Rapid Real-Time Nucleic Acid Sequence-Based Amplification-Molecular Beacon Platform To Detect Fungal and Bacterial Bloodstream Infections

Abstract

Bloodstream infections (BSIs) are a significant cause of morbidity and mortality. Successful patient outcomes are diminished by a failure to rapidly diagnose these infections and initiate appropriate therapy. A rapid and reliable diagnostic platform of high sensitivity is needed for the management of patients with BSIs. The combination of an RNA-dependent nucleic acid sequence-based amplification and molecular beacon (NASBA-MB) detection system in multiplex format was developed to rapidly detect medically important BSI organisms. Probes and primers representing pan-gram-negative, pan-gram-positive, pan-fungal, pan-Candida, and panAspergillus organisms were established utilizing 16S and 28S rRNA targets for bacteria and fungi, respectively. Two multiplex panels were developed to rapidly discriminate bacterial or fungal infections at the subkingdom/ genus level with a sensitivity of 1 to 50 genomes. A clinical study was performed to evaluate the accuracy of this platform by evaluating 570 clinical samples from a tertiary-care hospital group using blood bottle samples.

Multiplex Blood PCR in Combination with Blood Cultures for Improvement of Microbiological Documentation of Infection in Febrile Neutropenia

“…Homemade multiplex or broad-range PCR assays for the detection of bloodstream pathogens in cases of sepsis or febrile neutropenia have provided variable sensitivity and specificity compared with blood cultures (5,8,9,28,31,42,44). Their use is limited by the lack of standardized technical procedures and commercially available systems.…”

Biomarkers for Invasive Aspergillosis: The Challenges Continue

“…Nucleic acid-based tests (NATs) comprise a range of amplification technologies and sophisticated detection methods that are readily adaptable for use in the fungal diagnostic laboratory [134] The most commonly used NAT is real-time qPCR, although there are a few reports describing the use of nucleic acid sequencebased amplification, an isothermal technique for the amplification of RNA that is especially powerful when combined with molecular beacon chemistry [135][136][137][138]. However, the theoretical advantage of higher sensitivity and reduced likelihood of carry-over contamination must be balanced by the complexity of this technology: it requires a combination of three enzymes, the avian myeloblastosis virus, reverse transcriptase/ DNA polymerase, T7 RNA polymerase and a separate RNase H [134].…”

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Rapid Detection and Quantification of agr Functionality in Clinical Staphylococcus aureus Isolates

“…The artificial target RNA fragments for RNAIII and gyrB were synthesized by in vitro transcription as previously described (24). Primers used to generate specific PCR products as template for in vitro transcription are listed in Table 1.…”