Critical insight into recombinase polymerase amplification technology.

INTRODUCTION: Recombinase polymerase amplification (RPA) is a promising and emerging technology for rapidly amplifying target nucleic acid from minimally processed samples and through small portable instruments. RPA is suitable for point-of-care testing (POCT) and on-site field testing, and it is compatible with microfluidic devices. Several detection assays have been developed, but limited research has dug deeper into the chemistry of RPA to understand its kinetics and fix its shortcomings. AREAS COVERED: This review provides a detailed introduction of RPA molecular mechanism, kits formats, optimization, application, pros, and cons. Moreover, this critical review discusses the nonspecificity issue of RPA, highlights its consequences, and emphasizes the need for more research to resolve it. This review discusses the reaction kinetics of RPA in relation to target length, product quantity, and sensitivity. This critical review also questions the novelty of recombinase-aided amplification (RAA). In short, this review discusses many aspects of RPA technology that have not been discussed previously and provides a deeper insight and new perspectives of the technology. EXPERT OPINION: RPA is an excellent choice for pathogen detection, especially in low-resource settings. It has a potential to replace PCR for all purposes, provided its shortcomings are fixed and its reagent accessibility is improved.

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors.

DNA extraction can be lengthy and sometimes ends up with amplification inhibitors. We present the potential of recombinase polymerase amplification (RPA) to replace plant DNA extraction. In our rapid 'RPA-PCR couple' concept, RPA is tuned to slower reaction kinetics to promote amplification of long targets. RPA primers amplify target and some flanking regions directly from simple plant macerates. Then PCR primers exponentially amplify the target directly from the RPA reaction. We present the coupling of RPA with conventional, TaqMan and SYBR Green PCR assays. We applied the concept to strawberry Phytophthora pathogens and the Phytophthora identification marker atp9-nad9. We found RPA-PCR couple specific, sensitive and reliable. The approach may also benefit other difficult samples such as food, feces and ancient samples.

Expression of the GAF Sensor, Carbohydrate-Active Enzymes, Elicitins, and RXLRs Differs Markedly Between Two Phytophthora cactorum Isolates.

The phytopathogen Phytophthora cactorum infects economically important herbaceous and woody plant species. P. cactorum isolates differ in host specificity; for example, strawberry crown rot is often caused by a specialized pathotype. Here we compared the transcriptomes of two P. cactorum isolates that differ in their virulence to garden strawberry (Pc407: high virulence; Pc440: low virulence). De novo transcriptome assembly and clustering of contigs resulted in 19,372 gene clusters. Two days after inoculation of Fragaria vesca roots, 3,995 genes were differently expressed between the P. cactorum isolates. One of the genes that were highly expressed only in Pc407 encodes a GAF sensor protein potentially involved in membrane trafficking processes. Two days after inoculation, elicitins were highly expressed in Pc407 and lipid catabolism appeared to be more active than in Pc440. Of the carbohydrate-active enzymes, those that degrade pectin were often more highly expressed in Pc440, whereas members of glycosyl hydrolase family 1, potentially involved in the metabolism of glycosylated secondary metabolites, were more highly expressed in Pc407 at the time point studied. Differences were also observed among the RXLR effectors: Pc407 appears to rely on a smaller set of key RXLR effectors, whereas Pc440 expresses a greater number of RXLRs. This study is the first step toward improving understanding of the molecular basis of differences in the virulence of P. cactorum isolates. Identification of the key effectors is important, as it enables effector-assisted breeding strategies toward crown rot-resistant strawberry cultivars.

Deregulated expression of circadian clock and clock-controlled cell cycle genes in chronic lymphocytic leukemia.

Circadian rhythms are endogenous and self-sustained oscillations of multiple biological processes with approximately 24-h rhythmicity. Circadian genes and their protein products constitute the molecular components of the circadian oscillator that form positive/negative feedback loops and generate circadian rhythms. The circadian regulation extends from core clock genes to various clock-controlled genes that include various cell cycle genes. Aberrant expression of circadian clock genes, therefore, may lead to genomic instability and accelerated cellular proliferation potentially promoting carcinogenesis. The current study encompasses the investigation of simultaneous expression of four circadian clock genes (Bmal1, Clock, Per1 and Per2) and three clock-controlled cell cycle genes (Myc, Cyclin D1 and Wee1) at mRNA level and determination of serum melatonin levels in peripheral blood samples of 37 CLL (chronic lymphocytic leukemia) patients and equal number of age- and sex-matched healthy controls in order to indicate association between deregulated circadian clock and manifestation of CLL. Results showed significantly down-regulated expression of Bmal1, Per1, Per2 and Wee1 and significantly up-regulated expression of Myc and Cyclin D1 (P < 0.0001) in CLL patients as compared to healthy controls. When expression of these genes was compared between shift-workers and non-shift-workers within the CLL group, the expression was found more aberrant in shift-workers as compared to non-shift-workers. However, this difference was found statistically significant for Myc and Cyclin D1 only (P < 0.05). Serum melatonin levels were found significantly low (P < 0.0001) in CLL subjects as compared to healthy controls whereas melatonin levels were found still lower in shift-workers as compared to non-shift-workers within CLL group (P < 0.01). Our results suggest that aberrant expression of circadian clock genes can lead to aberrant expression of their downstream targets that are involved in cell proliferation and apoptosis and hence may result in manifestation of CLL. Moreover, shift-work and low melatonin levels may also contribute in etiology of CLL by further perturbing of circadian clock.