RESUMEN
A reverse transcription Linear-After-The-Exponential polymerase chain reaction (RT LATE-PCR) assay was evaluated for detection of foot-and-mouth disease virus (FMDV). This pan-serotypic assay targets highly conserved sequences within the 3D (RNA polymerase) region of the FMDV genome, and uses end-point hybridisation analysis of a single mismatch-tolerant low temperature probe to confirm the identity of the amplicons. An Armored RNA served as an internal control to validate virus negative results. The ability of the assay to identify FMDV was directly compared to a real-time RT-PCR assay routinely used by reference laboratories. The analytical sensitivity of the RT LATE-PCR assay was 10 genomic copies and the dynamic range of the test was identical to real-time RT-PCR based on decimal dilutions of an FMDV-positive sample. This pan-serotypic assay was able to detect FMDV in a broad range of clinical samples collected from field cases of FMD (n = 121), while samples of other viruses causing vesicular disease in livestock and genetic relatives of FMDV were negative. In addition to the laboratory-based utility of this diagnostic test, the RT LATE-PCR assay format has potential application for use in a portable ("point-of-care") device designed to achieve rapid detection of FMDV in the field.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Virus de la Fiebre Aftosa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas Virales/genética , Animales , ADN Complementario/química , ADN Complementario/genética , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/virología , Virus de la Fiebre Aftosa/clasificación , Virus de la Fiebre Aftosa/enzimología , Genoma Viral/genética , Datos de Secuencia Molecular , ARN Viral/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Serotipificación , Especificidad de la EspecieRESUMEN
Rapid identification of specific TEM-type ß-lactamase genes in bacterial infections is important for determining appropriate clinical treatment. We report here the design and initial testing of a molecular diagnostic assay capable of amplifying a large segment of the blaTEM gene, as well as detecting widely spaced extended-spectrum ß-lactamase (ESBL) mutations and inhibitor-resistant TEM (IRT) mutations (eg, clavulanic acid resistance). Single-stranded DNA is generated using linear-after-the-exponential PCR (LATE-PCR) and is analyzed at the endpoint, using a set of four fluorescently labeled and four quencher-labeled probes in a single closed tube. These lights-on/lights-off probes work in concert to generate sequence-specific fluorescence contours over a temperature range from 25°C to 75°C. Mutant sequences from synthetic TEM gene variants and from TEM gene variants in bacterial strains generated large increases in fluorescent signal relative to that from the reference sequence for TEM-1. Clinical use of this convenient, single-closed-tube assay would make it possible to rapidly distinguish ESBL from non-ESBL variants and thereby to begin early treatment with suitable antibiotics.