Dataset Information

Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses.

ABSTRACT: Enteroviruses are found in most environments and cause several diseases in humans. Loop-mediated isothermal amplification (LAMP) was adapted and evaluated for the rapid detection of enteroviruses. Based on the highly conserved 5' untranslated region (5'-UTR) of the human enteroviruses (HEVs), particularly human enterovirus A (HEV-A) and HEV-B, a set of universal primers was designed. The LAMP amplification was carried out under isothermal conditions at 61 °C, depending on the template concentration results were obtained within 45-90 min. The detection limits were found to be 10(1) copies of cloned enterovirus 71 fragments, more sensitive than conventional PCR. Nine water samples collected from drinking water sources during three seasons and 19 stool specimens collected from HFMD patients were analyzed. By using the LAMP assay, the majority of samples was tested positive, 9/9 (100 %) and 18/19 (94.7 %), respectively. LAMP is a practical method for the rapid detection of enteroviruses in environmental and clinical samples.

SUBMITTER: Zhao HB

PROVIDER: S-EPMC3889841 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses.

Zhao Hua-Bing HB Yin Guang-Ya GY Zhao Guo-Ping GP Huang Ai-Hua AH Wang Jun-Hong JH Yang Shuang-Feng SF Gao Hong-Sheng HS Kang Wei-Jun WJ

Indian journal of microbiology 20130402 1

Enteroviruses are found in most environments and cause several diseases in humans. Loop-mediated isothermal amplification (LAMP) was adapted and evaluated for the rapid detection of enteroviruses. Based on the highly conserved 5' untranslated region (5'-UTR) of the human enteroviruses (HEVs), particularly human enterovirus A (HEV-A) and HEV-B, a set of universal primers was designed. The LAMP amplification was carried out under isothermal conditions at 61 °C, depending on the template concentrat ...[more]

PMID: 24426171

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Project description:Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA.LAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification.The use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 10(3) parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica.The simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed.

Dataset Information

Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses.

Publications

Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses.

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