A One-Step, Triplex, Real-Time RT-PCR Assay for the Simultaneous Detection of Enterovirus 71, Coxsackie A16 and Pan-Enterovirus in a Single Tube

Abstract

The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001–0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.

Understanding Enterovirus 71 Neuropathogenesis and Its Impact on Other Neurotropic Enteroviruses

“…Overall, the rate of virus isolation from CSF is very low (<5%) compared to throat, vesicle and rectal swabs . Recently, several new molecular diagnostic methods for simple, rapid identification of EV‐A71 in clinical specimens have been described, including nested RT‐PCR, real‐time RT‐PCR, reverse transcription loop‐mediated isothermal amplification and droplet digital PCR .However, these new methods require specific primers (targeted to the VP1 gene) that have to be constantly revised accordingly to the prevalent EV‐A71 genogroups/variants in a new outbreak area.…”

Plasma virome of 781 Brazilians with unexplained symptoms of arbovirus infection include a novel parvovirus and densovirus

“…Samples negative for pan-flavivirus PCR were then submitted to a pan-Alphavirus multiplex qPCR assay [25]. The samples that showed negative results for the pan-Alphavirus assay were then submitted to a pan-Enterovirus real time PCR assay targeting the 5' UTR [26].…”

Children’s Caregivers and Public Playgrounds: Potential Reservoirs of Infection of Hand-foot-and-mouth Disease

“…The range of activities for adults is generally far greater than five-years-old children, and there is a greater chance that these caregivers are able to contact with enterovirus. Considering the infection of adults is mostly asymptomatic infection27, it makes them difficult to be identified, as preventing and controlling the spread of enterovirus-related diseases. The difficulty can also apply to the public playgrounds.…”

“…Sample cDNA that generated from the reverse transcription was stored under −80 °C temperature. Referring from previous studies in literature review273132, primers were designed (Invitrogen Custom Primers, shanghai). Details of the primers were listed in Table 3.…”