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Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease-Towards a Point-of-Care Test.

Beissner M, Phillips RO, Battke F, Bauer M, Badziklou K, Sarfo FS, Maman I, Rhomberg A, Piten E, Frimpong M, Huber KL, Symank D, Jansson M, Wiedemann FX, Banla Kere A, Herbinger KH, Löscher T, Bretzel G - PLoS Negl Trop Dis (2015)

Bottom Line: Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate.Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application.Both LAMP formats constitute equivalent alternatives to conventional PCR techniques.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT

Background: As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents.

Methodology/principal findings: Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays.

Conclusions/significance: Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.

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Related in: MedlinePlus

Binding sites of LAMP primers within the IS2404.Fig 1 shows binding sites of LAMP primers within the IS2404 of M. ulcerans strain Agy 99 (Genbank accession number: CP000325.1). Primer MU2-FIP consists of a first reverse complementary region “F1” and a second forward region “F2”. Primer MU2-BIP (first described by Ablordey et al. [28]) consists of a first forward region “B1” and a second reverse complementary region “B2”. Primers and corresponding regions are highlighted in colors; red: primer MU2-F3 (region “F3”); yellow: primer MU2-FIP (region “F2”); light blue: primer MU2-FIP (region “F1”); dark blue: primer MU2-BIP (region “B1”); green: primer MU2-BIP (region “B2”) and pink: primer MU2-B3 (region “B3”; first described by Ablordey et al. [28]).
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pntd.0004219.g001: Binding sites of LAMP primers within the IS2404.Fig 1 shows binding sites of LAMP primers within the IS2404 of M. ulcerans strain Agy 99 (Genbank accession number: CP000325.1). Primer MU2-FIP consists of a first reverse complementary region “F1” and a second forward region “F2”. Primer MU2-BIP (first described by Ablordey et al. [28]) consists of a first forward region “B1” and a second reverse complementary region “B2”. Primers and corresponding regions are highlighted in colors; red: primer MU2-F3 (region “F3”); yellow: primer MU2-FIP (region “F2”); light blue: primer MU2-FIP (region “F1”); dark blue: primer MU2-BIP (region “B1”); green: primer MU2-BIP (region “B2”) and pink: primer MU2-B3 (region “B3”; first described by Ablordey et al. [28]).

Mentions: A set of four primers was designed for amplification of the M. ulcerans specific IS2404 by manually analyzing the target sequence and designing the primers according to the needs for LAMP amplification. Specificity of the primers for M. ulcerans was confirmed in silico by means of the basic local alignment search tool (BLAST, GenBank, NCBI) [40]. The primer set consisted of two smaller oligonucleotides with forward (MU2-F3) and reverse complementary (MU2-B3) sequences and two larger oligonucleotides (MU2-FIP and MU2-BIP) with a complex sequence construction. Primer sequences are provided in Table 1 and binding sites within the IS2404 are displayed in Fig 1. Primers MU2-B3 and MU2-BIP were first published by Ablordey et al. [28].


Loop-Mediated Isothermal Amplification for Laboratory Confirmation of Buruli Ulcer Disease-Towards a Point-of-Care Test.

Beissner M, Phillips RO, Battke F, Bauer M, Badziklou K, Sarfo FS, Maman I, Rhomberg A, Piten E, Frimpong M, Huber KL, Symank D, Jansson M, Wiedemann FX, Banla Kere A, Herbinger KH, Löscher T, Bretzel G - PLoS Negl Trop Dis (2015)

Binding sites of LAMP primers within the IS2404.Fig 1 shows binding sites of LAMP primers within the IS2404 of M. ulcerans strain Agy 99 (Genbank accession number: CP000325.1). Primer MU2-FIP consists of a first reverse complementary region “F1” and a second forward region “F2”. Primer MU2-BIP (first described by Ablordey et al. [28]) consists of a first forward region “B1” and a second reverse complementary region “B2”. Primers and corresponding regions are highlighted in colors; red: primer MU2-F3 (region “F3”); yellow: primer MU2-FIP (region “F2”); light blue: primer MU2-FIP (region “F1”); dark blue: primer MU2-BIP (region “B1”); green: primer MU2-BIP (region “B2”) and pink: primer MU2-B3 (region “B3”; first described by Ablordey et al. [28]).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643924&req=5

pntd.0004219.g001: Binding sites of LAMP primers within the IS2404.Fig 1 shows binding sites of LAMP primers within the IS2404 of M. ulcerans strain Agy 99 (Genbank accession number: CP000325.1). Primer MU2-FIP consists of a first reverse complementary region “F1” and a second forward region “F2”. Primer MU2-BIP (first described by Ablordey et al. [28]) consists of a first forward region “B1” and a second reverse complementary region “B2”. Primers and corresponding regions are highlighted in colors; red: primer MU2-F3 (region “F3”); yellow: primer MU2-FIP (region “F2”); light blue: primer MU2-FIP (region “F1”); dark blue: primer MU2-BIP (region “B1”); green: primer MU2-BIP (region “B2”) and pink: primer MU2-B3 (region “B3”; first described by Ablordey et al. [28]).
Mentions: A set of four primers was designed for amplification of the M. ulcerans specific IS2404 by manually analyzing the target sequence and designing the primers according to the needs for LAMP amplification. Specificity of the primers for M. ulcerans was confirmed in silico by means of the basic local alignment search tool (BLAST, GenBank, NCBI) [40]. The primer set consisted of two smaller oligonucleotides with forward (MU2-F3) and reverse complementary (MU2-B3) sequences and two larger oligonucleotides (MU2-FIP and MU2-BIP) with a complex sequence construction. Primer sequences are provided in Table 1 and binding sites within the IS2404 are displayed in Fig 1. Primers MU2-B3 and MU2-BIP were first published by Ablordey et al. [28].

Bottom Line: Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate.Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application.Both LAMP formats constitute equivalent alternatives to conventional PCR techniques.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases and Tropical Medicine (DITM), University Hospital, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT

Background: As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents.

Methodology/principal findings: Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays.

Conclusions/significance: Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.

Show MeSH
Related in: MedlinePlus