Methylation-Specific Loop-Mediated Isothermal Amplification for Detecting Hypermethylated DNA in Simplex and Multiplex Formats

Zerilli, Francesco; Bonanno, Cinzia; Shehi, Erlet; Amicarelli, Giulia; Adlerstein, Daniel; Makrigiorgos, G. Mike

doi:10.1373/clinchem.2010.143545

Cited by 35 publications

(20 citation statements)

References 32 publications

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“…Primer concentration is an important factor in mLAMP assays, because it affects the detection time. mLAMP assays have been carried out at various primer concentrations (Zerilli et al ., ; Shao et al ., ). In this study, the detection time was shorter with the original concentration of each primer set than with concentrations different from the standard method (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products

Kasahara

Ishikawa

Sato

et al. 2014

FEMS Microbiol Lett

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Rapid detection of yeast contamination is important in the food industry. We have developed loop-mediated isothermal amplification (LAMP) assays to detect the emerging opportunistic pathogenic yeasts: Candida albicans, Candida glabrata, Candida tropicalis, the Candida parapsilosis group, Trichosporon asahii, and Trichosporon mucoides. These yeasts may cause deep-seated candidiasis or trichosporonosis. Four LAMP primer sets specific for Candida were designed to target the internal transcribed spacer 2 (ITS2) region between the 5.8S and 26S rRNA genes, and two LAMP primer sets specific for Trichosporon were designed to target the intergenic spacer 1 (IGS1) region between the 26S and 5S rRNA genes. The LAMP assays could detect these yeasts in a range between 10(0) and 10(3) cells mL(-1) in a contaminated dairy product within 1 h. We also developed multiplex LAMP assays to detect these Candida or Trichosporon species in a single reaction. Multiplex LAMP assays can detect contamination if at least one of the target species is present; they are more time- and cost-efficient than conventional methods and could detect target yeasts with sensitivity close to that of the LAMP assays. Multiplex LAMP assays established in this study can be used as a primary screening method for yeast contamination in food products.

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Section: Discussionmentioning

confidence: 99%

Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products

Kasahara

Ishikawa

Sato

et al. 2014

FEMS Microbiol Lett

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“…In addition, the sensitivity of LAMP reactions to carryover contamination is so great that manufacturer recommendations (Eiken Chemical, Tokyo, Japan) suggest not opening LAMP reaction vessels, or doing so in separate facilities with separate equipment, furtherdecreasing the desirability of post-LAMP manipulation. Previous real-time methods use nonspecific quenching, either through loss-of-signal guanine quenching (20) or gain-of-signal fluorescence using labeled primers and an intercalating dye (21). These methods can be less sensitive, and nonspecific quenching limits the selection of fluorophores available for multiplexing.…”

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confidence: 99%

Simultaneous Multiple Target Detection in Real-Time Loop-Mediated Isothermal Amplification

2012

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Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 14 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

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“…Some of these include multiplex ligation-dependent probe amplification (MLPA), bisulfite assisted genomic sequencing PCR (BSP), loop mediated isothermal amplification (MS-LAMP), nested MSP, fluorescence resonance energy transfer (FRET), padlock probes (PP), and multi-component nucleic acid enzymes (MNAzymes)2627282930313233343536. However, all these multiplex DNA methylation analysis methods have several disadvantages compared to MethyLight technology.…”

Section: Discussionmentioning

confidence: 99%

Novel Multiplex MethyLight Protocol for Detection of DNA Methylation in Patient Tissues and Bodily Fluids

Olkhov‐Mitsel

Zdravic

Kron

et al. 2014

Sci Rep

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Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings.

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Methylation-Specific Loop-Mediated Isothermal Amplification for Detecting Hypermethylated DNA in Simplex and Multiplex Formats

Cited by 35 publications

References 32 publications

Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products

Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products

Simultaneous Multiple Target Detection in Real-Time Loop-Mediated Isothermal Amplification

Novel Multiplex MethyLight Protocol for Detection of DNA Methylation in Patient Tissues and Bodily Fluids

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