A Homogeneous HLA-B*27 Genotyping Assay Using Dried Reagent Mixtures

Ilonen, Jorma; Lövgren, Timo

doi:10.1155/2009/849784

Cited by 6 publications

(1 citation statement)

References 18 publications

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“…87 Kiviniemi et al have reported using a combination of vacuum-and air-drying to stabilize PCR reagents. 88 Paraffinpassivation is another method of stabilizing reagents on-board microfluidic chips, in which reagents are added during assembly and then encapsulated in paraffin wax. 89 Glassification 90 is another method of reagent stabilization used in commercial products which includes the addition of a glass-forming filler material, such as glucose, sucrose, maltose or maltotriose, to produce material that is stable at room temperature using freeze-drying 91,92 or vacuum-drying.…”

Section: 81mentioning

confidence: 99%

Device Engineering for Infectious Disease Diagnosis using Isothermal DNA Amplification and Lateral Flow Detection

Roskos

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Technologies that enable infectious diseases diagnosis in low-resource settings could greatly facilitate effective treatment and containment of such diseases. Nucleic acid amplification testing can be used to identify pathogens, but typically requires highlytrained personnel and large, expensive lab equipment, neither of which is available in low-resource settings. Our overall goal is to develop a portable diagnostic system that utilizes a low-cost, disposable, mesofluidic cartridge and a handheld electronics unit to perform fully-integrated nucleic acid testing at the point of care in low-resource settings.As a first step toward this goal, we developed a subunit to execute isothermal nucleic acid amplification coupled with lateral flow detection, in parallel with the development of a sample preparation subunit by our collaborators at Claremont BioSolutions. Fluid handling inside the amplification and detection cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard, scalable manufacturing techniques, such as injection molding. Using an initial prototype system, we demonstrated detection of purified Mycobacterium tuberculosis genomic DNA. We then developed a refined amplification and detection cartridge in conjunction with an improved portable instrument, which automates pumping, heating, and timing, using a design format compatible with eventual integration with the sample preparation subunit.This refined cartridge incorporates a novel, inexpensive, stand-alone, passive valve, smaller, integrated pump components, a more complex injection molded polycarbonate cartridge core piece, and enhanced lateral flow chambers to improve visual detection. The independent valve component can be tailored for a variety of fluidic systems. We demonstrated appropriate fluidic and thermal control, and successful isothermal nucleic acid amplification within this refined amplification and detection subunit. We have developed a separate fluidic module for master-mix reagent storage and reconstitution that is designed to act as the interface between the amplification and detection subunit and the upstream sample preparation subunit. We envision that the merger of these two subunits into a fully-integrated cartridge will enable user-friendly, automated sample-in to answer-out diagnosis of infectious diseases in primary care settings of low-resource countries with high disease burden.

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Section: 81mentioning

confidence: 99%

Device Engineering for Infectious Disease Diagnosis using Isothermal DNA Amplification and Lateral Flow Detection

Roskos

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One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDX™ PCR system

Hirvonen

Lode²,

Nevalainen

et al. 2012

Eur J Clin Microbiol Infect Dis

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A method for the rapid detection of methicillin-sensitive and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) with a straightforward sample preparation protocol of blood cultures using an automated homogeneous polymerase chain reaction (PCR) assay, the GenomEra™ MRSA/SA (Abacus Diagnostica Oy, Turku, Finland), is presented. In total, 316 BacT/Alert (bioMérieux, Marcy l'Etoile, France) and 433 BACTEC (Becton Dickinson, Sparks, MD, USA) blood culture bottles were analyzed, including 725 positive cultures containing Gram-positive cocci in clusters (n = 419) and other Gram stain forms (n = 361), as well as 24 signal- and growth-negative bottles. Detection sensitivities for MSSA, MRSA, and MRCoNS were 99.4 % (158/159), 100.0 % (9/9), and 99.3 % (132/133), respectively. One false-positive MRSA result was detected from a non-staphylococci-containing bottle, yielding a specificity of 99.8 %. The lowest detectable amount of viable cells in the blood culture sample was 4 × 10(4) CFU/mL. The results were available within one hour after microbial growth detection and the two-step, time-resolved fluorometric (TRF) measurement mode employed by the GenomEra CDX™ instrument showed no interference from blood, charcoal, or culture media. The method described lacks all sample purification steps and allows reliable and simplified pathogen detection also in clinical microbiology laboratory settings without specialized molecular microbiology competence.

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Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes

Lehmusvuori¹,

Ilonen²,

Soukka³

2014

Analytical Biochemistry

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A Homogeneous HLA-B*27 Genotyping Assay Using Dried Reagent Mixtures

Cited by 6 publications

References 18 publications

Device Engineering for Infectious Disease Diagnosis using Isothermal DNA Amplification and Lateral Flow Detection

Device Engineering for Infectious Disease Diagnosis using Isothermal DNA Amplification and Lateral Flow Detection

One-step sample preparation of positive blood cultures for the direct detection of methicillin-sensitive and -resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci within one hour using the automated GenomEra CDX™ PCR system

Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes

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