Application of an asymmetric helical tube reactor for fast identification of gene transcripts of pathogenic viruses by micro flow-through PCR

Abstract

We have established a fast PCR-based micro flow-through process consisting of a helical constructed tube reactor. By this approach we can detect transcripts of measles and human papilloma virus (HPV) by continuous flow allowing for reverse transcription (RT) and amplification of cDNA. The micro reaction system consisted of two columnar reactors for thermostating the different reaction zones of the RT process and the amplification. The PCR reactor was built by asymmetric heating sections thus realizing different residence times and optimal conditions for denaturation, annealing and elongation. The system concept is based on low electrical power consumption (50-120 W) and is suited for portable diagnostic applications. The samples were applied in form of micro fluidic segments with single volumes between 65 and 130 nL injected into an inert carrier liquid inside a Teflon FEP tube with an inner diameter of 0.5 mm. Optimal amplification for template lengths of 292 bp (lambda-DNA), 127 bp (measles virus) and 95 bp (HPV) was achieved by maximal cycle times of 75 s.

Droplet-Based Microfluidics as a Biomimetic Principle: From PCR-Based Virus Diagnostics to a General Concept for Handling of Biomolecular Information

“…This disadvantage could be avoided by spiral-like arrangements [53]. An asymmetrical helical tube reactor was developed in order to realize optimal conditions of thermocycling with sufficient time for efficient elongation and a minimal thermal stress for the PCR mix during the denaturation phases [37]. The helical coiled microtube follows the periphery of a cylindric core and forms 42 loops of equal total length and constant length of the three temperature sections in each loop (Fig.…”

“…7.10a). The heating cylinder consists of three copper sections which are equipped with electrical heaters [37] and resistive temperature sensors for precise temperature control. The three sections can be heated individually in order to realize up to three different temperatures as required for denaturation, annealing, and primer extension.…”

“…7.11a). The complete microcontinuous-flow RT-PCR arrangement was applied for the detection of virus gene expression [37].…”

“…So, it can be applied, for example, for the implementation of PCR [34][35][36] and for the detection of virus transcripts by a RT-PCR [37]. In the following, principles of the handling and characterization of microfluid segments and the application of the technique for the detection of infective viruses will be explained and challenges for the future development of microsegmented flow devices to systems for handling of chemical and biological information will be discussed.…”

Analysis of Thermal Performance in a Bidirectional Thermocycler by Including Thermal Contact Characteristics

“…The mixture is kept static, and the temperature of the reaction chamber is cycled [2]. Some researchers have examined the continuous flow PCR (CFPCR) devices, and the reaction mixture moves through one or several different isothermal regions repetitively in a microchannel or tube [3]. There are three main construction types of the CFPCR reactors: unidirectional, closed-loop and oscillatory reactors.…”