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Loop-mediated isothermal amplification technology: towards point of care diagnostics.

Njiru ZK - PLoS Negl Trop Dis (2012)

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland, The School of Veterinary Sciences, Gatton Campus, Queensland, Australia. z.njiru@uq.edu.au

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The invention of the loop-mediated isothermal amplification (LAMP) method a decade ago has given new impetus towards development of point of care diagnostic tests based on amplification of pathogen DNA, a technology that has been the precinct of well-developed laboratories... The LAMP technology amplifies DNA with high sensitivity relying on an enzyme with strand displacement activity under isothermal conditions... Nucleic acid (DNA) amplification tests targeting pathogen markers have high sensitivity and specificity but generally fail to meet the ASSURED guidelines in terms of affordability, rapidity, and being equipment free... However, with the recent invention and advancement of isothermal technologies, development of ASSURED tests based on DNA amplification seems realistic... One such potential method is the LAMP technology, which has salient advantages over most DNA-based amplification tests (Box 1)... The LAMP method has the advantage of amplifying the target DNA from partially processed and/or non-processed samples... This inherent advantage of LAMP shortens the reaction time and eliminates the need for DNA extraction, a step that is prone to contamination and may result in significant loss of DNA... The preparation of template DNA is the least developed method associated with LAMP technology... A higher test specificity can be achieved by targeting an internal sequence of the amplicon through incorporating fluorescent molecular beacon probe, thus minimising non-specific signal, or by using a lateral flow dipstick (LFD) format (Figure 1G), though at a slightly higher cost... In general, the use of dyes is most preferred because they are cheap and most allow definite visual inspection of results based on colour change... However, the need for treatment and/or a decision on case management dictates unequivocal result interpretation... For example, it is worth noting that the human African trypanosomiasis case definition requires demonstration of trypanosomes in the body fluid, and that the LAMP test may not be relied upon to make a treatment decision... Nevertheless, the expected high specificity of LAMP may reduce costs associated with serological false positives that card agglutination test for trypanosomiasis (CATT) registers, of which only few end up being genuine HAT cases.

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A proposed three-step LAMP method for diagnosis of neglected tropical diseases.Step 1 includes processing of varied specimen (A) through boiling or use of kits to yield a stable and concentrated DNA template (B). In step 2, the lyophilised master mix (C) is reconstituted by the addition of water and the ideal amount of DNA template. In step 3, the amplification and the detection format are combined into a single step to avoid opening the tube (D), hence the results can be acquired in real time (E) through incubation of the reaction with a reporting dye (F) and through the use of a novel LAMP LFD format.
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pntd-0001572-g001: A proposed three-step LAMP method for diagnosis of neglected tropical diseases.Step 1 includes processing of varied specimen (A) through boiling or use of kits to yield a stable and concentrated DNA template (B). In step 2, the lyophilised master mix (C) is reconstituted by the addition of water and the ideal amount of DNA template. In step 3, the amplification and the detection format are combined into a single step to avoid opening the tube (D), hence the results can be acquired in real time (E) through incubation of the reaction with a reporting dye (F) and through the use of a novel LAMP LFD format.

Mentions: The LAMP method has the advantage of amplifying the target DNA from partially processed and/or non-processed samples [9]. This inherent advantage of LAMP shortens the reaction time and eliminates the need for DNA extraction, a step that is prone to contamination and may result in significant loss of DNA. The preparation of template DNA is the least developed method associated with LAMP technology. For example, the ideal specimens (biological fluids, tissue, swabs, scraping etc.) (Figure 1A) for LAMP reactions are yet to be determined. The direct use of native cerebrospinal fluid, serum, heat-treated blood [10], and addition of detergent [11] have yielded viable DNA templates; however, precise preparation protocols need to be defined and optimised. Nevertheless, these results offer exciting possibilities that definition of a simple field-based template preparation protocol is possible. To improve the performance of LAMP tests, methodologies for specimen collection and processing need to be simple, optimised, and ensure high target yields. In addition, selected buffers should not only stabilise the DNA (reduce degradation in case of storage), but should also enhance amplification whenever possible. An ideal template protocol will thus depend on the sample being tested. For example, whole blood, stool, and tissue specimens may include direct boiling followed by a single buffer-purification step to separate the template from the debris or application of a sample preparation kit that readily removes unwanted biological products followed by the collection and concentration of the template (Figure 1B). Such developments should be approached with the primary aim of reducing cost and potential LAMP inhibitors.


Loop-mediated isothermal amplification technology: towards point of care diagnostics.

Njiru ZK - PLoS Negl Trop Dis (2012)

A proposed three-step LAMP method for diagnosis of neglected tropical diseases.Step 1 includes processing of varied specimen (A) through boiling or use of kits to yield a stable and concentrated DNA template (B). In step 2, the lyophilised master mix (C) is reconstituted by the addition of water and the ideal amount of DNA template. In step 3, the amplification and the detection format are combined into a single step to avoid opening the tube (D), hence the results can be acquired in real time (E) through incubation of the reaction with a reporting dye (F) and through the use of a novel LAMP LFD format.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383729&req=5

pntd-0001572-g001: A proposed three-step LAMP method for diagnosis of neglected tropical diseases.Step 1 includes processing of varied specimen (A) through boiling or use of kits to yield a stable and concentrated DNA template (B). In step 2, the lyophilised master mix (C) is reconstituted by the addition of water and the ideal amount of DNA template. In step 3, the amplification and the detection format are combined into a single step to avoid opening the tube (D), hence the results can be acquired in real time (E) through incubation of the reaction with a reporting dye (F) and through the use of a novel LAMP LFD format.
Mentions: The LAMP method has the advantage of amplifying the target DNA from partially processed and/or non-processed samples [9]. This inherent advantage of LAMP shortens the reaction time and eliminates the need for DNA extraction, a step that is prone to contamination and may result in significant loss of DNA. The preparation of template DNA is the least developed method associated with LAMP technology. For example, the ideal specimens (biological fluids, tissue, swabs, scraping etc.) (Figure 1A) for LAMP reactions are yet to be determined. The direct use of native cerebrospinal fluid, serum, heat-treated blood [10], and addition of detergent [11] have yielded viable DNA templates; however, precise preparation protocols need to be defined and optimised. Nevertheless, these results offer exciting possibilities that definition of a simple field-based template preparation protocol is possible. To improve the performance of LAMP tests, methodologies for specimen collection and processing need to be simple, optimised, and ensure high target yields. In addition, selected buffers should not only stabilise the DNA (reduce degradation in case of storage), but should also enhance amplification whenever possible. An ideal template protocol will thus depend on the sample being tested. For example, whole blood, stool, and tissue specimens may include direct boiling followed by a single buffer-purification step to separate the template from the debris or application of a sample preparation kit that readily removes unwanted biological products followed by the collection and concentration of the template (Figure 1B). Such developments should be approached with the primary aim of reducing cost and potential LAMP inhibitors.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland, The School of Veterinary Sciences, Gatton Campus, Queensland, Australia. z.njiru@uq.edu.au

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

The invention of the loop-mediated isothermal amplification (LAMP) method a decade ago has given new impetus towards development of point of care diagnostic tests based on amplification of pathogen DNA, a technology that has been the precinct of well-developed laboratories... The LAMP technology amplifies DNA with high sensitivity relying on an enzyme with strand displacement activity under isothermal conditions... Nucleic acid (DNA) amplification tests targeting pathogen markers have high sensitivity and specificity but generally fail to meet the ASSURED guidelines in terms of affordability, rapidity, and being equipment free... However, with the recent invention and advancement of isothermal technologies, development of ASSURED tests based on DNA amplification seems realistic... One such potential method is the LAMP technology, which has salient advantages over most DNA-based amplification tests (Box 1)... The LAMP method has the advantage of amplifying the target DNA from partially processed and/or non-processed samples... This inherent advantage of LAMP shortens the reaction time and eliminates the need for DNA extraction, a step that is prone to contamination and may result in significant loss of DNA... The preparation of template DNA is the least developed method associated with LAMP technology... A higher test specificity can be achieved by targeting an internal sequence of the amplicon through incorporating fluorescent molecular beacon probe, thus minimising non-specific signal, or by using a lateral flow dipstick (LFD) format (Figure 1G), though at a slightly higher cost... In general, the use of dyes is most preferred because they are cheap and most allow definite visual inspection of results based on colour change... However, the need for treatment and/or a decision on case management dictates unequivocal result interpretation... For example, it is worth noting that the human African trypanosomiasis case definition requires demonstration of trypanosomes in the body fluid, and that the LAMP test may not be relied upon to make a treatment decision... Nevertheless, the expected high specificity of LAMP may reduce costs associated with serological false positives that card agglutination test for trypanosomiasis (CATT) registers, of which only few end up being genuine HAT cases.

Show MeSH