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

Poritz MA, Blaschke AJ, Byington CL, Meyers L, Nilsson K, Jones DE, Thatcher SA, Robbins T, Lingenfelter B, Amiott E, Herbener A, Daly J, Dobrowolski SF, Teng DH, Ririe KM - PLoS ONE (2011)

Bottom Line: FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time.Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens.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.

View Article: PubMed Central - PubMed

Affiliation: Idaho Technology, Inc., Salt Lake City, Utah, United States of America. mark_poritz@idahotech.com

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Real-time amplification and melt curves from the array.Respiratory Pathogen pouches were injected with viral transport medium spiked with 200 TCID50 FluA H1-seasonal (panels A and B), 4×106 cfu B. per and 200 TCID50 FluA-H1 (panels C and D), or 4×106 cfu B. per (panels E and F) and run on the FilmArray instrument. Real time amplification curves (panels A and C and E) and post-amplification melt curves (panels B and D and F) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red), FluA-H3 (black), B. per (Green), Yeast RNA process control (dark blue), Second stage PCR control (light blue). For clarity the controls are shown in panels E and F only.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198457&req=5

pone-0026047-g004: Real-time amplification and melt curves from the array.Respiratory Pathogen pouches were injected with viral transport medium spiked with 200 TCID50 FluA H1-seasonal (panels A and B), 4×106 cfu B. per and 200 TCID50 FluA-H1 (panels C and D), or 4×106 cfu B. per (panels E and F) and run on the FilmArray instrument. Real time amplification curves (panels A and C and E) and post-amplification melt curves (panels B and D and F) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red), FluA-H3 (black), B. per (Green), Yeast RNA process control (dark blue), Second stage PCR control (light blue). For clarity the controls are shown in panels E and F only.

Mentions: Typical amplification and melt curves generated using a research version of the FilmArray instrument and RP pouch are shown in Figure 4. FluA H1 virus (200 Tissue Culture Infectious Doses-50% (TCID50)) injected into an RP pouch produced amplification products in the wells of the array containing PCR primers specific for all of the FluA H1 specific targets (Figure 4A). Melting curves generated from wells for a given assay have Tms that are characteristic of the amplicons from those targets (Figure 4B). The FluA H3 assay, which should not be positive for this virus, does not show evidence of amplification or melt peaks. Figure 4E and F show the result of injecting a very high level of another organism, B. per, into a FilmArray pouch. The amplification curves for the B. per assay replicates have a Cq of 5. This is the earliest Cq observed in the system and represents dilution and second stage amplification of an outer amplicon that has fully entered plateau in the first stage reaction.


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

Poritz MA, Blaschke AJ, Byington CL, Meyers L, Nilsson K, Jones DE, Thatcher SA, Robbins T, Lingenfelter B, Amiott E, Herbener A, Daly J, Dobrowolski SF, Teng DH, Ririe KM - PLoS ONE (2011)

Real-time amplification and melt curves from the array.Respiratory Pathogen pouches were injected with viral transport medium spiked with 200 TCID50 FluA H1-seasonal (panels A and B), 4×106 cfu B. per and 200 TCID50 FluA-H1 (panels C and D), or 4×106 cfu B. per (panels E and F) and run on the FilmArray instrument. Real time amplification curves (panels A and C and E) and post-amplification melt curves (panels B and D and F) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red), FluA-H3 (black), B. per (Green), Yeast RNA process control (dark blue), Second stage PCR control (light blue). For clarity the controls are shown in panels E and F only.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198457&req=5

pone-0026047-g004: Real-time amplification and melt curves from the array.Respiratory Pathogen pouches were injected with viral transport medium spiked with 200 TCID50 FluA H1-seasonal (panels A and B), 4×106 cfu B. per and 200 TCID50 FluA-H1 (panels C and D), or 4×106 cfu B. per (panels E and F) and run on the FilmArray instrument. Real time amplification curves (panels A and C and E) and post-amplification melt curves (panels B and D and F) for selected wells on the array are shown. Assays are spotted in triplicate: FluA-pan1 (orange), FluA-pan2 (pink), FluA-H1-pan (red), FluA-H3 (black), B. per (Green), Yeast RNA process control (dark blue), Second stage PCR control (light blue). For clarity the controls are shown in panels E and F only.
Mentions: Typical amplification and melt curves generated using a research version of the FilmArray instrument and RP pouch are shown in Figure 4. FluA H1 virus (200 Tissue Culture Infectious Doses-50% (TCID50)) injected into an RP pouch produced amplification products in the wells of the array containing PCR primers specific for all of the FluA H1 specific targets (Figure 4A). Melting curves generated from wells for a given assay have Tms that are characteristic of the amplicons from those targets (Figure 4B). The FluA H3 assay, which should not be positive for this virus, does not show evidence of amplification or melt peaks. Figure 4E and F show the result of injecting a very high level of another organism, B. per, into a FilmArray pouch. The amplification curves for the B. per assay replicates have a Cq of 5. This is the earliest Cq observed in the system and represents dilution and second stage amplification of an outer amplicon that has fully entered plateau in the first stage reaction.

Bottom Line: FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time.Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens.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.

View Article: PubMed Central - PubMed

Affiliation: Idaho Technology, Inc., Salt Lake City, Utah, United States of America. mark_poritz@idahotech.com

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus