Test composition for screening cancers

Materials and Methods

[0030]First, DNA was extracted from the test sample and treated with bisulfate. Use the primer sets and probes (see Table 1 to Table 5) disclosed in Table 1 to Table 4 for amplification of PAX1, ZNF582, SOX1 and NKX6-1 as well as the internal control genes for detection. The main components of the test composition are shown in Table 6:

TABLE 1Primer sets and probes used for amplification ofthe methylation sites of the target gene PAX1ID No:primer sets and probes for PAX1SEQ ID No: 15′ attcgcgcgttttcggcgtga 3′SEQ ID No: 25′ gttaaattgattttcgtacgttgtag 3′SEQ ID No: 35′ tattttgggtttggggtcgc 3′SEQ ID No: 45′ ttattttgggtttggggtcgcg 3′SEQ ID No: 55′ gggcggtagcgcgtttcgtt 3′SEQ ID No: 65′ tagcggcggcggtaggttttgga 3′SEQ ID No: 75′ gtagtgacgggaattaatgagt 3′SEQ ID No: 85′ aacatcccacgaccacgccg 3′SEQ ID No: 95′ acgaccacgccgaaaaccgt 3′SEQ ID No: 105′ acaaacaacgaaaaatacgcg 3′SEQ ID No: 115′ acgacgaaaaaaacgacgacg 3′SEQ ID No: 125′ ttaaattgattttcgtacgttgtag 3′SEQ ID No: 135′ gcgacccca

Analysis of the Methylation Status of the Target Genes in Different Cancer Cell Lines

[0033]This test utilized the primer sets and probes disclosed in Table 1 to Table 4 for amplification of the methylated regions of PAX1, ZNF582, SOX1 and NKX6-1 in different cancer cell lines, and the results of the methylation status are shown in Table 7. The results indicate that methylation of PAX1, ZNF582, SOX1 and NKX6-1 was detected in cervical cancer cell line, Hela; methylation of PAX1, ZNF582, and SOX1 was detected in cervical cancer cell line, SiHa; methylation of PAX1, ZNF582, SOX1 and NKX6-1 was detected in cervical cancer cell line, CaSki; methylation of ZNF582, SOX1 and NKX6-1 was detected in cervical cancer cell line, C-33 A; methylation of PAX1, ZNF582, SOX1 and NKX6-1 was detected in colorectal cancer cell line, COLO 205; methylation of ZNF582, SOX1 and NKX6-1 was detected in colorectal cancer cell line, Caco-2; methylation of PAX1, ZNF582, SOX1 and NKX6-1 was detected in colorectal ca

Analysis of the Methylation Status of the Target Genes in Cervical Cancer Samples

[0035]The test was conducted on 279 diagnosed normal and cervical cancer samples collected in Taiwan and as shown in Table 8, 239 samples are normal (85.7%), 22 samples are CIN1 (7.9%), 2 samples are CIN2 (0.7%), 12 samples are CIN3 / CIS (4.3%), and 4 samples are squamous cell carcinoma (1.4%). The DNA of these samples was extracted and then treated with bisulfite, and the primer sets and probes disclosed in Table 1 to Table 4 for amplification of the methylated regions in PAX1, ZNF582, SOX1, and NKX6-1 were used for detection. As indicated in Table 9, when compared with the normal cervical samples, using PAX1, ZNF582, SOX1, and NKX6-1 as the target gene to examine the severe cervical dysplasia samples showed 85.45-fold (95% CI=33.95-215.11), 289.17-fold (95% CI=39.20˜2133.14), 67.69-fold (95% CI=20.55-223.01) and 2.56-fold (95% CI=1.36 to 4.82) increased the occurrence of severe cervical cancer, respecti