Genotyping of Human Platelet Antigens 1 to 6 and 15 by High-Resolution Amplicon Melting and Conventional Hybridization Probes

Liew, Michael; Nelson, Lesa; Margraf, Rebecca L.; Mitchell, Sheri; Erali, Maria; Mao, Rong; Lyon, Elaine; Wittwer, Carl T.

doi:10.2353/jmoldx.2006.050053

Cited by 54 publications

(31 citation statements)

References 30 publications

(26 reference statements)

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“…[33] since this is a closed-tube assay (does not require additional precautions to prevent crossover of PCR products), avoids sequencing processes, laborious procedures and high costs as well as offering robust and high efficiency in its results [25,34]. On average, this type of test has been calculated to be three times faster and five times cheaper than other types of analysis such as MLST and RFLPs [17].…”

Section: Resultsmentioning

confidence: 99%

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

Hernández

Álvarez

González

et al. 2014

Parasites & Vectors

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Background: Leishmaniases are tropical zoonotic diseases, caused by parasites from the genus Leishmania. New World (NW) species are related to sylvatic cycles although urbanization processes have been reported in some South American Countries such as Colombia. This eco-epidemiological complexity imposes a challenge to the detection of circulating parasite species, not only related to human cases but also infecting vectors and reservoirs. Currently, no harmonized methods have been deployed to discriminate the NW Leishmania species. Findings: Herein, we conducted a systematic and mechanistic High-Resolution Melting (HRM) assay targeted to HSP70 and ITS1. Specific primers were designed that coupled with a HRM analyses permitted to discriminate six NW Leishmania species. In order to validate the herein described algorithm, we included 35 natural isolates obtained from human cases, insect vectors and mammals. Our genotyping assay allowed the correct assignment of the six NW Leishmania species (L. mexicana, L. infantum (chagasi), L. amazonensis, L. panamensis, L. guyanensis and L. braziliensis) based on reference strains. When the algorithm was applied to a set of well-characterized strains by means of PCR-RFLP, MLEE and monoclonal antibodies (MA) we observed a tailored concordance between the HRM and PCR-RFLP/MLEE/MA (KI = 1.0). Additionally, we tested the limit of detection for the HRM method showing that this is able to detect at least 10 equivalent-parasites per mL. Conclusions: This is a rapid and reliable method to conduct molecular epidemiology and host-parasite association studies in endemic areas.

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Section: Resultsmentioning

confidence: 99%

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

Hernández

Álvarez

González

et al. 2014

Parasites & Vectors

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“…42 With small PCR products, homozygous variants are distinguished by Tm and heterozygotes are identified by melting curve shape. The method has been applied to many common clinical single-base variants 43 and compared against dual-hybridization probes 44 and unlabeled probes. 45 However, many small insertions/deletions and some single base changes can be difficult to distinguish from wild type when homozygous.…”

mentioning

confidence: 99%

LightCycler Technology in Molecular Diagnostics

Lyon

Wittwer

2009

The Journal of Molecular Diagnostics

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From the first report of the LightCycler as a real-time polymerase chain reaction (PCR) instrument 1 clinical laboratories quickly realized its potential as a diagnostic tool. Quantitative applications important for infectious disease and oncology applications were followed by genotyping assays that took advantage of accurate temperature control to melt DNA amplicons or probe/amplicon duplexes. The first Food and Drug Administration-cleared molecular genetic assays were developed for this platform, specifically genotyping single base variants in F5 and F2. According to a College of American Pathologists survey, 2 ϳ30% of clinical laboratories report using the LightCycler for these two assays. This article will review the history of LightCycler technology, focusing on those applications widely incorporated into molecular diagnostics. In addition we will review recent developments that may impact future clinical testing.Real-time PCR instruments simultaneously amplify and detect, eliminating the need to open tubes containing PCR amplicon and therefore reducing the risk of future contamination. Fluorescent dyes or probes allow continuous monitoring as template DNA is amplified.3 By monitoring fluorescence every cycle, the amount of original target can be calculated. By monitoring fluorescence as the temperature changes, genotyping and heterozygote scanning can be performed by melting analysis, often removing the need for downstream analysis.The first two platforms developed for real-time PCR were the LightCycler (Roche, Indianapolis, IN) and the ABI 7700 (Applied Biosystems, Foster City, CA). The instruments were as different as the groups that created them. At the time, ABI was the undisputed corporate leader of PCR technology and the 7700 was a large 96-well laser-based instrument focused on throughput.

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“…Amplicon melting monitored with a DNA dye uses temperature for multiplexing instead of probe color. Prior multiplex genotyping via amplicon melting has been limited to duplex assays (15,31 ). We have demonstrated that the method can be multiplexed to at least 4 amplicons.…”

Section: Discussionmentioning

confidence: 99%

Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting

Seipp¹,

Pattison²,

Durtschi³

et al. 2008

Clinical Chemistry

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Genotyping of Human Platelet Antigens 1 to 6 and 15 by High-Resolution Amplicon Melting and Conventional Hybridization Probes

Cited by 54 publications

References 30 publications

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay

LightCycler Technology in Molecular Diagnostics

Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting

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