ABSTRACT
The capacity of absorbent beads in BacT/ALERT® FA Plus and BACTEC® Aerobic/F Plus blood culture bottles to bind and neutralize antibiotics was compared. Binding was established using reverse-phase HPLC, and inactivation was based on the recovery of susceptible test stains from simulated blood cultures. The FA Plus medium demonstrated more rapid and better overall binding kinetics for each drug tested, resulting in significantly better overall recovery rates. Differences in time to detection favored the FA Plus medium for three drug/organism combinations and Aerobic/F Plus for two.
Subject(s)
Adsorption , Anti-Bacterial Agents/isolation & purification , Blood Culture/methods , Culture Media/chemistry , Specimen Handling/methods , Anti-Bacterial Agents/analysis , Chromatography, High Pressure Liquid , Humans , Kinetics , Time FactorsABSTRACT
Q beta replicase can replicate a single molecule of certain species of RNA to 10(14) copies in minutes. This replication ability has been used for in vitro studies of molecular evolution and is currently being utilized as a method of amplifying RNAs that contain probe sequences. It has been observed that Q beta replicase can produce replicatable RNA even in the absence of exogenously added template RNA. The origin of this RNA has been ascribed either to contamination with replicatable RNA or to an ability of Q beta replicase to synthesize RNA de novo from the nucleotides present in the reaction. Technologies that employ Q beta replicase require a thorough understanding of the conditions that lead to this so-called spontaneous RNA production. We have created an expression system and purification method with which we produce gram quantities of highly purified Q beta replicase, and we have identified reaction conditions that prevent the amplification of RNA in assays that do not contain added RNA. However, when these reaction conditions are relaxed, spontaneous RNA replication is seen in up to 100% of the assays. To understand the origin of this RNA, we have cloned several spontaneously produced RNAs. Sequence analysis of one of these RNAs shows that it arose by the evolution of Escherichia coli tRNA into a replicatable template and not by de novo synthesis from nucleoside triphosphates in the reaction.
Subject(s)
Q beta Replicase/metabolism , RNA, Bacterial/metabolism , Allolevivirus/genetics , Allolevivirus/metabolism , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Bacterial , Escherichia coli/genetics , Escherichia coli/metabolism , Metals/metabolism , Molecular Sequence Data , Nucleic Acid Conformation , Q beta Replicase/isolation & purification , RNA Probes , RNA, Bacterial/biosynthesis , RNA, Bacterial/chemistry , RNA, Transfer/metabolism , RNA, Viral/metabolism , Templates, GeneticABSTRACT
Antibodies specific for DNA:RNA hybrids were coated onto polystyrene test tubes and applied to hybridization assays involving DNA and RNA. Synthetic DNA probes complementary to 16S rRNA of Campylobacter were labeled with biotin and hybridized to ribosomal RNA directly in lysates of bacterial cells. After hybridization, DNA:RNA hybrids were captured with immobilized anti-DNA:RNA antibody, and the biotinylated probe was detected with streptavidin-horseradish peroxidase (EC 1.11.1.7) conjugate. The assay was optimized to detect as few as 70,000 Campylobacter cells in a sample. We compared the utility of this hybridization assay with that of conventional microbiology methods by examination of 1448 stool samples from hospital clinical laboratories. The DNA hybridization assay had a sensitivity of 98.7% (75/76) and a specificity of 98.2% (1347/1372) and overall agreed with 98.2% of the conventional results for a test population that had a 5.2% incidence (76/1448) of Campylobacter infection. The assay is simple to perform and yields results within 2.5 h.