Sensitive and rapid detection of viruses associated with hand foot and mouth disease using multiplexed MALDI-TOF analysis

Background: Human enterovirus (HEV) is the major cause of hand foot and mouth disease (HFMD). A powerful method for detecting HEVs associated with HFMD can provide results in a clinically relevant time frame. However, the limitations of the current enterovirus test make it difficult to identify multiple genotypes on the first pass. Objective: To develop a more sensitive and easy applicable assay for detecting 18 HFMD-associated HEV serotypes in multiplex PCR products. Study design: : A total of 241 clinical specimens were collected from HFMD patients during the 2010 outbreak in China. These samples were tested by DNA sequencing and MassARRAY analysis, respectively. Results: Analysis of a dilution series of plasmids revealed the detection limit per PCR reaction for the MassARRAY method was one copy for the tested HEVs. We compared results from 241 samples to those of the sequence analysis of the VP1 gene. The MassARRAY method detected all samples found positive by consensus PCR and sequencing method. Comparison of the results of MassARRAY and the DNA sequencing method found concordant results for 225 (93.4%) of the 241 samples. In 14 (5.8%) samples, the MassARRAY method detected multiple types, whereas the DNA sequencing method detected a single type. In another 2 (0.8%) samples, the MassARRAY method detected single types, whereas the DNA sequencing method detected no HEV. Conclusions: The MassARRAY assay is a highly sensitive and accurate method for the type-specific detection of 18 HEVs in HFMD and is a powerful complement to current detection methods. (C) 2012 Elsevier B. V. All rights reserved.