Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

Liu, Wei; Huang, Simo; Liu, Ningwei; Dong, Derong; Yang, Zhan; Tang, Yu Lan; Ma, Wei; He, Xiaoming; Ao, Da; Xu, Yanyu; Zou, Dayang; Huang, Liuyu

doi:10.1038/srep40125

Cited by 102 publications

(89 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…To improve an assay's analytical specificity and sensitivity, one strategy is to eliminate the detection of non-specific amplification. In bulk LAMP experiments, non-specific amplification can be excluded from detection by using probes, beacons, FRET or reporter-quencher schemes that show only specific amplification of the target (10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Although these methods improve the assay, they do not capture non-specific reactions and thus cannot give insights into the origin of non-specific amplification or the conditions that led to non-specific amplicons.…”

Section: Introductionmentioning

confidence: 99%

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Rolando

Jue

Barlow

et al. 2020

Nucleic Acids Research

142

View full text Add to dashboard Cite

Isothermal amplification assays, such as loopmediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.

show abstract

Section: Introductionmentioning

confidence: 99%

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Rolando

Jue

Barlow

et al. 2020

Nucleic Acids Research

142

View full text Add to dashboard Cite

show abstract

“…In a recent approach, MBc was used during the LAMP reaction for the specific and quantitative detection of NA (Liu and others ). MBc is a single‐chain oligonucleotide having a hairpin structure in such a manner that the fluorophore and quencher remain in close proximity (Figure ) which prevent the fluorescence to occur.…”

Section: Advances In Lamp Methods For Quality Assessment Of Meat Prodmentioning

confidence: 99%

“…The problem of nonspecific detection by indirect evaluation methods can be avoided by the use of molecular beacons (MBc), which produce fluorescence signals only after binding to the specific target DNA (Liu and others ). MBc is an oligonucleotide probe having self‐complementary ends and a single‐stranded hairpin structure in its native state.…”

Section: Loop‐mediated Isothermal Amplificationmentioning

confidence: 99%

Loop‐Mediated Isothermal Amplification (LAMP): A Rapid and Sensitive Tool for Quality Assessment of Meat Products

Kumar

Bansal

Jaiswal

2017

Comp Rev Food Sci Food Safe

View full text Add to dashboard Cite

Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies target nucleic acids under isothermal conditions. It is a rapid, specific, and sensitive method, which does not require costly thermal cyclers for the detection of nucleic acids. Thus, it is suitable for on-site detection assays under low-resource settings. It can also be integrated on compact lab-on-a-chip devices for the development of micro-total analysis systems. This review discusses LAMP-based methods, as well as LAMP-based centrifugal, microfluidic, and other fluid-handling devices, which have been developed for the assessment of meat quality parameters that are related to the presence or absence of nucleic acids, for example, animal species identification and microbiological quality. Advances in improving the rapidity, specificity, and sensitivity of LAMP techniques for the assessment of these meat quality parameters are also discussed in this review.

show abstract

“…The hairpin structure avoids the fluorescence because quencher is physically closed to fluorophore. Moreover, binding to LAMP product sequences changes the MB spatial configuration, which separate fluorophore and quencher at both the ends of a single strand of nucleic acids, and desorbing fluorescence (Liu et al 2017). Ascochyta blight disease (which is caused by Ascochyta rabiei L. fungi) in chickpeas has been diagnosed using LAMP method.…”

Section: Loop-mediated Isothermal Amplificationmentioning

confidence: 99%

Recent advances in molecular techniques for the identification of phytopathogenic fungi – a mini review

Aslam

Tahir

Aslam

et al. 2017

Journal of Plant Interactions

View full text Add to dashboard Cite

At present, 1.5 million species of fungi are estimated. Among these less than 5% have been described. Many fungal species cause disease in plants. These diseases cause major economic and production losses in the agricultural industry worldwide. Monitoring plant health and detecting the pathogen early are essential to reduce the disease spread, and facilitate effective management practices. DNA-based methods now provide essential tools for accurate plant disease diagnosis. Recently, effective amplification platforms, probe development, various quantitative PCR, DNA barcoding and RNA-Seq-based next-generation sequencing have revolutionized the research in fungal detection field, and differentiation area. Although the molecular diagnostics techniques have grown extensively over the last couple of decades but still there is a long way to go in the development and application of molecular diagnostics to assist the plant disease diagnosticians. Finally, molecular diagnostic techniques used in plant disease diagnostic clinics need to be robust, reliable, inexpensive and easy to be used that they can compete with, and complement traditional techniques. Challenge now remains residue with the researchers to develop the practical techniques used for diagnostic setting. Examples of the recent advancement in the molecular techniques for diagnosing the fungi causing plant disease are discussed in the review.

show abstract

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

Cited by 102 publications

References 23 publications

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Loop‐Mediated Isothermal Amplification (LAMP): A Rapid and Sensitive Tool for Quality Assessment of Meat Products

Recent advances in molecular techniques for the identification of phytopathogenic fungi – a mini review

Contact Info

Product

Resources

About