A simplified multiplex methylated DNA testing for early detection of colorectal cancer in stool DNA

BACKGROUND: ColoDefense1.0 assay has demonstrated its excellent sensitivity and specificity for early detection of colorectal cancer (CRC) by detecting the methylation levels of SDC2 and SEPT9, while exhibited limitations on relatively large sample capacity required and limited detection throughput by applying triplicate PCR reactions for each sample. In this study, ColoDefense1.0 was simplified and optimized into ColoDefense2.0 in a single PCR reaction. METHODS: A total 529 stool specimens were collected, and 244 CRC patients, 34 patients with advanced adenomas (AA), 64 with small polyps (SP) and 187 control subjects were divided in training and validation cohorts. Methylation levels of SEPT9 and SDC2 were examined by qPCR reactions in triplicate or single. RESULTS: The stool DNA quantity stored in preservative buffer at 37 °C up to 7 days exhibited no significant decrease. In the training cohort, when the number of replicates reduced from 3 to 1, the overall performance of ColoDefense2.0 was identical to that of ColoDefense1.0, showing sensitivities of 71.4% for AA and 90.8% for all stage CRC with a specificity of 92.9%. In the validation cohort, sensitivities of SP, AA and CRC using ColoDefense2.0 were 25.0%, 55.0% and 88.2%, increased from 14.1% (20.3%), 40.0% (40.0%) and 79.4% (67.6%) using SDC2 (SEPT9) alone; along with an overall specificity of 90.2%, decreased from 94.1% (95.1%) using SDC2 (SEPT9) alone. CONCLUSION: The simplified ColoDefense test maintained the overall performance while reduced the number of PCR reactions to 1/3, and provided an effective and convenient tool to detect early CRC and precancerous lesions and potentially improve the compliance of screening. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12876-022-02512-6.