DNA/RNA preparation for molecular detection.

BACKGROUND: Effective upstream preparation of nucleic acid (NA) is important for molecular techniques that detect unique DNA or RNA sequences. The isolated NA should be extracted efficiently and purified away from inhibitors of a downstream molecular assay. CONTENT: Many NA sample preparation techniques and commercial kits are available. Techniques for cell lysis and isolation or purification of NA were discovered in early NA characterization studies, evolved in the 20th century with molecular techniques, and still serve as the foundation for current methods. Advances in solid phase extraction methods with nonhazardous chemicals and automated systems have changed the way NA is prepared. Factors to consider when selecting NA preparation methods for molecular detection include lysis (from sources as diverse as human cells, viruses, bacterial spores, or protozoan oocysts), DNA vs RNA, sample background, appropriate preparation chemicals, and required detection limits. Methods are also selected on the basis of requirements for a particular application, such as sample volume or removal of inhibitors. Sometimes tradeoffs are made. SUMMARY: Good automated and manual methods are available to effectively prepare NA for molecular detection in under an hour. Numerous systems are available for various applications, including techniques that are flexible for multiple sample types, are capable of processing large batches, can be performed in <10 min, or that can yield high-purity NA. When methods are selected using the most applicable combination of lysis isolation efficiency and concentration, NA preparation can be very effective, even for molecular detection of multiple targets from the same sample.

Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system.

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.

RAZOR EX anthrax air detection system.

The RAZOR EX Anthrax Air Detection System, developed by Idaho Technology, Inc. (ITI), is a qualitative method for the detection of Bacillus anthracis spores collected by air collection devices. This system comprises a DNA extraction kit, a freeze-dried PCR reagent pouch, and the RAZOR EX real-time PCR instrument. Each pouch contains three assays, which distinguish potentially virulent B. anthracis from avirulent B. anthracis and other Bacillus species. These assays target the pXO1 and pXO2 plasmids and chromosomal DNA. When all targets are detected, the instrument makes an "anthrax detected" call, meaning that virulence genes of the anthrax bacillus are present. This report describes results from AOAC Method Developer (MD) and Independent Laboratory Validation (ILV) studies, which include matrix, inclusivity/exclusivity, environmental interference, upper and lower LOD of DNA, robustness, product consistency and stability, and instrument variation testing. In the MD studies, the system met the acceptance criteria for sensitivity and specificity, and the performance was consistent, stable, and robust for all components of the system. For the matrix study, the acceptance criteria of 95/96 expected calls was met for three of four matrixes, clean dry filters being the exception. Ninety-four of the 96 clean dry filter samples tested gave the expected calls. The nucleic acid limit of detection was 5-fold lower than AOAC's acceptable minimum detection limit. The system demonstrated no tendency for false positives when tested with Bacillus cereus. Environmental substances did not inhibit accurate detection of B. anthracis. The ILV studies yielded similar results for the matrix and inclusivity/exclusivity studies. The ILV environmental interference study included environmental substances and environmental organisms. Subsoil at a high concentration was found to negatively interfere with the pXO1 reaction. No interference was observed from the environmental organisms. The nucleic acid LOD, however, was 10 times higher (1 pg/reaction, equivalent to about 200 spores) than that found in the MD study. These results indicate that the RAZOR System is a sensitive and specific system that accurately identifies B. anthracis in aerosol matrixes and in the presence of interfering substances, and that the method can be performed by an independent laboratory and achieve similar results.

FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection.

The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the "FilmArray", which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.