Repeated DNA Sequences Research Articles

Maximization of micropropagule production in banana cultivars Grand naine (AAA) and Elakki (AB)

Shoot tips from two different cultivars of banana known as Grand Naine (AAA) and Elakki (AB), were evaluated for their in vitro response in different concentrations of cytokinin and auxin which included 6-benzylaminopurine (6-BAP; 2, 3, and 4 mg L−1) and α-naphthalene acetic acid (NAA; 1 mg L−1) which was added to Murashige and Skoog (MS) basal media. The mean survival rates for cultured Grand Naine and Elakki explants in the varied growth regulator treatments were 66.25% and 52.5%, respectively. Rooting was initiated in the seventh passage using half-strength MS medium supplemented with 200 mg L−1 activated charcoal and 20 mg L−1 adenine sulfate (AS). The highest mean number of shoots generated per explant at the end of the seventh subculture from Grand Naine was 20.10 ± 0.73, and in Elakki, it was 11.20 ± 0.53. MS media supplemented with 2 mg L−1 6-BAP, and 1 mg L−1 NAA was found to be the best combination for shoot proliferation in both the cultivars. The in vitro-regenerated plants were hardened primarily under shade net conditions using cocopeat substrate and were later transferred to a 1:1:1 ratio of cocopeat, red soil, and sand mix before exposure to field conditions. The survival rate for Grand Naine and Elakki cultivars was 98.5% and 96.5%, respectively, during the acclimatization period of the micropropagules under shade net conditions. Polymorphism was not observed between the mother plant and the in vitro-regenerated micropropagules when analyzed for specific randomly amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR).

A Polyphasic Approach to Compare the Genomic Profiles of Aflatoxigenic and Non-Aflatoxigenic Isolates of Aspergillus Section Flavi.

Aflatoxins (AF) are highly toxic compounds produced by Aspergillus section Flavi. They spoil food crops and present a serious global health hazard to humans and livestock. The aim of this study was to examine the phylogenetic relationships among aflatoxigenic and non-aflatoxigenic Aspergillus isolates. A polyphasic approach combining phylogenetic, sequence, and toxin analyses was applied to 40 Aspergillus section Flavi isolates collected from eight countries around the world (USA, Philippines, Egypt, India, Australia, Indonesia, China, and Uganda). This allows one to pinpoint the key genomic features that distinguish AF producing and non-producing isolates. Based on molecular identification, 32 (80%) were identified as A. flavus, three (7.5%) as A. parasiticus, three (7.5%) as A. nomius and one (2.5%) as A. tamarii. Toxin analysis showed that 22 (55%) Aspergillus isolates were aflatoxigenic. The majority of the toxic isolates (62.5%) originated from Egypt. The highest aflatoxin production potential was observed in an A. nomius isolate which is originally isolated from the Philippines. DNA-based molecular markers such as random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) were used to evaluate the genetic diversity and phylogenetic relationships among these 40 Aspergillus isolates, which were originally selected from 80 isolates. The percentage of polymorphic bands in three RAPD and three ISSR primers was 81.9% and 79.37%, respectively. Analysis of molecular variance showed significant diversity within the populations, 92% for RAPD and 85% for ISSR primers. The average of Polymorphism Information Content (PIC), Marker Index (MI), Nei’s gene diversity (H) and Shannon’s diversity index (I) in ISSR markers are higher than those in RAPD markers. Based on banding patterns and gene diversities values, we observed that the ISSR-PCR provides clearer data and is more successful in genetic diversity analyses than RAPD-PCR. Dendrograms generated from UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cluster analyses for RAPD and ISSR markers were related to the geographic origin.

TDZ-induced axillary shoot proliferation of Rhododendron mucronulatum Turcz and assessment of clonal fidelity using DNA-based markers and flow cytometry

In order to induce in vitro axillary shoot proliferation from single-node explants of Rhododendron mucronulatum Turcz., two techniques of thidiazuron (TDZ) application were tested: (i) two-step procedure including cultivation on Anderson medium (AM) supplemented with varying TDZ concentrations (0.1 μM; 0.25 μM; 0.5 μM; 1.0 μM; 2.5 μM) for 8 wk followed by cultivation on hormone-free medium (AM0) for 6 wk and (ii) 4-h liquid-pulse treatment with different TDZ concentrations (7.5 μM, 15.0 μM, or 30.0 μM) followed by cultivation on AM0 for 8 wk. The highest number of axillary shoots per explant was achieved with 0.1-μM TDZ after the two-step procedure. The best response in terms of percent regeneration (87%), shoot length (13 mm), absence of structure anomalies, and the shortest shoot production cycle (8 wk) was obtained with 30.0-μM TDZ liquid-pulse treatment for 4 h. The clonal fidelity of regenerated shoots was evaluated by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers and flow cytometry. Genetic similarity of all regenerants between themselves and with the mother seedlings was 99%. Flow cytometric analysis revealed that all samples studied were diploid. The nuclear DNA content of microshoots obtained under the TDZ treatments varied from 1.26 to 1.32 pg per 2C. There were no significant differences in DNA content among mother seedlings and in vitro developed shoots triggered by 0.1- and 2.5-μM TDZ nor by those triggered by the 30.0-μM TDZ pulse treatment.

The association of telomere length and telomerase activity with adverse outcomes in older patients with non-ST-elevation acute coronary syndrome.

Non-ST elevation acute coronary syndrome (NSTEACS) occurs more frequently in older patients with an increased occurrence of recurrent cardiac events following the index presentation. Telomeres are structures consisting of repeated DNA sequences as associated shelterin proteins at the ends of chromosomes. We aim to determine whether telomere length (TL) and telomerase activity (TA) predicted poor outcomes in older patients presenting with NSTEACS undergoing invasive care. Older patients undergoing invasive management for NSTEACS were recruited to the ICON-1 biomarker study (NCT01933581). Peripheral blood mononuclear cells (PBMC) were recovered on 153 patients. DNA was isolated and mean TL was measured by quantitative PCR expressed as relative T (telomere repeat copy number) to S (single copy gene number) ratio (T/S ratio), and a telomere repeat amplification assay was used to assess TA during index presentation with NSTEACS. Primary clinical outcomes consisted of death, myocardial infarction (MI), unplanned revascularisation, stroke and significant bleeding recorded at 1 year. TL and TA were divided into tertile groups for analysis. Cox proportional hazards regression was performed. Ordinal regression was performed to evaluate the relationship between TL and TA and traditional cardiovascular risk factors at baseline. 298 patients were recruited in the ICON-1 study of which 153 had PBMC recovered. The mean age was 81.0 ± 4.0 years (64% male). Mean telomere length T/S ratio was 0.47 ± 0.25 and mean TA was 1.52 ± 0.61 units. The primary composite outcome occurred in 44 (28.8%) patients. There was no association between short TL or low TA and incidence of the primary composite outcome (Hazard Ratio [HR] 1.50, 95% Confidence Interval [CI] 0.68-3.34, p = 0.32 and HR 1.33, 95% CI 0.52-3.36, p = 0.51 respectively). TL and TA are not found to be associated with the incidence of adverse outcomes in older patients presenting with NSTEACS undergoing invasive care. URL: https://www.clinicaltrials.gov Unique identifier: NCT01933581.

Genetic Diversity of Potato Based on Random Amplified Polymorphic DNA and Simple Sequence Repeat Marker

Various potato clones are cultivated by farmers in Banjarnegara and Wonosobo , Central Java, Indonesia such as MZ, NH1, NH2, Vega, Gareta, Granola, Bliss, Merah (Red Potato), Ungu (Purple Potato), Klon_17 (K17), Local Dieng, Margahayu, and X. This encourages the importance of genetic diversity and genetic similarities. This study aimed to analyze genetic diversity and genetic similarities between 13 accessions of potatoes based on seven Random Amplified Polymorphic DNA (RAPD) primer s and nine pairs of Simple Sequence Repeat Marker (SSR) primer s. The results showed that RAPD and SSR primers could be used to analyze genetic diversity and genetic similarities of 13 potatoes accessions from Banjarnegara and Wonosobo. The PLP value was 80.9% in the RAPD primer and 65.41% in the SSR primer. Four RAPD primer s were informative based on PIC value: OPG 08, OPM 19, OPP 13, and OPX 04. Three SSR primer were informative: STM 2005, RGH- SSR 8, and S tI 035. Genetic similarities presented by Phylogenetic tree analysis resulted in two main clusters. The first cluster consisted of Granola, MZ, Ungu (Purple potato), Merah (Red Potato), Local Dieng, Margahayu, Gareta, Vega, NH2, NH1, Klon_17 (K17), and Bliss. The second cluster consisted of X. Granola and MZ having a high genetic similarity with a genetic distance of 0.07 and 0.132. Meanwhile, K17 and X had a low genetic similarity with a genetic distance of 0.31 and 0.987.

Molecular characterization of jasmine genotypes using RAPD and ISSR markers

Understanding the genetic background of existing germplasm represents a value-added component of managing collections. Molecular approaches collectively represent a potential tool for gathering information on genetic improvement. Keeping in view, the present investigation was carried out to explore the molecular diversity among 18 jasmine genotypes by employing two DNA based molecular marker techniques, viz., random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR). A total of 33 polymorphic primers (20 RAPD and 13 ISSR) were used. Amplification of genomic DNA of 18 genotypes, using RAPD analysis resulted in a total of 248 products, out of which 234 (94.35%) products were polymorphic. The polymorphism percentage ranged from 80–100% with an average of 94.24% polymorphism per primer. In ISSR analysis, among thirteen primers tested, a total of 595 products were produced out of which 562 (94.45%) products were polymorphic. The polymorphism ranged from 82.60–100% per cent, with an average of 94.45% polymorphism per primer. Based on the similarity matrix data, dendrograms were generated using UPGMA method. The genotypes were categorized into several groups and subgroups which varied distinctly with J. primulinum forming a separate cluster.

ISSR markers to assess genetic diversity of cultivated populations from artificial selection of Stevia rebaudiana (Bert.) Bertoni.

Artificial selection related with important agronomic characteristics of Stevia rebaudiana may cause genetic divergence and formation of genetically structured populations with genetic uniformity or diversity within cultivars. Current study employed inter simple sequence repeats of DNA (ISSR markers) to assess genetic diversity within and among a single cultivated population maintained through sexual propagation (SR1) and four cultivated populations generated by artificial selection and maintained by vegetative propagation (SR2–SR5). Highest polymorphism rate was reported in SR1 (89.24%), whilst the lowest rate of polymorphism occurred in SR2 (60.13%). ISSR markers revealed that selection of plants with traits of vegetative-propagated interest may lead towards the generation of genetically more uniform DNA-level populations, while plants maintained by sexual propagation have high genetic variability. High estimated genetic divergence level indicated that the five areas of stevia form genetically structured populations. SR2 and SR4 are constituted by plants more homogeneous at DNA level for the selected characteristics than plants of SR3 and SR5 populations. Predominant and homogeneous genotypes selected at SR2 and SR4 areas could be valuable for tracing strategies to obtain stevia plants with the desirable agronomic characteristics through crosses between contrasting individuals in future breeding programs.

Intra- and inter-cultivar genetic variability in Bulgarian tobacco

PURPOSE: The genus Nicotiana is a well-defined group of species of which tobacco (Nicotiana tabacum L.) is the most important crop plant and plays a significant role in the economies of many countries. Recent advances in molecular genetics of the crop allowed the identification of many important genes and their location on chromosomes. However, the genetic analysis of this inbreeding, highly homozygous plant that serves as a model in many other studies is still very much work in progress. METHODS: A large proportion of the tobacco genome is represented by highly repeated DNA sequences, which makes molecular markers, based on them, an obvious first choice in diversity studies. Here we present an assessment with Inter-Simple Sequence Repeat (ISSR) markers of the intra- and inter-cultivar variability in a set of Bulgarian tobacco varieties. RESULTS: The screening of the 4 primers revealed a varying number of bands generated by different primers. The total number of bands varied between 4 and 13. No correlation between the total number of bands and the number of polymorphic bands was observed. Testing with ISSR primers revealed that plants with different DNA profiles can be found in varieties used. The highly similar profiles when E7 primer was used in two of the tested varieties from different production types indicate that the DNA fingerprinting technique may present a particular challenge in this crop. CONCLUSIONS: The present study demonstrates the power of the ISSR technique as a tool for diversity identification in tobacco.

Accounting for phylogenetic relatedness in cross-species analyses of telomere shortening rates

AbstractTelomeres are repeating DNA sequences found on the ends of chromosomes, which shorten with age and are implicated in senescence. Cross-species analyses of telomere shortening rates (TSR) and telomere lengths are important for understanding mechanisms underlying senescence, lifespan and life-history strategies of different species. Whittemore et al. (2019) generated a new dataset on variation in TSR, lifespan and body mass. In phylogenetically uncorrected analyses they found that TSR negatively correlates with lifespan. We re-ran analyses of their dataset using appropriate phylogenetic corrections. We found a strong phylogenetic signal in the association between TSR and body mass. We were able to corroborate Whittemore et al.’s major findings, including while correcting for body mass in a multivariate analysis. Since laboratory mice have different telomere lengths and potentially different telomere dynamics than wild mice, we removed mice from the analysis, which attenuates most associations.

Heterochromatin and microsatellites detection in karyotypes of four sea turtle species: Interspecific chromosomal differences.

The wide variation in size and content of eukaryotic genomes is mainly attributed to the accumulation of repetitive DNA sequences, like microsatellites, which are tandemly repeated DNA sequences. Sea turtles share a diploid number (2n) of 56, however recent molecular cytogenetic data have shown that karyotype conservatism is not a rule in the group. In this study, the heterochromatin distribution and the chromosomal location of microsatellites (CA)n, (GA)n, (CAG)n, (GATA)n, (GAA)n, (CGC)n and (GACA)n in Chelonia mydas, Caretta caretta, Eretmochelys imbricata and Lepidochelys olivacea were comparatively investigated. The obtained data showed that just the (CA)n, (GA)n, (CAG)n and (GATA)n microsatellites were located on sea turtle chromosomes, preferentially in heterochromatic regions of the microchromosomes (mc). Variations in the location of heterochromatin and microsatellites sites, especially in some pericentromeric regions of macrochromosomes, corroborate to proposal of centromere repositioning occurrence in Cheloniidae species. Furthermore, the results obtained with the location of microsatellites corroborate with the temperature sex determination mechanism proposal and the absence of heteromorphic sex chromosomes in sea turtles. The findings are useful for understanding part of the karyotypic diversification observed in sea turtles, especially those that explain the diversification of Carettini from Chelonini species.

Study of genetic diversity in Tunisian local cattle populations using ISSR markers

The inter-simple DNA sequence repeat (ISSR) method was used to study genetic diversity in three local cattle from the north, northeast and north west of Tunisia. Twenty samples were analysed using three ISSR primers. In total, 22 bands were amplified of which 15 are polymorphic (68.18%). The total genetic diversity (Ht), genetic diversity within populations (Hs), coefficient of gene differentiation (Gst) and gene flow (Nm) were 0.2706, 0.01314, 0.8841 and 0.0656. To better visualize the structure of the population, a UPGMA dendrogram constructed from the genetic distances of NEI shows that the populations of North (Bizerte) and Northeast (Nabeul) are genetically closest while that of Northwest (Jendouba and Siliana) is the furthest from the two others.

Alternative Lengthening of Telomeres (ALT) in Tumors and Pluripotent Stem Cells.

A telomere consists of repeated DNA sequences (TTAGGG)n as part of a nucleoprotein structure at the end of the linear chromosome, and their progressive shortening induces DNA damage response (DDR) that triggers cellular senescence. The telomere can be maintained by telomerase activity (TA) in the majority of cancer cells (particularly cancer stem cells) and pluripotent stem cells (PSCs), which exhibit unlimited self-proliferation. However, some cells, such as telomerase-deficient cancer cells, can add telomeric repeats by an alternative lengthening of the telomeres (ALT) pathway, showing telomere length heterogeneity. In this review, we focus on the mechanisms of the ALT pathway and potential clinical implications. We also discuss the characteristics of telomeres in PSCs, thereby shedding light on the therapeutic significance of telomere length regulation in age-related diseases and regenerative medicine.

Molecular analysis of genetic diversity in population of Silybum marianum (L.) Gaertn in Egypt

BackgroundSilybum marianum L. Gaertn is a medicinal plant of unique pharmaceutical properties in the treatment of liver disorders and diabetic nephropathy. Biochemical (SDS-PAGE) and molecular markers such as randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) technologies were used in this work to detect genetic diversity of 14 collections of Silybum marianum population in Egypt. ResultsThe electrophoretic pattern of seed protein gave different molecular weight bands, ranging from 24 to 111 KDa with the presence of unique bands. RAPD results revealed a high level of polymorphism (73.2%) using 12 RAPD primers, but only eight of them gave reproducible polymorphic DNA pattern. Sixteen primers were used in the ISSR method; only ten of them yielded clearly identifiable bands. The percentage of polymorphism is about 80% of the studied samples. ConclusionThe obtained data confirmed that SDS-protein, RAPD, and ISSR markers are important tools for genetic analysis for Silybum marianum and recommended to give accurate results.

DNA sequence repeats identify numerous Type I restriction‐modification systems that are potential epigenetic regulators controlling phase‐variable regulons; phasevarions

Over recent years several examples of randomly switching methyltransferases, associated with Type III restriction‐modification (R‐M) systems, have been described in pathogenic bacteria. In every case examined, changes in simple DNA sequence repeats result in variable methyltransferase expression and result in global changes in gene expression, and differentiation of the bacterial cell into distinct phenotypes. These epigenetic regulatory systems are called phasevarions, phase‐variable regulons, and are widespread in bacteria, with 17.4% of Type III R‐M system containing simple DNA sequence repeats. A distinct, recombination‐driven random switching system has also been described in Streptococci in Type I R‐M systems that also regulate gene expression. Here, we interrogate the most extensive and well‐curated database of R‐M systems, REBASE, by searching for all possible simple DNA sequence repeats in the hsdRMS genes that encode Type I R‐M systems. We report that 7.9% of hsdS, 2% of hsdM, and of 4.3% of hsdR genes contain simple sequence repeats that are capable of mediating phase variation. Phase variation of both hsdM and hsdS genes will lead to differential methyltransferase expression or specificity, and thereby the potential to control phasevarions. These data suggest that in addition to well characterized phasevarions controlled by Type III mod genes, and the previously described Streptococcal Type I R‐M systems that switch via recombination, approximately 10% of all Type I R‐M systems surveyed herein have independently evolved the ability to randomly switch expression via simple DNA sequence repeats.

Profile of secondary metabolites and genetic stability analysis in new lines of Echinacea purpurea (L.) Moench micropropagated via somatic embryogenesis

Echinacea purpurea (L.) Moench is a plant species important for the phytopharmaceutical industry and in horticulture. Currently, there is lack of standardized plant material with an increased content of secondary metabolites in purple coneflower. The following research meets the expectations of the industry, as new selected lines of purple coneflower were micropropagated by somatic embryogenesis. The plant lines were analyzed both in terms of the content of main secondary metabolites by High Performance Liquid Chromatography (HPLC), as well as the genetic stability within the line and the genetic distance between lines using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) genetic markers. Significant differences were found in the relative percentage composition of individual phenolic acids in the tested plant material. Among six selected lines of Echinacea purpurea, three were characterized by a higher content of cichoric acid in relation to the other lines studied. A higher mean polymorphism rate (>90%) was found with the RAPD technique, with a total of 1427 scorable bands produced (142.7 products per one primer). Unlike the RAPD analysis, ISSRs detected mostly monomorphic loci (63.4%), followed by polymorphic ones (36.6%), while there were no specific loci present. Cluster analysis of both marker systems showed that the tested genotypes were grouped according to their respective lines.

Telomerase-triggered DNAzyme spiders for exponential amplified assay of cancer cells

A highly flexible electrochemical assay based on target-triggered DNAzyme spiders was proposed for the detection of telomerase. The DNAzyme-telomerase substrate primers (D-TSP) containing Cu2+-dependent DNAzymes serve as recognition elements, and primers of telomerase. Telomerase extracted from Hela cells recognize the D-TSP and elongated with DNA sequence repeats. A synthetic telomerase product hybridized with scaffold sequences of two DNAzyme-tethered probes on the basis of the mechanism of the proximity-ligation assay. The three-leg DNAzyme spiders has been assembled and initiated the autonomous hybridization/nicking/displacement cycles on substrate modified surface. The cleaved ferrocene-labeled fragements are adsorbed on gold surface leading to an increase in the electrochemical signal. As a result, the one input target, telomerase, release large amount of ferrocene-labeled DNA strands, achieving an exponential signal amplification and an excellent improvement in sensitivity over single molecule or two-component ‘sandwich’ binding complexes. Our proposed biosensor showed a nonlinear dependence with Hela cell numbers, ranging from 25 to 2000 with a detection limit of 10 cells. Telomerase activities from different cell lines were also successfully evaluated. Our electrochemical strategy based on target-triggered DNAzyme spiders was enzyme-free, PCR-free, simple in operation which indicated that it expected to expand the scope of DNA nanotechnology in the areas of clinical diagnosis.

RNase H amplified RNA probe and graphene oxide system for highly sensitive detection of (CAG)n DNA repeat sequences

Huntington’s disease is a chronic progressive neurodegeneration which is caused by CAG repeat sequences expanding in the huntingtin gene. There is currently no disease-modifying treatment for the disease, and its progression can only be slowed down before the onset of symptoms. A novel fluorescent platform which contains an RNA probe and graphene oxide for detection of the biomarker of Huntington’s disease, CAG repeat sequences, was constructed in this investigation. In addition, RNase H was employed in the fluorescent system to enhance the sensitivity of the detection capability. The fluorescent signal was increased through the cyclic amplified reaction, which results from RNase H, specifically digestion of the RNA strand in the complement of the RNA–DNA duplex. The designed measurement method can detect CAG repeat sequences with a detection limit of 108 pM (R2 = 0.968) under which we optimized assay conditions. Furthermore, the detection limit is approximately 18 times lower than the traditional DNA and graphene oxide detection method without assistance of RNase H. Additionally, the probing platform also shows stronger ability to discriminate between the fluorescence of the target sequence and that of other non-target sequences. The results of our studies demonstrate that the RNase H amplified RNA probe and graphene oxide system exhibited excellent sensitivity and selectivity to the target of CAG repeats sequences.

An Advanced Algorithm for Finding Tandem Repeats in DNA Sequencing based on Text Mining

In this paper a new searching technique based on pattern matching is proposed. In bioinformatics, finding Tandem Repeats (TR) in DNA sequences is an critical issue. There exist many pattern matching algorithms and KMP (Knuth Morris Pratt) is one of the pattern matching algorithm that undergo deficiencies of runtime complexity and cost when size of the data set increases. The main aim of the paper is to generate an effective algorithm for detecting and identifying Tandem Repeats over a DNA sequence more efficiently. By introducing the concept of 2Dimensional matrix to minimize the purview scope and optimizing the problem, Tandem Repeat finding algorithm makes the detecting or identifying process more efficient and effective that improves the quality of results. The theoretical analysis and experimental results concludes that tandem repeat finding algorithm get equivalent results in less runtime. This algorithm is better to KMP for determining results, and it also reduces or weaken the runtime cost which is beneficial when DNA data becomes greater.

A novel photoelectrochemical strategy based on an integrative photoactive heterojunction nanomaterial and a redox cycling amplification system for ultrasensitive determination of microRNA in cells

An ultrasensitive photoelectrochemical (PEC) bioassay for determination of microRNA was proposed based on an integrative photoactive heterojunction nanomaterial to provide the basis of excellent PEC responses and an efficient redox cycling amplification system to improve the detection performances. To establish the bioassay system, the biosensor was firstly modified with Bi2WO6@Bi2S3 and alkaline phosphatase (ALP). The detection solution was composed of ascorbic acid phosphate (AAP) and ferrocenecarboxylic acid (FcA), where ALP converted AAP into ascorbic acid (AA) to trigger a process of redox cycling amplification by reducing FcA+ to FcA, resulting in enhanced photocurrent responses of Bi2WO6@Bi2S3. In the presence of microRNA 21, it could trigger a hybridization chain reaction via the special designed hairpin DNA to produce a long repeated DNA sequences to inhibit ALP activity. Thus the reduced ALP activity and consequently decreased photocurrent signal could be obtained for detection of microRNA 21. As expected, this bioassay system performed the satisfactory performances for the ultrasensitive detection of microRNA 21 in the range from 1 fM to 1 nM with an experimental detection limit of 0.26 fM and acceptable practical applicability. Collectively, an efficient PEC bioassay for microRNA 21 is established and this strategy can be expanded to detect other microRNAs, even other molecules in cells.

Targeting a highly repeated germline DNA sequence for improved real-time PCR-based detection of Ascaris infection in human stool.

BackgroundWith the expansion of soil transmitted helminth (STH) intervention efforts and the corresponding decline in infection prevalence, there is an increased need for sensitive and specific STH diagnostic assays. Previously, through next generation sequencing (NGS)-based identification and targeting of non-coding, high copy-number repetitive DNA sequences, we described the development of a panel of improved quantitative real-time PCR (qPCR)-based assays for the detection of Necator americanus, Ancylostoma duodenale, Ancylostoma ceylanicum, Trichuris trichiura, and Strongyloides stercoralis. However, due to the phenomenon of chromosome diminution, a similar assay based on high copy-number repetitive DNA was not developed for the detection of Ascaris lumbricoides. Recently, the publication of a reference-level germline genome sequence for A. lumbricoides has facilitated our development of an improved assay for this human pathogen of vast global importance.Methodology/Principal findingsRepurposing raw DNA sequence reads from a previously published Illumina-generated, NGS-based A. lumbricoides germline genome sequencing project, we performed a cluster-based repeat analysis utilizing RepeatExplorer2 software. This analysis identified the most prevalent repetitive DNA element of the A. lumbricoides germline genome (AGR, Ascaris germline repeat), which was then used to develop an improved qPCR assay. During experimental validation, this assay demonstrated a fold increase in sensitivity of ~3,100, as determined by relative Cq values, when compared with an assay utilizing a previously published, frequently employed, ribosomal internal transcribed spacer (ITS) DNA target. A comparative analysis of 2,784 field-collected samples was then performed, successfully verifying this improved sensitivity.Conclusions/SignificanceThrough analysis of the germline genome sequence of A. lumbricoides, a vastly improved qPCR assay has been developed. This assay, utilizing a high copy-number repeat target found in eggs and embryos (the AGR repeat), will improve prevalence estimates that are fundamental to the programmatic decision-making process, while simultaneously strengthening mathematical models used to examine STH infection rates. Furthermore, through the identification of an optimal target for PCR, future assay development efforts will also benefit, as the identity of the optimized repeat DNA target is likely to remain unchanged despite continued improvement in PCR-based diagnostic technologies.