Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16

Landaverde, Lena; Wong, Winnie; Hernández, Gabriela; Fan, Andy; Klapperich, Catherine M.

doi:10.1007/s00216-020-02702-9

Cited by 12 publications

(23 citation statements)

References 22 publications

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“…There have been many efforts to reduce LAMP false-positives caused by nonspecific amplification. These include using fluorescently tagged primers to minimize false-positives, 45 , 46 mathematical modeling to predict desired LAMP amplicon sizes, 47 and the inclusion of dimethyl sulfoxide (DMSO) in the amplification. 48 , 49 DMSO is a hazardous chemical, and its application in a LAMP reaction requires significant optimization.…”

Section: Resultsmentioning

confidence: 99%

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Electrochemical Strategy for Low-Cost Viral Detection

et al. 2021

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Sexually transmitted infections, including the human immunodeficiency virus (HIV) and the human papillomavirus (HPV), disproportionally impact those in low-resource settings. Early diagnosis is essential for managing HIV. Similarly, HPV causes nearly all cases of cervical cancer, the majority (90%) of which occur in low-resource settings. Importantly, infection with HPV is six times more likely to progress to cervical cancer in women who are HIV-positive. An inexpensive, adaptable point-of-care test for viral infections would make screening for these viruses more accessible to a broader set of the population. Here, we report a novel, cost-effective electrochemical platform using gold leaf electrodes to detect clinically relevant viral loads. We have combined this platform with loop-mediated isothermal amplification and a CRISPR-based recognition assay to detect HPV. Lower limits of detection were demonstrated down to 10 4 total copies of input nucleic acids, which is a clinically relevant viral load for HPV DNA. Further, proof-of-concept experiments with cervical swab samples, extracted using standard extraction protocols, demonstrated that the strategy is extendable to complex human samples. This adaptable technology could be applied to detect any viral infection rapidly and cost-effectively.

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Section: Resultsmentioning

confidence: 99%

“…The BD SurePath samples were prepared as previously reported. 46 In short, clinical samples were taken from the container and aliquoted into 1.7-mL tubes. In order to pellet the sample, all tubes were spun down at 13 000 rpm.…”

Section: Materials and Methodsmentioning

confidence: 99%

Electrochemical Strategy for Low-Cost Viral Detection

et al. 2021

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“…Results demonstrated the expected wide distribution of fragment sizes as described by others. 1,2,17,18 Qubit quantification of Qiagen-purified amplicon product exceeded 6 mg total, whereas NTCs were below 100-300 ng (data not shown).…”

Section: T a B L Ementioning

confidence: 94%

Molecular Characterization of Increased Amplicon Lengths in SARS-CoV-2 Reverse Transcription Loop-Mediated Isothermal Amplification Assays

et al. 2021

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Loop-mediated isothermal amplification (LAMP) is a power tool for the amplification of specific RNA and DNA targets. Much like PCR, LAMP requires primers that surround a target amplification region and generates exponential product through a unique highly specific daisy-chain single-temperature amplification reaction. However, until recently, attempts to amplify targets of greater than 200 base pairs (bp) have been mostly unsuccessful and limited to short amplicon targets of less than 150 bp. Although short amplicons have the benefit of a rapid detection (,40 min), they do not allow for the prediction of RNA integrity based on RNA length and possible intactness. In this study, 8 primer sets were developed using 2 LAMP primer-specific software packages against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid gene with insert lengths ranging from 262 to 945 bp in order to amplify and infer the integrity of viral RNA. Because these amplification lengths are greater than the current methods that use an insert length of 130 or less, they require a longer incubation, modified primer and temperature strategies, and the addition of specific adjuncts to prevent nonspecific amplification. This proof of concept study resulted in successful reverse transcription LAMP reactions for amplicon targets of 262, 687, 693, and 945 bp using a clinical nasopharyngeal NP sample, purified SARS-CoV-2 RNA, and crude lysate containing inactivated virus.

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“…The polymerase chain reaction (PCR) is one of the most commonly used methods in clinical practice, which can amplify a target sequence more than a million times in a brief period and requires small sample volumes [ 3 ]. Other available techniques include the rolling circle amplification reaction (RCA) [ 4 , 5 ], the hybridisation chain reaction (HCR) [ 6 ], and loop-mediated isothermal amplification (LAMP) [ 7 , 8 , 9 ]. However, these techniques present a series of limitations.…”

Section: Introductionmentioning

confidence: 99%

Single-Molecule Detection of Nucleic Acids via Liposome Signal Amplification in Mass Spectrometry

Lin

Zhao

et al. 2022

Sensors

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A single-molecule detection method was developed for nucleic acids based on mass spectrometry counting single liposome particles. Before the appearance of symptoms, a negligible amount of nucleic acids and biomarkers for the clinical diagnosis of the disease were already present. However, it is difficult to detect extremely low concentrations of nucleic acids using the current methods. Hence, the establishment of an ultra-sensitive nucleic acid detection technique is urgently needed. Herein, magnetic beads were used to capture target nucleic acids, and liposome particles were employed as mass tags for single-particle measurements. Liposomes were released from magnetic beads via photocatalytic cleavage. Hence, one DNA molecule corresponded to one liposome particle, which could be counted using mass spectrometric measurement. The ultrasensitive detection of DNA (10–18 M) was achieved using this method.

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Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16

Cited by 12 publications

References 22 publications

Electrochemical Strategy for Low-Cost Viral Detection

Electrochemical Strategy for Low-Cost Viral Detection

Molecular Characterization of Increased Amplicon Lengths in SARS-CoV-2 Reverse Transcription Loop-Mediated Isothermal Amplification Assays

Single-Molecule Detection of Nucleic Acids via Liposome Signal Amplification in Mass Spectrometry

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