title: Accurate and easy-to-use assessment of contiguous DNA methylation sites based on proportion competitive quantitative-PCR and lateral flow nucleic acid biosensor.
abstract: Many types of diagnostic technologies have been reported for DNA methylation, but they require a standard curve for quantification or only show moderate accuracy. Moreover, most technologies have difficulty providing information on the level of methylation at specific contiguous multi-sites, not to mention easy-to-use detection to eliminate labor-intensive procedures. We have addressed these limitations and report here a cascade strategy that combines proportion competitive quantitative PCR (PCQ-PCR) and lateral flow nucleic acid biosensor (LFNAB), resulting in accurate and easy-to-use assessment. The P16 gene with specific multi-methylated sites, a well-studied tumor suppressor gene, was used as the target DNA sequence model. First, PCQ-PCR provided amplification products with an accurate proportion of multi-methylated sites following the principle of proportionality, and double-labeled duplex DNA was synthesized. Then, a LFNAB strategy was further employed for amplified signal detection via immune affinity recognition, and the exact level of site-specific methylation could be determined by the relative intensity of the test line and internal reference line. This combination resulted in all recoveries being greater than 94%, which are pretty satisfactory recoveries in DNA methylation assessment. Moreover, the developed cascades show significantly high usability as a simple, sensitive, and low-cost tool. Therefore, as a universal platform for sensing systems for the detection of contiguous multi-sites of DNA methylation without external standards and expensive instrumentation, this PCQ-PCR-LFNAB cascade method shows great promise for the point-of-care diagnosis of cancer risk and therapeutics.
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China; Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Wentao
authors.initials: W
authors.lastname: Xu
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Cheng
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China; Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Kunlun
authors.initials: K
authors.lastname: Huang
authors.affiliation: School of Mechanical and Materials Engineering, Washington State University, Pullman 99164, USA.
authors.forename: Yuehe
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Chenguang
authors.initials: C
authors.lastname: Wang
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Yuancong
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China.
authors.forename: Longjiao
authors.initials: L
authors.lastname: Zhu
authors.affiliation: School of Mechanical and Materials Engineering, Washington State University, Pullman 99164, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Du
authors.affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, China. Electronic address: lyb@cau.edu.cn.
authors.forename: Yunbo
authors.initials: Y
authors.lastname: Luo
mesh_terms: D001483:Base Sequence / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D019941:Cyclin-Dependent Kinase Inhibitor p16; D019175:DNA Methylation / Q000235:genetics*; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification*; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques
chemical_list: C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009363:Neoplasm Proteins
keywords: DNA methylation; Lateral flow nucleic acid biosensor; Proportion competitive quantitative PCR