One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei

Tzeling, Jilien Michelle Wong; Syafirah, E.A.R. Engku Nur; Irekeola, Ahmad Adebayo; Yusof, Wardah; Baki, Nurul Najian Aminuddin; Zueter, AbdelRahman; Harun, Azian; Chan, Yean Yean

doi:10.1016/j.aca.2021.338682

Cited by 13 publications

(6 citation statements)

References 32 publications

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“…The specificity of the crBP34-DETECTR may arise from the designed crRNA—crBP34, which was a result of large-scale phylogenetics and bioinformatics analyses to remove cross-reactivity ( Fig 1B and 1C ). This contrasts with previously reported detection assays using real-time PCR [ 21 – 25 ], LAMP [ 26 , 27 ] or RPA [ 28 – 30 ], in which primers were designed based on selected genomic loci or limited bioinformatics analysis on few reference strains. Testing crBP34 and the other crRNAs with B .…”

Section: Discussionmentioning

confidence: 75%

“…Although these methods showed high sensitivity and specificity, real-time PCR-based assays require expensive real-time PCR machines, which may not be readily available in many laboratories. As alternatives, isothermal DNA amplifications such as loop-mediated isothermal amplification (LAMP) [26,27] and recombinase polymerase amplification (RPA) [28][29][30] have been developed to overcome this dependency on expensive equipment. Both assays exhibit very sensitive detection.…”

Section: Introductionmentioning

confidence: 99%

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Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

et al. 2022

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Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.

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Section: Discussionmentioning

confidence: 75%

Section: Introductionmentioning

confidence: 99%

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

et al. 2022

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“…This design reduces crosstalk, enhances amplification, and diminishes background noise. 19 In addition, the use of FQ probes does not require a complex pre-processing procedure. Due to the complexity of distinguishing non-specific LAMP amplicons from target amplicons, FQ probes are more specific than conventional LAMP assays using DNA intercalating dyes and can significantly reduce background signals.…”

Section: Discussionmentioning

confidence: 99%

Digital Nanofluidic Chip for Simple and Highly Quantitative Detection of HPV Target

Liu,

Dollery,

Tobin

et al. 2024

Preprint

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Quantitative analysis of human papillomavirus (HPV)-infected cervical cancer is essential for early diagnosis and timely treatment of cervical cancer. Here, we introduce a novel energy transfer-labeled oligonucleotide probe to enhance the loop-mediated isothermal amplification (LAMP) assay for highly sensitive and specific detection of HPV 16. Conducted as a single-step assay within a digital nanofluidic chip featuring numerous reaction reservoirs, our method facilitates target amplification under isothermal conditions. Targeting an HPV 16 gene, our chip demonstrates the capability to detect HPV DNA at concentrations as low as 1 fM, spanning a dynamic range of five orders of magnitude. Importantly, our digital chip allows for highly quantitative detection of target genes at low concentrations, with the correlation between target concentration and the number of microwells exhibiting fluorescence signals. Furthermore, we have developed a computer vision method for automated and 100% accurate quantification of target concentrations. This research holds promising applications in clinical diagnosis and is poised for seamless integration into both hospital and point-of-care settings.

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“…IBFQ and IBRQ have been paired with fluorescein, TYE 665, and Cy5.5, respectively, and these FRET pairs have been used in the construction of DNA logic gates, [172] evaluation of anti‐CMV and anti‐HSV‐2 antibodies, [173] and the detection of MSCs, osteogenic differentiation, [174] and B. pseudomallei genomic DNA [175] …”

Section: Structure–property Relationship and Application Of Quenchers...mentioning

confidence: 99%

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

et al. 2022

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For fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state nonradiatively, and are promising alternatives to fluorescent FRET acceptors. Small-molecule (dark) quenchers have been widely used as acceptors in FRET-based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small-molecule quenchers that are used in FRET-based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophorequencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

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One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei

Cited by 13 publications

References 32 publications

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

Digital Nanofluidic Chip for Simple and Highly Quantitative Detection of HPV Target

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

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