One-step multiplex real-time RT-PCR for detection and typing of dengue virus.

Previous studies reported that severity of dengue is associated with multiple factors, including secondary infection, age, viral load and infecting serotype and genotype. In addition, other studies have reported that a dengue virus-2 (DENV-2) infection is associated with a prognosis of more severe clinical manifestations than DENV-1 and DENV-4 infections. For these reasons, the ability to identify the DENV serotypes is critical for optimal patient diagnosis and epidemiological studies. In this study, we developed a TaqMan probe-based, one-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) system for detection and serotyping DENV. Our linear dynamic range (101 to 107 copies/reaction) showed the R2 values of DENV-1, 2, 3 and 4 as 0.998, 0.998, 0.994, and 0.998, respectively. The detection limits of DENV-1, 2, 3, and 4, were 10 copies/reaction, 100 copies/reaction, 10 copies/reaction, and 100 copies/reaction, respectively. Specificity test results indicated that this system is specific for DENV-1, 2, 3, and 4 and does not react with other viruses. Finally, we validated our results with five different real-time PCR instruments. Our results showed that the Ct values of the four serotype templates were similar in five real-time PCR instruments. Thus, this system provides an accurate method for detection and serotyping of DENV, which can be applied in diagnostics, surveillance, and epidemiology. Dengue can be found in many nations with varying socioeconomic and monetary resources. The results of our validation analyses using five different real-time PCR instruments suggest that this method can easily and confidently be used world-wide.

Unilateral extrapedicular vertebroplasty and kyphoplasty in lumbar compression fractures : technique, anatomy and preliminary results.

OBJECTIVE: A single balloon extrapedicular kyphoplasty has been introduced as one of the unilateral approaches for thoracic compression fractures; however, the unilateral extrapedicular technique in the lumbar area needs a further understanding of structures in the lumbar area. The purpose of the present study is to describe methods and pitfalls of this procedure based on the anatomy of the lumbar area and to analyze clinical outcome and complications. METHODS: Anatomical evaluation was performed with 2 human cadavers. A retrospective review of unilateral extrapedicular approaches yielded 74 vertebral levels in 55 patients that were treated with unilateral extrapedicular vertebroplasty and kyphoplasty. Radiographic assessment included the restoration rate of vertebral height and correction of kyphosis. RESULTS: Anatomical evaluation indicates that the safe needle entry zone of bone for the extrapedicular approach was located in the supero-lateral aspect of the junction between the pedicle and vertebral body. The unilateral extrapedicular procedure achieved adequate pain relief with a mean decreases in pain severity of 7.25±1.5 and 2.0±1.4, respectively. Complications were 1 retroperitoneal hematoma, 6 unilateral fillings and 3 epidural leak of the polymethylmethacrylate. CONCLUSION: The method of a unilateral extrapedicular approach in kyphoplasty and vertebroplasty in the lumbar area might be similar to that in thoracic approach using a route via the extrapedicular space. However, different anatomical characteristics of the lumbar area should be considered.

Rapid detection of Enterobacter sakazakii using TaqMan real-time PCR assay.

Enterobacter sakazakii is an emerging food pathogen, which induces severe meningitis and sepsis in neonates and infants, with a high fatality rate. The disease is generally associated with the ingestion of contaminated infant formula. In this study, we describe the development of a real-time PCR protocol to identify E. sakazakii using a TaqMan probe, predicated on the nucleotide sequence data of the 16S rRNA gene obtained from a variety of pathogens. To detect E. sakazakii, four primer sets and one probe were designed. Five strains of E. sakazakii and 28 non-E. sakazakii bacterial strains were used in order to ensure the accuracy of detection. The PCR protocol successfully identified all of the E. sakazakii strains, whereas the 28 non-E. sakazakii strains were not detected by this method. The detection limits of this method for E. sakazakii cells and purified genomic DNA were 2.3 CFU/assay and 100 fg/assay, respectively. These findings suggest that our newly developed TaqMan real-time PCR method should prove to be a rapid, sensitive, and quantitative method for the detection of E. sakazakii.

PCR identification of ruminant tissue in raw and heat-treated meat meals.

To control the spread of bovine spongiform encephalopathy in cattle through contaminated animal feedstuffs, screening of feed products is essential. We designed five pairs of primers to identify specifically raw and heat-treated tissue from cattle, sheep, goat, deer, and ruminants in general. A forward common primer was designed based on a conserved DNA sequence in the mitochondrial 12S rRNA-tRNA(val)-16S rRNA gene, and reverse primers were designed to hybridize with a species-specific DNA sequence for each species considered. All primers were developed to create a specific PCR product small enough (less than 200 bp) to be suitable for heat-treated material. To evaluate the effect of heat treatment, a severe sterilization condition (133 degrees C at 300 kPa for 20 min) was chosen. Species-specific amplicons were obtained from all types of heat-treated meat meals. Analysis of laboratory-contaminated vegetable meals revealed that the detection limit of the assay was 0.05% for each species analyzed. This PCR-based analysis can be used as a routine method for detecting banned animal-derived ingredients in raw and heat-treated feedstuffs.