Citation: LIU Lin-Lin, QI Xie-Min, ZOU Bing-Jie, SONG Qin-Xin, ZHOU Guo-Hua. Quantitative Detection of Methylated Gene in Stool Samples Based on Invader Assay Coupled with Real-time Polymerase Chain Reaction and Its Application in Non-invasive Screening of Colorectal Cancer[J]. Chinese Journal of Analytical Chemistry, ;2018, 46(10): 1552-1559. doi: 10.11895/j.issn.0253-3820.181117 shu

Quantitative Detection of Methylated Gene in Stool Samples Based on Invader Assay Coupled with Real-time Polymerase Chain Reaction and Its Application in Non-invasive Screening of Colorectal Cancer

1.
Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education, China Pharmaceutical University, Nanjing 210009, China;
2.
Department of Pharmacology, Jinling Hospital, Nanjing 210002, China

Received Date: 14 February 2018
Revised Date: 17 July 2018

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos. 81673390, 81603219), the Key Research Program of Jiangsu Province of China (Social Development) (No. BE2016745), the Basic Research Program of Jiangsu Province, China (Natural Science Foundation) (No. BK20151445), the Program of MOE Key Laboratory of Drug Quality Control and Pharmacovigilance of China (No. DQCP2017MS01) and the Qing Lan Project of Jiangsu Province of China.

Bone morphogenetic protein 3 (BMP3) gene methylation in stool DNA is an effective biomarker for non-invasive screening of colorectal cancer. However, the detection of BMP3 gene methylation in stool DNA requires a highly sensitive and specific method. Here, we developed a BMP3 gene methylation quantification method by combining real-time polymerase chain reaction (PCR) with invasive reaction using Beta-actin (ACTB) gene as a reference. After optimizing the concentration of detection probes, FEN1 enzyme and Taq polymerase, both amplification efficiencies of BMP3 and ACTB genes were close to 100%, and the accurate relative quantification of methylation of BMP3 gene was achieved with ΔCT algorithms. The method could detect as low as 10 copies of BMP3 fragment, and 0.01% methylated templates could be picked up from unmethylated templates, indicating that the method was highly sensitive and specific for the detection of BMP3 gene methylation. The method was successfully applied to detect BMP3 methylation in stool DNA samples from 16 colorectal cancer patients, 7 adenoma patients and 19 healthy volunteers. The result showed that methylation of BMP3 occurred in 5 cases of 16 cancer patients and 2 cases of 7 adenoma patients, but was not observed in 19 cases of healthy volunteers. Therefore, this method could be used to quantify the gene methylation in stool samples, providing an effective technique for non-invasive screening of colorectal cancer.
- DNA methylation,
- Real-time polymerase chain reaction,
- Invasive reaction,
- Colorectal cancer,
- Stool DNA
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