RESUMEN
In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis.
Asunto(s)
ADN de Plantas/aislamiento & purificación , Ricinus/genética , Manejo de Especímenes/métodos , Automatización de Laboratorios , Ricinus communis/química , Sustancias para la Guerra Química/análisis , Extractos Vegetales/genética , Extractos Vegetales/aislamiento & purificación , Polvos/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Ricina/análisisRESUMEN
Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C T ) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4) colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.
Asunto(s)
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/aislamiento & purificación , ADN Bacteriano/sangre , ADN Bacteriano/aislamiento & purificación , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , ADN Bacteriano/genética , HumanosRESUMEN
The use of Bacillus anthracis as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of B. anthracis spores. This study determined the gamma irradiation dose for inactivating virulent B. anthracis spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of B. anthracis were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 10(7) CFU/ml to a dose of 2.5 x 10(6) rads resulted in a 6-log-unit reduction of spore viability. TaqMan real-time PCR analysis of untreated versus irradiated Ames strain (K1694) spores showed that treatment significantly enhanced the detection of B. anthracis chromosomal DNA targets but had no significant effect on the ability to detect targets on the pXO1 and pXO2 plasmids of B. anthracis. When analyzed by an enzyme-linked immunosorbent assay (ELISA), irradiation affected the detection of B. anthracis spores in a direct ELISA but had no effect on the limit of detection in a sandwich ELISA. The results of this study showed that gamma irradiation-inactivated spores can be tested by real-time PCR or sandwich ELISA without decreasing the sensitivity of either type of assay. Furthermore, the results suggest that clinical and public health laboratories which test specimens for B. anthracis could potentially incorporate gamma irradiation into sample processing protocols without compromising the sensitivity of the B. anthracis assays.
Asunto(s)
Bacillus anthracis/efectos de la radiación , Rayos gamma , Esporas Bacterianas/efectos de la radiación , Cromosomas Bacterianos , ADN Bacteriano/aislamiento & purificación , Relación Dosis-Respuesta en la Radiación , Ensayo de Inmunoadsorción Enzimática , Viabilidad Microbiana , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , VirulenciaRESUMEN
A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection.
Asunto(s)
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Animales , Secuencia de Bases , Chlorocebus aethiops , Cartilla de ADN/genética , ADN Viral/genética , Humanos , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Sensibilidad y Especificidad , Síndrome Respiratorio Agudo Grave/diagnóstico , Síndrome Respiratorio Agudo Grave/epidemiología , Síndrome Respiratorio Agudo Grave/virología , Células VeroAsunto(s)
Bacillus anthracis/genética , Bacillus anthracis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , Carbunco/diagnóstico , Carbunco/microbiología , Bacillus anthracis/clasificación , Bioterrorismo , Monitoreo del Ambiente , Genes Bacterianos , Genotipo , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Sequence analysis was conducted on the hemagglutinin (H) and nucleoprotein (N) genes from nine wild-type measles viruses (MV) isolated during an outbreak that occurred in Morocco during 1998 and 1999. The sequence data showed that all the viruses were closely related to each other and were members of genotype C2. Genotype C2 has been shown to be circulating in Europe, and the sequences of the Moroccan isolates were most closely related to the sequences of recent viral isolates from western Europe. This report presents the first molecular epidemiological study of circulating wild-type measles viruses in Morocco. Knowledge of the indigenous strain of measles virus circulating in Morocco will help to describe viral transmission pathways and should contribute to efforts to evaluate the effectiveness of future vaccination campaigns.