Details

Autor(en) / Beteiligte

• Du, Wen-Fang
• Ge, Jian-Hui
• Li, Jun-Jie
• Tang, Li-Juan
• Yu, Ru-Qin
• Jiang, Jian-Hui

Titel

Single-step, high-specificity detection of single nucleotide mutation by primer-activatable loop-mediated isothermal amplification (PA-LAMP)

Ist Teil von

• Analytica chimica acta, 2019-03, Vol.1050, p.132-138

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2019

Quelle

MEDLINE

Beschreibungen/Notizen

• Loop-mediated isothermal amplification (LAMP) is a useful platform for nucleic acids detection in point-of-care (POC) situations, and development of single-step, close-tube LAMP reactions for specific detection of single nucleotide mutations (SNMs) remains a challenge. We develop a novel primer-activatable LAMP (PA-LAMP) strategy that enables highly specific and sensitive SNM detection using single-step, close-tube reactions. This strategy designs a terminal-blocked inner primer with a ribonucleotide insertion, which is cleaved and activated specifically to perfectly matched targets by ribonuclease (RNase) H2, to realize efficient amplification of mutant genes. It has shown dynamic responses of mutant target in a linear range from 220 aM to 22 pM with a lowest detectable concentration of 22 aM. It also demonstrates very high specificity in identifying the mutant in a large excess of the wild-type with a discrimination ratio as high as ∼10,000. It has been successfully applied to mutation detection of genomic DNA in tumor cells. The PA-LAMP strategy provides a useful, portable and affordable POC platform for highly sensitive and specific detection of genetic mutations in clinical applications. [Display omitted] •A novel primer-activatable loop-mediated isothermal amplification (PA-LAMP) strategy was constructed.•The strategy enables highly specific and sensitive single nucleotide mutation detection.•This method can be used to detect single nucleotide mutation of genomic DNA in tumor cells.