A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of St. Louis encephalitis and eastern equine encephalitis viruses

Abstract

A duplex TaqMan real-time RT-PCR assay was developed for the detection of St. Louis encephalitis virus (SLEV) and Eastern equine encephalitis virus (EEEV), for use in human and vector surveillance. The respective targets selected for the assay were the conserved NS5 and E1 genes of the two viruses. Due to the insufficient number of NS5 sequences from SLEV strains in the GenBank database, we determined the sequence of an approximately 1-kb region for each of 25 strains of SLEV in order to select primers and probes in a conserved region. Our assay has a sensitivity of 5 gene copies/reaction for EEEV and 10 gene copies/reaction for SLEV, and it’s performance is linear over at least 6 log10 gene copies. The assay is specific and detected all strains of SLEV (69) and EEEV (12) that were tested. An internal control ensures detection of efficient nucleic acid extraction and possible PCR inhibition.

Características gerais e epidemiologia dos arbovírus emergentes no Brasil

“…Os relatos de casos fatais podem variar de 5% a 20%, entretanto, os números são ainda maiores entre a população idosa 24 . Um surto de SLEV ocorreu simultaneamente ao de DENV-3, em São José do Rio Preto, Estado de São Paulo.…”

Molecular detection of viral causes of encephalitis and meningitis in New York State

“…As an internal extraction and RT control and in order to detect PCR inhibition, each clinical specimen was spiked with a known quantity of GFP transcript during the lysis step of the nucleic acid extraction process [Tavakoli et al, 2007;Hull et al, 2008]. The GFP transcript was made as described above from plasmid pTU65 [Chalfie et al, 1994] and subsequently quantified.…”

A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of California serogroup and Cache Valley viruses

“…As an internal nucleic acid extraction control and for detection of potential PCR inhibition, each clinical specimen was spiked during the lysis step of the extraction process with a known quantity of green fluorescent protein (GFP) RNA transcript (2200 gc/μL) (Hull et al, 2008). After completion of the extraction, the nucleic acid was analyzed by a 1-step real-time RT-PCR with a primer and probe set specific for GFP (Table 1) Streptococcus sanguis; and Streptococcus pneumoniae serotypes 10A, 11A, and 18F were obtained from the Bacteriology Department at the Wadsworth Center.…”