Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye.

Xiang Wang,Fang Zhang,Wenzhi Zheng,Fenlian Ma,Lishu Zheng,Yinglong Bai,Qian Zhang,Zhihui Zhao

doi:10.1186/1743-422x-9-138

Abstract

BackgroundHuman metapneumovirus (hMPV) is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide. The objective of this study is to develop a visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of hMPV and applied to the clinical samples.ResultsIn this study, visual RT-LAMP assay for hMPV was performed in one step with the addition of hydroxynaphthol blue (HNB), and were used to detect respiratory samples. Six primers, including two outer primers (F3 and B3), two inner primers (FIP, BIP) and two loop primers (LF and LB), were designed for hMPV N gene by the online software. Moreover, the RT-LAMP assay showed good specificity and no cross-reactivity was observed with human rhinovirus (HRV), human respiratory syncytial Virus (RSV), or influenza virus A/PR/8/34 (H1N1). The detection limit of the RT-LAMP assay was approximately ten viral RNA copies, lower than that of traditional reverse transcriptase polymerase chain reaction (RT-PCR) 100 RNA copies. In the 176 nasopharyngeal samples, 23 (13.1%) were conformed as hMPV positive by RT-LAMP, but 18 (10.2%) positive by RT-PCR.ConclusionCompared with conventional RT-PCR, the visual hMPV RT-LAMP assay performed well in the aspect of detect time, sensitivity, specificity and visibility. It is anticipated that the RT-LAMP will be used for clinical tests in hospital or field testing during outbreaks and in emergency.

Highlights

Human metapneumovirus is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide
The most common Human metapneumovirus (hMPV) detection methods are by virus isolation, enzyme-linked immunoassays, McAb immuno-fluorescent assays and molecular biology methods based on the polymerase chain reaction (PCR, reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, and real-time PCR)
Sequencing and restriction endonuclease digestion of reverse transcription loop-mediated isothermal amplification (RT-Loop-mediated isothermal amplification assay (LAMP)) products As shown in Figure 1A, RT-LAMP products of hMPV RNA showed a ladder-like pattern upon agarose gel electrophoresis

Summary

Introduction

Human metapneumovirus (hMPV) is a major cause of acute respiratory infections ranging from wheezing to bronchiolitis and pneumonia in children worldwide. Osterhaus first reported and named the human metapneumovirus (hMPV) [1]; it was detected in Europe, America, Australia, and other countries [2,3,4]. Sensitive cell lines for hMPV are limited. The cell line LLCMK2 can reach a 100% infection within 5 days of isolation [10], but this is far from fulfilling the requirements in terms of time efficiency for positive identification during outbreaks. HMPV antigen detection requires the preparation of monoclonal antibodies; this entails significant costs and can be time-consuming. RT-PCR, qRT-PCR, and microarrays are suitable for high-throughput analyses and can produce rapid, specific, and sensitive results, the expensive instruments required for such tests restrict their application

Objectives

Methods

Results

Discussion

Conclusion