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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.

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Tm data used to establish assay specific melt windows.Histograms of the theoretical or observed Tms of the hMPV assay are shown. Tm data for the FilmArray runs includes each of the three replicates of the second stage PCR. A: Tms calculated from 13 sequence variants published in the NCBI databases. B: Tm data generated during the system beta-testing with 37 banked hMPV-positive patient samples. C: Tm data generated during the inclusivity testing with 10 hMPV strains representing subtypes A1, A2, B1 and B2. Multiple FilmArray runs of these strains are included in this data set. D: Tm data from 74 hMPV-positive patient samples collected during the clinical evaluation.
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pone-0026047-g007: Tm data used to establish assay specific melt windows.Histograms of the theoretical or observed Tms of the hMPV assay are shown. Tm data for the FilmArray runs includes each of the three replicates of the second stage PCR. A: Tms calculated from 13 sequence variants published in the NCBI databases. B: Tm data generated during the system beta-testing with 37 banked hMPV-positive patient samples. C: Tm data generated during the inclusivity testing with 10 hMPV strains representing subtypes A1, A2, B1 and B2. Multiple FilmArray runs of these strains are included in this data set. D: Tm data from 74 hMPV-positive patient samples collected during the clinical evaluation.

Mentions: To maximize the specificity of melt curve analysis, we determined the range of possible Tms for amplicons from each different organism assay. The theoretical melting temperature of a DNA sequence on the FilmArray instrument was calculated using the model (modified from [66]):(1)where GC is the mole fraction of G and C bp in the sequence, L is the length of the amplicon, and To, TGC, and TL are empirically fit parameters estimated using FilmArray data from samples of known sequence. GenBank was searched for sequence variants of each organism and these data were trimmed to the inner PCR product of the nmPCR. Predicted Tms for these variants were calculated using the mathematical model determined above (the data for hMPV are shown in Figure 7A). This distribution of Tms was used to establish the expected melt range for each assay. These ranges were expanded beyond the minimum and maximum predicted Tms to account for system variability (determined by the Tms of the control assays) and the Tms obtained from initial clinical testing (Figure 7B). To validate these predictions, the distributions of Tm data from testing of reference strains (Figure 7C) and from further clinical evaluations (Figure 7D) were compared to the initial melt range. The overlap in distribution of Tms between the different sample sets suggested that the melt ranges adequately, capture the full diversity of amplicon Tms for this organism. Narrowing the melt window in this way eliminates some false positives due to nonspecific amplification (data not shown).


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)

Tm data used to establish assay specific melt windows.Histograms of the theoretical or observed Tms of the hMPV assay are shown. Tm data for the FilmArray runs includes each of the three replicates of the second stage PCR. A: Tms calculated from 13 sequence variants published in the NCBI databases. B: Tm data generated during the system beta-testing with 37 banked hMPV-positive patient samples. C: Tm data generated during the inclusivity testing with 10 hMPV strains representing subtypes A1, A2, B1 and B2. Multiple FilmArray runs of these strains are included in this data set. D: Tm data from 74 hMPV-positive patient samples collected during the clinical evaluation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198457&req=5

pone-0026047-g007: Tm data used to establish assay specific melt windows.Histograms of the theoretical or observed Tms of the hMPV assay are shown. Tm data for the FilmArray runs includes each of the three replicates of the second stage PCR. A: Tms calculated from 13 sequence variants published in the NCBI databases. B: Tm data generated during the system beta-testing with 37 banked hMPV-positive patient samples. C: Tm data generated during the inclusivity testing with 10 hMPV strains representing subtypes A1, A2, B1 and B2. Multiple FilmArray runs of these strains are included in this data set. D: Tm data from 74 hMPV-positive patient samples collected during the clinical evaluation.
Mentions: To maximize the specificity of melt curve analysis, we determined the range of possible Tms for amplicons from each different organism assay. The theoretical melting temperature of a DNA sequence on the FilmArray instrument was calculated using the model (modified from [66]):(1)where GC is the mole fraction of G and C bp in the sequence, L is the length of the amplicon, and To, TGC, and TL are empirically fit parameters estimated using FilmArray data from samples of known sequence. GenBank was searched for sequence variants of each organism and these data were trimmed to the inner PCR product of the nmPCR. Predicted Tms for these variants were calculated using the mathematical model determined above (the data for hMPV are shown in Figure 7A). This distribution of Tms was used to establish the expected melt range for each assay. These ranges were expanded beyond the minimum and maximum predicted Tms to account for system variability (determined by the Tms of the control assays) and the Tms obtained from initial clinical testing (Figure 7B). To validate these predictions, the distributions of Tm data from testing of reference strains (Figure 7C) and from further clinical evaluations (Figure 7D) were compared to the initial melt range. The overlap in distribution of Tms between the different sample sets suggested that the melt ranges adequately, capture the full diversity of amplicon Tms for this organism. Narrowing the melt window in this way eliminates some false positives due to nonspecific amplification (data not shown).

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