Highly Sensitive Assay for Detection of Enterovirus in Clinical Specimens by Reverse Transcription-PCR with an Armored RNA Internal Control

Abstract

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics ( The human enteroviruses (EVs) are members of the family Picornaviridae, are ubiquitous, and are mainly enterically transmitted. EVs have traditionally been identified by serotype-specific antisera in a virus-neutralizing test, and 66 EV types are known to infect humans (19). The 66 EV serotypes were initially recognized and divided into five major groups: polioviruses (PV; types 1 to 3), coxsackieviruses A (CVAs; types 1 to 22 and 24), coxsackieviruses B (CVBs; types 1 to 6), echoviruses (types 1 to 7, 9, 11 to 27, and 29 to 33), and EV types 68 to 71 (17). Recent molecular analyses have proved that echovirus types 22 and 23 are genetically distinct from the members of the genus Enterovirus and have been reclassified in a separate genus, Parechovirus, in the family Picornaviridae (13,20,23).Infections with EVs cause a wide range of clinical outcomes, such as asymptomatic infections, aseptic meningitis (meningeal inflammation in the absence of a bacterial pathogen), encephalitis, paralytic poliomyelitis, and myocarditis. Although the majority of EV infections do not cause significant disease, infection can cause serious illness, especially in infants and immune-compromised patients. EV infections are the most common cause of aseptic meningitis and account for 80 to 90% of all cases of central nervous system infections for which a possible causative agent is identified (24). In the neonate, aseptic meningitis-induced complications and poor outcomes of EV infections generally occur within the first 2 days of life (1, 2). Aseptic meningitis in immune-competent adults is characterized by sudden onset of fever, but neurological abnormalities are rare, and both short-term and long-term outcomes are generally good. Encephalitis caused by EV infections is a less common but a more severe disease than aseptic meningitis (18,29,30). Immune-compromised children and adults who are infected with EV may develop chronic meningitis and encephalitis, which may last for years before becoming fatal (16).The early clinical symptoms of meningitis caused by viruses, bacteria, and fungi are quite similar and are difficult to distinguish, but the diagnosis, therapy, and outcome of disease caused by these pathogens vary considerably. A reli...

Large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in Vietnam

“…Reverse transcription using SuperScript III reverse transcriptase (Invitrogen, UK) was performed according to the manufacturer's instruction. EVs were screened by a real-time PCR protocol (Beld et al, 2004). Typing of EVs from 177 positive samples, including 10 from diarrhoeic pigs, representing ranges of geography, C t values and ages of infected pigs, was performed by amplification and sequencing of the VP1 region as described previously (Van Dung et al, 2014).…”

Use of an armored RNA standard to measure microcystin synthetase E gene expression in toxic Microcystis sp. by reverse‐transcription QPCR

“…Both oligonucleotides overlap over a sequence stretch of 24 nucleotides. The mutagenesis cassette was constructed according to Beld et al (2004) by hybridization and elongation of 1 ng of sense and antisense oligonucleotide in a 50 µL reaction containing 6 mM MgSO 4 , 0.2 mM dNTPs (Roche Diagnostics), 3.0 U of proofreading Platinum Pfx DNA Polymerase (Invitrogen), 1× PCR reaction buffer, and 1 µg non-acetylated BSA (Sigma). The reaction was incubated for 10 min at 95°C, 5 min at 55°C, and 20 min at 68°C.…”

Human Parechoviruses, New Players in the Pathogenesis of Viral Meningitis

“…Therefore, PCR is the preferred method for detection of viruses in this material (Espy et al, 2006;Read et al, 1997;Romero 1999;Rotbart HA & Romero JR, 1995;van Doornum et al, 2007). But while with cell culture, both EVs and HPeVs can be diagnosed, PCR specific for EVs will fail to detect HPeVs because the targeted 5'UTR is too diverse between HPeVs and EVs (Beld et al, 2004;Benschop et al, 2006a;Hyypia, 1989;Hyypia et al, 1989;Oberste et al, 1999). Therefore a separate RT-PCR specifically targeting the 5'UTR of HPeVs is required.…”