LAVA: An Open-Source Approach To Designing LAMP (Loop-Mediated Isothermal Amplification) DNA Signatures

Torres, Clinton; Vitalis, Elizabeth; Baker, Brian R.; Gardner, Shea N.; Torres, Matthew P.; Dzenitis, John M.

doi:10.1186/1471-2105-12-240

Cited by 73 publications

(51 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…One of our assays, HMS Assay 1, performed particularly well compared to the others (data not shown for others). We then modified the forward inner primer (FIP) and backward inner primer (BIP) of this assay to include a "TTTT" linker between the F1c and F2 regions to create HMS Assay 1e, as this has been reported to further improve the reaction (for HMS Assay 1 and HMS Assay 1e oligo sequences, see Table 1) 5 . HMS Assay 1/1e is designed within the ORF1ab of SARS-CoV-2 in a region that is not highly conserved with either SARS or Bat SARS-like coronavirus isolate Rs4084, two closely related coronaviruses ( Figure 1).…”

Section: Introduction/backgroundmentioning

confidence: 99%

SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification

Rabe

Cepko

2020

Preprint

View full text Add to dashboard Cite

As the current SARS-CoV-2 pandemic spreads, the need for more diagnostic capabilities is great. In order to address this need, we have developed a highly sensitive RT-LAMP assay compatible with current reagents, that utilizes a colorimetric readout in as little as 30 minutes. In addition to this, we have developed an inexpensive pipeline to further increase sensitivity without requiring highly specialized equipment. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was also developed to increase sensitivity and sample stability. This protocol, combined with our RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with our RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. We hope that this inactivation and purification pipeline, which costs approximately $0.07 per sample and which uses readily available reagents, will increase the availability of SARS-CoV-2 testing, as well as expand the settings in which this testing can be performed.

show abstract

Section: Introduction/backgroundmentioning

confidence: 99%

SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification

Rabe

Cepko

2020

Preprint

View full text Add to dashboard Cite

show abstract

“…21 A combination of LAMP primers has been used in multiplex reactions 22,23 and various approaches to designing the primers exist. 24 Considering the various time points and processes used to prepare DNA from blood culture for the PCR process, certain aspects can be observed. For example, for the majority of positive samples, two pre-processing steps and LAMP technology for amplification gave the best results.…”

Section: Discussionmentioning

confidence: 99%

Loop Mediated Isothermal PCR (LAMP) for the Detection of Borrelia from Blood Cultures

Wood¹

2015

JMEN

View full text Add to dashboard Cite

Borrelia organisms were harvested from blood culture and tested using single primers and loop mediated isothermal PCR (LAMP). Samples were harvested at either 6 days or at 8 weeks and DNA was extracted using one of three methods. In addition, pre-processing steps included Liberase treatments to release organisms from any residual attached collagen and human DNA removal. Combinations of processes were used to determine the best procedures for harvesting organisms, processing the pellet, and extracting DNA prior to using PCR. Of the total number of positive signals obtained, 9 of 45 (20%) gave positive signals when using plasmid primers specific for B. burgdorferi, 14 of 40 (35%) were positive when using OspC primers, and 25 of 47 (53%) were positive when using LAMP primers for the DNA that codes for 16S rRNA.

show abstract

“…LAMP is more resistant to these inhibitors than standard PCR, which would make LAMP a more ideal method for detecting DNA in body fluid samples and would require less prior sample preparation such as DNA/RNA extraction [98]. However, the use of LAMP is limited by the difficult design of primers, requiring the use of software kits [99], as well as being more restricted in its range of designs compared to conventional PCR markers. This is particularly a concern for forensic applications as some biomarkers may have transcriptional variants, where the design of primers would have to be centred around a common sequence.…”

Section: Development Of Reliable and Rapid Isothermal Pcr Would Be Ofmentioning

confidence: 99%

Field-based detection of biological samples for forensic analysis: Established techniques, novel tools, and future innovations

Morrison

Watts

Hobbs

et al. 2018

Forensic Science International

View full text Add to dashboard Cite

Field based forensic tests commonly provide information on the presence and identity of biological stains and can also support the identification of species. Such information can support downstream processing of forensic samples and generate rapid intelligence. These approaches have traditionally used chemical and immunological techniques to elicit the result but some are known to suffer from a lack of specificity and sensitivity. The last 10 years has seen the development of field-based genetic profiling systems, with specific focus on moving the mainstay of forensic genetic analysis, namely STR profiling, out of the laboratory and into the hands of the non-laboratory user. In doing so it is now possible for enforcement officers to generate a crime scene DNA profile which can then be matched to a reference or database profile. The introduction of these novel genetic platforms also allows for further development of new molecular assays aimed at answering the more traditional questions relating to body fluid identity and species detection. The current drive for field-based molecular tools is in response to the needs of the criminal justice system and enforcement agencies, and promises a step-change in how forensic evidence is processed. However, the adoption of such systems by the law enforcement community does not represent a new strategy in the way forensic science has integrated previous novel approaches. Nor do they automatically represent a threat to the quality control and assurance practices that are central to the field. This review examines the historical need and subsequent research and developmental breakthroughs in field-based forensic analysis over the past two decades with particular focus on genetic methods Emerging technologies from a range of scientific fields that have potential applications in forensic analysis at the crime scene are identified and associated issues that arise from the shift from laboratory into operational field use are discussed.

show abstract

LAVA: An Open-Source Approach To Designing LAMP (Loop-Mediated Isothermal Amplification) DNA Signatures

Cited by 73 publications

References 12 publications

SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification

SARS-CoV-2 Detection Using an Isothermal Amplification Reaction and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification

Loop Mediated Isothermal PCR (LAMP) for the Detection of Borrelia from Blood Cultures

Field-based detection of biological samples for forensic analysis: Established techniques, novel tools, and future innovations

Contact Info

Product

Resources

About