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Colorimetric detection of coxsackievirus A6 by reverse transcription loop mediated isothermal amplification / 中华预防医学杂志
Chinese Journal of Preventive Medicine ; (12): 1009-1013, 2015.
Article in Chinese | WPRIM | ID: wpr-296645
ABSTRACT
<p><b>OBJECTIVE</b>To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB).</p><p><b>METHODS</b>The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 °C for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease.</p><p><b>RESULTS</b>A positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%.</p><p><b>CONCLUSION</b>The established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.</p>
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Virology / Chemistry / Sensitivity and Specificity / DNA Primers / Colorimetry / Enterovirus / Nucleic Acid Amplification Techniques / Reverse Transcription / Coloring Agents / Real-Time Polymerase Chain Reaction Type of study: Diagnostic study Limits: Humans Language: Chinese Journal: Chinese Journal of Preventive Medicine Year: 2015 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Virology / Chemistry / Sensitivity and Specificity / DNA Primers / Colorimetry / Enterovirus / Nucleic Acid Amplification Techniques / Reverse Transcription / Coloring Agents / Real-Time Polymerase Chain Reaction Type of study: Diagnostic study Limits: Humans Language: Chinese Journal: Chinese Journal of Preventive Medicine Year: 2015 Type: Article