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Methylated SEPT9 showed relatively low sensitivity in detecting early stage colorectal cancer (CRC) and advanced adenomas (AA) in plasma. Combination of multiple biomarkers was an effective strategy to improve sensitivity in early stage cancer diagnosis and screening. A new qPCR‐based assay combining the detection of methylated SEPT9 and SDC2 (ColoDefense test) was used. Methylation statuses of SEPT9 and SDC2 were examined in 40 sets of cancer tissues and paired adjacent tissues, 10 adenomatous polyps and 3 hyperplastic polyps (HP). Then evaluated with 384 plasma samples, including 117 CRC patients, 23 AA patients, 78 small polyps patients, and 166 normal individuals. The limit of detection of ColoDefense was about 25 pg per reaction. Both SEPT9 and SDC2 were shown by ColoDefense to be heavily methylated in CRC tissues when compared to paired paracancerous tissues and HP …