Expanding Archaeal Diversity and Phylogeny: Past, Present, and Future.

The discovery of the Archaea is a major scientific hallmark of the twentieth century. Since then, important features of their cell biology, physiology, ecology, and diversity have been revealed. Over the course of some 40 years, the diversity of known archaea has expanded from 2 to about 30 phyla comprising over 20,000 species. Most of this archaeal diversity has been revealed by environmental 16S rRNA gene amplicon sequencing surveys using a broad range of universal and targeted primers. Of the few primers that target a large fraction of known archaeal diversity, all display a bias against recently discovered lineages, which limits studies aiming to survey overall archaeal diversity. Induced by genomic exploration of archaeal diversity, and improved phylogenomics approaches, archaeal taxonomic classification has been frequently revised. Due to computational limitations and continued discovery of new lineages, a stable archaeal phylogeny is not yet within reach. Obtaining phylogenetic and taxonomic consensus of archaea should be a high priority for the archaeal research community.

Genome-wide simple sequence repeat analysis and specific molecular marker development of rye.

BACKGROUND: Rye (Secale cereale L.) is the most widely used related species in wheat genetic breeding, and the introduction of its chromosome fragments into the wheat genome through distant hybridization is essential for enriching the genetic diversity of wheat. Rapid and accurate detection of rye chromatin in the wheat genome is important for distant hybridization. Simple sequence repeats (SSRs) are widely distributed in the genome, and SSRs of different species often exhibit species-specific characteristics. RESULTS: In this study, genome-wide SSRs in rye were identified, and their characteristics were outlined. A total of 997,027 SSRs were selected, with a density of 115.97 SSRs/Mb on average. There was no significant difference in the number of SSRs on each chromosome. The number of SSRs on 2R was the highest (15.29%), and the number of SSRs on 1R was the lowest (13.02%). The number of SSRs on each chromosome is significantly correlated with chromosome length. The types of SSR motifs were abundant, and each type of SSR was distributed on 7 chromosomes of rye. The numbers of mononucleotide simple sequence repeats (MNRs), dinucleotide simple sequence repeats (DNRs), and trinucleotide simple sequence repeats (TNRs) were the greatest, accounting for 46.90%, 18.37%, and 22.64% of the total number, respectively. Among the MNRs, the number of G/C repeats and the number of 10 bp motifs were the greatest, accounting for 26.24% and 31.32% of the MNRs, respectively. Based on the SSR sequences, a total of 657 pairs of primers were designed. The PCR results showed that 119 pairs of these primers were rye-specific and could effectively detect rye chromatin in the wheat genome. Moreover, 86 pairs of the primers could also detect one or more specific rye chromosomes. CONCLUSION: These results lay a foundation for both genomic evolution studies of rye and molecular breeding in wheat.

Marker-assisted introgression to improve the oleic acid content in the TMV 7 groundnut (Arachis hypogaea L.) variety suitable for the oil industry.

BACKGROUND: Improving the quality and shelf life of groundnut oil is one of the foremost objectives of groundnut breeding programmes. This can be achieved by marker-assisted introgression, a technique that efficiently and precisely enables breeders to develop plants with enhanced qualities. This study focused on improving the oleic acid content of an elite groundnut variety, TMV 7, by introgressing a recessive mutation responsible for the increase in oleic acid from ICG 15419. Hybridization was performed between the donor and recurrent parents to develop the F1, BC1F1, BC2F1 and BC2F2 populations. Introgressed lines with increased oleic acid in the genetic background of TMV 7 were identified using allele-specific marker, F435-F, F435SUB-R and a set of SSR markers were employed to recover the genome of the recurrent parent. RESULTS: With two backcrosses, a total of ten homozygous plants in the BC2F2 population were identified with oleic acid content ranging from 54.23 to 57.72% causing an increase of 36% over the recurrent parent. Among the ten lines, the line IL-23 exhibited the highest level of recurrent parent genome recovery of 91.12%. CONCLUSIONS: The phenotypic evaluation of 10 homozygous introgressed lines indicated fewer differences for all other traits under study compared to the recurrent parent, except for oleic acid and linoleic acid content confirming the genetic background of the recurrent parent. The identified lines will be subjected to multilocation trials before their commercial release.

Environmental DNA: The next chapter.

Molecular tools are an indispensable part of ecology and biodiversity sciences and implemented across all biomes. About a decade ago, the use and implementation of environmental DNA (eDNA) to detect biodiversity signals extracted from environmental samples opened new avenues of research. Initial eDNA research focused on understanding population dynamics of target species. Its scope thereafter broadened, uncovering previously unrecorded biodiversity via metabarcoding in both well-studied and understudied ecosystems across all taxonomic groups. The application of eDNA rapidly became an established part of biodiversity research, and a research field by its own. Here, we revisit key expectations made in a land-mark special issue on eDNA in Molecular Ecology in 2012 to frame the development in six key areas: (1) sample collection, (2) primer development, (3) biomonitoring, (4) quantification, (5) behaviour of DNA in the environment and (6) reference database development. We pinpoint the success of eDNA, yet also discuss shortfalls and expectations not met, highlighting areas of research priority and identify the unexpected developments. In parallel, our retrospective couples a screening of the peer-reviewed literature with a survey of eDNA users including academics, end-users and commercial providers, in which we address the priority areas to focus research efforts to advance the field of eDNA. With the rapid and ever-increasing pace of new technical advances, the future of eDNA looks bright, yet successful applications and best practices must become more interdisciplinary to reach its full potential. Our retrospect gives the tools and expectations towards concretely moving the field forward.

Facilitating taxonomy and phylogenetics: An informative and cost-effective protocol integrating long amplicon PCRs and third-generation sequencing.

Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide.

LAMPrimers iQ: New primer design software for loop-mediated isothermal amplification (LAMP).

Nucleic acids amplification is a widely used technique utilized for different manipulations with DNA and RNA. Although, polymerase chain reaction (PCR) remains the most popular amplification method, isothermal approaches are gained more attention last decades. Among these, loop-mediated isothermal amplification (LAMP) became an excellent alternative to PCR. LAMP requires an increased number of primers and, therefore, is considered a highly specific amplification reaction compared to PCR. LAMP primers design is still a non-trivial task, and all niceties should be taken into account during their selection. Here, we report on a new program called LAMPrimers iQ destined for high-quality LAMP primers design. LAMPrimers iQ is based on an original algorithm considering rigorous criteria for primers selection. Unlike alternative programs, LAMPrimers iQ can process long DNA or RNA sequences, and completely avoid primers that can form homo- and heterodimers. The quality of the primers designed was checked using SARS-CoV-2 coronavirus RNA as a model target. It was shown that primers selected with LAMPrimers iQ provide higher specificity and reliable detection of viral RNA compared to those obtained by alternative programs. The program is available at https://github.com/Restily/LAMPrimers-iQ.

Simple and field-adapted species identification of biological specimens combining multiplex multienzyme isothermal rapid amplification, lateral flow dipsticks, and universal primers for initial rapid screening without standard PCR laboratory.

Species identification of biological specimens can provide the valuable clues and accelerate the speed of prosecution material processing for forensic investigation, especially when the case scene is inaccessible and the physical evidence is cumbersome. Thus, establishing a rapid, simple, and field-adapted species identification method is crucial for forensic scientists, particularly as first-line technology at the crime scene for initial rapid screening. In this study, we established a new field-adapted species identification method by combining multiplex multienzyme isothermal rapid amplification (MIRA), lateral flow dipstick (LFD) system, and universal primers. Universal primers targeting COX I and COX II genes were used in multiplex MIRA-LFD system for seven species identification, and a dedicated MIRA-LFD system primer targeting CYT B gene was used to detect the human material. DNA extraction was performed by collecting DNA directly from the centrifuged supernatant. Our study found that the entire amplification process took only 15 min at 37 °C and the results of LFDs could be visually observed after 10 min. The detection sensitivity of human material could reach 10 pg, which is equivalent to the detection of single cell. Different common animal samples mixed at the ratio of 1 ng:1 ng, 10 ng:1 ng, and 1 ng:10 ng could be detected successfully. Furthermore, the damaged and degraded samples could also be detected. Therefore, the convenient, feasible, and rapid approach for species identification is suitable for popularization as first-line technology at the crime scene for initial rapid screening and provides a great convenient for forensic application.

A study on waterlogging tolerance in sugarcane: a comprehensive review.

Sugarcane (Saccharum officinarum) is an important crop, native to tropical and subtropical regions and it is a major source of sugar and Bioenergy in the world. Abiotic stress is defined as environmental conditions that reduce growth and yield below the optimum level. To tolerate these abiotic stresses, plants initiate several molecular, cellular, and physiological changes. These responses to abiotic stresses are dynamic and complex; they may be reversible or irreversible. Waterlogging is an abiotic stress phenomenon that drastically reduces the growth and survival of sugarcane, which leads to a 15-45% reduction in cane's yield. The extent of damage due to waterlogging depends on genotypes, environmental conditions, stage of development and duration of stress. An improved understanding of the physiological, biochemical, and molecular responses of sugarcane to waterlogging stress could help to develop new breeding strategies to sustain high yields against this situation. The present review offers a summary of recent findings on the adaptation of sugarcane to waterlogging stress in terms of growth and development, yield and quality, as well as biochemical and adaptive-molecular processes that may contribute to flooding tolerance.

Development of novel species-specific and genus-specific primers for the detection of Babaco Mosaic Virus (BabMV).

Babaco is a hybrid cultivar native to the Andean region of Ecuador and Colombia, commercially attractive for its fruit. Babaco production in Ecuador faces losses from plant pathogens like babaco mosaic virus (BabMV), an RNA virus that causes chlorosis, leaf mottling, and deformation. Phylogenetic studies link BabMV to papaya mosaic virus (PapMV), alternanthera mosaic virus, and senna mosaic virus. To address this threat, we developed novel species-specific primers to detect BabMV targeting a 165 bp region of the coat protein (CP). Genus-specific primers were designed to validate the species-specific primers and attest their ability to discriminate between BabMV and its closest relatives. These primers targeted a 175 bp fragment of the CP region. The most effective sets of primers were chosen for reverse transcription polymerase chain reaction (RT-PCR) and SYBR® Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) in symptomatic and asymptomatic babaco plants. Among 28 plants tested, 25 were positive and 3 were negative for BabMV using species-specific and genus-specific primers in RT-PCR and RT-qPCR, while the PapMV positive control was detected with the genus-specific primers and was negative for the species-specific primers. These primers represent a valuable molecular tool for detecting BabMV, potentially enhancing crop management.

An Inhibitor-Monitorable Single-Tube Duplex Quantitative Real-Time PCR Assay for the Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is a citrus infectious disease caused by 'Candidatus Liberibacter' spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3'-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of 'Candidatus Liberibacter asiaticus' (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.

Increasing marine trophic web knowledge through DNA analyses of fish stomach content: a step towards an ecosystem-based approach to fisheries research.

Multispecies and ecosystem models, which are key for the implementation of ecosystem-based approaches to fisheries management, require extensive data on the trophic interactions between marine organisms, including changes over time. DNA metabarcoding, by allowing the simultaneous taxonomic identification of the community present in hundreds of samples, could be used for speeding up large-scale stomach content data collection. Yet, for DNA metabarcoding to be routinely implemented, technical challenges should be addressed, such as the potentially complicated sampling logistics, the detection of a high proportion of predator DNA, and the inability to provide reliable abundance estimations. Here, we present a DNA metabarcoding assay developed to examine the diet of five commercially important fish, which can be feasibly incorporated into routinary samplings. The method is devised to speed up the analysis process by avoiding the stomach dissection and content extraction steps, while preventing the amplification of predator DNA by using blocking primers. Tested in mock samples and in real stomach samples, the method has proven effective and shows great effectiveness discerning diet variations due to predator ecology or prey availability. Additionally, by applying our protocol to mackerel stomachs previously analyzed by visual inspection, we showcase how DNA metabarcoding could complement visually based data by detecting overlooked prey by the visual approach. We finally discuss how DNA metabarcoding-based data can contribute to trophic data collection. Our work reinforces the potential of DNA metabarcoding for the study and monitoring of fish trophic interactions and provides a basis for its incorporation into routine monitoring programs, which will be critical for the implementation of ecosystem-based approaches to fisheries management.

Multiplexing LAMP Assays: A Methodological Review and Diagnostic Application.

The loop-mediated isothermal amplification (LAMP) technique is a great alternative to PCR-based methods, as it is fast, easy to use and works with high sensitivity and specificity without the need for expensive instruments. However, one of the limitations of LAMP is difficulty in achieving the simultaneous detection of several targets in a single tube, as the methodologies that allow this rely on fluorogenic probes containing specific target sequences, complicating their adaptation and the optimization of assays. Here, we summarize different methods for the development of multiplex LAMP assays based on sequence-specific detection, illustrated with a schematic representation of the technique, and evaluate their practical application based on the real-time detection and quantification of results, the possibility to visualize the results at a glance, the prior stabilization of reaction components, promoting the point-of-care use, the maximum number of specific targets amplified, and the validation of the technique in clinical samples. The various LAMP multiplexing methodologies differ in their operating conditions and mechanism. Each methodology has its advantages and disadvantages, and the choice among them will depend on specific application interests.

First report of Meloidogyne hapla on hemp (Cannabis sativa) in Oregon.

Hemp is a crop that has gained interest in Washington and Oregon. As with other crops, hemp production faces challenges due to biotic factors, including plant-parasitic nematodes. During a survey for plant-parasitic nematodes associated with hemp, Meloidogyne sp. was found in a composite root sample collected in Oregon. Morphological characterization of second-stage juveniles identified the nematode as Meloidogyne hapla. Molecular identification confirmed the population as M. hapla. To our knowledge, this is the first report of M. hapla on hemp in the Pacific Northwest of the United States.

intI1 gene abundance from septic tanks in Thailand using validated intI1 primers.

IMPORTANCE: Antimicrobial resistance is a global crisis, and wastewater treatment, including septic tanks, remains an important source of antimicrobial resistance (AMR) genes. The role of septic tanks in disseminating class 1 integron, and by extension AMR genes, in Thailand, where antibiotic use is unregulated remains understudied. We aimed to monitor gene abundance as a proxy to infer potential AMR from septic tanks in Thailand. We evaluated published intI1 primers due to the lack of consensus on optimal Q-PCR primers and the absence of standardization. Our findings confirmed septic tanks are a source of class 1 integron to the environment. We highlighted the significance of intI1 primer choice, in the context of interpretation of risk associated with AMR spread from septic tanks. We recommend the validated set (F3-R3) for optimal intI1 quantification toward the goal of achieving standardization across studies.

In silico evaluation of the impact of Omicron variant of concern sublineage BA.4 and BA.5 on the sensitivity of RT-qPCR assays for SARS-CoV-2 detection using whole genome sequencing.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VoC) Omicron (B.1.1.529) has rapidly spread around the world, presenting a new threat to global public human health. Due to the large number of mutations accumulated by SARS-CoV-2 Omicron, concerns have emerged over potentially reduced diagnostic accuracy of reverse-transcription polymerase chain reaction (RT-qPCR), the gold standard diagnostic test for diagnosing coronavirus disease 2019 (COVID-19). Thus, we aimed to assess the impact of the currently endemic Omicron sublineages BA.4 and BA.5 on the integrity and sensitivity of RT-qPCR assays used for coronavirus disease 2019 (COVID-19) diagnosis via in silico analysis. We employed whole genome sequencing data and evaluated the potential for false negatives or test failure due to mismatches between primers/probes and the Omicron VoC viral genome. METHODS: In silico sensitivity of 12 RT-qPCR tests (containing 30 primers and probe sets) developed for detection of SARS-CoV-2 reported by the World Health Organization (WHO) or available in the literature, was assessed for specifically detecting SARS-CoV-2 Omicron BA.4 and BA.5 sublineages, obtained after removing redundancy from publicly available genomes from National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian Influenza Data (GISAID) databases. Mismatches between amplicon regions of SARS-CoV-2 Omicron VoC and primers and probe sets were evaluated, and clustering analysis of corresponding amplicon sequences was carried out. RESULTS: From the 1164 representative SARS-CoV-2 Omicron VoC BA.4 sublineage genomes analyzed, a substitution in the first five nucleotides (C to T) of the amplicon's 3'-end was observed in all samples resulting in 0% sensitivity for assays HKUnivRdRp/Hel (mismatch in reverse primer) and CoremCharite N (mismatch in both forward and reverse primers). Due to a mismatch in the forward primer's 5'-end (3-nucleotide substitution, GGG to AAC), the sensitivity of the ChinaCDC N assay was at 0.69%. The 10 nucleotide mismatches in the reverse primer resulted in 0.09% sensitivity for Omicron sublineage BA.4 for Thai N assay. Of the 1926 BA.5 sublineage genomes, HKUnivRdRp/Hel assay also had 0% sensitivity. A sensitivity of 3.06% was observed for the ChinaCDC N assay because of a mismatch in the forward primer's 5'-end (3-nucleotide substitution, GGG to AAC). Similarly, due to the 10 nucleotide mismatches in the reverse primer, the Thai N assay's sensitivity was low at 0.21% for sublineage BA.5. Further, eight assays for BA.4 sublineage retained high sensitivity (more than 97%) and 9 assays for BA.5 sublineage retained more than 99% sensitivity. CONCLUSION: We observed four assays (HKUnivRdRp/Hel, ChinaCDC N, Thai N, CoremCharite N) that could potentially result in false negative results for SARS-CoV-2 Omicron VoCs BA.4 and BA.5 sublineages. Interestingly, CoremCharite N had 0% sensitivity for Omicron Voc BA.4 but 99.53% sensitivity for BA.5. In addition, 66.67% of the assays for BA.4 sublineage and 75% of the assays for BA.5 sublineage retained high sensitivity. Further, amplicon clustering and additional substitution analysis along with sensitivity analysis could be used for the modification and development of RT-qPCR assays for detecting SARS-CoV-2 Omicron VoC sublineages.

Development of PCR based SSR markers for Wilsonomyces carpophilus and a PCR based diagnosis protocol for the early detection of shot hole disease in stone fruit crops.

BACKGROUND: The conidial Ascomycota fungus Wilsonomyces carpophilus causing shot hole in stone fruits is a major constraint in the production of stone fruits worldwide. Shothole disease symptoms appear on leaves, fruits, and twigs. Successful isolation of the pathogen from different hosts on synthetic culture medium is a time consuming and tedious procedure for identification of the pathogen based on morpho-cultural characterization. METHODS AND RESULTS: The present research was carried out to develop a successful PCR based early detection protocol for the shot hole disease of stone fruits, viz., peach, plum, apricot, cherry, and almond using the pathogen specific SSR markers developed from the Wilsonomyces carpophilus genome using Genome-wide Microsatellite Analysing Tool package (GMATA) software. Diseased leaf samples of different stone fruits were collected from the SKUAST-K orchard and the pathogen was isolated on potato dextrose agar (PDA) medium and maintained on Asthana and Hawkers' medium with a total of 50 pathogen isolates comprised of 10 isolates each from peach, plum, apricot, cherry and almond. The DNA was extracted from both healthy and infected leaf samples of different stone fruits. The DNA was also extracted from the isolated pathogen cultures (50 isolates). Out of 2851 SSR markers developed, 30 SSRs were used for the successful amplification of DNA extracted from all the 50 pathogen isolates. These SSRs were used for the amplification DNA from shot hole infected leaf samples of different stone fruits, but the amplification was not observed in the control samples (DNA from healthy leaves), thus confirming the detection of this disease directly from the shot hole infected samples using PCR based SSR markers. To our knowledge, this forms the first report of SSR development for the Wilsonomyces carpophilus and their validation for the detection of shot hole disease directly from infected leaves. CONCLUSION: PCR based SSR makers were successfully developed and used for the detection of Wilsonomyces carpophilus causing shot hole disease in stone fruits including almond in nuts for the first time. These SSR markers could successfully detect the pathogen directly from the infected leaves of stone fruits namely peach, plum, apricot and cherry including almond from the nuts.

Identification, Diversity, and Distribution of Meloidogyne spp. in Vegetable Fields of South Georgia, U.S.A.

Root-knot nematode (RKN; Meloidogyne spp.) is the most prevalent plant-parasitic nematode in vegetable fields of Georgia, with an incidence of 67.3%. Because aggressive RKN species are reported in the southeastern United States, molecular-based identification of RKN species was conducted on soil samples taken from a nematode surveillance study in 2018 from 292 RKN-infested vegetable fields in southern Georgia. The RKN-infested soil was potted with tomato cultivar Rutgers, and individual nematode females were isolated from galled roots and subjected to species-specific PCR and mitochondrial haplotype-based RKN species identification. The incidence (%), mean, and maximum relative abundance (second-stage juveniles per 100 cm3 of soil) of the five RKN species identified consisted of M. incognita (91.9, 486, 14,144), M. arenaria (36.0, 707, 14,144), M. floridensis (2.2, 909, 5,264), M. javanica (5.5, 352, 1,488), and M. haplanaria (0.7, 8, 14). A large proportion of fields (29%) had mixed populations of M. incognita and M. arenaria, which may reflect the region's long history of cotton and peanut cultivation. For unknown reasons, mixed populations of M. incognita and M. arenaria were associated with higher population densities. M. incognita is the most important RKN species in vegetable fields, followed by M. arenaria; therefore, pure or mixed populations of these species should be addressed in nematode management programs. Although at a lower incidence, the newly detected species, M. floridensis and M. haplanaria, have the potential to become a major threat since they reproduce on vegetables with Mi-resistant genes.

Detection of Specific RNA Targets by Multimerization.

Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to essential role of RNA in transfer of genetic information and development of diseases. Here, we report on an approach for detection of RNA targets based on the particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase, and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was verified by using genetic material of the SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate the SARS-CoV-2 RNA-positive samples from the SARS-CoV-2 negative samples with high reliability. The proposed technique allows detection of RNA even in the samples, which were subjected to multiple freezing-thawing cycles.

Development of a remineralizing calcium phosphate nanoparticle-containing self-etching system for orthodontic bonding.

OBJECTIVES: This study aimed to incorporate hydroxyapatite nanoparticles (nHA) or amorphous calcium phosphate nanoparticles (nACP) into a self-etch primer (SEP) to develop a simplified orthodontic bonding system with remineralizing and enamel preserving properties. MATERIALS AND METHODS: nHA and nACP were incorporated into a commercial SEP (Transbond™ plus) in 7% weight ratio and compared with the plain SEP as a control. Shear bond strengths (SBS), enamel damage, and adhesive remnant index (ARI) scores were evaluated at 24 h and post 5000 thermocycling. Field-emission scanning electron microscope (FESEM) was used to inspect the distribution of the nanoparticles in the experimental SEPs and evaluate the enamel surface integrity both before bracket bonding and post bracket debonding. Phase determination and remineralizing capability of the modified SEP were characterized by X-ray diffraction and Raman spectroscopy, respectively. RESULTS: The addition of nHA or nACP to the SEP significantly reduced the SBS, ARI, and enamel damage (p < 0.05) as compared to the control SEP; however, only nHA-SEP survived the thermocycling protocol and yielded acceptable SBS (13.38 MPa). Enamel remineralizing ability of the developed nHA-SEP was confirmed by both FESEM images and Raman phosphate map. CONCLUSIONS: Incorporating nHA into SEP resulted in clinically acceptable bond strengths with remineralizing ability. CLINICAL RELEVANCE: The newly developed nHA-SEP has unprecedented ability to simultaneously etch, prime, and remineralize the enamel in a single step leaving immaculate enamel surface with the potential of saving cost and time at the post-debonding step.

Rhodophyta DNA Barcoding: Ribulose-1, 5-Bisphosphate Carboxylase Gene and Novel Universal Primers.

Red algae (Rhodophyta) are a heterogeneous group of marine algal species that have served as a source of high-value molecules, including antioxidants and scaffolds, for novel drug development. However, it is challenging to identify Rhodophytes through morphological features alone, and in most instances, that has been the prevailing approach to identification. Consequently, this study undertook the identification of red algae species in Kenton-on-Sea, South Africa, as a baseline for future research on red algae biodiversity and conservation. The identification was achieved by designing, analysing, and using a set of universal primers through DNA barcoding of the rbcL gene. The PCR products of the rbcL gene were sequenced, and 96% of the amplicons were successfully sequenced from this set and matched with sequences on BOLD, which led to these species being molecularly described. Amongst these species are medicinally essential species, such as Laurencia natalensis and Hypnea spinella, and potential cryptic species. This calls for further investigation into the biodiversity of the studied region. Meanwhile, the availability of these primers will ease the identification process of red algae species from other coastal regions.