Validation of the Loop-Mediated Isothermal Amplification Method for Single Nucleotide Polymorphism Genotyping with Whole Blood

Iwasaki, Masaomi; Yonekawa, Toshihiro; Otsuka, Kimihiko; Suzuki, Wataru; Nagamine, Kentaro; Hase, Tetsu; Tatsumi, Ke-ita; Horigome, Tsuneyoshi; Notomi, Tsugunori; Kanda, Hidetoshi

doi:10.1166/gl.2003.028

Cited by 66 publications

(39 citation statements)

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“…Poon et al estimated that the cost of running a LAMP assay is about 1/10 of that of the normal PCR method for P. falciparum detection (21). The biggest reduction in cost and time came from simple sample preparation, with no requirement for previous DNA extraction (6). Briefly, simple heating of the infected blood at 99°C for 10 min was enough to prepare the DNA template for LAMP without any inhibition of the reaction (21).…”

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confidence: 99%

Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis

Watanabe²,

et al. 2007

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Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.

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Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis

Watanabe²,

et al. 2007

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“…More importantly, it appears to make the enzyme more prone to inhibitors found in biological samples. DNA amplification methods that utilize single temperature regimes (isothermal) are more robust than PCR and are capable of amplifying from fairly crude DNA templates (Hosono et al 2003;Iwasaki et al 2003;Mitani et al 2007; Lee et al 2009a). Likewise, isothermal PCR, where a helicase unwinds the two strands allowing Taq pol to make more copies of the target without denaturation, is able to amplify from crude samples (Vincent et al 2004).…”

Section: Introductionmentioning

confidence: 99%

“…LAMP is robust, can be used to amplify DNA from crude biological samples, and is highly specific, with near single-copy sensitivity (Lee et al 2009a(Lee et al , 2009b. The main application of LAMP has been for the detection of target DNA, though the assay can be modified to differentiate between single nucleotide variants for genotyping (Iwasaki et al 2003). Here we have designed a LAMP assay to amplify a simple sequence repeat (SSR) motif in rice.…”

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confidence: 99%

Genotyping SSR length variants by isothermal DNA amplification

Lee

Ialicicco

Akkinepalli

et al. 2012

Genome

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Loop-mediated isothermal DNA amplification (LAMP) is an alternative method for the amplification of DNA sequences. It has been applied primarily for the detection of specific targets. We demonstrate the novel use of LAMP to amplify SSR alleles in a set of rice varieties and show the results to be consistent with analysis performed by PCR. Furthermore, we test the sensitivity of the assay and show it to amplify from near single copy target.

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“…Numerous strategies have been developed for SNP discrimination (Table 1) (23)(24)(25)(26)(27)(28)(29)(30)(31)(32), but no method has eliminated background amplification or DNA purification so that CYP2C9 and VKORC1 genotypes can be diagnosed within 1 h of blood collection. Recently, we reported the SMart-Amplification Process version 2 (SMAP 2), which can detect SNPs after about 30 min of incubation under isothermal conditions (33 ).…”

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Rapid Single-Nucleotide Polymorphism Detection of Cytochrome P450 (CYP2C9) and Vitamin K Epoxide Reductase (VKORC1) Genes for the Warfarin Dose Adjustment by the SMart-Amplification Process Version 2

et al. 2009

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Background: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (−1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. Methods: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 −1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. Results: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. Conclusions: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2–based diagnostics have key advantages.

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Validation of the Loop-Mediated Isothermal Amplification Method for Single Nucleotide Polymorphism Genotyping with Whole Blood

Cited by 66 publications

References 0 publications

Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis

Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis

Genotyping SSR length variants by isothermal DNA amplification

Rapid Single-Nucleotide Polymorphism Detection of Cytochrome P450 (CYP2C9) and Vitamin K Epoxide Reductase (VKORC1) Genes for the Warfarin Dose Adjustment by the SMart-Amplification Process Version 2

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