Novel one step real time RT-PCR assay for the detection of Enterovirus, A

PosterThis poster describes the development of a novel real time assay that utilizes primers and an Eclipse probe in an overlapping format upstream of the Rotbart amplicon

Real time PCR assay for the detection of CMV using eclipse hybridization probes

PosterWe developed a qualitative real time PCR assay to detect CMV in patient samples, using a novel hybridization probe termed the Eclipse probe (Epoch Biosciences). These are single probes with a fluorescent molecule attached to the 3' end and a dark quencher on the 5' end. Also conjugated to the 5' end of the probe is a minor groove binding moiety that stabilizes probe binding and allows much shorter probe sequences to be used. The assay is run on the HT7900 Sequence Detection System (Applied Biosystems) in a 96-well plate. We incorporated a heterologous internal control in the assay to monitor the extraction process and detect PCR inhibition. The internal control target is a plasmid, containing a fusion of GFP and C. elegans DNA. We validated ten sample types (spinal fluid, plasma, serum, amniotic fluid, ocular fluid, buffy coat, tissue, urine, bone marrow and bronchioalveolar lavage), and demonstrated the dependence that the limit of detection has on sample type. Since this assay went on-line last year, we have identified a number of patient samples that generated discrepant results between our qualitative test and the Roche CMV Amplicor Quantitative assay. Here we present the validation data for our real time assay, and results of the discrepant analysis

Description and validation of a novel real-time RT-PCR enterovirus assay

Journal ArticleEnteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5' nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry

Ultrasonic enhancement of antibiotic action on several species of bacteria

Journal ArticleThe effect of the antibiotics gentamicin, streptomycin, kanamycin, tetracycline, and ampicillin on planktonic cultures of Enterobacter aerogenes, Serratia marcescens, Salmonella derby, Streptococcus mitis, and Staphylococcus epidermidis with and without an application of 70 kHz ultrasound was studied. The ultrasound was applied at levels that had no inhibitory effect on planktonic cultures of bacteria. Measurements of viability at, above, and below the minimum inhibitory concentration of the above antibiotics on the planktonic cultures of these bacteria showed that a simultaneous application of 70 kHz ultrasound and antibiotic significantly increased the effectiveness of selected antibiotics. Bacterial viability was reduced several orders of magnitude when harmless levels of ultrasound were combined with some antibiotics, especially the aminoglycosides. Similar synergistic effects of combined ultrasound and antibiotic treatment were seen in both Gram-positive and Gram-negative bacteria with several classes of antibiotics. These results may have application in the treatment of bacterial infections normally resistant to some antibiotics

Performance of diagnostic tests to detect respiratory viruses in older adults.

The performance of 4 laboratory methods for diagnosis of viral respiratory tract infections (RTI) in older adults was evaluated. Seventy-four nasopharyngeal (NP) swab specimens were obtained from 60 patients with RTI at a long-term care facility over 2 respiratory seasons. Sixteen specimens were positive for a respiratory virus by at least 1 method. Multiplex reverse transcriptase polymerase chain reaction (RT-PCR) by the Luminex xTAG Respiratory Viral Panel (RVP) detected 16 (100%) of the positive specimens, RVP of 24-h culture supernatant detected 8 (50%), direct fluorescent antibody testing detected 4 (25%), rapid culture detected 2 (12.5%), and rapid antigen testing detected none. For a comparison group, RVP was performed on NP swabs from 20 outpatient children with RTI. The mean fluorescence intensity by RVP was significantly lower for positive adult patients than pediatric patients (P = 0.0373). Our data suggest that older adult patients shed lower titers of viruses, necessitating a highly sensitive assay such as RT-PCR to reliably detect respiratory viral pathogens

Real time RT-PCR assay for norovirus detection with eclipse probes and a non-competitive internal control.

PosterNoroviruses are the major causative agents of nonbacterial gastroenteritis worldwide and are estimated to cause approximately 23 million cases in the United States annually. Real time RT-PCR is rapidly becoming the principle diagnostic means for norovirus detection due to its enhanced sensitivity and specificity, but generating broadly reactive primers and probes for amplification and detection of genogroups I and II has proven to be a challenge. The aim of this study was to develop a broadly reactive one step real time RT-PCR assay to detect norovirus genogroups I and II. Primers and MGB Eclipse hybridization probes used in this assay target the ORF1-ORF2 junction. Utilization of neutral and super bases along with MGB probes allowed for shorter probe lengths and increased specificity, which is particularly important for highly polymorphic norovirus strains. An RNA internal control was incorporated to monitor nucleic acid extraction and amplification inhibition. Assay optimization was accomplished by testing clinical specimens previously confirmed as positive. By using a cloned amplicon, sensitivity for genogroups I and II was determined to be approximately 10 copies per reaction

Novel one step real time RT-PCR Assay for the detection of Enterovirus

Posternteroviruses (EV) are the leading cause of aseptic meningitis in pediatric and adult populations and can be associated with severe disease such as myocarditis, encephalitis, and paralytic poliomyelitis. RT-PCR has rapidly become the diagnostic methodology of choice due to its sensitivity and rapid turn-around-time allowing significant improvement in patient care and management. Most molecular assays target the highly conserved regions within the 5' nontranslated region (NTR) described by Rotbart el al. We describe the development of a novel real time assay that amplifies and detects a conserved region upstream of the Rotbart amplicon utilizing primers and an Eclipse probe. As a further enhancement, an RNA internal control (IC) is integrated into the reaction. The analytical sensitivity was determined and a comparison to the Chemicon Oligodetect Panenterovirus kit was made during the 2005 enterovirus season

Quantitative real time PCR assay for the detection of human herpes 6.

PosterHuman herpes virus type 6 is a beta virus that occurs as two variants (A & B) and infects nearly 100% of the population before two years of age. Primary infections in children can produce fever and a unique lacy rash that gives the disease its name: roseola. The rare primary infection in adults can produce infectious mononucleosis-like disease and atypical lymphocytosis. In immunocompromised hosts, HHV6 reactivation can cause localized organ disease, organ rejection, encephalitis, and leukopenia

Limited Utility of Culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae for Diagnosis of Respiratory Tract Infections ▿

We assessed the utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae to diagnose respiratory tract infections. Compared to PCR and IgM serology, culture was less sensitive and had extremely low yield. Culture is not recommended for these pathogens, and this method should be eliminated from routine practice