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

Schematic of second stage PCR mix entering the array.The layers of film and adhesive attaching the array to the pouch are separated to show the flow of liquid into the cells of the array (figure is not to scale). From the top the layers are: 2nd pouch film, 1st pouch film, array adhesive layer (orange), pricked cover film, array (black, with wells), and array cover film. All of the actual layers are transparent except for the array itself. Second stage PCR primers are spotted into the cells during manufacture and air-dried (Methods). Arrows show the flow of PCR master mix (without primers) entering the array through a hole cut in the 1st pouch film.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198457&req=5

pone-0026047-g002: Schematic of second stage PCR mix entering the array.The layers of film and adhesive attaching the array to the pouch are separated to show the flow of liquid into the cells of the array (figure is not to scale). From the top the layers are: 2nd pouch film, 1st pouch film, array adhesive layer (orange), pricked cover film, array (black, with wells), and array cover film. All of the actual layers are transparent except for the array itself. Second stage PCR primers are spotted into the cells during manufacture and air-dried (Methods). Arrows show the flow of PCR master mix (without primers) entering the array through a hole cut in the 1st pouch film.

Mentions: The second stage PCR array is manufactured from 0.5 mm thick black polycarbonate plastic. 102 wells of 1 µl volume each are drilled into the array (“I” of Figure 1B and Figure 2). Laminating film is heat-sealed to the back of each array and 96 arrays are placed on a platen on the bed of a piezo-electric microarraying instrument (Nano-Plotter NP2.1e, GeSiM, Großerkmannsdorf, Germany). The second stage primer sets are dispensed into the wells of the array using the standard GeSiM Nano-Tip. After spotting, the arrays are sealed with a second layer of laminating film containing a matching array of holes (Figure 2) and then attached to the outside of the pouch (“I” in Figure 1) using pressure sensitive adhesive film.


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)

Schematic of second stage PCR mix entering the array.The layers of film and adhesive attaching the array to the pouch are separated to show the flow of liquid into the cells of the array (figure is not to scale). From the top the layers are: 2nd pouch film, 1st pouch film, array adhesive layer (orange), pricked cover film, array (black, with wells), and array cover film. All of the actual layers are transparent except for the array itself. Second stage PCR primers are spotted into the cells during manufacture and air-dried (Methods). Arrows show the flow of PCR master mix (without primers) entering the array through a hole cut in the 1st pouch film.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026047-g002: Schematic of second stage PCR mix entering the array.The layers of film and adhesive attaching the array to the pouch are separated to show the flow of liquid into the cells of the array (figure is not to scale). From the top the layers are: 2nd pouch film, 1st pouch film, array adhesive layer (orange), pricked cover film, array (black, with wells), and array cover film. All of the actual layers are transparent except for the array itself. Second stage PCR primers are spotted into the cells during manufacture and air-dried (Methods). Arrows show the flow of PCR master mix (without primers) entering the array through a hole cut in the 1st pouch film.
Mentions: The second stage PCR array is manufactured from 0.5 mm thick black polycarbonate plastic. 102 wells of 1 µl volume each are drilled into the array (“I” of Figure 1B and Figure 2). Laminating film is heat-sealed to the back of each array and 96 arrays are placed on a platen on the bed of a piezo-electric microarraying instrument (Nano-Plotter NP2.1e, GeSiM, Großerkmannsdorf, Germany). The second stage primer sets are dispensed into the wells of the array using the standard GeSiM Nano-Tip. After spotting, the arrays are sealed with a second layer of laminating film containing a matching array of holes (Figure 2) and then attached to the outside of the pouch (“I” in Figure 1) using pressure sensitive adhesive film.

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