Isothermal solid-phase amplification system for detection of Yersinia pestis

Abstract

DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.

Recombinase polymerase amplification: Basics, applications and recent advances

“…Whilst it was initially believed that specifically designed primers of 30e35 bases in length were necessary for RPA, there are several reports demonstrating that normal PCR primers can be used and efficient amplification achieved [5,6]. Longer primers (up to 45 nucleotides) can be used, but they could lead to secondary structures and potential primer artifacts.…”

“…In solution-phase, due to the unimpeded diffusion of primers and reaction reagents, amplification kinetics are favoured and the achieved limit of detection is subsequently usually better and amplification is achieved in a faster time than solid-phase. Nevertheless, solidphase and bridge amplification present some advantages, such as the potential for spatially resolved multiplexed amplification or the possibility to couple the amplification with diverse detection techniques including ring resonators [27e29], electrochemical [30e36] and colorimetric detection [5,6,30,33,37,38]. Several methods have been developed with solid phase amplification with performances usually inferior to that achieved with solution phase amplification [5,27e30,39] as primer accessibility is more restricted impeding amplification efficiency, and future work will need to focus on strategies to decrease amplification time.…”

“…In some strategies RPA is carried out in solution and the product captured by magnetic beads [33] or on a microtitre plate following denaturation of the duplex RPA amplicon [37]. Other strategies involve immobilising one of the primers on a substrate and performing solid phase amplification [5,30] followed by denaturation, hybridization with enzyme labelled reporter probe and optical/ electrochemical detection. In an alternative approach, chemiluminescence detection is achieved via the use of a biotinylated primer, and post-amplification incubation with streptavidinhorseradish peroxidase, luminol and H 2 O 2 [43].…”

Isothermal Nucleic Acid Amplification Techniques and Their Use in Bioanalysis

“…Despite the high rate of homogenous amplifica tion, heterogeneous amplification is used more widely, because in the majority of cases, it prevents the matrix effect and allows to develop more sensitive analytical techniques. It has been also reported that heterogeneous RPA reduces non specific amplification [55].…”

“…RPA can be used in combination with various signal readout methods for detection of different pathogens, e.g., Yersinia pestis [55]. In this study, the forward primer was immobilized in the wells of enzyme immunoassay microplate, and the reverse primer was modified with biotin for binding the peroxidase streptavidin conjugate.…”