets rid of dependence on a standard curve, directly gives out copy numbers of target sequences, improves the stability of experimental results in batches and among batches, realizes absolute quantification of initial samples, improves the sensitivity of a nucleic acid detection method, effectively reduces the occurrence of false negatives, and further realizes early screening of esophageal cancer.

Description

Esophageal cancer gene methylation detection primer probe combination, kit and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an esophageal cancer gene methylation detection primer probe combination, a kit and application thereof.

Background

Esophageal cancer is a malignant tumor of the digestive tract from the hypopharynx to the esophageal epithelium between the joints of the esophagus and the stomach, is one of the ten common malignant tumors of human beings, and has the sixth death rate in all cancers. China is a high-incidence area of esophageal cancer, and the average death of the disease is about 15 ten thousand people each year. The research shows that the five-year survival rate of early patients with esophageal cancer can reach more than 95%, but the early symptoms of most patients with esophageal cancer are atypical, if progressive dysphagia is usually in middle and late stages, the prognosis of the patients with middle and late stages is poor, the five-year survival rate is lower, and the total survival rate is not more than 15%, so that the establishment of the method for early warning and early screening of esophageal cancer is very important for preventing and treating esophageal cancer, and early discovery, early diagnosis, early treatment and early operation are effective means for preventing and controlling esophageal cancer.

The existing detection and screening methods for esophagus cancer comprise imaging detection technology, tissue biopsy, tumor serum marker detection and the like. The method has the advantages of high equipment cost, strong operation technical requirements and high detection cost; or is invasive and not suitable for screening early cancers; or the sensitivity is very low, and the clinical detection needs cannot be met. Therefore, there is a need to develop a novel sensitive and specific esophageal cancer marker and detection technology, which can improve early esophageal cancer detection rate, improve esophageal cancer treatment effect and reduce esophageal cancer mortality.

Epigenetic is a hot field of recent tumor research, and methylation of DNA, histone modification, chromatin remodeling, non-coding RNA regulation and other epigenetic changes are considered to have close relation with tumor occurrence, wherein the methylation of DNA is the most common epigenetic change, can regulate cell proliferation, apoptosis and differentiation, and the level is closely related with the biological characteristics of the tumor. Various studies have demonstrated that the esophageal cancer disease process is a complex process of multiple genetic variation accumulation involving abnormal methylation of multiple oncogenes and cancer suppressor genes, most of which are hypermethylated for cancer suppressor genes, which often lead to transcriptional silencing of cancer suppressor genes. DNA methylation abnormalities usually occur in early cancer and throughout the course of cancer development and progression, the methylation state of which changes once it is established that it requires prolonged continuous stimulation by the external environment, so that the detection of DNA methylation indicators can be used as important biological indicators for diagnosis, early screening and prognosis of cancer.

The main detection methods for DNA methylation are numerous and can be broadly divided into two categories: whole genome methylation analysis and specific site methylation detection. The whole genome methylation analysis has high detection cost and is often used as a means for screening and finding target genes with high flux. The specific site methylation detection method comprises a restriction enzyme analysis method (COBRA), a methylation specificity PCR Method (MSP), a methylation fluorescence quantification method (Methyllight), a methylation sensitivity high-resolution melting curve analysis method and the like which are combined with sodium bisulphite, the restriction enzyme analysis method can only obtain the methylation condition of a specific enzyme cutting site, the methylation specificity PCR method is complicated in operation and easy to cause sample pollution based on common PCR and electrophoresis analysis, the methylation sensitivity high-resolution melting curve analysis method has high requirements on instruments, a fluorescent quantitative PCR instrument with a High Resolution Melting (HRM) module is needed, the methylation fluorescence quantification method has high flux and sensitivity based on the fluorescent quantitative PCR instrument, electrophoresis, hybridization and other operations are not needed, pollution and operation errors are reduced, the methylation fluorescence quantification method based on the fluorescent quantitative PCR is widely applied to DNA methylation detection, but an additional preparation standard curve is needed to quantify sample nucleic acid, meanwhile, the sensitivity is insufficient when detecting a low-concentration nucleic acid sample, false negative is easy to cause early diagnosis of cancer, and early diagnosis of cancer is easy to cause, and the DNA methylation detection challenges are brought. Compared with fluorescent quantitative PCR, the digital PCR has higher detection sensitivity and accuracy, the digital PCR method distributes the nucleic acid sample into the micro-reaction unit by diluting the nucleic acid sample and following the poisson distribution rule, completes the PCR amplification of the target nucleic acid fragment in the micro-reaction unit efficiently and sensitively, acquires fluorescent signals for statistical analysis, thoroughly gets rid of the dependence on a standard curve and directly gives out the copy number of the target sequence, improves the stability of experimental results in batches and between batches, realizes the absolute quantification of the initial sample, improves the sensitivity of the nucleic acid detection method, and effectively reduces the occurrence of false negative.

In view of the above, the invention establishes a digital PCR-based methylation detection method of esophageal cancer genes by screening esophageal cancer related methylation genes, and is expected to obtain detection reagents with higher sensitivity, specificity and accuracy so as to realize early screening and diagnosis of esophageal cancer.

Disclosure of Invention

In order to achieve the purpose, the invention provides a composition for detecting esophageal cancer, a kit and application thereof, 2 methylation detection sites of DACH1, ZNF132 and the like are screened out through a TCGA database, and the esophageal cancer detection kit with higher sensitivity is obtained by establishing digital PCR-based esophageal cancer gene methylation detection, so that early screening and diagnosis of esophageal cancer are realized, and early diagnosis and early treatment of esophageal cancer are facilitated.

In a first aspect, the invention provides an esophageal cancer gene methylation detection site, wherein the gene methylation detection site comprises DACH1 and/or ZNF132.

The second aspect of the invention provides a PCR primer probe combination for detecting esophageal cancer gene methylation, which comprises the following nucleic acid sequence combination shown in 1) and/or 2):

1) The PCR primer and the probe for DACH1 methylation detection comprise one of a primer probe combination 1 and a primer probe combination 3, wherein the primer probe combination 1 comprises an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2 and a fluorescent probe shown as SEQ ID NO.3, and the primer probe combination 3 comprises an upstream primer shown as SEQ ID NO.7, a downstream primer shown as SEQ ID NO.8 and a fluorescent probe shown as SEQ ID NO. 9;

2) The PCR primer and the probe for ZNF132 methylation detection comprise one of a primer probe combination 5 and a primer probe combination 6, wherein the primer probe combination 5 comprises an upstream primer shown as SEQ ID NO.13, a downstream primer shown as SEQ ID NO.14 and a fluorescent probe shown as SEQ ID NO.15, and the primer probe combination 6 comprises an upstream primer shown as SEQ ID NO.16, a downstream primer shown as SEQ ID NO.17 and a fluorescent probe shown as SEQ ID NO. 18.

In an embodiment of the invention, the PCR primer probe combination for esophageal cancer gene methylation detection further comprises a PCR primer for detecting an internal reference gene GAPDH and a probe, wherein the primer comprises an upstream primer shown as SEQ ID NO.19 and a downstream primer shown as SEQ ID NO.20, and the probe comprises a fluorescent probe shown as SEQ ID NO. 21.

In an embodiment of the present invention, the 5' end of the fluorescent probe includes a fluorescent reporter group, and the fluorescent reporter group includes any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY and CY 5.

In one embodiment of the invention, the 3' end of the fluorescent probe comprises a fluorescence quenching group, and the fluorescence quenching group comprises any one of MGB, BHQ-1, BHQ-2 and BHQ-3.

In a preferred embodiment of the invention, the fluorescence quenching group is MGB.

The third aspect of the invention provides an esophageal cancer gene methylation detection kit, which comprises the PCR primer probe combination according to the second aspect of the invention, and further comprises a positive quality control product and a negative quality control product.

In one embodiment of the invention, the positive quality control is human esophageal cancer cell line DNA.

In an embodiment of the invention, the negative quality control product is human peripheral blood leukocyte DNA.

In one embodiment of the present invention, the final concentration composition of the esophageal cancer gene methylation detection kit reaction system comprises: 0.1-1. Mu.M PCR primer and 0.1-1. Mu.M probe.

In a preferred embodiment of the present invention, the final concentration composition of the esophageal cancer gene methylation detection kit reaction system comprises: 0.1-0.5. Mu.M PCR primer, 0.1-0.5. Mu.M probe.

In one embodiment of the invention, the digital PCR reaction conditions of the esophageal cancer gene methylation detection kit are as follows:

in a preferred embodiment of the present invention, the digital PCR reaction conditions of the esophageal cancer gene methylation detection kit are as follows:

the fourth aspect of the present invention provides a method for detecting methylation of esophageal cancer genes, comprising the steps of:

1) Isolating nucleic acid of a target gene in a biological sample to be tested;

2) Subjecting the nucleic acid obtained in step 1) to bisulfite conversion treatment to obtain bisulfite-converted DNA (Bis-DNA);

3) Detecting the methylation state of the Bis-DNA obtained in the step 2) by adopting a digital PCR technology.

In one embodiment of the invention, the biological sample of step 1) comprises peripheral blood, fresh pathological tissue, paraffin-embedded tissue, and esophageal cast-off cells.

In a preferred embodiment of the invention, the biological sample of step 1) is esophageal cast-off cells.

In a fifth aspect, the invention provides the application of the esophageal cancer gene methylation detection site disclosed in the first aspect, the PCR primer probe combination disclosed in the second aspect, the esophageal cancer gene methylation detection kit disclosed in the third aspect or the detection method disclosed in the fourth aspect in preparation of the esophageal cancer detection kit.

The invention has the following beneficial effects:

1) Can be used as an important index for early screening, progress monitoring and prognosis evaluation of esophageal cancer: the esophageal cancer gene methylation detection kit provided by the invention takes DNA methylation abnormality as a detection object, the DNA methylation abnormality usually occurs in early cancer and penetrates through the occurrence and development processes of the cancer, and the methylation state of the kit can be changed once the kit is formed and needs to be continuously stimulated by the external environment for a long time, so that the detection of the DNA methylation index can be used as an important biological index for early screening, process monitoring and prognosis evaluation of esophageal cancer;

2) Non-invasive detection is possible: the esophageal cancer gene methylation detection kit provided by the invention can detect various samples, and can realize noninvasive detection by detecting the methylation state of esophageal cast-off cells and peripheral blood genes;

3) The accuracy is high: the esophageal cancer gene methylation detection kit provided by the invention is based on a digital PCR technology, can efficiently and sensitively complete PCR amplification of target nucleic acid fragments in a micro-reaction unit, acquire fluorescent signals for statistical analysis, thoroughly get rid of dependence on a standard curve and directly give out copy numbers of target sequences, improve the stability of experimental results in batches and among batches, realize absolute quantification of initial samples, improve the sensitivity of a nucleic acid detection method, and effectively reduce the occurrence of false negative.

Drawings

FIG. 1 is a diagram of the primer probe combination digital PCR detection result of the target gene DACH1 methylation detection provided by the embodiment of the invention;

fig. 2 is a diagram of a primer probe combination digital PCR detection result of the target gene ZNF132 methylation detection provided by the embodiment of the invention.

Detailed Description

The present invention is described in detail below by way of specific examples to enable those skilled in the art to readily practice the invention in light of the present disclosure. The embodiments described below are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified. Experimental methods, in which specific conditions are not specified, are generally performed under conventional conditions, or under conditions recommended by the manufacturer.

According to the invention, bisulphite conversion is carried out on a nucleic acid sample of esophageal cancer to be detected by adopting a bisulphite modification method, a digital PCR technology is combined, a TCGA database is used for screening a high methylation candidate gene of the esophageal cancer, a specific esophageal cancer gene methylation detection primer and a specific probe are designed, a DNA sample to be detected which is modified by bisulphite and prepared by microdroplet is amplified, the methylation condition of a target gene in the sample to be detected is determined according to the number of positive microdroplets amplified by PCR, and auxiliary diagnosis is provided for the esophageal cancer.

The gene methylation detection site of the esophageal cancer gene methylation detection kit provided by the invention comprises DACH1 and/or ZNF132.

The PCR primer probe combination of the esophageal cancer gene methylation detection kit provided by the invention comprises the following nucleic acid sequence combinations shown in 1) and/or 2):

The invention provides a PCR primer probe combination of an esophageal cancer gene methylation detection kit, which also comprises a PCR primer for detecting an internal reference gene GAPDH and a probe, wherein the primer comprises an upstream primer shown as SEQ ID NO.19 and a downstream primer shown as SEQ ID NO.20, and the probe comprises a fluorescent probe shown as SEQ ID NO. 21.

Preferably, the 5' end of the fluorescent probe comprises a fluorescent reporter group, including any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY and CY 5.

Preferably, the 3' end of the fluorescent probe comprises a fluorescence quenching group, including any one of MGB, BHQ-1, BHQ-2 and BHQ-3.

Further preferably, the fluorescence quenching group is MGB.

In an exemplary embodiment of the present invention, digital PCR employs a droplet PCR technique (PCR) that is performed after 10000-20000 micro-reaction droplets are generated by adding a digital PCR mixture to a droplet generator. After PCR amplification reaction, judging whether the sample to be detected contains methylated DNA target molecules according to the type of fluorescent signals, and setting internal reference genes simultaneously according to the formula to determine the quantity and the content of the methylated DNA target molecules: methylation ratio= [ methylation copy number/methylation copy number+reference gene copy number ] ×100%, and the experimental research result determines that: the methylation proportion of the target gene is more than or equal to 4%, and the interpretation result is positive; the methylation proportion of the target gene is less than 4%, and the result is negative.

In the present invention, a DNA sample to be tested comprises: peripheral blood, fresh pathological tissue, paraffin embedded tissue and esophageal cast-off cells.

Example 1: sample DNA extraction and bisulfite conversion

1. Sample processing and DNA extraction

1) DNA extraction: the esophageal cast-off cell DNA is extracted by using a nucleic acid extraction or purification reagent (general type) produced by Anhuida medical science and technology Co., ltd, specifically comprising the following steps:

a) Taking a proper amount of cell samples or tissue samples, adding 500 mu L of lysate and 30 mu L of proteinase K, and performing cleavage at 70 ℃ for 40min after fully mixing.

b) After brief centrifugation, 200. Mu.L of isopropanol was added, thoroughly mixed, transferred to an adsorption column, centrifuged at 12000rpm for 1min, and the waste liquid was discarded.

d) 600. Mu.L of rinse solution I was added for rinsing, and the mixture was centrifuged at 12000rpm for 30 seconds, and the waste liquid was discarded.

e) 600. Mu.L of rinse solution II was added for rinsing, and the mixture was centrifuged at 12000rpm for 30 seconds, and the waste liquid was discarded.

f) 600. Mu.L of rinse solution II was again added for rinsing, centrifuged at 12000rpm for 30s, the waste solution was discarded, and centrifuged at 12000rpm for 3min.

g) Uncapping, and airing in a fume hood for 2min.

h) Adding 50-100 mu L of eluent into the adsorption column, standing at room temperature for 3min, and centrifuging at 12000rpm for 2min.

i) Repeating the step h), collecting DNA into a centrifuge tube, and preserving at-20 ℃.

2) Bisulfite conversion: the genomic DNA obtained in the step was subjected to bisulfite conversion by using a nucleic acid extraction or purification reagent (centrifugation column) produced by Anhuida medical science, inc., having the steps of:

(a) Taking 45 mu L of DNA sample to be detected in a new 1.5mL centrifuge tube, adding 5 mu L of conversion buffer solution, and placing in a metal bath for incubation at a constant temperature of 37 ℃ for 15min;

(b) After the incubation is completed, 100 mu L of a pre-prepared conversion solution is added into each sample, the mixture is uniformly mixed and centrifuged for a short time, and the metal bath is incubated for 12 to 16 hours at 50 ℃ in a dark place;

(c) Incubating the sample on ice (0-4deg.C) for 10min;

(d) Placing the adsorption column in a collecting pipe, and adding 400 mu L of binding solution into the adsorption column;

(e) C, adding the sample in the step into an adsorption column (containing a binding solution), covering a tube cover, uniformly mixing the sample with the binding solution in an upside down manner for a plurality of times, centrifuging the sample at full speed (14000 rpm) for 30s, and discarding waste liquid;

(f) Adding 100 mu L of rinsing liquid into the adsorption column, centrifuging at full speed for 30s, and discarding waste liquid;

(g) Adding 200 mu L of desulfonation liquid into an adsorption column, incubating for 20min at room temperature (20-30 ℃), centrifuging for 30s at full speed, and discarding waste liquid;

(h) Adding 200 mu L of rinsing liquid into the adsorption column, centrifuging at full speed for 30s, repeatedly adding 200 mu L of rinsing liquid, centrifuging at full speed for 30s, discarding waste liquid and collecting the tube;

(i) Placing the adsorption column into a 1.5mL sterile centrifuge tube, suspending and dripping 30 mu L of eluent into the middle part of the adsorption film, eluting and transforming DNA, centrifuging at full speed for 1min, collecting Bis-DNA, and preserving at-20 ℃.

Example 2: esophageal cancer tissue hypermethylation candidate gene, specific primer and probe screening

1. Screening of candidate genes for hypermethylation of esophageal cancer tissues

Methylation chip data related to esophageal cancer and transcriptome sequencing data corresponding to the methylation chip data are obtained through a TCGA database (http:// cancerargenome.nih.gov /), methylation sites with obvious differences are screened out, and finally DACH1 and ZNF132 are screened out and used as high methylation candidate genes of esophageal cancer tissues.

2. Specific primer and probe screening for methylation detection of esophageal cancer

1) Specific primer and probe screening:

according to the nucleic acid sequences of DACH1 and ZNF132, the design of methylation primers and probes is carried out on Methyl primer Express v1.0 software, the digital PCR probes and primers for related gene methylation are obtained through screening by repeated design and push of the applicant, and the designed primers and probes are sent to Beijing Rui Boxing family biotechnology Co., ltd for synthesis, and the specific sequences are shown in the following table:

meanwhile, a specific primer and a probe aiming at the reference gene GAPDH are arranged, and the specific sequence is as follows:

name of the name	Sequence (5 '-3')
		METHY-GAPDH-F	GTGGAGAGAAATTTGGGAGGTTAG(SEQ ID NO.19)
METHY-GAPDH-R	CAACACAAACACATCCAACCTACA(SEQ ID NO.20)
		METHY-GAPDH-P	ATGGTTTGAAGGTGGTAGGG(SEQ ID NO.21)

Wherein the 5 'end of the probe sequence is modified with a fluorescent group selected from any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY and CY5, and the 3' end is marked with a fluorescence quenching group selected from any one of MGB, BHQ-1, BHQ-2 and BHQ-3.

2) PCR amplification further primer probe combinations were screened (for example, berle QX200 microdroplet digital PCR):

the reaction system: 10. Mu.L of 2 Xdigital PCR reaction premix, 0.1. Mu.L of each GAPDH primer and probe of 10. Mu.M, 0.5. Mu.L of each primer and probe of 10. Mu.M of each primer probe combination, 0.2. Mu.L of each probe, and 6. Mu.L of Bis-DNA, and water was added to 20. Mu.L.

Droplet preparation: the 20. Mu.L of each reaction system was loaded onto a droplet-generating card containing 70. Mu.L of droplet-generating oil and placed on a droplet generator to generate droplets, which were typically completed within 2 minutes, and the generated droplets were transferred to a 96-well plate, which was then sealed with a preheated PX1 heat sealer, and after sealing, PCR reactions were performed within 30 minutes.

Reaction conditions:

the annealing temperature was chosen at 54-60℃depending on the TM value of each primer combination.

Droplet reading and signal analysis: the PCR 96-well plate with the reaction completed was placed in a microdroplet reader and QuantaSoft was opened ^TM And the software establishes sample module information according to the sample quantity and the sample layout, and runs after the setting is completed. After the data reading is completed, the threshold is automatically adjusted for positive and negative droplet assignment for each detection channel. Sample adjustment partitioning threshold, methylation data collection and analysis are shown in QuantaSoft ^TM In a software interface.

The DNA samples of esophageal cancer confirmed patients and healthy people are taken as templates, the primer probe combination screened in the step 1) is screened through PCR amplification, wherein the detection result of the primer probe combination of the target gene DACH1 is shown in figure 1, under the condition that the detection of a control sample is negative and can be clearly distinguished, the positive amplification point number of the primer probe combination 2 is obviously less than that of the primer probe combination 1 and the primer probe combination 3, the detection result of the primer probe combination of the target gene ZNF132 is shown in figure 2, and under the condition that the detection of the control sample is negative and can be clearly distinguished, the positive amplification point number of the primer probe combination 4 is obviously less than that of the primer probe combination 5 and the primer probe combination 6, so that the primer probe combination 1, the primer probe combination 3, the primer probe combination 5 and the primer probe combination 6 are screened as primer probe combinations for further testing.

Example 3: clinical sample detection and verification kit effect

The test procedure described in examples 1 and 2 above was used to verify the effect of the kit on clinical samples, and the test results were interpreted as follows: the total number of droplets in the test sample is greater than 15,000 to determine that the reaction is effective. The sum of the methylation copy number of the detected sample and the copy number of the reference gene is more than or equal to 100, so that the detection result of the sample is effective, and the analysis of the result can be continued; if less than 100, the sample detection is not effective and a re-detection is required. The methylation ratio of the sample is calculated from the following formula: methylation ratio= [ methylation copy number/methylation copy number+reference gene copy number ] ×100%, and the experimental research result determines that: the methylation proportion of the target gene is more than or equal to 4%, and the interpretation result is positive; the methylation proportion of the target gene is less than 4%, and the result is negative.

1) Esophageal desquamation cell gene methylation detection result:

in order to evaluate that the primer probe combination provided by the invention can be used for detecting the methylation of the esophageal cancer DACH1 and ZNF132 genes in a sample by digital PCR, a DNA template from the same sample is divided into 8 parts, and the digital PCR detection is completed under the primer probe combinations with different T1-T8, wherein the digital PCR detection system is as follows:

detection system	Primer probe combination
		T1	DACH1-1
T2	DACH1-3
		T3	ZNF132-2
T4	ZNF132-3
		T5	DACH1-1、ZNF132-2
T6	DACH1-1、ZNF132-3
		T7	DACH1-3、ZNF132-2
T8	DACH1-3、ZNF132-3

The methylation detection of genes of esophageal cancer cells of 79 cases of patients with confirmed esophageal cancer and 21 cases of normal human esophageal cast-off cells is completed in a T1-T8 digital PCR detection system, and the results show that 66-74 cases of positive esophageal cancer gene methylation samples can be detected by T1-T8 in 79 cases of patients with confirmed esophageal cancer, the detection rate is 83-95%, and the total detection rate under all detection conditions is more than 83%. In 21 healthy person control samples, only 1 case of the T6 detection system is higher than a set threshold value (but the result value is obviously lower than the result of positive esophageal cancer), and the specificity of the detection method is 95%; in other detection systems, no 1 case was above the set threshold, and the specificity of the detection method was 100%. For the stage I esophageal cancer, the detection rate of T1-T8 is more than 72%, which shows that the esophageal cancer gene methylation detection kit provided by the invention has a good detection effect on early esophageal cancer samples, and the detailed table is shown below:

clinical staging of esophageal cancer patients	Sample size/number of samples detected	Detection rate (%)	Detection system
				Phase I	13/18	72.22	T1
Stage II	16/20	80.00	T1
				Stage III	20/22	90.91	T1
Stage IV	18/19	94.74	T1
				All patients diagnosed	67/79	84.81	T1
Normal person control	0/21	0	T1

Clinical staging of esophageal cancer patients	Sample size/number of samples detected	Detection rate (%)	Detection system
				Phase I	14/18	77.78	T2
Stage II	16/20	80.00	T2
				Stage III	20/22	90.91	T2
Stage IV	18/19	94.74	T2
				All patients diagnosed	68/79	86.08	T2
Normal person control	0/21	0	T2

Clinical staging of esophageal cancer patients	Sample size/number of samples detected	Detection rate (%)	Detection system
				Phase I	13/18	72.22	T3
Stage II	16/20	80.00	T3
				Stage III	19/22	86.36	T3
Stage IV	18/19	94.74	T3
				All patients diagnosed	66/79	83.54	T3
Normal person control	0/21	0	T3

Clinical staging of esophageal cancer patients	Sample size/number of samples detected	Detection rate (%)	Detection system
				Phase I	14/18	77.78	T4
Stage II	17/20	85.00	T4
				Stage III	20/22	90.91	T4
Stage IV	18/19	94.74	T4
				All patients diagnosed	69/79	87.34	T4
Normal person control	0/21	0	T4

Clinical staging of esophageal cancer patients	Sample size/number of samples detected	Detection rate (%)	Detection system
				Phase I	15/18	83.33	T5
Stage II	17/20	85.00	T5
				Stage III	21/22	95.45	T5
Stage IV	19/19	100.00	T5
				All patients diagnosed	72/79	91.14	T5
Normal person control	0/21	0	T5

Clinical staging of esophageal cancer patients	Sample size/number of samples detected	Detection rate (%)	Detection system
				Phase I	16/18	88.89	T6
Stage II	18/20	90.00	T6
				Stage III	21/22	95.45	T6
Stage IV	19/19	100.00	T6
				All patients diagnosed	74/79	93.67	T6
Normal person control	1/21	4.76	T6

Clinical staging of esophageal cancer patients	Sample size/number of samples detected	Detection rate (%)	Detection system
				Phase I	15/18	83.33	T7
Stage II	18/20	90.00	T7
				Stage III	21/22	95.45	T7
Stage IV	19/19	100.00	T7
				All diagnosed patientsPerson(s)	73/79	92.41	T7
Normal person control	0/21	0	T7

Clinical staging of esophageal cancer patients	Sample size/number of samples detected	Detection rate (%)	Detection system
				Phase I	16/18	88.89	T8
Stage II	18/20	90.00	T8
				Stage III	22/22	100.00	T8
Stage IV	19/19	100.00	T8
				All patients diagnosed	74/79	94.94	T8
Normal person control	0/21	0	T8

2) Esophageal cancer peripheral blood gene methylation detection results:

the methylation of the peripheral blood genes of the esophageal cancer is detected by adopting a detection system T1-T8 in the detection of the methylation of the esophageal cast-off cell genes, 30 peripheral blood genes of patients diagnosed with the esophageal cancer and 10 peripheral blood genes of normal people are detected, and the results are shown in the following table:

the result shows that 24-28 cases of esophageal cancer gene methylation positive samples are detected in 30 cases of patients with esophageal cancer diagnosis, the detection rate is 80-93%, and the total detection rate is more than 80% under all detection conditions; the specificity of the detection method was 100% in 10 healthy human control samples. The result shows that the esophageal cancer gene methylation detection kit provided by the invention has higher detection sensitivity and detection specificity when detecting esophageal cancer peripheral blood gene methylation, and can be used for early screening of esophageal cancer.

While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.

Sequence listing

<110> Guangzhou Dajian Biotechnology Co., ltd

ANHUI DAJIAN MEDICAL TECHNOLOGY Co.,Ltd.

<120> esophageal cancer gene methylation detection primer probe combination, kit and application thereof

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

tcgcggacga aatggaat 18

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

cgaaaacaca aaaataacga tcga 24

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

acggtagtag tagaagagaa 20

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

cgtttcgggt gagggtttc 19

<210> 5

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

cgaatcattt ccctcccga 19

<210> 6

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

aggcgaggga gcggattagt attcga 26

<210> 7

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gtattgggat gtttagcggt tttg 24

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

cctcttaata aatcctcccc cact 24

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tcgttcgtcg ttttcggt 18

<210> 10

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ttgggttaag attttttaat gcgtag 26

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tatccgcgac tctcgaaaat c 21

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ttggtttcgg tttcgcgt 18

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tttcgtttga taacgcggtt t 21

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tccgaacgaa atataaaacg ctaa 24

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tcgaggacga cgacggaagt ttcgt 25

<210> 16

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cgtacgtttt atgtttatag tcgggc 26

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gaaaatatcc gcgactctcg a 21

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ttggtttcgg tttcgcgt 18

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

gtggagagaa atttgggagg ttag 24

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

caacacaaac acatccaacc taca 24

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

atggtttgaa ggtggtaggg 20

Claims

1. A PCR primer probe combination for detecting methylation of esophageal cancer genes, which is characterized by comprising a nucleic acid sequence combination shown in the following 1) and 2):

2. The combination of PCR primers for methylation detection of esophageal cancer genes according to claim 1, further comprising a PCR primer for detecting GAPDH of a reference gene and a probe, wherein the primer comprises an upstream primer shown as SEQ ID No.19 and a downstream primer shown as SEQ ID No.20, and the probe comprises a fluorescent probe shown as SEQ ID No. 21.

3. The PCR primer probe combination for esophageal cancer gene methylation detection of claim 1 or claim 2, wherein the 5' end of the fluorescent probe comprises a fluorescent reporter group comprising any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY, CY 5.

4. The PCR primer probe combination for esophageal cancer gene methylation detection of claim 1 or claim 2, wherein the 3' end of the fluorescent probe comprises a fluorescence quenching group comprising any one of MGB, BHQ-1, BHQ-2, BHQ-3.

5. An esophageal cancer gene methylation detection kit, which comprises the PCR primer probe combination according to claim 1, and further comprises a positive quality control product and a negative quality control product.

6. The esophageal cancer gene methylation detection kit of claim 5, wherein the final concentration composition of the esophageal cancer gene methylation detection kit reaction system comprises: 0.1-1. Mu.M PCR primer and 0.1-1. Mu.M probe.

7. The esophageal cancer gene methylation detection kit of claim 5, wherein the digital PCR reaction conditions of the kit are as follows: 1) 95 ℃ for 2 minutes; 2) Cycling for 30-50 times at 94 ℃ for 30 seconds and 54-60 ℃ for 60 seconds; 3) 98 ℃ for 10 minutes; 4) Cooled to 4 ℃.

8. The use of the PCR primer probe combination for esophageal cancer gene methylation detection of claim 1 in the preparation of a kit for esophageal cancer detection.