Dynamic Second Harmonic Imaging of Proton Translocation Through Water Needles in Lipid Membranes.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we report apparently universal transmembrane potential fluctuations in lipid membrane systems. Molecular simulations and free energy calculations suggest that H+ permeation proceeds predominantly across a thin, membrane-spanning water needle and that the transient transmembrane potential drives H+ ions across the water needle. This mechanism differs from the transport of other cations that require completely open pores for transport and follows naturally from the well-known Grotthuss mechanism for proton transport in bulk water. Furthermore, SH imaging and conductivity measurements reveal that the rate of proton transport depends on the structure of the hydrophobic core of bilayer membranes.

Biocontrol of bacterial wilt in tomato with a cocktail of lytic bacteriophages.

Bacteriophages (phages) have been proposed as promising alternative pesticides against various bacterial diseases of crops. However, the efficacy of phages in managing plant bacterial diseases is variable and poorly understood in natural settings. In this study, two lytic phages, RpT1 and RpY2, were investigated for their biocontrol potential against bacterial wilt by Ralstonia pseudosolanacearum invasion in tomato plants. The two phages possess similar morphology and genome organization to those of the Autographiviridae family with a broad host range. Treatment with the two phages (alone or in combination) resulted in a significant reduction in bacterial wilt incidence. Three days post-treatment with phages, which was performed after R. pseudosolanacearum inoculation with a specified density of 108 PFU (plaque forming units)/g of soil, led to the most effective biocontrol activity compared to other treatments and a lower density of phage. A phage cocktail containing both RpT1 and RpY2 suppressed disease symptoms in agricultural soils, mimicking their ability to control diseases in natural settings. Furthermore, supplementation with specific adjuvants enhanced the biocontrol potential of both phages. The persistence of the two phages under various environmental conditions indicates their stable activity in soils. Consequently, the consistent biocontrol activity of these phages provides insights into the proper application, timing, and density of phages for effective phage therapy in bacterial wilt control in tomato. KEY POINTS: • Biocontrol potential of phages in natural settings individually and as a cocktail. • Apparent long-term persistence of phages in natural soils, various temperatures, and pH. • An effective approach for developing phages for biocontrol.

A Novel Valued Tolerance Rough Set and Decision Rules Method for Indoor Positioning Using WiFi Fingerprinting.

In recent years, due to the ubiquitous presence of WiFi access points in buildings, the WiFi fingerprinting method has become one of the most promising approaches for indoor positioning applications. However, the performance of this method is vulnerable to changes in indoor environments. To tackle this challenge, in this paper, we propose a novel WiFi fingerprinting method that uses the valued tolerance rough set theory-based classification method. In the offline phase, the conventional received signal strength (RSS) fingerprinting database is converted into a decision table. Then a new fingerprinting database with decision rules is constructed based on the decision table, which includes the credibility degrees and the support object set values for all decision rules. In the online phase, various classification levels are applied to find out the best match between the RSS values in the decision rules database and the measured RSS values at the unknown position. The experimental results compared the performance of the proposed method with those of the nearest-neighbor-based and the random statistical methods in two different test cases. The results show that the proposed method greatly outperforms the others in both cases, where it achieves high accuracy with 98.05% of right position classification, which is approximately 50.49% more accurate than the others. The mean positioning errors at wrong estimated positions for the two test cases are 1.71 m and 1.99 m, using the proposed method.

Flavobacterium dauae sp. nov., isolated from rhizosphere soil of a tomato plant.

A bacterial strain, designated TCH3-2T, was isolated from the rhizosphere of tomato plant grown at Dong-A University Agricultural Experiment Station, Republic of Korea. The strain was Gram-stain-negative, obligate aerobic, orange yellow-coloured, motile by gliding and short rod-shaped. Strain TCH3-2 T only grew on 1/2 tryptic soy agar and Luria-Bertani agar among the media tested, with optimum growth at 28 °C and pH 7. Salt of 1 % NaCl was necessary to support the growth of TCH3-2T. Strain TCH3-2T produced flexirubin-type pigments. The predominant cellular fatty acids were iso-C15 : 0 (55.6 %), iso-C17 : 0 3-OH (17.9 %), summed feature 9 (comprising C16 : 0 10-methyl and/or iso-C17 : 1 ω9c; 10.5 %), iso-C15 : 0 3-OH (4.8 %) and anteiso-C15 : 0 (2.3 %). The major menaquinone was menaquinone-6 and the major polar lipids were phosphatidylethanolamine, five unknown aminolipids and three unknown lipids. Phylogenetic analysis based on 16S rRNA sequences indicated that TCH3-2T was closely related to Flavobacterium ummariense DS-12T (95.16 %), Flavobacterium marinum SW105T (95.14 %) and Flavobacterium viscosus YIM 102796T (94.54 %). The draft genome of TCH3-2T comprised ca. 2.8 Mb with a G+C content of 34.61 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between TCH3-2T and closely related Flavobacterium species showed that it belongs to a distinct species. Furthermore, the results of morphological, physiological and biochemical tests allowed further phenotypic differentiation of TCH3-2T from its closest relatives. Thus, chemotaxonomic characteristics together with phylogenetic affiliation illustrate that TCH3-2T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium dauae sp. nov. (type strain TCH3-2T=KACC 19054T=JCM 34025T) is proposed.

Enhancement of electrode performance through surface modification using carbon nanotubes and porous gold nanostructures.

Recently, the demand for the sensitive detection of nanomaterials and biomolecules has been increasing for evaluating the toxicity of nanomaterials and early diagnosis of diseases. Although many studies have developed new detection assays, these are heavily influenced by the capabilities of the detection equipment. Therefore, the aim of the present study was to improve electrode performance by modifying the surface of the detection electrode using a simple method. Electrode surface modification was performed using carbon nanotubes (CNT) and porous gold nanostructures (NS) with excellent electrical and chemical properties. Through the simple physical deposition of CNT and electrochemical reduction of NS, the increasement of the electrode surface area was achieved. Because of the CNTs attached to the electrodes at the first step, the metal ions constituting the NS can adhere well to the electrodes. Nanoparticles with a porous structure can be generated through electrochemical reduction (cyclic voltammetry) of metal ions attached to electrodes. Consequently, the surface area of the electrode increased and electrochemical performance was improved (confirmed by atomic force microscopy, Nyquist plot and Bode plot). To quantitatively confirm the improvement of electrode performance according to the surface change through the proposed treatment technique, DNA was detected. Unlike previous surface modification studies, the developed surface treatment technique can be applied to a variety of detection equipment. To confirm this, the detection was performed using two detection devices with different operating principles. DNA detection using the two types of equipment confirmed that the detection limit was increased by approximately 1000-fold through applying a simple surface treatment. In addition, this method is applicable to detect various sizes of nanomaterials. The method proposed in this study is simple and has the advantage that it can be applied to various devices and various materials.

Imaging Cu2+ binding to charged phospholipid membranes by high-throughput second harmonic wide-field microscopy.

The interaction of divalent copper ions (Cu2+) with cell membranes is crucial for a variety of physiological processes of cells, such as hormone synthesis and cellular energy production. These interactions would not be possible without membrane hydration. However, the role of water has not received a lot of attention in membrane studies. Here, we use high-throughput wide-field second harmonic (SH) microscopy to study the interaction between Cu2+ and hydrated freestanding Montal-Müller lipid membranes. The symmetric lipid membranes are composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and either 1,2-diphytanoyl-sn-glycero-3-phosphate or 1,2-diphytanoyl-sn-glycero-3-phospho L-serine and are brought into contact with divalent Cu2+, which are added to one leaflet while maintaining the ionic strength balance. We observe transient domains of high SH intensity. In these areas, Cu2+ ions bind to the charged head groups, leading to charge neutralization on one side of the membrane. This exposes the ordered water at the non-interacting side of the membrane interface, which can be used to compute the interfacial membrane potential difference. We find that the domains of lipids with phosphatidic acid head groups display a higher interfacial membrane potential than those with phosphatidylserine head groups, which converts into higher dynamic electrostatic free energies and binding constants.

Complete Genome Sequence of a Novel Bacteriophage RpY1 Infecting Ralstonia solanacearum Strains.

Ralstonia solanacearum species complex is deleterious plant pathogenic bacteria causing bacterial wilt in the members of solanaceous crops and the bacterial wilt is difficult to control. Bacteriophages-based biocontrol is an environmentally friendly and promising strategy to control bacterial plant diseases. In this study, we isolated 72 phages from the various crop cultivated soils in Korea using five different strains of R. solanacearum. Among 72 phages, phage RpY1 was selected for further study based on the specificity of the targeted host. This phage was identified as a member of Podoviridae with a head measuring 60-70 nm in length and short tail according to the morphology of transmission electron microscopy images. The genome size of phage RpY1 is 43,284 bp with G + C content of 61.4% and 53 open reading frames (ORFs), including 18 annotated ORFs and 35 hypothetical proteins. This phage genome showed no homology to the genome of known phages except for the DU_RP_II phage infecting R. solanacearum; however, the host range of phage RpY1 is much narrower than that of DU_RP_II.

Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase.

Enoyl-acyl carrier protein reductases (ENR), such as FabI, FabL, FabK, and FabV, catalyze the last reduction step in bacterial type II fatty acid biosynthesis. Previously, we reported metagenome-derived ENR homologs resistant to triclosan (TCL) and highly similar to 7-α hydroxysteroid dehydrogenase (7-AHSDH). These homologs are commonly found in Epsilonproteobacteria, a class that contains several human-pathogenic bacteria, including the genera Helicobacter and Campylobacter Here we report the biochemical and predicted structural basis of TCL resistance in a novel 7-AHSDH-like ENR. The purified protein exhibited NADPH-dependent ENR activity but no 7-AHSDH activity, despite its high homology with 7-AHSDH (69% to 96%). Because this ENR was similar to FabL (41%), we propose that this metagenome-derived ENR be referred to as FabL2. Homology modeling, molecular docking, and molecular dynamic simulation analyses revealed the presence of an extrapolated six-amino-acid loop specific to FabL2 ENR, which prevented the entry of TCL into the active site of FabL2 and was likely responsible for TCL resistance. Elimination of this extrapolated loop via site-directed mutagenesis resulted in the complete loss of TCL resistance but not enzyme activity. Phylogenetic analysis suggested that FabL, FabL2, and 7-AHSDH diverged from a common short-chain dehydrogenase reductase family. This study is the first to report the role of the extrapolated loop of FabL2-type ENRs in conferring TCL resistance. Thus, the FabL2 ENR represents a new drug target specific for pathogenic Epsilonproteobacteria.

Thickness-Dependent Phonon Renormalization and Enhanced Raman Scattering in Ultrathin Silicon Nanomembranes.

We report on the thickness-dependent Raman spectroscopy of ultrathin silicon (Si) nanomembranes (NMs), whose thicknesses range from 2 to 18 nm, using several excitation energies. We observe that the Raman intensity depends on the thickness and the excitation energy due to the combined effects of interference and resonance from the band-structure modulation. Furthermore, confined acoustic phonon modes in the ultrathin Si NMs were observed in ultralow-frequency Raman spectra, and strong thickness dependence was observed near the quantum limit, which was explained by calculations based on a photoelastic model. Our results provide a reliable method with which to accurately determine the thickness of Si NMs with thicknesses of less than a few nanometers.

Comparative bacterial community analysis in relatively pristine and anthropogenically influenced mangrove ecosystems on the Red Sea.

Mangrove habitats are ecologically important ecosystems that are under severe pressure worldwide because of environmental changes and human activities. In this study, 16S rRNA gene amplicon deep-sequencing was used to compare bacterial communities in Red Sea mangrove ecosystems at anthropogenically influenced coastal sites with those at a relatively pristine island site. In total, 32 phyla were identified from the mangrove rhizospheres, with Proteobacteria predominating at each of the studied sites; however, the relative abundance was significantly decreased at the coastal sites (Mastorah, MG-MS; Ar-Rayis, MG-AR) compared with the pristine island site near Dhahban (MG-DBI). The phyla Actinobacteria, Firmicutes, Acidobacteria, Chloroflexi, Spirochetes, and Planctomycetes were present at a relative abundance of >1% at the MG-MS and MG-AR sites, but their concentration was <1% at the MG-DBI site. A total of 1659 operational taxonomic units (OTUs) were identified at the species level, and approximately 945 OTUs were shared across the different sampling sites. Multivariate principal coordinate data analysis separated the MG-DBI site from the MG-AR and MG-MS cluster. Specific bacterial taxa were enriched at each location, and in particular, the genera Pseudoalteromonas and Cobetia were predominantly identified in the MG-DBI site compared with the anthropogenically influenced coastal sites.

Synergistic Effects of Oxygen on Phosphine and Ethyl Formate for the Control of Phthorimaea operculella (Lepidoptera: Gelechiidae).

Phosphine (PH3) and ethyl formate (EF) are two potentially powerful postharvest fumigant insecticides. We investigated the effectiveness of both PH3 and EF as fumigants at all developmental stages of the potato tuber moth Phthorimaea operculella Zeller, and we also studied the synergistic effects of these fumigants under controlled atmospheres of 50 and 80% oxygen (O2). The larval stage of P. operculella was the most susceptible to fumigation with PH3 at both 5°C and 20°C. All of the developmental stages showed greater susceptibility to PH3 at 20°C than at 5°C, whereas the susceptibility of adult P. operculella to this fumigant was not affected by temperature. The toxicity of EF did not differ with temperature for any of the P. operculella developmental stages. The atmospheric oxidation of PH3 increased the toxicity of this fumigant toward all developmental stages at both temperatures. In contrast, no differences in toxicity were observed for oxidized EF compared with EF alone at any developmental stage. In conclusion, using fumigation tests, we showed that atmospherically oxidized PH3 was much more effective against P. operculella than PH3 alone, demonstrating a synergistic effect for this fumigant and O2. Therefore, treatment with PH3 and high concentrations of O2, as described in this study, could be useful for managing the postharvest pest P. operculella.

The Plant-Associated Flavobacterium: A Hidden Helper for Improving Plant Health.

Flavobacterium is a genus within the phylum Bacteroidota that remains relatively unexplored. Recent analyses of plant microbiota have identified the phylum Bacteroidota as a major bacterial group in the plant rhizosphere. While Flavobacterium species within the phylum Bacteroidota have been recognized as pathogens in the aquatic habitats, microbiome analysis and the characterization of novel Flavobacterium species have indicated the great diversity and potential of their presence in various environments. Many Flavobacterium species have positively contribute to plant health and development, including growth promotion, disease control, and tolerance to abiotic stress. Despite the well-described beneficial interactions of the Flavobacterium species with plants, the molecular mechanisms and bacterial determinants underlying these interactions remain unclear. To broaden our understanding of the genus Flavobacterium's role in plant health, we review the recent studies focusing on their ecological niche, functional roles, and determinants in plant-beneficial interactions. Additionally, this review discusses putative mechanisms explaining the interactions between plants and Flavobacterium. We have also introduced the importance of future research on Flavobacterium spp. and its potential applications in agriculture.

The MYB-CC Transcription Factor PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) Functions in Phosphate Homeostasis and Affects Salt Stress Tolerance in Rice.

Inorganic phosphate (Pi) homeostasis plays an important role in plant growth and abiotic stress tolerance. Several MYB-CC transcription factors involved in Pi homeostasis have been identified in rice (Oryza sativa). PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) is a class II MYC-CC protein, in which the MYC-CC domain is located at the N terminus. In this study, we established that OsPHL7 is localized to the nucleus and that the encoding gene is induced by Pi deficiency. The Pi-responsive genes and Pi transporter genes are positively regulated by OsPHL7. The overexpression of OsPHL7 enhanced the tolerance of rice plants to Pi starvation, whereas the RNA interference-based knockdown of this gene resulted in increased sensitivity to Pi deficiency. Transgenic rice plants overexpressing OsPHL7 produced more roots than wild-type plants under both Pi-sufficient and Pi-deficient conditions and accumulated more Pi in the shoots and roots. In addition, the overexpression of OsPHL7 enhanced rice tolerance to salt stress. Together, these results demonstrate that OsPHL7 is involved in the maintenance of Pi homeostasis and enhances tolerance to Pi deficiency and salt stress in rice.

Rhizobacterial syntrophy between a helper and a beneficiary promotes tomato plant health.

Microbial interactions impact the functioning of microbial communities. However, microbial interactions within host-associated communities remain poorly understood. Here, we report that the beneficiary rhizobacterium Niallia sp. RD1 requires the helper Pseudomonas putida H3 for bacterial growth and beneficial interactions with the plant host. In the absence of the helper H3 strain, the Niallia sp. RD1 strain exhibited weak respiration and elongated cell morphology without forming bacterial colonies. A transposon mutant of H3 in a gene encoding succinate-semialdehyde dehydrogenase displayed much attenuated support of RD1 colony formation. Through the subsequent addition of succinate to the media, we found that succinate serves as a public good that supports RD1 growth. Comparative genome analysis highlighted that RD1 lacked the gene for sufficient succinate, suggesting its evolution as a beneficiary of succinate biosynthesis. The syntrophic interaction between RD1 and H3 efficiently protected tomato plants from bacterial wilt and promoted tomato growth. The addition of succinate to the medium restored complex II-dependent respiration in RD1 and facilitated the cultivation of various bacterial isolates from the rhizosphere. Taken together, we delineate energy auxotrophic beneficiaries ubiquitous in the microbial community, and these beneficiaries could benefit host plants with the aid of helpers in the rhizosphere.

Effect of Gallotannins Derived from Sedum takesimense on Tomato Bacterial Wilt.

In the process of searching antibacterial agents from plants, we discovered that the methanol extract of Sedum takesimense showed potent antibacterial activity against Ralstonia solanacearum in vitro and in vivo. Eight antibacterial gallotannins were isolated from the aerial parts of S. takesimense and identified as gallic acid, methyl gallate, 4,6-di-O-galloylarbutin, 2,6-di-O-galloylarbutin, 2,4,6-tri-O-galloyl-glucose, 1,3,4,6-tetra-O-galloyl-ß-glucose, 1,2,4,6-tetra-O-galloyl-ß-glucose, and 1,2,3,6-tetra-O-galloyl-ß-glucose based on electrospray ionization mass spectrometry and proton nuclear magnetic resonance spectroscopy. These gallotannins displayed broad-spectrum activity against various plant-pathogenic bacteria, and the strongest in vitro antibacterial activities of these gallotannins were against R. solanacearum minimum inhibitory concentration = 0.02 to 0.10 g/liter). Among these gallotannins, methyl gallate and 1,2,3,6-tetra-O-galloyl-ß-glucose showed the strongest activities. In addition, synergistic or partial synergistic effects were observed in most combinations between major antibacterial compounds. The wettable powder formulation of the S. takesimense crude extract effectively reduced the development of tomato bacterial wilt caused by R. solanacearum under greenhouse conditions for 14 days after infection. This is the first report on the isolation of antibacterial compounds from S. takesimense. These results suggest that the extract from S. takesimense or the isolated gallotannins could be used as natural bactericides for the control of tomato bacterial wilt.

A Mutation of a Putative NDP-Sugar Epimerase Gene in Ralstonia pseudosolanacearum Attenuates Exopolysaccharide Production and Bacterial Virulence in Tomato Plant.

Ralstonia solanacearum species complex (RSSC) is a soil borne plant pathogen causing bacterial wilt on various important crops, including Solanaceae plants. The bacterial pathogens within the RSSC produce exopolysaccharide (EPS), a highly complicated nitrogen-containing heteropolymeric polysaccharide, as a major virulence factor. However, the biosynthetic pathway of the EPS in the RSSC has not been fully characterized. To identify genes in EPS production beyond the EPS biosynthetic gene operon, we selected the EPS-defective mutants of R. pseudosolanacearum strain SL341 from Tn5-inserted mutant pool. Among several EPS-defective mutants, we identified a mutant, SL341P4, with a Tn5-insertion in a gene encoding a putative NDP-sugar epimerase, a putative membrane protein with sugar-modifying moiety, in a reverse orientation to EPS biosynthesis gene cluster. This protein showed similar to other NDP-sugar epimerases involved in EPS biosynthesis in many phytopathogens. Mutation of the NDP-sugar epimerase gene reduced EPS production and biofilm formation in R. pseudosolanacearum. Additionally, the SL341P4 mutant exhibited reduced disease severity and incidence of bacterial wilt in tomato plants compared to the wild-type SL341 without alteration of bacterial multiplication. These results indicate that the NDP-sugar epimerase gene is required for EPS production and bacterial virulence in R. pseudosolanacearum.

Draft genome sequence of the antifungal-producing plant-benefiting bacterium Burkholderia pyrrocinia CH-67.

Burkholderia pyrrocinia CH-67 was isolated from forest soil as a biocontrol agent to be utilized in agriculture. Here, we report the 8.05-Mb draft genome sequence of this bacterium. Its genome contains genes involved in biosynthesis of secondary metabolites and plant growth promotion, which may contribute to probiotic effects on plants.

Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase.

Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.

Exploration for the salt stress tolerance genes from a salt-treated halophyte, Suaeda asparagoides.

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse transcription-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S. asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides would contribute for the accumulating genetic resources to improve osmotic tolerance in plants.

Ultrahigh sensitive and selective detection of single nucleotide polymorphism using peptide nucleic acid and ribonuclease H assembled DNA amplification (PRADA).

Early diagnosis and monitoring of cancer is the best way to increase the survival rate among patients with cancer. However, the current cancer screening technology is expensive and time-consuming; hence, cancer screening remains challenging. Therefore, developing a relatively inexpensive and high-performance analytical method is necessary. Especially, mutations in KRAS can be characterized as single nucleotide polymorphism mutations. Therefore, discrimination of single nucleotide polymorphism is essential to detect cancer mutations. This study introduces a method with high sensitivity and selectivity of real-time PCR using peptide nucleic acid (PNA) and RNase H II to detect KRAS single nucleotide polymorphism. This method was developed via the fusion of the existing PNA clamping PCR technique and the RNase H-dependent PCR technique. A synergistic effect was realized by mitigating the shortcomings of each method. Our method had a detection limit of 1 aM and selectivity of 0.01%. This study demonstrated completed validation tests with DNA-spiked plasma sample analysis, cell culture, and analysis of blood samples collected from patients with cancer. Furthermore, we demonstrated the applicability of this method for breath biopsy.