Design of a single-tube, endpoint, linear-after-the-exponential-PCR assay for 17 pathogens associated with sepsis.

AIMS: The goal of this study was to construct a single-tube multiplex molecular diagnostic assay using linear-after-the-exponential (LATE)-PCR for the detection of 17 microbial pathogens commonly associated with septicaemia. METHODS AND RESULTS: The assay described here detects 17 pathogens associated with sepsis via amplification and analysis of gene-specific sequences. The pathogens and their targeted genes were: Klebsiella spp. (phoE); Acinetobacter baumannii (gyrB); Staphylococcus aureus (spa); Enterobacter spp. (thdF); Pseudomonas aeruginosa (toxA); coagulase-negative staphylococci (tuf), Enterococcus spp. (tuf); Candida spp. (P450). A sequence from an unidentified gene in Lactococcus lactis, served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were analysed at endpoint over a wide range of temperatures in four fluorescent colours. Each target was detected by its pattern of hybridization to a sequence-specific low-temperature fluorescent probe derived from molecular beacons. CONCLUSIONS: All 17 microbial targets were detected in samples containing low numbers of pathogen genomes in the presence of high levels of human genomic DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay used new technology to achieve an advance in the field of molecular diagnostics: a single-tube assay for detection of pathogens commonly responsible for septicaemia.

Design and verification of a highly reliable Linear-After-The-Exponential PCR (LATE-PCR) assay for the detection of African swine fever virus.

African swine fever virus (ASFV) is a highly pathogenic DNA virus that is the causative agent of African swine fever (ASF), an infectious disease of domestic and wild pigs of all breeds and ages, causing a range of syndromes. Acute disease is characterized by high fever, haemorrhages in the reticuloendothelial system, and a high mortality rate. A powerful novel diagnostic assay based on the Linear-After-The-Exponential-PCR (LATE-PCR) principle was developed to detect ASFV. LATE-PCR is an advanced form of asymmetric PCR which results in direct amplification of large amount of single-stranded DNA. Fluorescent readings are acquired using endpoint analysis after PCR amplification. Amplification of the correct product is verified by melting curve analysis. The assay was designed to amplify the VP72 gene of ASFV genome. Nineteen ASFV DNA cell culture virus strains and three tissue samples (spleen, tonsil, and liver) from infected experimental pigs were tested. Virus was detected in all of the cell culture and tissue samples. None of five ASFV-related viruses tested produced a positive signal, demonstrating the high specificity of the assay. The sensitivity of the LATE-PCR assay was determined in two separate real-time monoplex reactions using samples of synthetic ASFV and synthetic control-DNA targets that were diluted serially from 109 to 1 initial copies per reaction. The detection limit was 1 and 10 copies/reaction, respectively. The sensitivity of the assay was also tested in a duplex end-point reactions comprised of a constant level of 150 copies of synthetic control-DNA and a clinical sample of spleen tissue diluted serially from 10⁻¹ to 10⁻5. The detection limit was 10⁻5 dilution which corresponds to approximately 1 copy/reaction. Since the assay is designed to be used in either laboratory settings or in a portable PCR machine (Bio-Seeq Portable Veterinary Diagnostics Laboratory; Smiths Detection, Watford UK), the LATE-PCR provides a robust and novel tool for the diagnosis of ASF both in the laboratory and in the field.