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

Abstract

This application discloses a primer and probe combination for detecting esophageal cancer gene methylation, along with its kit and application. The kit includes a TLX2 primer set and / or a HOXD3 primer set, a TLX2 probe set and / or a HOXD3 probe set. The TLX2 primer set includes an upstream primer TLX2-F and a downstream primer TLX2-R, with sequences listed as SEQ ID NO. 3 and SEQ ID NO. 4, respectively. The HOXD3 primer set includes an upstream primer HOXD3-F and a downstream primer HOXD3-R, with sequences listed as SEQ ID NO. 18 and SEQ ID NO. 19, respectively. The TLX2 probe set includes a TLX2-P6 probe, with a sequence listed as SEQ ID NO. 10. The HOXD3 probe set includes a HOXD3-P6 probe, with a sequence listed as SEQ ID NO. 25. This application can improve the sensitivity and specificity of detection.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, and in particular to a primer-probe combination for detecting esophageal cancer gene methylation, its kit, and its application. Background Technology

[0003] Currently, methods for detecting and screening esophageal cancer include imaging techniques, tissue biopsies, and tumor serum marker detection. However, imaging techniques are costly, require highly skilled operators, and are expensive to perform; tissue biopsies are highly invasive and unsuitable for early cancer screening; and tumor serum marker detection has low sensitivity, failing to meet clinical needs. Therefore, it is necessary to develop sensitive and specific novel esophageal cancer markers and detection technologies to improve the early detection rate of esophageal cancer, enhance treatment outcomes, and reduce mortality.

[0004] Epigenetics has become a hot topic in cancer research in recent years. DNA methylation is the most common epigenetic alteration, which can regulate cell proliferation, apoptosis, and differentiation, and its level is closely related to the biological characteristics of tumors. Abnormal DNA methylation usually occurs in the early stages of cancer and continues throughout the occurrence and development of cancer. Therefore, the detection of DNA methylation markers can serve as an important biomarker for cancer diagnosis, early screening, and prognosis.

[0005] In view of this, this application establishes a new method for detecting esophageal cancer gene methylation by screening esophageal cancer-related methylation genes, so as to achieve early screening and diagnosis of esophageal cancer. Summary of the Invention

[0006] To improve the sensitivity and specificity of esophageal cancer detection, this application provides a primer-probe combination for esophageal cancer gene methylation detection, along with its kit and application.

[0007] In a first aspect, this application provides a primer-probe combination for detecting esophageal cancer gene methylation, employing the following technical solution: A primer-probe combination for detecting esophageal cancer gene methylation includes a TLX2 primer set and / or a HOXD3 primer set, a TLX2 probe set and / or a HOXD3 probe set. The TLX2 primer set includes an upstream primer TLX2-F and a downstream primer TLX2-R, whose sequence listings are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The HOXD3 primer set includes an upstream primer HOXD3-F and a downstream primer HOXD3-R, whose sequence listings are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively. The TLX2 probe set includes a TLX2-P6 probe, whose sequence listing is shown in SEQ ID NO.10. The HOXD3 probe set includes a HOXD3-P6 probe, whose sequence listing is shown in SEQ ID NO.25.

[0008] By adopting the above-mentioned technical solution, this application detects esophageal cancer genes based on methylation sites (TLX2 and HOXD3), which can improve the sensitivity and specificity of detection, thus facilitating the diagnosis and early screening of esophageal cancer. Furthermore, this detection method is applicable to circulating cell-free DNA in peripheral blood samples, enabling non-invasive detection and greatly improving screening compliance and applicability.

[0009] Optionally, the 5' end of the TLX2-P6 probe is marked with FAM, and the 3' end of the TLX2-P6 probe is marked with MGB; the 5' end of the HOXD3-P6 probe is marked with VIC, and the 3' end of the HOXD3-P6 probe is marked with MGB.

[0010] By employing the above technical solution, the TLX2-P6 probe and the HOXD3-P6 probe utilize FAM and VIC fluorescent reporter groups, respectively, allowing their emission spectra to be detected independently by the instrument. This enables simultaneous detection of the TLX2 and HOXD3 genes within the same PCR reaction tube, significantly improving detection efficiency and throughput. Furthermore, both probes are modified with MGB quencher groups at their 3' ends. This modification effectively enhances the hybridization stability and specificity of the probes and reduces fluorescence background, thereby improving overall detection sensitivity and accuracy. This design simplifies the detection process and is more economical, facilitating the standardization of this primer-probe combination and its clinical application.

[0011] Optionally, the base sequences of the TLX2 probe set and / or HOXD3 probe set are modified with locked nucleic acids.

[0012] By employing the above-mentioned technical solutions, the introduction of locked nucleic acid modifications into TLX2 and / or HOXD3 probes can significantly improve the thermal stability of the probe binding to complementary target sequences. This enhanced stability allows the probe to more strictly distinguish between perfectly matched methylated sequences and single-base mismatched unmethylated sequences, thereby greatly improving the detection's discriminative power and signal-to-noise ratio, ultimately achieving a significant improvement in detection specificity.

[0013] Optionally, the locked nucleic acid modification involves a three-base locked nucleic acid modification targeting the core CG site, and the probe length of the TLX2 probe set and / or HOXD3 probe set is 12-16 bp.

[0014] By employing the above-mentioned technical solution, a localized "ultra-stable domain" is constructed through continuous triplet nucleic acid modification in the key region containing the core CG site. This domain maximizes the binding force between the probe and the perfectly matched methylated sequence, while strongly rejecting unmethylated sequences with single-base mismatches, thereby improving detection specificity. This application controls the probe length of the locked nucleic acid-modified probe to 12-16 bp, ensuring efficient hybridization kinetics and fluorescence resonance energy transfer efficiency, which is beneficial for obtaining earlier and stronger fluorescence signals in PCR amplification, thus improving detection sensitivity.

[0015] Secondly, this application provides an esophageal cancer gene methylation detection kit, which adopts the following technical solution: An esophageal cancer gene methylation detection kit, comprising a primer-probe combination for esophageal cancer gene methylation detection.

[0016] By adopting the above technical solution, this kit pre-configures and standardizes the optimized specific primer-probe combination (TLX2 primer-probe combined with HOXD3 primer-probe) and necessary reaction components, ensuring a high degree of uniformity in component concentration and reaction conditions. This effectively eliminates variables introduced by manual preparation or batch-to-batch differences, greatly improving the repeatability, consistency, and accuracy of detection results. Simultaneously, this design significantly simplifies the operation process, lowers the technical threshold, and facilitates its widespread application in clinical settings.

[0017] Optionally, the PCR primers and probes for the internal reference gene ACTB are also included. The PCR primers for the internal reference gene ACTB include upstream primer AB-F and downstream primer AB-R, the sequence listings of which are shown in SEQ ID NO.33 and SEQ ID NO.34, respectively. The PCR probes for the internal reference gene ACTB include the AB-P1 probe, the sequence listing of which is shown in SEQ ID NO.35.

[0018] By adopting the above technical solution, this application uses ACTB as an internal reference gene. Its stable amplification curve provides a benchmark for the interpretation of the Ct value of the target gene, effectively identifying and excluding invalid detections caused by sample degradation, insufficient DNA quantity, or experimental operation failure, thereby greatly reducing the risk of false negatives.

[0019] Optionally, the 5' end of the AB-P1 probe is marked with ROX, and the 3' end of the AB-P1 probe is marked with MGB.

[0020] By adopting the above technical solution, this application uses the FAM channel to detect the TLX2 gene and the VIC channel to detect the HOXD3 gene, allowing the internal reference gene to be detected in a third independent optical channel. Furthermore, the 3' end of the AB-P1 probe is also modified with MGB, ensuring its efficient and accurate binding to the conserved sequence of the ACTB gene, thus truly reflecting the actual state of the DNA in the sample and avoiding quality control failure due to poor performance of the internal reference probe itself.

[0021] Optionally, the final concentration composition of the reaction system of the kit includes: 12.5 μL of premix, 0.4 μL of upstream primer of the target sequence, 0.4 μL of downstream primer of the target sequence, 0.2 μL of target sequence detection probe, 0.4 μL of upstream primer of internal reference gene, 0.4 μL of downstream primer of internal reference gene, 0.2 μL of internal reference gene probe, 10 μL of transformed DNA template, and 0.5 μL of pure water.

[0022] Optionally, the reaction conditions of the kit are: pre-denaturation at 95°C for 5 min; followed by denaturation at 95°C for 15 s and annealing extension at 60°C for 40 s, for 45 cycles.

[0023] Thirdly, this application provides an application of a primer-probe combination for detecting esophageal cancer gene methylation in the detection of esophageal cancer.

[0024] In summary, this application includes at least one of the following beneficial technical effects: 1. This application focuses on the detection of esophageal cancer gene methylation sites (TLX2 and HOXD3), which can improve the sensitivity and specificity of the detection, thus benefiting the diagnosis and early screening of esophageal cancer. Furthermore, this detection method is applicable to circulating cell-free DNA in peripheral blood samples, enabling non-invasive detection and greatly improving screening compliance and applicability. 2. The TLX2-P6 and HOXD3-P6 probes of this application employ FAM and VIC fluorescent reporter groups, respectively, and their emission spectra can be independently detected by the instrument. This enables simultaneous detection of the TLX2 and HOXD3 genes in the same PCR reaction tube, significantly improving detection efficiency and throughput. Simultaneously, the 3' ends of both probes are modified with MGB quencher groups. This modification effectively enhances the hybridization stability and specificity of the probes and reduces fluorescence background, thereby improving overall detection sensitivity and accuracy. This design simplifies the detection process and is more economical, facilitating the standardization of this primer-probe combination and its clinical application. 3. This application significantly improves the thermal stability of probe binding to complementary target sequences by introducing locked nucleic acid modifications into TLX2 and / or HOXD3 probes. This enhanced stability allows the probes to more strictly distinguish between perfectly matched methylated sequences and single-base mismatched unmethylated sequences, thereby greatly improving the detection's discriminative power and signal-to-noise ratio, ultimately achieving a significant improvement in detection specificity; 4. This application constructs a localized "ultra-stable domain" by performing continuous triplet nucleic acid modification in a key region containing the core CG site. This domain maximizes the binding affinity of the probe to perfectly matched methylated sequences while strongly rejecting unmethylated sequences with single-base mismatches, thereby improving detection specificity. This application controls the probe length of the locked nucleic acid-modified probe to 12-16 bp, ensuring efficient hybridization kinetics and fluorescence resonance energy transfer efficiency, which is beneficial for obtaining earlier and stronger fluorescence signals in PCR amplification, thus improving detection sensitivity. Attached Figure Description

[0025] Figure 1 This is an amplification curve of the kit of Example 12 of this application for methylation detection of clinical blood samples from patients clinically diagnosed with esophageal squamous cell carcinoma; Figure 2 This is an amplification curve of methylation detection in clinical blood samples from patients clinically diagnosed with esophagitis using the kit of Example 12 of this application; Figure 3 This is an amplification curve of methylation detection in clinical blood samples from patients clinically diagnosed with colorectal cancer, based on the kit of Example 12 of this invention. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] This application designs a primer-probe combination for detecting esophageal cancer gene methylation, including a TLX2 primer set and / or a HOXD3 primer set, a TLX2 probe set and / or a HOXD3 probe set; the TLX2 primer set includes an upstream primer TLX2-F and a downstream primer TLX2-R, the sequence listings of which are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively; the HOXD3 primer set includes an upstream primer HOXD3-F and a downstream primer HOXD3-R, the sequence listings of which are shown in SEQ ID NO. 18 and SEQ ID NO. 19, respectively; the TLX2 probe set includes a TLX2-P6 probe, the sequence listing of which is shown in SEQ ID NO. 10; the HOXD3 probe set includes a HOXD3-P6 probe, the sequence listing of which is shown in SEQ ID NO. 19. As shown in NO.25; the 5' end of the TLX2-P6 probe is labeled with FAM, and the 3' end of the TLX2-P6 probe is labeled with MGB; the 5' end of the HOXD3-P6 probe is labeled with VIC, and the 3' end of the HOXD3-P6 probe is labeled with MGB; the base sequences of the TLX2-P6 probe group and / or the HOXD3-P6 probe group contain three consecutive bases with the CG site as the core, which are modified with locked nucleic acids, and the probe length of the TLX2 probe group and / or the HOXD3 probe group is 12-16 bp.

[0028] This application designs an esophageal cancer gene methylation detection kit, comprising an esophageal cancer gene methylation detection primer-probe combination, and PCR primers and probes for the internal reference gene ACTB. The PCR primers for the internal reference gene ACTB include upstream primer AB-F and downstream primer AB-R, whose sequence listings are shown in SEQ ID NO.33 and SEQ ID NO.34, respectively. The PCR probe for the internal reference gene ACTB includes the AB-P1 probe, whose sequence listing is shown in SEQ ID NO.35. The 5' end of the AB-P1 probe is labeled with ROX, and the 3' end of the AB-P1 probe is labeled with MGB. The final concentration composition of the kit reaction system includes: 12.5 μL of premix, 0.4 μL of the target sequence upstream primer, 0.4 μL of the target sequence downstream primer, 0.2 μL of the target sequence detection probe, 0.4 μL of the internal reference gene upstream primer, 0.4 μL of the internal reference gene downstream primer, 0.2 μL of the internal reference gene probe, 10 μL of transformed DNA template, and 0.5 μL of pure water. The reaction conditions for the kit were: pre-denaturation at 95°C for 5 min; followed by denaturation at 95°C for 15 s and annealing extension at 60°C for 40 s, for 45 cycles.

[0029] The reagents and instruments used in the embodiments of this application are all commercially available, wherein: DNA extraction kit, VAMNE MagUltra Circulating Cell-free DNA Isolation Kit (N913), Nanjing Novizan Biotechnology Co., Ltd.; EpiArt Ultrafast Magnetic DNA Methylation Bisulfite Kit (EM113), Nanjing Novizan Biotechnology Co., Ltd. qPCR reaction reagent for methylation detection, Robustart Premix OmniⅢ (Probe qPCR), Baorui Biotechnology Co., Ltd.; Real-time PCR instrument, model SLAN-96P, Shanghai Hongshi Medical Technology Co., Ltd.

[0030] Example 1: Screening of target genes Methylation microarray data and gene expression data of esophageal cancer were obtained from the TCGA database, and differentially expressed methylation sites, namely the TLX2 and HOXD3 genes, were screened. The chromosomal location of the TLX2 gene is: chr2:74515592-74516211 (negative strand). Specifically, the DNA strand of TLX2 (chr2:74515592-74516211) was selected from the DNA strand of GRCh38.p14 primary assembly: chr2:74515592-74516211 (SEQ ID NO.1). The chromosomal location of the HOXD3 gene is: chr2:176164685-176165213 (positive strand).Specifically, the DNA strand of HOXD3 (chr2:176164685-176165213) is selected from GRCh38.p14 primary assembly: chr2:176164685-176165213 (SEQ ID NO.2).

[0031] Example 2: Primer and probe design TLX2 and HOXD3 genes were selected as target regions, and methylation-specific primer pairs and probes were designed respectively. The primers and probes were designed using Methyl primer Express v1.0 software. All primers were synthesized by Shanghai Sangon Biotech Co., Ltd. The primer and probe combination sequences for TLX2 and HOXD3 genes are shown in Table 1-2.

[0032] Table 1. Primer and probe sequence listings for the TLX2 and HOXD3 genes (SEQ ID NO. 3-32)

[0033] Note: The underlined base sequence is the base sequence modified with locked nucleic acid.

[0034] Table 2 Primer and probe combinations for TLX2 and HOXD3 genes

[0035] Example 3: Quantitative Real-Time PCR Detection Method Blood samples were collected, and cell-free DNA (cfDNA) was extracted from the plasma using a cell-free DNA extraction kit. The cfDNA was then converted to bisulfite using a magnetic bead-based bisulfite conversion kit to obtain transformed DNA. Using the transformed DNA as a template and ACTB as an internal control gene, quantitative real-time PCR was performed using the aforementioned primer and probe combination and methylation detection qPCR reagents. Fluorescence detection results were obtained. The ACTB primer and probe sequences are shown in Table 3. Appropriate fluorescence thresholds were determined based on the amplification curves. The ACTB threshold was set at the inflection point of the initial stage of the exponential amplification phase of the positive control. The threshold for sample methylation status was set when the threshold exceeded the highest point of the negative control but was at the inflection point of the initial stage of the exponential amplification phase of the positive control. After setting the baseline and fluorescence thresholds, the intersection of the threshold line and the amplification curve was defined as the Ct value.

[0036] The real-time PCR reaction system consisted of 25 μL, including: 12.5 μL premixed buffer, 0.4 μL (25 μmol / L) upstream primer of the target sequence, 0.4 μL (25 μmol / L) downstream primer of the target sequence, 0.2 μL (25 μmol / L) target sequence detection probe, 0.4 μL (25 μmol / L) upstream primer of the internal reference gene, 0.4 μL (25 μmol / L) downstream primer of the internal reference gene, 0.2 μL (25 μmol / L) internal reference gene probe, 10 μL transformed DNA template, and 0.5 μL pure water. The conditions were: 95℃ pre-denaturation for 5 min, followed by 95℃ denaturation for 15 s and 60℃ annealing extension for 40 s, for 45 cycles.

[0037] Table 3. Primer and probe sequence listing for the internal reference gene ACTB (SEQ ID NO. 33-35)

[0038] The results of the test samples are determined based on the internal control Ct value of the internal control gene ACTB: if the internal control Ct value of the test sample is ≤34, the sample is valid; otherwise, the sample is invalid. After the negative control, positive control, and internal control are qualified, the sample test results can be judged according to Tables 4 and 5.

[0039] Table 4 Interpretation of Gene Testing Results

[0040] Table 5. Judgment of Sample Detection Results

[0041] Example 4: The effect of different locked nucleic acid modifications of the TLX2 gene on the detection of esophageal cancer using primer-probe combinations Blood samples were collected from 80 patients with clinically and pathologically confirmed high-grade esophageal squamous cell carcinoma, 60 patients with advanced esophageal squamous cell carcinoma, 60 patients with early-stage esophageal squamous cell carcinoma, and 80 healthy individuals from a cancer hospital in Anyang. The sample collection process was approved by the ethics committee, and all patients signed informed consent forms. All samples were anonymized. Methylation-based quantitative PCR was performed on primer-probe combinations TLX2-A to TLX2-H according to the method provided in Example 3. The PCR results were analyzed to determine the positivity of the samples, and the sensitivity and specificity of different combinations for detecting esophageal cancer plasma samples were calculated. The results are shown in Table 6. Specificity = PCR negative / (PCR negative + false positive) × 100%, sensitivity = PCR positive / (PCR positive + false negative) × 100%.

[0042] Table 6. Sensitivity and specificity of different primer-probe combinations in detecting esophageal cancer plasma samples.

[0043] Table 6 shows that compared to the primer-probe combination TLX2-A without locked nucleic acid modification, the specificity and sensitivity of primer-probe combinations TLX2-B~H were significantly improved, and the TLX2-F combination exhibited the best overall diagnostic efficacy. Among them, TLX2-F showed the highest specificity (98.75%) in normal human blood. The sensitivities of TLX2-F for high-grade lesions, early esophageal cancer, and advanced esophageal cancer were as high as 85.00%, 93.33%, and 96.67%, respectively. These results indicate that continuous triplet nucleic acid modification of the core methylation CG site and adjacent bases in the probe can significantly improve the sensitivity and specificity of TLX2 gene methylation detection.

[0044] Example 5: The effect of different probe lengths on primer-probe combinations for esophageal cancer detection based on TLX2 gene Blood samples were collected from 80 patients with clinically and pathologically confirmed high-grade esophageal squamous cell carcinoma, 60 patients with advanced esophageal squamous cell carcinoma, 60 patients with early-stage esophageal squamous cell carcinoma, and 80 healthy individuals from a cancer hospital in Anyang. The sample collection process was approved by the ethics committee, and all patients signed informed consent forms. All samples were anonymized. Methylation-based quantitative PCR was performed on primer-probe combinations TLX2-I to TLX2-M according to the method in Example 3, and the sensitivity and specificity of each combination for detecting esophageal cancer were calculated. The results are shown in Table 7.

[0045] Table 7. Sensitivity and specificity of different primer-probe combinations for detecting esophageal cancer plasma samples.

[0046] As shown in Table 7, among all primer-probe combinations from TLX2-I to TLX2-M, the TLX2-L combination exhibited the best overall diagnostic performance. TLX2-L demonstrated the highest specificity (98.75%) in normal human samples. The TLX2-L primer-probe combination showed extremely high sensitivity for high-grade lesions, early-stage esophageal cancer, and advanced-stage esophageal cancer, at 86.25%, 95.00%, and 96.67%, respectively. These results indicate that a probe length of 16 bp for the TLX2 gene resulted in the most efficient capture of methylation sites.

[0047] Example 6: The effect of HOXD3 gene-locked nucleic acid modification on primer-probe combination detection of esophageal cancer Blood samples were collected from 70 patients with clinically and pathologically confirmed high-grade esophageal squamous cell carcinoma, 60 patients with advanced-stage esophageal squamous cell carcinoma, 80 patients with early-stage esophageal squamous cell carcinoma, and 60 healthy individuals from a cancer hospital in Anyang. The sample collection process was approved by the ethics committee, and all patients signed informed consent forms. All samples were anonymized. All esophageal cancer patients had esophageal squamous cell carcinoma. Methylation-based quantitative PCR was performed on the primer-probe combinations HOXD3-A to HOXD3-H according to the method in Example 3. The sensitivity and specificity for detecting esophageal cancer plasma samples were calculated, and the results are shown in Table 8.

[0048] Table 8. Sensitivity and specificity of different primer-probe combinations for detecting esophageal cancer plasma samples.

[0049] Table 8 shows that compared to the primer-probe combination HOXD3-A without locked nucleic acid modification, the specificity and sensitivity of primer-probe combinations HOXD3-B~H were significantly improved, and the HOXD3-F combination exhibited the best overall diagnostic efficacy. Among them, HOXD3-F showed the highest specificity (96.67%) in normal human blood. The sensitivity of HOXD3-F for high-grade lesions, early esophageal cancer, and advanced esophageal cancer was as high as 88.57%, 95.00%, and 96.67%, respectively. These results indicate that continuous triplet nucleic acid modification of the core methylation CG site and adjacent bases in the probe can significantly improve the sensitivity and specificity of HOXD3 gene methylation detection.

[0050] Example 7: Investigating the effect of different probe lengths on primer-probe combinations for esophageal cancer detection based on the HOXD3 gene. Blood samples were collected from 70 patients with clinically and pathologically confirmed high-grade esophageal squamous cell carcinoma, 60 patients with advanced-stage esophageal squamous cell carcinoma, 80 patients with early-stage esophageal squamous cell carcinoma, and 60 healthy individuals from a cancer hospital in Anyang. The sample collection process was approved by the ethics committee, and all patients signed informed consent forms. All samples were anonymized. All esophageal cancer patients had esophageal squamous cell carcinoma. Methylation-based quantitative PCR was performed on primer-probe combinations HOXD3-I to HOXD3-M according to the method in Example 3. The sensitivity and specificity for detecting esophageal cancer plasma samples were calculated, and the results are shown in Table 9.

[0051] Table 9. Sensitivity and specificity of different primer-probe combinations for detecting esophageal cancer plasma samples.

[0052] As shown in Table 9, among all primer-probe combinations from HOXD3-I to HOXD3-M, the HOXD3-L combination exhibited the best overall diagnostic performance. Specifically, HOXD3-L demonstrated the highest specificity (96.67%) in normal human samples. The HOXD3-L primer-probe combination showed extremely high sensitivity for high-grade lesions, early-stage esophageal cancer, and advanced-stage esophageal cancer, at 87.14%, 95.00%, and 96.67%, respectively. These results indicate that a probe length of 16 bp for the HOXD3 gene resulted in the most efficient capture of methylation sites on the HOXD3 gene.

[0053] Example 8: Detection of esophageal cancer using TLX2-F combined with OXD3-F primer and probe combination. Blood samples were collected from 60 patients with clinically and pathologically confirmed high-grade esophageal squamous cell carcinoma, 80 patients with advanced esophageal squamous cell carcinoma, 90 patients with early-stage esophageal squamous cell carcinoma, and 60 healthy individuals from an affiliated hospital in Xinxiang. The sample collection process was approved by the ethics committee, and all patients signed informed consent forms. All samples were anonymized. Methylation-based quantitative PCR was performed using the primer-probe combination TLX2-F and HOXD3-F according to the method in Example 3. The sensitivity and specificity for detecting esophageal cancer plasma samples were calculated, and the results are shown in Table 10.

[0054] Table 10 Sensitivity and specificity of different primer-probe combinations for detecting esophageal cancer plasma samples

[0055] As shown in Table 10, the combination of TLX2-F and OXD3-F primers and probes for detecting esophageal cancer achieved a specificity of 98.33% and a sensitivity of 90.00%-97.50%, indicating that the combined target detection of TLX2 and OXD3 gene methylation is beneficial to improving specificity and sensitivity, and is helpful for the diagnosis and early screening of esophageal cancer.

[0056] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A primer-probe combination for detecting esophageal cancer gene methylation, characterized in that, The TLX2 primer set includes a TLX2 primer set and / or a HOXD3 primer set, a TLX2 probe set and / or a HOXD3 probe set. The TLX2 primer set includes an upstream primer TLX2-F and a downstream primer TLX2-R, whose sequence listings are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The HOXD3 primer set includes an upstream primer HOXD3-F and a downstream primer HOXD3-R, whose sequence listings are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively. The TLX2 probe set includes a TLX2-P6 probe, whose sequence listing is shown in SEQ ID NO.

10. The HOXD3 probe set includes a HOXD3-P6 probe, whose sequence listing is shown in SEQ ID NO.

25.

2. The primer-probe combination for detecting esophageal cancer gene methylation according to claim 1, characterized in that, The 5' end of the TLX2-P6 probe is marked with FAM, and the 3' end of the TLX2-P6 probe is marked with MGB; the 5' end of the HOXD3-P6 probe is marked with VIC, and the 3' end of the HOXD3-P6 probe is marked with MGB.

3. The primer-probe combination for detecting esophageal cancer gene methylation according to claim 1, characterized in that, The base sequences of the TLX2 probe set and / or HOXD3 probe set are modified with locked nucleic acids.

4. The primer-probe combination for detecting esophageal cancer gene methylation according to claim 3, characterized in that, The locked nucleic acid modification involves a three-base modification of the core CG site, and the probe lengths of the TLX2 probe set and / or HOXD3 probe set are 12-16 bp.

5. An esophageal cancer gene methylation detection kit, characterized in that, Includes the primer-probe combination for detecting esophageal cancer gene methylation as described in any one of claims 1-4.

6. The esophageal cancer gene methylation detection kit according to claim 5, characterized in that, It also includes PCR primers and probes for the internal reference gene ACTB. The PCR primers for the internal reference gene ACTB include upstream primer AB-F and downstream primer AB-R, the sequence listings of which are shown in SEQ ID NO.33 and SEQ ID NO.34, respectively. The PCR probes for the internal reference gene ACTB include the AB-P1 probe, the sequence listing of which is shown in SEQ ID NO.

35.

7. The esophageal cancer gene methylation detection kit according to claim 5, characterized in that, The 5' end of the AB-P1 probe is marked with ROX, and the 3' end of the AB-P1 probe is marked with MGB.

8. The esophageal cancer gene methylation detection kit according to claim 5, characterized in that, The final concentration composition of the reaction system of the kit includes: 12.5 μL of premix, 0.4 μL of upstream primer of the target sequence, 0.4 μL of downstream primer of the target sequence, 0.2 μL of target sequence detection probe, 0.4 μL of upstream primer of internal reference gene, 0.4 μL of downstream primer of internal reference gene, 0.2 μL of internal reference gene probe, 10 μL of transformed DNA template, and 0.5 μL of pure water.

9. The esophageal cancer gene methylation detection kit according to claim 5, characterized in that, The reaction conditions for the kit are: pre-denaturation at 95°C for 5 min; followed by denaturation at 95°C for 15 s and annealing extension at 60°C for 40 s, for 45 cycles.

10. The use of the primer-probe combination for detecting esophageal cancer gene methylation as described in any one of claims 1-4 in the detection of esophageal cancer.

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