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The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA--random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method.
Common effluent treatment plant (CETP) is employed for treatment of tannery effluent. However, the performance of CETP for reducing the genotoxic substances from the raw effluent is not known. In this study, phytotoxic and genotoxic effects of tannery effluents were investigated in mung bean (Vigna radiata (L.) Wilczek). For this purpose, untreated and treated tannery effluents were collected from CETP Unnao (UP), India. Seeds of mung bean were grown in soil irrigated with various concentrations of tannery effluents (0, 25, 50, 75, and 100%) for 15 days. Inhibition of seed germination was 90% by 25% untreated effluent and 75% treated effluent, compared to the control. Plant growth was inhibited by 51% and 41% when irrigated with untreated and treated effluents at 25% concentration. RAPD technique was used to evaluate the genotoxic effect of tannery effluents (untreated and treated) irrigation on the mung bean. The RAPD profiles obtained showed that both untreated and treated were having genotoxic effects on mung bean plants. This was discernible with appearance/disappearance of bands in the treatments compared with control plants. A total of 87 RAPD bands were obtained using eight primers and 42 (48%) of these showed polymorphism. Irrigating plants with untreated effluent caused 12 new bands to appear and 18 to disappear. Treated effluent caused 8 new bands and the loss of 15 bands. The genetic distances shown on the dendrogram revealed that control plants and those irrigated with treated effluent were clustered in one group (joined at distance of 0.28), whereas those irrigated with untreated effluent were separated in another cluster at larger distance (joined at distance of 0.42). This indicates that treated effluent is less genotoxic than the untreated. Nei's genetic similarity indices calculated between the treatments and the control plants showed that the control and the plants irrigated with treated tannery effluent had a similarity index of 0.75, the control and plants irrigated with untreated 0.65, and between the treatments 0.68. We conclude that both untreated and treated effluents contain genotoxic substances that caused DNA damage to mung beans. CETP Unnao removes some, but not all, genotoxic substances from tannery effluent. Consequently, use of both untreated and treated wastewater for irrigation poses health hazard to human and the environment.
Elizabethkingia species are ubiquitous bacteria that uncommonly cause human infection. Elizabethkingia anophelis was first identified in 2011 from the mosquito Anopheles gambiae. The currently available bacterial typing systems vary greatly with respect to labour, cost, reliability, and ability to discriminate among bacterial strains. Polymerase chain reaction (PCR)-based fingerprinting using random amplified polymorphic DNA (RAPD) is commonly used to identify genetic markers. To our knowledge, no system coupling RAPD-PCR and capillary gel electrophoresis (CGE) has been utilized for the epidemiological typing of E. anophelis. Thus, the aim of the present study was to establish a reliable and reproducible molecular typing technique for E. anophelis isolates based on a multi-centre assessment of bacteraemia patients. Here, we used a rapid CGE-light-emitting diode-induced fluorescence (LEDIF)-based method in conjunction with RAPD-PCR to genotype E. anophelis with a high level of discrimination. All clinical isolates of E. anophelis were found to be typeable, and isolates from two hospitals formed two distinct clusters. The results demonstrated the potential of coupling RAPD and CGE as a rapid and efficient molecular typing tool, providing a reliable method for surveillance and epidemiological investigations of bacterial infections. The proposed method shows promise as a novel, cost-effective, high-throughput, first-pass typing method.
Cancer is genetically heterogeneous regarding to molecular genetic characteristics and pathogenic pathways. A wide spectrum of biomarkers, including DNA markers, is used in determining genomic instability, molecular subtype determination and disease prognosis, and estimating sensitivity to different drugs in clinical practice. In a previous study, we developed highly effective DNA markers using improved random amplified polymorphic DNA (RAPD) with high-GC primers, which is a valuable approach for the genetic authentication of medicinal plants. In this study, we applied this effective DNA marker technique to generate genetic fingerprints that detect genomic alterations in human breast cancer tissues and then developed sequence-characterized amplified region (SCAR) markers. Three SCAR markers (BC10-1, BC13-4 and BC31-2) had high levels of genomic DNA amplification in breast cancer. The PHKG2 and RNF40 genes are either overlapping or close to the sequences of SCAR marker BC13-4, while SCAR marker BC10-1 is in the intron and overlap the DPEP1 gene, suggesting that alterations in the expression of these genes could contribute to cancer progression. Screening of breast cancer cell lines showed that the mRNA expression levels for the PHKG2 and DPEP1 were lower in non-tumorigenic mammary epithelial cell MCF10A, but elevated in other cell lines. The DPEP1 mRNA level in invasive ductal carcinoma specimens was significantly higher than that of the adjacent normal tissues in women. Taken together, high-GC RAMP-PCR provides greater efficacy in measuring genomic DNA amplifications, deletion or copy number variations. Furthermore, SCAR markers BC10-1 and BC13-4 might be useful diagnostic markers for breast cancer carcinomas.
Food consumers make decisions primarily on the basis of a product's nutritional, functional, and sensorial aspects. In this context, this study evaluated the persistence in sourdough of a multistrain starter culture from laboratory to bakery plant production and the effect of the starter on antioxidant and rheological properties of sourdoughs and derived bread. Lactobacillus sanfranciscensis B450, Leuconostoc citreum B435, and Candida milleri L999 were used as a multispecies starter culture to produce a sourdough subsequently used to modify two traditional sourdoughs to make novel bread with improved health and rheological properties. Both these novel bakery sourdoughs showed the persistence of L. sanfranciscensis B450 and C. milleri L999, and showed a significantly different lactic acid bacteria (LAB) concentration from the traditional sourdoughs. The novel sourdough PF7 M had a higher phenolic content (170% increase) and DPPH (8% increase) than the traditional bakery sourdough PF7 F. The novel sourdough PF9 M exhibited an improvement in textural parameters. Further research would be useful on the bioavailability of bio-active compounds to obtain bread with improved characteristics.
Target spot, a recently observed citrus disease that is caused by Pseudofabraea citricarpa, can cause substantial economic losses in citrus production. In this study, a 797 bp marker specific to Ps. citricarpa was identified via random amplified polymorphic DNA (RAPD) technique. The primer pair Pc-SFP/Pc-SRP, which was designed from RAPD amplicons, was utilized as a sequence-characterized amplified region (SCAR) marker. This marker identified Ps. citricarpa with a single and distinct band of 389 bp but did not amplify DNA from other tested fungal species. The PCR assay was highly sensitive to the target DNA at picogram levels and could reliably amplify Ps. citricarpa sequences with the Pc-SFP/Pc-SRP primer pair. The SCAR marker that was identified in the present study can facilitate rapid decision-making and precise disease forecasting and management.
The Lactic Acid Bacteria (LAB) are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD) are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two Lactobacillus strains, and also tracked the fate of these two administered strains using a RAPD technique.
The common split-gilled mushroom, Schizophyllum commune is found throughout the world on woody plants. This study was initiated to evaluate conditions for favorable vegetative growth and to determine molecular phylogenetic relationship in twelve different strains of S. commune. A suitable temperature for mycelial growth was obtained at 30℃. This mushroom grew well in acidic conditions and pH 5 was the most favorable. Hamada, glucose peptone, Hennerberg, potato dextrose agar and yeast malt extract were favorable media for growing mycelia, while Lilly and glucose tryptone were unfavorable. Dextrin was the best and lactose was the less effective carbon source. The most suitable nitrogen sources were calcium nitrate, glycine, and potassium nitrate, whereas ammonium phosphate and histidine were the least effective for the mycelial growth of S. commune. The genetic diversity of each strain was investigated in order to identify them. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR. The size of the ITS1 and ITS2 regions of rDNA from the different strains varied from 129 to 143 bp and 241 to 243 bp, respectively. The sequence of ITS1 was more variable than that of ITS2, while the 5.8S sequences were identical. A phylogenetic tree of the ITS region sequences indicated that the selected strains were classified into three clusters. The reciprocal homologies of the ITS region sequences ranged from 99 to 100%. The strains were also analyzed by random amplification of polymorphic DNA (RAPD) with 20 arbitrary primers. Twelve primers efficiently amplified the genomic DNA. The number of amplified bands varied depending on the primers used or the strains tested. The average number of polymorphic bands observed per primer was 4.5. The size of polymorphic fragments was obtained in the range of 0.2 to 2.3 kb. These results indicate that the RAPD technique is well suited for detecting the genetic diversity in the S. commune strains tested.
The genus Chrysodracon has six endemic species in the Hawaii Islands. Chrysodracon hawaiiensis is endemic to Hawaii Island and was described as a distinct species in 1980. It was listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List in 1997. This woody plant species was, at one time, common in exposed dry forests, but it became very rare due to grazing pressure and human development. The tree species Chrysodracon auwahiensis (C. auwahiensis), endemic to Maui and Molokai, still has large adult populations in dry lands of the islands, but unfortunately no regeneration from seed has been reported in those areas for many years. The two endemic species were examined using the molecular technique of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) to determine the genetic structure of the populations and the amount of variation. Both species possess similar genetic structure. Larger and smaller populations of both species contain similar levels of genetic diversity as determined by the number of polymorphic loci, estimated heterozygosity, and Shannon's index of genetic diversity. Although population diversity of Chrysodracon hawaiiensis (C. hawaiiensis) is thought to have remained near pre-disturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Conservation recommendations for both species are suggested.
Fimbristylis dichotoma, Ammannia baccifera and their co-plantation consortium FA independently degraded Methyl Orange, simulated dye mixture and real textile effluent. Wild plants of F. dichotoma and A. baccifera with equal biomass showed 91% and 89% decolorization of Methyl Orange within 60h at a concentration of 50ppm, while 95% dye removal was achieved by consortium FA within 48h. Floating phyto-beds with co-plantation (F. dichotoma and A. baccifera) for the treatment of real textile effluent in a constructed wetland was observed to be more efficient and achieved 79%, 72%, 77%, 66% and 56% reductions in ADMI color value, COD, BOD, TDS and TSS of textile effluent, respectively. HPTLC, GC-MS, FTIR, UV-vis spectroscopy and activated oxido-reductive enzyme activities confirmed the phytotrasformation of parent dye in to new metabolites. T-RFLP analysis of rhizospheric bacteria of F. dichotoma, A. baccifera and consortium FA revealed the presence of 88, 98 and 223 genera which could have been involved in dye removal. Toxicity evaluation of products formed after phytotransformation of Methyl Orange by consortium FA on bivalves Lamellidens marginalis revealed less damage of the gills architecture when analyzed histologically. Toxicity measurement by Random Amplification of Polymorphic DNA (RAPD) technique revealed bivalve DNA banding pattern in treated Methyl Orange sample suggesting less toxic nature of phytotransformed dye products.
A total of 37 bacterial isolates were obtained from dye-contaminated soil samples at a textile processing factory in Nakhon Ratchasima Province, Thailand, and the potential of the isolates to decolorize and biotransform azo dye Reactive Red 141 (RR141) was investigated. The most potent bacterium was identified as Paenibacillus terrigena KKW2-005, which showed the ability to decolorize 96.45% of RR141 (50 mg/l) within 20 h under static conditions at pH 8.0 and a broad temperature range of 30-40°C. The biotransformation products were analyzed by using UV-Vis spectrophotometry and Fourier-transform infrared spectroscopy. Gas chromatography-mass spectroscopy analysis revealed four metabolites generated from the reductive biodegradation, namely sodium 3-diazenylnaphthalene-1,5-disulfonate (I), sodium naphthalene-2-sufonate (II), 4-chloro-1,3,5-triazin-2-amine (III) and N1-(1,3,5-triazin-2-yl) benzene-1,4-diamine (IV). Decolorization intermediates reduced phytotoxicity as compared with the untreated dye. However, they had phytotoxicity when compared with control, probably due to naphthalene and triazine derivatives. Moreover, genotoxicity testing by high annealing temperature-random amplified polymorphic DNA technique exhibited different DNA polymorphism bands in seedlings exposed to the metabolites. They compared to the bands found in seedlings subjected to the untreated dye or distilled water. The data from this study provide evidence that the biodegradation of Reactive Red 141 by P. terrigena KKW2-005 was genotoxic to the DNA seedlings.
Recent circumstantial evidence suggests that an increasing number of Iranian patients with cutaneous leishmaniasis are unresponsive to meglumine antimoniate (Glucantime), the first line of treatment in Iran. This study was designed to determine whether the clinical responses (healing, or non-healing) were correlated with the susceptibility of Leishmania parasites to Glucantime.
Mitochondrial DNA (mtDNA) analysis has displayed an important role and been considered as a powerful tool in various fields of forensic science applications. Nowadays, single nucleotide polymorphisms (SNPs) on mtDNA have become additional DNA markers when conventional STR typing practically fails. mtDNA sequencing of polymerase chain reaction (PCR) products from the hypervariable region I (HVRI) and II (HVRII) is the standard method of mtDNA analysis. However, mtDNA sequencing is rather expensive, time consuming and technically complex. This study aims to develop the SNPs minisequencing for screening of Thai populations. For this purpose, sixteen SNPs that possess high discriminating power in hypervariable regions were selected. The DNA samples were obtained from 100 buccal swab samples of Thai healthy individuals. All DNA samples were extracted and were subsequently amplified by single duplex PCR technique. The duplex PCR products were genotyped by SNPs minisequencing. Based on 16 SNPs, a total of 63 haplotypes were observed of which 46 haplotypes were unique. The haplotype diversity, discriminating power and random match probability were calculated to be 0.9830, 0.9732 and 0.0268, respectively. The SNPs at 150, 199, 489, 16129, 16189, 16223, and 16304 were highly polymorphic in the studied population. Our results suggested that the SNPs minisequencing can be an alternative method of SNPs genotyping. This method can be used for an exclusion of a large number of mismatch samples and as a presumptive test prior to do confirmatory mtDNA sequencing.
Southwest China is an important biodiversity hotspot. The interactions among the complex topography, climate change, and ecological factors in the dry-hot valley areas in southwest China may have profoundly affected the genetic structure of plant species in this region. In this study, we determined the effects of the Tanaka Line on genetic variation in the wild Bombax ceiba tree in southwest China. We sampled 224 individuals from 17 populations throughout the dry-hot valley regions. Six polymorphic expressed sequence tag-simple sequence repeat primers were employed to sequence the PCR products using the first-generation Sanger technique. The analysis based on population genetics suggested that B. ceiba exhibited a high level of gene diversity (HE: 0.2377-0.4775; I: 0.3997-0.7848). The 17 populations were divided into two groups by cluster analysis, which corresponded to geographic characters on each side of the Tanaka Line. In addition, a Mantel test indicated that the phylogeographic structure among the populations could be fitted to the isolation-by-distance model (r2 = .2553, p < .001). A barrier test indicated that there were obstacles among populations and between the two groups due to complex terrain isolation and geographic heterogeneity. We inferred that the Tanaka Line might have promoted the intraspecific phylogeographic subdivision and divergence of B. ceiba. These results provide new insights into the effects of the Tanaka Line on genetic isolation and population differentiation of plant species in southwest China.
Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL(-1) of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.
Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, thereby offering the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as palindromic sequence-targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5' tail attached to the sequence-specific primer and the other anneals to a different 5' tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2-3 h to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific random amplified polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display (TD), which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the maize (Zea mays) genome was used as a sequence tag during the TD procedure to characterize the Ac integration sites.
With the increased use of metal nanoparticles (NPs), their access to the food chain has become a main concern to scientists and holds controversial social implications. This research particularly sheds light on copper nanoparticles (CuNP), as they have been commonly used in several industries nowadays. In this study, we investigated the phytotoxicity of CuNP on cucumber (Cucumis sativus) plants grown hydroponically. Atomic Absorption Spectroscopy (AAS), X-Ray Fluorescence (XRF), and Scanning Electron Microscopy (SEM) analysis confirmed that C. sativus treated with CuNP accumulated CuNP in the plant tissues, with higher levels in roots, with amounts that were concentration dependent. Furthermore, genotoxicity was assessed using Random amplified polymorphic DNA (RAPD) technique, and our results showed that CuNP caused genomic alterations in C. sativus. Phenotypical, physiological, and biochemical changes were assessed by determining the CuNP treated plant's total biomass, chlorophyll, H2O2 and MDA contents, and electrolyte leakage percentage. The results revealed notable adverse phenotypical changes along with decreased biomass and decreased levels of the photosynthetic pigments (Chlorophyll a and b) in a concentration-dependent manner. Moreover, CuNP induced damage to the root plasma membrane as determined by the increased electrolyte leakage. A significant increase in H2O2 and MDA contents were detected in C. sativus CuNP treated plants. Additionally, copper-zinc superoxide dismutase (Cu-Zn SOD) gene expression was induced under CuNP treatment. Overall, our results demonstrated that CuNP of 10-30 nm size were toxic to C. sativus plants. This finding will encourage the safe production and disposal NPs. Thus, reducing nano-metallic bioaccumulation into our food chain through crop plants; that possesses a threat to the ecological system.
Genetic variability and identification of some molecular markers were studied in twenty promising lines of wheat using agronomic traits, ISSR (inter simple sequences repeats) and RAPD (random amplified polymorphic DNA) markers. Significant variation was evidenced in all agronomic traits. The lines proved to be superior to the check cultivar Sahel1 in yield and its component traits. Lines L2, L7 and L8 were the best in most yield component traits in both seasons. Moreover, Lines L2, L4, L5, L7 and L8 showed drought tolerance by which they displayed high performance in agronomic traits as well as a low drought susceptibility index. The percentage of polymorphism was 39.3% and 53.2% for ISSRs and RAPDs, respectively. UBC-881 belonged to penta-nucleotide repeat sequences (GGGTG) that produced the highest level of polymorphism, while UBC-846 belonged to di-nucleotide repeat sequences (CA) that produced the lowest level of polymorphism. Genetic similarities among wheat lines based on ISSR and RAPD markers ranged from 0.81 to 1.00 and from 0.86 to 0.98, respectively. There was a low average of PIC (polymorphism information content) values which were 0.10 (ISSR) and 0.15 (RAPD). The RAPD technique exhibited a higher marker index (MI = 0.69) compared to ISSR (MI = 0.43). There was insignificant correlation between ISSR and RAPD data (0.168, p > 0.05). There were two markers (UBC-881450bp and OPF-10540bp), on each of which two traits regressed significantly. The associated markers each explained a maximum regression of 18.92-34.95% of the total available variation for individual associated traits.
Lycium schweinfurthii is a Mediterranean wild shrub rich in plant secondary metabolites. In vitro propagation of this plant may support the production of valuable dietary supplements for humanity, introduction of it to the world market, and opportunities for further studies. The presented study aimed to introduce an efficient and reproducible protocol for in vitro micropropagation of L. schweinfurthii and assess the genetic stability of micropropagated plants (MiPs) as well as to estimate phenolic, flavonoid, ferulic acid contents, and the antioxidant activity in leaves of micropropagated plants. Two DNA-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), and one biochemical technique, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were used to assess the genetic stability in MiPs. Spectrophotometric analysis was performed to estimate total phenolic and flavonoid contents and antioxidant activity of MiPs leaves, while ferulic acid content was estimated using high-performance thin-layer chromatography (HPTLC). Sufficient shoot proliferation was achieved at MS (Murashige and Skoog) medium supplemented with 0.4 mg L-1 kinetin and rooted successfully on half-strength MS medium fortified with 0.4 mg L-1 Indole-3-butyric acid (IBA). The Jaccard's similarity coefficients detected in MiPs reached 52%, 55%, and 82% in the RAPD, ISSR, and SDS-PAGE analyses, respectively. In the dried leaves of MiPs, the phenolic, flavonoid, and ferulic acid contents of 11.53 mg gallic acid equivalent, 12.99 mg catechin equivalent, and 45.52 mg were estimated per gram, respectively. However, an IC50 of 0.43, and 1.99 mg mL-1 of MiP dried leaves' methanolic extract was required to scavenge half of the DPPH, and ABTS free radicals, respectively. The study presented a successful protocol for in vitro propagation of a valued promising plant source of phenolic compounds.
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