Amplification Of Polymorphic DNA Research Articles

Investigating Safety and Technological Traits of a Leading Probiotic Species: Lacticaseibacillus paracasei.

Lacticaseibacillus spp. are genetically close lactic acid bacteria species widely used in fermented products for their technological properties as well as their proven beneficial effects on human and animal health. This study, the first to include such a large collection of heterogeneous isolates (121) obtained from international collections belonging to Lacticaseibacillus paracasei, aimed to characterize the safety traits and technological properties of this important probiotic species, also making comparisons with other genetically related species, such as Lacticaseibacillus casei and Lacticaseibacillus zeae. These strains were isolated from a variety of heterogeneous sources, including dairy products, sourdoughs, wine, must, and human body excreta. After a preliminary molecular characterization using repetitive element palindromic PCR (Rep-PCR), Random Amplification of Polymorphic DNA (RAPD), and Sau-PCR, particular attention was paid to safety traits, evaluating antibiotic resistance profiles, biogenic amine (BA) production, the presence of genes related to the production of ethyl carbamate and diaminobenzidine (DAB), and multicopper oxidase activity (MCO). The technological characteristics of the strains, such as the capability to grow at different NaCl and ethanol concentrations and different pH values, were also investigated, as well as the production of bacteriocins. From the obtained results, it was observed that strains isolated from the same type of matrix often shared similar genetic characteristics. However, phenotypic traits were strain-specific. This underscored the vast potential of the different strains to be used for various purposes, from probiotics to bioprotective and starter cultures for food and feed production, highlighting the importance of conducting comprehensive evaluations to identify the most suitable strain for each purpose with the final aim of promoting human health.

Linking preterm infant gut microbiota to nasograstric enteral feeding tubes: exploring potential interactions and microbial strain transmission

IntroductionPreterm birth is a growing problem worldwide. Staying at a neonatal intensive care unit (NICU) after birth is critical for the survival of preterm infants whose feeding often requires the use of nasogastric enteral feeding tubes (NEFT). These can be colonized by hospital-associated pathobionts that can access the gut of the preterm infants through this route. Since the gut microbiota is the most impactful factor on maturation of the immune system, any disturbance in this may condition their health. Therefore, the aim of this study is to assess the impact of NEFT-associated microbial communities on the establishment of the gut microbiota in preterm infants.Material and methodsA metataxonomic analysis of fecal and NEFT-related samples obtained during the first 2 weeks of life of preterm infants was performed. The potential sharing of strains isolated from the same set of samples of bacterial species involved in NICU's outbreaks, was assessed by Random Amplification of Polymorphic DNA (RAPD) genotyping.ResultsIn the samples taken 48 h after birth (NEFT-1 and Me/F1), Staphylococcus spp. was the most abundant genera (62% and 14%, respectively) and it was latter displaced to 5.5% and 0.45%, respectively by Enterobacteriaceae. Significant differences in beta diversity were detected in NEFT and fecal samples taken at day 17 after birth (NEFT-3 and F3) (p = 0.003 and p = 0.024, respectively). Significant positive correlations were found between the most relevant genera detected in NEFT-3 and F3. 28% of the patients shared at least one RAPD-PCR profile in fecal and NEFT samples and 11% of the total profiles were found at least once simultaneously in NEFT and fecal samples from the same patient.ConclusionThe results indicate a parallel bacterial colonization of the gut of preterm neonates and the NEFTs used for feeding, potentially involving strain sharing between these niches. Moreover, the same bacterial RAPD profiles were found in neonates hospitalized in different boxes, suggesting a microbial transference within the NICU environment. This study may assist clinical staff in implementing best practices to mitigate the spread of pathogens that could threaten the health of preterm infants.

Molecular typing of clinical and non-clinical <i>Escherichia coli</i> from Rivers State, Nigeria using amplification-based RAPD and ERIC-PCR typing methods

Due to the diverse nature of Escherichia coli, typing of these organisms is essential. This can provide key epidemiological information not just on the strains in circulation and their relationship, but also on the development and spread of drug resistant clones. Affordable PCR-based typing techniques have resulted in an increase in typing studies particularly in resource limited settings. But information from Nigeria on strain typing is limited. This study was therefore aimed at typing clinical and non-clinical strains of E. coli from Nigeria using the PCR based Random Amplification of Polymorphic DNA (RAPD) and Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) typing methods. 
 E. coli was isolated from clinical and non-clinical sources using Eosin Methylene Blue agar. Characteristic green metallic sheen colonies associated with E. coli were purified, identities determined using standard biochemical tests and confirmed using molecular methods. Susceptibility testing was carried out using the modified Kirby-Bauer disc diffusion test and RAPD and ERIC-PCR typing carried out on 48 confirmed E. coli isolates as previously described. ERIC-PCR typing resulted in the generation of 38 unique patterns with a 0.94 diversity. Nine of these were singletons comprised of only a single isolate, while the remaining 29 patterns were grouped to 10 clusters. RAPD gave similar results of 37 patterns, 9 singletons, 0.94 diversity and 13 clusters. For ERIC-PCR, all isolates with identical patterns were from the same source. RAPD, however, had identical patterns present in isolates from different sources. 
 Using the ERIC-PCR typing method, this study was able to identify non-clinical isolates as distinct from the clinical E. coli isolates as evidenced by 100% identical ERIC-PCR profiles of isolates from the same source and a lack of 100% relatedness in isolates from different sources.

Innovative approach for high-throughput exploiting sex-specific markers in Japanese parrotfish Oplegnathus fasciatus.

The use of sex-specific molecular markers has become a prominent method in enhancing fish production and economic value, as well as providing a foundation for understanding the complex molecular mechanisms involved in fish sex determination. Over the past decades, research on male and female sex identification has predominantly employed molecular biology methodologies such as restriction fragment length polymorphism, random amplification of polymorphic DNA, simple sequence repeat, and amplified fragment length polymorphism. The emergence of high-throughput sequencing technologies, particularly Illumina, has led to the utilization of single nucleotide polymorphism and insertion/deletion variants as significant molecular markers for investigating sex identification in fish. The advancement of sex-controlled breeding encounters numerous challenges, including the inefficiency of current methods, intricate experimental protocols, high costs of development, elevated rates of false positives, marker instability, and cumbersome field-testing procedures. Nevertheless, the emergence and swift progress of PacBio high-throughput sequencing technology, characterized by its long-read output capabilities, offers novel opportunities to overcome these obstacles. Utilizing male/female assembled genome information in conjunction with short-read sequencing data survey and long-read PacBio sequencing data, a catalog of large-segment (>100bp) insertion/deletion genetic variants was generated through a genome-wide variant site-scanning approach with bidirectional comparisons. The sequence tagging sites were ranked based on the long-read depth of the insertion/deletion site, with markers exhibiting lower long-read depth being considered more effective for large-segment deletion variants. Subsequently, a catalog of bulk primers and simulated PCR for the male/female variant loci was developed, incorporating primer design for the target region and electronic PCR (e-PCR) technology. The Japanese parrotfish (Oplegnathus fasciatus), belonging to the Oplegnathidae family within the Centrarchiformes order, holds significant economic value as a rocky reef fish indigenous to East Asia. The criteria for rapid identification of male and female differences in Japanese parrotfish were established through agarose gel electrophoresis, which revealed 2 amplified bands for males and 1 amplified band for females. A high-throughput identification catalog of sex-specific markers was then constructed using this method, resulting in the identification of 3,639 (2,786 INS/853 DEL, ♀ as reference) and 3,672 (2,876 INS/833 DEL, ♂ as reference) markers in conjunction with 1,021 and 894 high-quality genetic sex identification markers, respectively. Sixteen differential loci were randomly chosen from the catalog for validation, with 11 of them meeting the criteria for male/female distinctions. The implementation of cost-effective and efficient technological processes would facilitate the rapid advancement of genetic breeding through expediting the high-throughput development of sex genetic markers for various species. Our study utilized assembled genome information from male and female individuals obtained from PacBio, in addition to data from short-read sequencing data survey and long-read PacBio sequencing data. We extensively employed genome-wide variant site scanning and identification, high-throughput primer design of target regions, and e-PCR batch amplification, along with statistical analysis and ranking of the long-read depth of the variant sites. Through this integrated approach, we successfully compiled a catalog of large insertion/deletion sites (>100bp) in both male and female Japanese parrotfish.

Molecular characterization and differential effects of levofloxacin and ciprofloxacin on the potential for developing quinolone resistance among clinical Pseudomonas aeruginosa isolates.

Fluoroquinolones are some of the most used antimicrobial agents for the treatment of Pseudomonas aeruginosa. This study aimed at exploring the differential activity of ciprofloxacin and levofloxacin on the selection of resistance among P. aeruginosa isolates at our medical center. 233 P. aeruginosa clinical isolates were included in this study. Antimicrobial susceptibility testing (AST) was done using disk diffusion and broth microdilution assays. Random Amplification of Polymorphic DNA (RAPD) was done to determine the genetic relatedness between the isolates. Induction of resistance against ciprofloxacin and levofloxacin was done on 19 isolates. Fitness cost assay was done on the 38 induced mutants and their parental isolates. Finally, whole genome sequencing was done on 16 induced mutants and their 8 parental isolates. AST results showed that aztreonam had the highest non-susceptibility. RAPD results identified 18 clusters. The 19 P. aeruginosa isolates that were induced against ciprofloxacin and levofloxacin yielded MICs ranging between 16 and 256 μg/mL. Levofloxacin required fewer passages in 10 isolates and the same number of passages in 9 isolates as compared to ciprofloxacin to reach their breakpoints. Fitness cost results showed that 12 and 10 induced mutants against ciprofloxacin and levofloxacin, respectively, had higher fitness cost when compared to their parental isolates. Whole genome sequencing results showed that resistance to ciprofloxacin and levofloxacin in sequenced mutants were mainly associated with alterations in gyrA, gyrB and parC genes. Understanding resistance patterns and risk factors associated with infections is crucial to decrease the emerging threat of antimicrobial resistance.

Comparison of non-aureus staphylococcal and mammaliicoccal species found in both composite milk and bulk-tank milk samples of dairy cows collected in tandem

Non-aureus staphylococci and the closely related mammaliicoccal species (NASM) are the most common causes of bovine subclinical mastitis on modern dairy farms and are highly prevalent in bulk-tank milk. The purpose of this study was to determine the distribution of NASM in both composite cow milk (CCM) and bulk-tank milk (BTM) samples collected in tandem in commercial Flemish dairy herds and to estimate the origin of the different (subgroups of) NASM species present in BTM by applying strain typing (random amplification of polymorphic DNA or random amplified DNA [RAPD]). A single cross-sectional sampling was performed over 5 herds that volunteered to participate in the study. Composite cow milk samples (n = 356) were collected from all lactating cows (except those with clinical mastitis) during a milking in tandem with 6 BTM samples per herd sequentially collected immediately post that milking (n = 30). In total, 421 and 80 NASM isolates were recovered and identified by MALDI-TOF mass spectrometry from the CCM and BTM samples, respectively and a total of 21 and 12 different NASM species were identified from CCM and BTM samples, respectively. Staphylococcus cohnii was the most prevalent NASM species found in BTM followed by Staphylococcus haemolyticus, Staphylococcus epidermidis, Mammaliicoccus lentus, and Staphylococcus equorum, whereas from CCM samples the most common species were S. hemolyticus, S. cohnii, S. equorum, S. epidermidis, and Staphylococcus chromogenes. The prevalent NASM species in both CCM and BTM samples was distinct for each herd, corroborating other studies observing a herd-specific NASM microbiota. Random amplified DNA analysis was performed on 9 NASM species (S. chromogenes, S. epidermidis, S. haemolyticus, S. equorum, Mammaliicoccus sciuri, Staphylococcus xylosus, S. cohnii, Staphylococcus debuckii, and M. lentus) because these species were isolated from both sample types in a herd. The same RAPD types were found in both sample types for all NASM species selected for strain typing in varying degrees. When assessing the distribution of NASM species, differences within NASM species should be examined meaning a closer look should be taken at the strain level rather than at the species level only.

Genetic relationships and genome verification of Thai banana cultivars using Random Amplification of Polymorphic DNA (RAPD) markers

Abstract. Boonsrangsom T, Fuenghoi C, Premjet D, Suvittawat K, Ratanasut K, Sujipuli K. 2023. Genetic relationships and genome verification of Thai banana cultivars using Random Amplification of Polymorphic DNA (RAPD) markers. Biodiversitas 24: 3758-3765. Edible bananas and plantains, belonging to the family Musaceae, genus Musa, represent one of the most important fruit crops, with an annual production of more than 65 million tons worldwide. Bananas have several hybrid variations since they are descended from the two species Musa acuminata Colla (AA genome) and Musa balbisiana Colla (BB genome). Different morphological traits divide almost hybrid bananas into various genomic groupings. Banana genome categorization and identification, however, have always been challenging issues. This study aimed to assess the genetic relationships and verify the genomes of Thai banana cultivars using random amplification of polymorphic DNA (RAPD) markers. Using the 15 selected RAPD markers, 149 RAPD bands were found, with sizes ranging from 0.2 to 3.2 kb, and 88.6% were polymorphic. Polymorphic information content (PIC) values ranged from 0.18 to 0.42, averaging 0.30. Based on the Jaccard coefficient, the unweighted pair-group method arithmetic average (UPGMA) analysis showed that the banana samples had a similarity range of 0.27 to 1.00 with a mean of 0.56, demonstrating an abundance of viability across six banana genomes. The dendrogram generated from RAPD data revealed that all 18 Musa samples could be divided into two main groups (Group I and II). Three additional subgroups were created for each primary group (A, B, and C). The accurate identification and genetic data on the available genetic resources for bananas will be beneficial for breeding and conservation programs.

Phytochemical Analysis and Genetic Characterization of Genus Blepharis in Rajasthan, India

Nature has been a source of therapeutic agents for thousands of years and a remarkable number of modern medications have been secluded from ordinary sources. Higher plants, as sources of therapeutic compounds, have sustained to play a leading role in the conservation of human health since pre-historic times. Blepharis is an Afro-asiatic genus including 129 species, belonging to the family Acanthaceae, widely distributed in dry and semi-dry habitats. The aim of this study was to identify primary metabolites quantitatively and flavonoid compounds qualitatively using thin-layer chromatography (TLC) from the leaves of two species of the Blepharis genus, and also to investigate the genetic analysis of these species using the random amplification of polymorphic DNA (RAPD). In quantitative analysis, primary metabolites such as protein, carbohydrates, lipid and phenol were found with significant values. In TLC characterization, Kaempferol and Rutin were identified as flavonoid compounds from both leaves samples. A total of 4 and 3 polymorphic bands were observed from OPA-2 primer in RAPD Profile of B. repens and B. repens var. rajasthanensis (a new varieties) leaves. The result concluded that these two different varieties of genus Blepharis can be used as an alternative to the conventional medicines due to having wide range of phytocemical activities. This study suggests that there is a need to know the possibility of using extracts and bio-active mixtures derived from Blepharis genus for the control of lasting ailments as claimed by traditional therapists.

DETECTION OF GENOMIC MARKERS BY RANDOM AMPLIFIED POLYMORPHIC DNA ANALYSIS IN LEUKEMIA PATIENTS

One of the hematological malignancies brought on by the abnormal proliferation of immature leukocytes is leukemia. Numerous epigenetic and genetic changes in hematopoietic progenitor cells are the cause of it. The Random Amplification of Polymorphic DNA (RAPD) technique has been utilized in the presented work in order to assess DNA polymorphisms and discover genomic markers in diverse leukemia types. The analysis utilizing a random primer regarding decamer oligonucleotides OPJ-04 produced distinct profiles of amplified fragments of DNA in the genomic DNA of 3 different types of the leukemia patients. The unique band (2178 bp), which is present in 60 and 66.6% of chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), respectively, exhibited a significant difference (P ≤ 0.01). While detected in only 20% of cases chronic lymphoid leukemia (CLL), and absent in the remaining leukemia patients and healthy controls. The results showed that the distinct bands (1737 and 1513 bp), which were present in all leukemia patients, however absent in healthy controls, showed extremely significant differences (P ≤ 0.01). Similarly, results have revealed highly significant differences (P ≤ 0.010) in fourth distinct band (1202 bp) that detected in all AML, 86.6% of CML, and 90% of CLL, and lacking in the remaining leukemia patients and in all healthy controls The results also showed that there have been significant differences (P ≤ 0.01) in the amplified band (750 bp), which was absent in 73.3% of CML patients, but present in the remaining CML patients, along with all AML patients, CLL patients, and healthy controls. The 4 amplified DNA fragments (1031, 870, 600, and 300 bp) that detected in all healthy controls and leukemia patients, at the same time, showed no significant differences in the results. The recognized DNA polymorphisms through the arbitrary primer OPJ-04 may be used as a genetic marker to design a successful RAPD primer for the diagnosis of leukemia patients

Mapping the Landscape of Indian Genomics Research: A Scientometric Analysis

This Scientometric study aimed to provide state-of-the-art information on research growth and trends, areas of potential growth and development in genomics in India, and identify the key players (organizations or institutions, and funding agencies). It was found that the number of publications and citations related to genomics research has been steadily increasing over the years, indicating a growing interest and investment in the field as the Indian Council of Agricultural Research was the leading contributor to the field. Among the 159 contributing countries from 2012 to 2021, India contributed 4.46% of publications. The Department of Biotechnology (Ministry of Science and Technology, India) provided the most funds for genomics research. In the last decade, research was primarily focused on "Genetic Diversity," "Polymorphism," "Comparative Genomics," "Phylogeny," " Random amplification of polymorphic DNA (RAPD)," "Single nucleotide polymorphism (SNP)," "Polymerase chain reaction (PCR)," "Gene Expression," etc. The study's findings may shed light on the strengths and weaknesses of the country's research infrastructure, as well as the effectiveness of government policies and funding mechanisms.

Pseudomonas Aeruginosa Colonising Patients with Cystic Fibrosis: Antibiotic Resistance and Growth Conditions Determined by in vivo and in vitro Gene Expression

Background: Pseudomonas aeruginosa is the main pathogen responsible for lung destruction in cystic fibrosis, becoming difficult to eradicate in chronic infection. Aims: To describe antibiotic resistance among strains of P.aeruginosa isolated from sputa of patients with cystic fibrosis. To investigate in vivo and in vitro expression of genes related to antibiotic resistance and anaerobic growth. Methods: Sputa (in vivo) and strains (in vitro) from 26 patients were obtained during 17 months. Genotypes were compared by random polymorphic DNA amplification. Expression of nirS (anaerobic respiration) and mexY (MexXY efflux pump) were measured by quantitative real time polymerase chain reaction. Expression levels of nirS in aerobiosis and anaerobiosis were compared to estimate oxygenation status within lungs. Mutations in the regulator gene mexZ were investigated in sputa expressing mexY and were correlated with strains’ antibiotic resistance. Results: Nine patients and 56 sputa were finally analysed. Seven patients carried a single genotype. Gene mexY was detected in all the sputa; expression levels were higher in sputa with mexZ mutations. Multi-resistance was frequent. Resistance profiles not always correlated with mexY expression levels or mexZ mutations. Comparison of in vivo and in vitro nirS expression indicated mainly aerobic and microaerophilic environments within sputa. Discussion: Mutations in e mexZ are frequent in strains of P.aeruginosa colonising patients with cystic fibrosis. Presence of these mutations correlates with increased expression of mexY in vivo and in vitro, but no with in vivo antibiotic resistance. Results of nirS expression suggest that the lungs represent heterogeneous environments regarding oxygenation status. This complexity explains that mechanisms of growth and antibiotic resistance within the lungs of these patients are still largely unknown. Conclusions: After many years of research few studies, including the present, revealed different aspects of in vivo growth of P. aeruginosa. We determined a cut-off to discriminate between sputa containing mexZ wild type and mutated alleles and showed that comparison of in vivo and in vitro nirS expression allows to predict oxygenation status. So far, none of the studies can explain all the factors influencing the behaviour of P.aeruginosa colonising cystic fibrosis patients making it difficult to design new therapeutic strategies.

RAPD MARKER TO SCREENING GENETIC DIVERSITY OF LOCAL CHICKEN

A total of 500 local chickens from different regions of Iraq, which were divided into five groups according to the regions from which they were taken, namely, the East (E), West (W), North (N), South (S), and Central (C) regions of Iraq. The blood samples were collected into tubes containing an EDTA, DNA extraction was carried. Using 20 primers from Gen Script USA, random amplification of polymorphic DNA (RAPD-PCR) was carried out. The reaction of PCR had a final volume of 25 μl. A total of 473 loci ranging between 200 and 1800 bp were amplified. The higher numbers of bands shown in north(N) local chicken group (101) than the other groups. East (E) and South (S) groups phenotypic revealed higher numbers of the polymorphic bands. The Polymorphism percent (%6.2) in East (E). Between the north (N) and west (W) groups, the genetic similarity score was found to be the highest (0.893). dendogram It was clear that the South(S) appeared to be most distant from the other groups whereas the West (W), North (N) were related closely together whose genetic closeness is highest. The main aim of present study is to assess the genetic specificity chickens in Iraq independs on RAPD analysis, To assess phylogenetic relatedness between groups of chickens, is to understand the extent of genetic variation between local chickens groups in Iraq.

In Vitro Activity, Stability and Molecular Characterization of Eight Potent Bacteriophages Infecting Carbapenem-Resistant Klebsiella pneumoniae.

Members of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment, including bacteriophage therapy. Bacteriophages are considered very safe and effective in treating bacterial infections. In this study, we characterize eight lytic bacteriophages that were previously isolated by our team against carbapenem-resistant Klebsiella pneumoniae. The one-step-growth curves, stability and lytic ability of eight bacteriophages were characterized. Restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) typing analysis and protein profiling were used to characterize the microbes at the molecular level. Phylogenetic trees of four important proteins were constructed for the two selected bacteriophages. All eight bacteriophages showed high efficiency for reducing bacterial concentration with high stability under different physical and chemical conditions. We found four major protein bands out of at least ten 15-190 KDa bands that were clearly separated by SDS-PAGE, which were assumed to be the major head and tail proteins. The genomes were found to be dsDNA, with sizes of approximately 36-87 Kb. All bacteriophages reduced the optical density of the planktonic K. pneumoniae abruptly, indicating great potential to reduce K. pneumoniae infection. In this study, we have found that tail fiber protein can further distinguished closely related bacteriophages. The characterised bacteriophages showed promising potential as candidates against carbapenem-resistant Klebsiella pneumoniae via bacteriophage therapy.

Determination of Some Morphological Traits and Genetic Factor of Two Species of Liriomyza spp (Diptera: Agromyzidae) on the Climbing Beans and Tomato Crops

This study was carried out to study some morphological parts of adult male of Liriomyza spp, including the antenna, the femur and the middle vein (Cu Al), as well as the shape of mines that larvae made them on the leaves. Results showed that there were two species of L.bryoinae on tomato crop and L.sativae on climbing beans crop, this was agreed with the molecular study that it was conducted using the RAPD Random Amplification of Polymorphic DNA based on the PCR technique to determine genetic variability of DNA that it was extraction from adults and pupae and obtained a quantity of it ranged from (35-556.9) mg and purity ranged between (1.7-2).
 The RAPD markers were studied using 19 random primers, 13 of which showed a bands and 6 of them didn’t show any bands, the total number of bands loci were 53 loci, 23 of which were main bands and 30 were polymorphism and genetic analysis was performed based on these results. The genetic distance was found and lowest was (0.125) among adults and pupae of bean crop and higher distance was (0.323) the among the adults and the pupae of the tomato crop and the genetic distance between the tomato pupae and bean pupae was (0.185), according to this values the genetic relationship was found which was the tomato adult's species was independent and the genetic relationship between Adults and pupae have closest. Results showed that the pupae which were taken from the crops, there are two species of Liriomyza spp .
 

DETECCIÓN CLONAL DE CEPAS PARENTALES DE Streptococcus agalactiae Lehmann y Neumann POR AMPLIFICACIÓN ALEATORIA DE ADN POLIMÓRFICO

Streptococcus agalactiae (GBS) causes invasive infections in newborns, being the most frequent the maternal transmission. Epidemiological studies use molecular techniques that assess genetic diversity, including random amplification of polymorphic DNA (RAPD) that is found to be accessible, sensitive and uses arbitrary primers to amplify polymorphic segments of DNA by PCR. The objective was to determine the clonal relationship between GBS strains recovered from mothers and their respective newborns. Four pairs of GBS isolates obtained from vaginal-rectal swabs of mothers and blood cultures of their newborns were studied with RAPD. Primers OPS11, OPB17 and OPB18 were used to select one with the ability to discriminate between non-genetically related strains. The Hunter-Gaston formula that establishes the discrimination index (D) was used; when D>0.90, it is considered that the isolates belong to different clones. The amplification profiles for the eight isolates, using each primer independently, allowed to calculate a D=1 for OPS11, and D=0.84 for OPB17 and OPB18. Therefore, OPS11 was selected for the study of the clonal relationship of the isolates, and similar amplification profiles were found by RAPD for each mother-newborn pair of GBS isolates. Different amplification profiles were observed between pairs of mother-newborn strains, which reveals the discrimination between unrelated strains, confirmed by pulsating field electrophoresis (PFGE). These results indicated vertical transmission for each studied case and robustness of the OPS11 primer. Appropriate conditions of the RAPD trial were found, which is useful for epidemiological studies. Key words: Streptococcus agalactiae, neonatal disease, molecular epidemiology, RAPD technique, vertical transmission

Clonal fidelity investigation of micropropagated hardened plants of jackfruit tree (Artocarpus heterophyllus L.) with RAPD markers

BackgroundArtocarpus heterophyllus is an important tropical agroforestry species that bears multiple applications. However, the population of this species is reduced due to various anthropogenic activities. For this reason, in vitro approach is needed to propagate or conserve this species as in vivo propagation methods face various obstacles. In this respect, the present investigation was undertaken to produce genetically stable jackfruit trees through in vitro technology. In vivo grew shoot tips were harvested on Murashige and Skoog (MS) medium containing several plant growth regulators to achieve this. ResultsThe 6-benzylaminopurine (BAP) at the concentration of 1.5 mg L-1, indole-3-butyric acid (IBA) at 0.5 mg L-1, and α-naphthaleneacetic acid (NAA) at 0.1 mg L-1 in combination on MS media yielded superior shoot response (94.44%), longest shoot length (4.02 cm), and the maximum number of shoots per explant (4.78). They were further multiplied by repeated subculturing on the same media composition and the third subculture resulted in a maximum number of shoots (5.92) with the largest shoot length (5.85 cm). Among the different media screened for rooting, the ¼ MS media yielded 94.44% rooting response, the longest root length (3.78 cm), and the maximum number of roots per shoot (8.44) with 0.1 mg L-1 NAA, 0.5 mg L-1 IBA and 0.1 mg L-1 BAP in combination. Primary hardening showed 88.89% of plant survival under greenhouse conditions after 4 weeks of incubation having a sterilized mixture of garden soil and vermiculite mixture (1:1, w/w). It increased to 90.60% after the secondary hardening process in a vermiculite-soil mixture (2:1; w/w). No polymorphism was detected on random amplification of polymorphic DNA (RAPD) profiling between the mother plant and hardened plants, indicating high genetic stability among the clones. ConclusionsThis is the first report of the genetic fidelity study of in vitro grown regenerants of A. heterophyllus. This study established a micropropagation protocol for genetically uniform in vitro regeneration of this species to supply plant resources to various industries or conservation of elite germplasm.

Genetic diversity of Aedes aegypti in the central-upper Paraná Cross-Border axis in Paraguay.

To determine the genetic diversity of Aedes aegypti in the Central-Alto Paraná cross-border road corridor of Paraguay, an area that has reports of dengue cases. Twenty adult females were selected from hatching Ae. aegypti eggs from households geolocated in the departments of Alto Paraná, Caaguazú, Cordillera and Central, between 2018 and 2019. DNA was extracted from the tissue of females for amplifying their polymorphic patterns by random amplification of polymorphic DNA by PCR (RAPD-PCR), using primers H3 and B03 in order to identify genetic parameters of population diversity. The relationships between mosquito populations according to locality were observed by unpaired arithmetic mean analysis. We used DIVA-GIS 7.3.0 and MAXENT to analyze the suitable areas of actual and potential geographic distribution of these Ae. aegypti populations. Forty loci were identified by RAPD-PCR profiling, with moderate gene differentiation (Gst = 0.12). The cross-border corridor presented bioclimatic conditions for the presence of variant populations of Ae. aegypti, with precipitation in the warmest quarter and mean temperature in the driest quarter being determinant in the distribution. There is evidence of moderate genetic diversity in Ae. aegypti populations from areas that have reported dengue cases in the cross-border road corridor linking the Central and Alto Paraná departments of Paraguay. The study of genetic variability of Ae. aegypti is very useful for entomo-epidemiological surveillance and evaluation of possible resistance to chemical control.

Interrelationship amongst varieties of edible mushroom through Molecular marker Study

The information regarding the DNA sequences of plant is very limited. When knowledge of the DNA sequence of the targeted genome is unavailable several PCR based techniques (RAPD, ITS, AFLP, SSR markers, SNP markers) are utilized to accumulate information about the genetic adaptability. Random Amplification of Polymorphic DNA is a technique by which the random segments of DNA are amplified. Short nucleotide primers (8–12 nucleotides) are used to proceed with the PCR using genomic DNA, for fragments to amplify. The primers bind somewhere in the sequence. By comparing the banding matrix the DNA patterns can be ascertained. RAPD has been utilized to characterize, trace the phylogeny of diverse plant species. Internal transcribed spacer (ITS) is to a non-functional RNA situated between structural ribosomal RNAs (rRNA) on a transcript. The sequence of rRNA precursor transcript is the 5' external transcribed sequence (5' ETS), 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA and the 3'ETS read from 5' to 3' direction. The complete sequence repeats themselves in tandem array for thousands of copies separated by regions of non-transcribed DNA termed non-transcribed spacer (NTS) or intergenic spacer (IGS). There is the presence of two ITS regions in eukaryotes. The first one located between 18S and 5.8S rRNA genes is called ITS1, while the next in the sequence is ITS2 that lies between 5.8S and 26S rRNA genes in plants. Each eukaryotic ribosomal cluster follows the sequence of the 5' external transcribed sequence (5' ETS), trailed by the 18S rRNA gene, the ITS1 and the 5.8S rRNA gene, the ITS2, the 28S rRNA gene, and finally the 3' ETS. ETS and ITS pieces are excised and rapidly degraded during rRNA maturation. In our study four different gilled mushrooms were analyzed for RAPD and ITS study.

Forensic Application of Genetic and Toxicological Analyses for the Identification and Characterization of the Opium Poppy (Papaver somniferum L.).

Simple SummaryThe identification and characterization of illicit plant species such as Papaver somniferum L. (opium poppy) are essential in order for law enforcement agencies to obtain information about the origin of seized drugs, and to improve the monitoring of illegal drug trafficking routes. This study focuses on the genetic and toxicological analyses of some poppy capsules seized by the Italian Police Forces in 2017 and 2018. The detection of specific morphological traits, the chemical study of the alkaloid profile by gas chromatography coupled with mass spectrometry (GC/MS), and the genetic analysis performed by the DNA barcode approach showed that the seized poppy capsules belonged to P. somniferum. The data collected also suggested that the opium capsules originated from Afghanistan.Background: A reliable and science-based taxonomic determination of Papaver somniferum L. (opium poppy), the illegal species of the genus Papaver, may have practical and legal implications for law enforcement. P. somniferum is a controlled plant because of its narcotic substances, such as morphine and codeine. As poppy plants have rather similar morphological features, both chemical and genetic analysis are required in order to achieve an accurate characterization of such species. The chemical structures of alkaloids are extremely variable even within the same species, which is why the genetic approach may lead to a more scientific Papaver sp. differentiation. The aim of our study was the taxonomic identification of poppy capsules seized by the Italian Police Forces being considered as potential P. somniferum derivatives. Methods: The alkaloids detected using gas chromatography/mass-spectrometry (GC/MS) were morphine, codeine, thebaine, noscapine, meconin, hydrocotarnine, and traces of papaverine. Further genetic analysis was carried out simultaneously using three plastid DNA barcoding regions (matK, trnH-psbA, and rbcL) for the samples’ identification. Results: The Random Amplification of Polymorphic DNA (RAPD) method showed that the analysed samples were genetically identical. Conclusions: The morphological, toxicological, and genetic profile of the samples revealed that they belonged to P. somniferum species. Furthermore, the alkaloid content of dried poppy capsules might be used to investigate and track their origin.

Assessment of Genetic Variation of the Genus Gymnema in Vietnam, Using RAPD and ITS-rDNA Markers

ABSTRACT Gymnema is a genus of the Apocynaceae with more than 50 species. Some of its species such as G. sylvestre and G. latifolium have been reported for their medicinal value. In this study, the genetic diversity of 20 samples of four species of Gymnema in Vietnam was evaluated using the Random Amplification of Polymorphic DNA (RAPD) and the internal transcribed spacer of nuclear ribosomal DNA (ITS-rDNA). Amplified bands obtained with primers UBC701, UBC709, and OPC4 were the highest polymorphic in RAPD analysis. The phylogenetic trees were built grouping the 20 samples into four clusters corresponding to the four Gymnema species according to morphological classification. A number of characteristic SNPs were identified for each species which can be used for authentication and differentiation. The results showed the consensus and usefulness of the two barcoding techniques in the molecular identification and characterization of the Gymnema genus.