Thermally multiplexed polymerase chain reaction

Abstract

Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 ll, oil-encapsulated reactions, and closed-loop pulsewidth modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 C and 68 C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. V C 2015 AIP Publishing LLC.

Rapid modeling of space structure of rock mass and the method of equivalent assignment

“…The number of networks will directly affect the efficiency of numerical computation. The algorithm used by COMSOL software is the finite element method . Similarly, most algorithm‐based finite element method is discrete (the computational domain is discretized into a finite element calculation, and the number of meshes directly affects the computational complexity of the solution).…”

Recent progress in the development of ultra high-sensitive and rapid detection systems for pathogens of emerging infectious diseases

“…PCR based on infrared (IR) heating: The idea of IR heating to drive PCR has been reported many years ago using a conventional tungsten lamp [41][42][43][44]. In this method, thermal cycling in the microfluidic system is usually carried out on the stage of the microscope by using IR light through an IR long-pass filter (Figure 1), so that the simultaneous fluorescence measurement for the confirmation and/or quantification of the PCR is successfully carried out during the thermal cycling (Figure 2).…”

“…An incoherent light source such as a classical tungsten lamp or a near-IR LED is more effective than the near-IR laser for this kind of application. The demonstration of the simple PCR in the microfluidic chip in the initial studies on this methodology, as well as that of the quantitative PCR and practical diagnostic assays of the influenza virus based on RT-PCR, have been reported [43,44]. The virus assay of a mock nasal swab sample at clinically relevant concentrations was carried out using a carefully designed, integrated two-chip system consisting of solid-phase extraction (SPE) and RT-PCR systems [43].…”

Towards Multiplex Molecular Diagnosis—A Review of Microfluidic Genomics Technologies

“…Son et al [ 108 ] achieved thermal ramping from 55 °C (annealing) to 95 °C (denaturation) within 5 min by employing thin Au films that convert incident LED light into heat through plasmon-assisted optical absorption ( Figure 7 ). Phaneuf et al [ 109 ] presented thermal multiplexing for multiplexed PCR using a laser diode, a single radiative heat source that is selectively modulated for temperature control in an array of PCR reactions. The system is able to generate different annealing temperatures in a single microchip suitable for simultaneous amplification 500 bp and 600 bp amplicons.…”