Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease

Abstract

Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

Fig. 1

Schematic description of the primer design for the RTN RT-PCR assay. F1 and R1 are the outer primers with 40 nt in length, which has an annealing temperature of 64 °C. F2 and R2 are the inner primers with 21 nt in length, which has an annealing temperature of 52 °C. For primer sequences, see Table 1

Fig. 2

A. Dissociation plots of the RTN RT-PCR products. The ordinate shows the fluorescence intensity, and the abscissa shows the temperature. 1, the melting curve of the negative control; 2, the melting curve of the positive control; B, the size analysis of the PCR products of the assay; M100, 100-bp DNA ladder marker; Lane 1, negative control; lane 2, 144-bp positive control. The low-molecular-weight bands in lanes L1 and L2 are primer dimers and products of nonspecific amplification

Fig. 3

Standard curve of the RTN RT-PCR assay. Samples 1-8 represent serial tenfold dilutions (log ⁻¹ to ⁻⁸) of viral RNA containing amounts equivalent to 10⁶ - 0.01 TCID₅₀/ml