Design and construction of a single tube, quantitative endpoint, LATE-PCR multiplex assay for ventilator-associated pneumonia.

AIMS: The goal of this study was to develop a molecular diagnostic multiplex assay for the quantitative detection of microbial pathogens commonly responsible for ventilator-associated pneumonia (VAP) and their antibiotic resistance using linear-after-the-exponential polymerase chain reaction (LATE-PCR). METHOD AND RESULTS: This multiplex assay was designed for the quantitative detection and identification of pathogen genomic DNA of methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, Pseudomonas aeruginosa, plus a control target from Lactococcus lactis. After amplification, the single-stranded amplicons were detected simultaneously in the same closed tube by hybridization to low-temperature molecular beacon probes labelled with four differently coloured fluorophores. The resulting hybrids were then analysed by determining the fluorescence intensity of each of the four fluorophores as a function of temperature. CONCLUSIONS: This LATE-PCR single tube multiplex assay generated endpoint fluorescent contours that allowed identification of all microbial pathogens commonly responsible for VAP, including MRSA. The assay was quantitative, identifying the pathogens present in the sample, no matter whether there were as few as 10 or as many 100 000 target genomes. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay is rapid, reliable and sensitive and is ready for preclinical testing using samples recovered from patients suffering from ventilator-associated pneumonia.

Design and optimization of a novel reverse transcription linear-after-the-exponential PCR for the detection of foot-and-mouth disease virus.

AIMS: A novel molecular assay for the detection of foot-and-mouth disease virus (FMDV) was developed using linear-after-the-exponential polymerase chain reaction (LATE-PCR). METHODS AND RESULTS: Pilot experiments using synthetic DNA targets demonstrated the ability of LATE-PCR to quantify initial target concentration through endpoint detection. A two-step protocol involving reverse transcription (RT) followed by LATE-PCR was then used to confirm the ability of the assay to detect FMDV RNA. Finally, RT and LATE-PCR were combined in a one-step duplex assay for co-amplification of an FMDV RNA segment and an internal control comprised of an Armored RNA. In that form, each of the excess primers in the reaction mixture hybridize to their respective RNA targets during a short pre-incubation, then generate cDNA strands during a 3-min RT step at 60°C, and the resulting cDNA is amplified by LATE-PCR without intervening sample processing. CONCLUSIONS: The RT-LATE-PCR assay generates fluorescent signals at endpoint that are proportional to the starting number of RNA targets and can detect a range of sequence variants using a single mismatch-tolerant probe. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to offering improvements over current laboratory-based molecular diagnostic assays for FMDV, this new assay is compatible with a novel portable ('point-of-care') device, the BioSeeq II, designed for the rapid diagnosis of FMD in the field.

Differential killing of pre-B acute lymphoblastic leukaemia cells by activated NK cells and the NK-92 ci cell line.

The use of NK cells in adoptive therapy for malignant disease is an area of great potential. Currently the only NK cell line in clinical trials is NK-92, an activated NK cell line with a broad range of cytotoxicity against malignant cells. The activity of NK-92 against pre-B acute lymphoblastic leukaemias, however, is highly variable. In this study we compare the cytotoxic mechanisms and signalling pathways utilized by NK-92 ci and IL-2 activated NK cells to mediate killing of pre-B acute lymphoblastic leukaemia cell lines. Deficiencies in TNF family mediated apoptosis, phosphoinositide-3 kinase dependent and phosphoinositide-3 kinase independent killing limit the efficiency of NK-92 ci against pre-B acute lymphoblastic leukaemia cells. Importantly, treatment of the poorly killed leukaemia cells with TNF-alpha augmented both phosphoinositide-dependent and -independent cytolysis.