An enclosed in-gel PCR amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics

Abstract

This work describes a self-contained, simple, disposable, and inexpensive gel capillary cassette for DNA amplification in near point of care settings. The cassette avoids the need for pumps or valves during raw sample delivery or polymerase chain reaction (PCR) amplification steps. The cassette contains capillary reaction units that can be stored at room temperature for up to 3 months. The current cassette configuration format simultaneously tests up to 16 patients for two or more targets, accommodates different sample types on the same cassette, has integrated positive and negative controls and allows flexibility for multiple geometries. PCR reagents in the cassette are desiccated to allow storage at room temperature with rehydration by raw sample at the time of testing. The sample is introduced to the cassette via a transfer pipette simply by capillary force. DNA amplification was carried out in a portable prototype instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis (MCA). To demonstrate performance, raw genital swabs and urine were introduced to the same cassette to simultaneously detect four sexually transmitted infections. Herpes Simplex Viruses (HSV-1 and HSV-2) were detected from raw genital swabs. Ureaplasma urealyticum (UU) and Mycoplasma homonis (MH) were detected from raw urine. Results for multiple patients were obtained in as little as 50 min. This platform allows multiparameter clinical testing with a pre-assembled cassette that requires only the introduction of raw sample. Modification of the prototype device to accommodate larger cassettes will ultimately provide high throughput simultaneous testing of even larger numbers of samples for many different targets, as is required for some clinical applications. Combinations of wax and/or polymer cassettes holding capillary reaction units are feasible. The components of the cassette are suited to mass production and robotic assembly to produce a readily manufactured disposable reaction cassette that can be configured for disease-specific testing panels. Rapid testing with a disposable reaction cassette on an inexpensive instrument will enable on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices.

Towards autonomous lab-on-a-chip devices for cell phone biosensing

“…Another interesting concept for molecular diagnostics supporting different type of samples utilizes modular in-gel PCR amplification cassettes configurable for multi-target detection (Figure 3e) (Manage et al, 2013). Modularity is achieved by isolating the detection elements, which consist of 7 mm-long capillary tubes filed with the PCR reaction mix and gel reagents.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…In this method RCA starts in the presence of circular DNA and other reaction components. The detection of the RCA products can be performed using surface associated methods such as microbead assisted diffractometry detection [78] and surface enhanced Raman spectroscopy [72]. Thus single protein recognition events are amplified thousands of times reaching a system sensitivity of 10 zeptomolar (1000 protein molecules) [72].…”

“…The rapidly growing field of soft-lithography driven microfluidic technology has resulted in the development a plethora of micro-devices for extraction [75] and amplification and analysis [31,[76][77][78][79][80][81] of nucleic acids. The main idea of microarray technology, namely spatial separation of microarray elements and their simultaneous subjection to one sample of interest can be easily realized using highly parallel architectures of micrometresized channels available/affordable due to microfluidics.…”

“…In general imaging of isothermal amplification products can be achieved by using all the various DNA visualization methods employed in microarray technologies such as hybridization with fluorescently labelled probes [63,64], chemiluminescent detection [65,66] and unspecific fluorescent dyes [11,78].…”

Advancements in Capillary-Assembled Microchip (CAs-CHIP) Development for Multiple Analyte Sensing and Microchip Electrophoresis

“…In general, potential applications for microchips have been demonstrated through various analytes, including viruses, 10,11 disease biomarkers, [12][13][14] environmental pollutants, [15][16][17] proteins and enzymes, [18][19][20][21][22] mammalian cells, [23][24][25] and antibiotics. 26 These involve various bioanalytical assays such as hybridization, immunoassay, and cell/viral analysis.…”