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

Measurement of substrate thermal resistance using DNA denaturation temperature

Heat Transfer and Thermal Management have become important aspects of the developing field of uTAS systems particularly in the application of the the uTAS philosophy to thermally driven analysis techniques such as PCR. Due to the development of flowing PCR thermocyclers in the field of uTAS, the authors have previously developed a melting curve analysis technique that is compatible with these flowing PCR thermocyclers. In this approach a linear temperature gradient is induced along a sample carrying microchannel. Any flow passing through the microchannel is subject to linear heating. Fluorescent monitoring of DNA in the flow results in the generation of DNA melting curve plots. This works presents an experimental technique where DNA melting curve analysis is used to measure the thermal resistance of microchannel substrates. DNA in solution is tested at a number of different ramp rates and the di®erent apparent denaturation temperatures measured are used to infer the thermal resistance of the microchannel substrates. The apparent variation in denaturation temperature is found to be linearly proportional to flow ramp rate. Providing knowledge of the microchannel diameter and a non-varying cross-section in the direction of heat flux the thermal resistance measurement technique is independent of knowledge of substrate dimensions, contact surface quality and substrate composition/material properties. In this approach to microchannel DNA melting curve analysis the difference between the measured and actual denaturation temperatures is proportional to the substrate thermal resistance and the ramp-rate seen by the sample. Therefore quantitative knowledge of the substrate thermal resistance is required when using this technique to measure accurately DNA denaturation temperatur

High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer

BACKGROUND: The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterisation of cancers. We describe here the application of high-resolution melting analysis (HRM) to screen for KRAS mutations in clinical cancer samples. In non-small cell lung cancer, KRAS mutations have been shown to identify a group of patients that do not respond to EGFR targeted therapies and the identification of these mutations is thus clinically important. METHODS: We developed a high-resolution melting (HRM) assay to detect somatic mutations in exon 2, notably codons 12 and 13 of the KRAS gene using the intercalating dye SYTO 9. We tested 3 different cell lines with known KRAS mutations and then examined the sensitivity of mutation detection with the cell lines using 189 bp and 92 bp amplicons spanning codons 12 and 13. We then screened for KRAS mutations in 30 non-small cell lung cancer biopsies that had been previously sequenced for mutations in EGFR exons 18–21. RESULTS: Known KRAS mutations in cell lines (A549, HCT116 and RPMI8226) were readily detectable using HRM. The shorter 92 bp amplicon was more sensitive in detecting mutations than the 189 bp amplicon and was able to reliably detect as little as 5–6% of each cell line DNA diluted in normal DNA. Nine of the 30 non-small cell lung cancer biopsies had KRAS mutations detected by HRM analysis. The results were confirmed by standard sequencing. Mutations in KRAS and EGFR were mutually exclusive. CONCLUSION: HRM is a sensitive in-tube methodology to screen for mutations in clinical samples. HRM will enable high-throughput screening of gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer

A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis

BACKGROUND: Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection

Fluorescent Duplex Allele-Specific PCR and Amplicon Melting for Rapid Homogeneous mtDNA Haplogroup H Screening and Sensitive Mixture Detection

BACKGROUND:For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA), sequence variation in particular mt haplogroups (hgs) and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS) assay were scrutinized in the light of these requirements. METHODOLOGY/PRINCIPAL FINDINGS:A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples) or the derived 7028C allele (hg H samples) in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA). The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed approximately 2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification. CONCLUSIONS:Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the analysis of mixtures. Homogeneous ARMS-DCA is a valuable tool for large-volume studies targeting small numbers of single nucleotide polymorphisms (SNPs)

Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR

BACKGROUND: Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of +/-25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4-8 determinations from each amplification reaction. FINDINGS: Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp) is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy. CONCLUSIONS: Founded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the potential to fundamentally transform how real-time qPCR is conducted

High-resolution (mtDNA) melting analysis for simple and efficient characterization of Africanized honey bee Apis mellifera (Hymenoptera:Apidae)

Analysis of the mtDNA variation in Apis mellifera L. has allowed distinguishing subspecies and evolutionary lineages by means of different molecular methods; from RFLP, to PCR-RFLP and direct sequencing. Likewise, geometric morphometrics (GM) has been used to distinguish Africanized honey bees with a high degree of consistency with studies using molecular information. High-resolution fusion analysis (HRM) allows one to quickly identify sequence polymorphisms by comparing DNA melting curves in short amplicons generated by real-time PCR (qPCR). The objective of this work was to implement the HRM technique in the diagnosis of Africanization of colonies of A. mellifera from Argentina, using GM as a validation method. DNA was extracted from 60 A. mellifera colonies for mitotype identification. Samples were initially analyzed by HRM, through qPCRs of two regions (485 bp/385 bp) of the mitochondrial cytochrome b gene (cytb). This technique was then optimizing to amplify a smaller PCR product (207 bp) for the HRM diagnosis for the Africanization of colonies. Of the 60 colony samples analyzed, 41 were classified as colonies of European origin whereas 19 revealed African origin. All the samples classified by HRM were correctly validated by GM, demonstrating that this technique could be implemented for arapid identification of African mitotypes in Apis mellifera samples.Fil: Porrini, Leonardo Pablo. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Brasesco, Maria Constanza. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Maggi, Matías Daniel. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Eguaras, Martin Javier. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Quintana, Silvina. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; Argentin

The development of molecular assays for the detection and identification of inherited disorders of haemoglobin in a highly heterogeneous population

Inherited disorders of haemoglobin are the most common monogenic diseases in the world. These conditions arise as a result of deletions or point mutations affecting the globin genes. Gap polymerase chain reaction (Gap-PCR) is commonly used to detect globin gene deletions, however this technique is not ideal since it is time consuming and only able to detect known deletions with well-defined breakpoints. To address these issues an alternative technique called Gene Ratio Assay Copy Enumeration (GRACE) PCR was developed. In contrast to Gap-PCR, GRACE-PCR is a rapid, closed tube technique, requiring no further hands on time after the initial PCR setup. Furthermore, since it is based on copy number determination, GRACE-PCR does not require prior knowledge of the breakpoints and can thus be used for the detection of both known and novel deletions. GRACE-PCR methods were validated for the most clinically significant globin genes, HBA1, HBA2 and HBB. The large number of point mutations associated with the HBB gene make gene scanning by High Resolution Melting (HRM) PCR a potentially attractive diagnostic method. HRM-PCR assays have been previously described, however interference from certain Single Nucleotide Polymorphisms (SNPs) limited their ability to detect some mutations. Through the use of unlabelled probes and primers incorporating universal bases, it was possible to develop a more universally applicable HRM-PCR assay that was not affected by common SNPs. Additionally, a number of examples of two very rare haemoglobins, Hb Fontainebleau and Hb Handsworth were encountered during the course of this work. Each of these variants had previously been reported in just ten individuals worldwide. The diagnostic features of these two rare variants are described