Journal article
One-PCR-tube approach for in situ DNA isolation and detection
Analyst (London), v 136(20), pp 4254-4259
01 Jan 2011
PMID: 21879029
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Traditional real-time polymerase chain reaction (PCR) requires a purified DNA sample for PCR amplification and detection. This requires PCR tests be conducted in clean laboratories, and limits its applications for field tests. This work developed a method that can carry out DNA purification, amplification and detection in a single PCR tube. The polypropylene PCR tube was first treated with chromic acid and peptide nucleic acids (PNA) as DNA-capturer were immobilized on the internal surface of the tube. Cauliflower mosaic virus 35S (CaMV-35S) promoter in the crude extract was hybridized with the PNA on the tube surface, and the inhibitors, interfering agents and irrelevant DNA in the crude extract were effectively removed by rinsing with buffer solutions. The tube that has captured the target DNA can be used for the following real-time PCR (RT-PCR). By using this approach, the detection of less than 2500 copies of 35S plasmids in a complex sample could be completed within 3 hours. Chocolate samples were tested for real sample analysis, and 35S plasmids in genetically modified chocolate samples have been successfully identified with this method in situ. The novel One-PCR-tube method is competitive for commercial kits with the same time and simpler operation procedure. This method may be widely used for identifying food that contains modified DNA and specific pathogens in the field.
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Details
- Title
- One-PCR-tube approach for in situ DNA isolation and detection
- Creators
- Xin Huang - Chinese Academy of Inspection and QuarantineLihua Hou - Chinese Academy of Inspection and QuarantineXiaohe Xu - Drexel UniversityHongjun Chen - Chinese Academy of Inspection and QuarantineHaifeng Ji - Drexel UniversityShuifang Zhu - Chinese Academy of Inspection and Quarantine
- Publication Details
- Analyst (London), v 136(20), pp 4254-4259
- Publisher
- Royal Soc Chemistry
- Number of pages
- 6
- Grant note
- 1R01NS057366 / National Institutes of Health (NIH); United States Department of Health & Human Services; National Institutes of Health (NIH) - USA R01NS057366 / NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Neurological Disorders & Stroke (NINDS) 2008ZX08012-001; 2011ZX08012-001; 2009ZX08012-001B / Important National Science & Technology Specific Projects
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Chemistry
- Web of Science ID
- WOS:000295334000025
- Scopus ID
- 2-s2.0-80053331476
- Other Identifier
- 991019168669804721
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- Collaboration types
- Domestic collaboration
- International collaboration
- Web of Science research areas
- Chemistry, Analytical