Short CommunicationComparative evaluation of Taqman real-time PCR and semi-nested VP1 PCR for detection of enteroviruses in clinical specimens
Section snippets
Background
Enteroviruses (EV) (genus Enterovirus, family Picornaviridae) are among the most common viruses infecting humans. Most infections are asymptomatic or result in only mild symptoms, such as non-specific febrile illness or mild upper respiratory symptoms (common cold). However, enteroviruses can also cause a wide variety of other clinical illnesses, including acute hemorrhagic conjunctivitis, aseptic meningitis, undifferentiated rash, acute flaccid paralysis, myocarditis, and neonatal sepsis-like
Objectives
To investigate the performance of two common enterovirus assays, we compared the relative sensitivity of real-time RT-PCR (rRT-PCR) and a VP1 semi-nested PCR (RT-snPCR). Sequencing of the VP1 amplicon also provides typing information, whereas the sequences in the rRT-PCR target do not correlate with serotype.
Study design
We tested 371 clinical specimens, received from a variety of sources, from February 2007 through November 2009. The samples tested included examples of all specimen types commonly tested for enterovirus in clinical laboratories (Table 1). RNA was extracted from 140 μl of each specimen, by using the QIAamp viral RNA minikit (Qiagen, Valencia, CA) according to the manufacturer's procedure, and eluted in 50 μl nuclease-free water. To ensure comparability, the same RNA preparation was used for both
Results
A total of 114 specimens were positive in at least one of the two assays, 101 (89% of positive specimens) in the VP1 assay and 87 (76% of positive specimens) in the Taqman assay (Fig. 1A). Overall, there was 89% concordance between the two methods (Fig. 1A). In the 27 specimens that were VP1 RT-snPCR-positive and rRT-PCR-negative, sequencing of the VP1 amplicon identified 9 coxsackie A (CVA) viruses (six CVA6, one CVA4, one CVA9, and one CVA11), two coxsackie B (CVB) viruses (one CVB3 and one
Discussion
For concordant positive samples, the range of CT values was 13–45 (median = 30), whereas the 13 rRT-PCR-positive, VP1-negative samples gave CT values of 32–39 (median = 35) (Fig. 1B). The difference in the CT value distributions was statistically significant (p = 5.5 × 10−4, Wilcoxon rank-sum test), but the overlap in the CT values suggests that the VP1-negative results cannot be fully explained by a very low genome copy number in the sample. The VP1 assay first PCR product is ∼750 bp and the second is
Conflict of interest statement
MS Oberste and WA Nix hold patents for the VP1 assay described here (US patent no. 7,247,457 and 7,714,122). The authors declare no other potential conflicts of interest.
References (10)
- et al.
Coxsackievirus, echovirus, and other enteroviruses
Reverse-transcription polymerase chain reaction detection of the enteroviruses
Arch Pathol Lab Med
(1999)- et al.
Impact of rapid polymerase chain reaction results on management of pediatric patients with enteroviral meningitis
Pediatr Infect Dis J
(2002) - et al.
Clinical utility of polymerase chain reaction testing for enteroviral meningitis
Pediatr Infect Dis J
(1999) - et al.
Five years’ experience of reverse-transcriptase polymerase chain reaction in daily diagnosis of enterovirus and rhinovirus infections
Clin Infect Dis
(2003)
Cited by (57)
Laboratory diagnosis of nonpolio enteroviruses: A review of the current literature
2023, Biosafety and HealthChanging epidemiology of human enteroviruses (HEV) in a hand, foot and mouth disease outbreak in Vellore, south India
2022, Indian Journal of Medical MicrobiologyCitation Excerpt :The tubes were then centrifuged at 2000 rpm for 10 min at 4 °C after which the supernatant was stored immediately at −80 °C in multiple aliquots. Two different real time PCRs (EV1& EV2) amplifying the 5′UTR of enterovirus was performed to identify the causative agent of this HFMD outbreak [15,16]. All samples with a Ct value of less than or equal to 37 in EV1&EV2 were considered positive and sequencing of the VP1–2C region was undertaken to identify the enterovirus serotype.
Discord between presence of follicular conjunctivitis and Chlamydia trachomatis infection in a single Torres Strait Island community: a cross-sectional survey
2022, Australian and New Zealand Journal of Public HealthPicornavirus etiology of acute infections among hospitalized infants
2019, Journal of Clinical VirologyCitation Excerpt :Available residual CSF and serum specimens collected during hospitalization were shipped to CDC for laboratory testing. All specimens were screened using pan-EV and pan-PeV real-time RT-PCR tests [5,6]. Additionally, all CSF specimens were tested using EV and PeV VP1 RT-PCR assays, followed by Sanger sequencing of amplicons to determine type [7,8].
A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus
2018, Diagnostic Microbiology and Infectious DiseaseWastewater contamination in Antarctic melt-water streams evidenced by virological and organic molecular markers
2017, Science of the Total Environment