Random Amplified Polymorphic DNA-polymerase Chain Reaction Research Articles

Characterization of Newly Isolated Bacteriophages Targeting Carbapenem-Resistant Klebsiella pneumoniae.

Klebsiella pneumoniae, a Gram-negative opportunistic pathogen, is increasingly resistant to carbapenems in clinical settings. This growing problem necessitates the development of alternative antibiotics, with phage therapy being one promising option. In this study, we investigated novel phages targeting carbapenem-resistant Klebsiella pneumoniae (CRKP) and evaluated their lytic capacity against clinical isolates of CRKP. First, 23 CRKP clinical isolates were characterized using Multi-Locus Sequence Typing (MLST), carbapenemase test, string test, and capsule typing. MLST classified the 23K. pneumoniae isolates into 10 sequence types (STs), with the capsule types divided into nine known and one unknown type. From sewage samples collected from a tertiary hospital, 38 phages were isolated. Phenotypic and genotypic characterization of these phages was performed using Random Amplification of Polymorphic DNA-PCR (RAPD-PCR), transmission electron microscopy (TEM), and whole genome sequencing (WGS) analysis. Host spectrum analysis revealed that each phage selectively lysed strains sharing the same STs as their hosts, indicating ST-specific activity. These phages were subtyped based on their host spectrum and RAPD-PCR, identifying nine and five groups, respectively. Fourteen phages were selected for further analysis using TEM and WGS, revealing 13 Myoviruses and one Podovirus. Genomic analysis grouped the phages into three clusters: one closely related to Alcyoneusvirus, one to Autographiviridae, and others to Straboviridae. Our results showed that the host spectrum of K. pneumoniae-specific phages corresponds to the STs of the host strain. These 14 novel phages also hold promise as valuable resources for phage therapy against CRKP.

Investigation of clonal relationship in hospital-associated Candida parapsilosis isolates.

We aimed to investigate the clonal relationship and antifungal susceptibility of C. parapsilosis isolated from hospitalized patients and to determine whether it is due to transmission or not and the spread status of resistant isolates. Between January 2017 and June 2019, totally 277 C parapsilosis isolated from blood, urine and catheter samples of adult or pediatric in-patient (intensive care and service) who applied to Mycology laboratory in our hospital were included in the study. All isolates were identified using conventional methods, API 20C AUX (Biomerieux, France) semi-automated system and confirmed by MALDI-TOF MS Biotyper Smart (Bruker Daltonik GmbH, Germany). Randomly amplified polymorphic DNA (RAPD) PCR method was used for molecular genotyping of isolates. MIC values for fluconazole, anidulafungin and amphotericin B were determined according to the M27-A3 CLSI broth microdilution reference method guideline. Seven different band patterns (A-G) were detected in 277 isolates by RAPD PCR method. According to the rank order of the isolates, 170 (61.37%) C, 65 (23.47%) A, 18 (6.50%) G, 11 (3.97%) B, six (2.17%) E, two (0.72%) F and one (0.36%) D patterns were determined. When the band patterns of the isolates were evaluated according to the years, it was detected that C pattern continued between 2017 and 2019 and that all isolates continued to spread only as C pattern in 2019. While 211 (76.17%) of the isolates were resistant to fluconazole (≥ 8µg/ml), two (0.72%) were resistant to amphotericin B (≥ 2µg/ml) and two (0.72%) were intermediate to anidulafungin. It is noteworthy that the spread of the C pattern in C. parapsilosis strains has increased over the years and is the main pattern isolated from the whole hospital. The detection of high fluconazole resistance in C. parapsilosis isolates in our hospital may also be related to the dominant pattern.

Genetic diversity and virulence properties of caprine Trueperella pyogenes isolates

BackgroundTrueperella pyogenes is an opportunistic pathogen that causes suppurative infections in various animal species, including goats. So far, only limited knowledge of phenotypic and genotypic properties of T. pyogenes isolates from goats has been gathered. In our study, we characterized the phenotypic and genotypic properties of caprine T. pyogenes isolates and established their relationship by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR).ResultsFrom 2015 to 2023, 104 T. pyogenes isolates were obtained from 1146 clinical materials. In addition, two T. pyogenes isolates were obtained from 306 swabs collected from healthy goats. A total of 51 T. pyogenes isolates were subjected to detailed phenotypic and genotypic characterization. The virulence genotype plo/nanH/nanP/fimA/fimC/luxS was predominant. All of the tested isolates showed the ability to form a biofilm but with different intensities, whereby most of them were classified as strong biofilm formers (72.5%). The high level of genetic diversity among tested caprine T. pyogenes isolates (19 different RAPD profiles) was observed. The same RAPD profiles were found for isolates obtained from one individual, as well as from other animals in the same herd, but also in various herds.ConclusionsThis study provided important data on the occurrence of T. pyogenes infections in goats. The assessment of virulence properties and genetic relationships of caprine T. pyogenes isolates contributed to the knowledge of the epidemiology of infections caused by this pathogen in small ruminants. Nevertheless, further investigations are warranted to clarify the routes of transmission and dissemination of the pathogen.

Determining the Genetic Markers of Schizophrenia using Molecular Diagnosis

Schizophrenia poses a considerable disease burden, notwithstanding its relatively low incidence rate. The escalation in population size and mean age has led to a substantial rise in the incidence of illness linked to schizophrenia, particularly in countries with moderate levels of prosperity. Therefore, it is necessary to find a way to detect and recognize the illness as fast as possible to stop the consequences from occurring and stop symptoms from appearing before they even start. Within this study’s scope, fifteen distinct primers were selected randomly to determine whether or not they could recognize schizophrenia. To accomplish this, a Randomly Amplified Polymorphic DNA-polymerase chain reaction (RAPD-PCR) was conducted utilizing DNA samples obtained from individuals afflicted with the condition and those in good health. The outcomes of the gel electrophoresis and subsequent analysis revealed that solely three primers including C12, E02 and E03 exhibited monomorphic characteristics. However, it was observed that 80% of the primers, precisely eleven out of fifteen, exhibited polymorphism. The results indicated that among the 15 primers tested, the primer labeled as H01 failed to generate any detectable bands. Subsequent examination of the polymorphic primers revealed that the primer efficiency varied between 0 and 0.0755, while the discrimination power ranged from 0 to 20 percent. The study observed variations in the frequency of disease occurrence between patients and healthy individuals. H05 primer utilization yielded three distinct polymorphic bands exhibiting diverse lengths. The band exhibiting the greatest length measured 1190 bp, whereas the other two bands had 480 and 430 bp lengths respectively. The two polymorphic bands exhibited 100% prevalence in the patient population while being completely absent in the healthy control group. Based on the available data, it appears that utilizing this primer to identify schizophrenia is a valid and valuable claim. Overall, DNA-based diagnostics may overlook a number of difficulties as laboratories have the ability to extract DNA from various sources easily. In this scenario, molecular and genetic techniques may be crucial for diagnosing some diseases.

Limited genetic variability and spatial population structure in grasshoppers between natural and metal-contaminated areas in Egypt.

Pollutants in an environment can have long-term implications for the species living there, resulting in local adaptations with implications for their genetic structure. Heavy metal pollutants infiltrate soils and groundwater, bioaccumulate in food webs, and negatively impact biota. In this study, we investigated the degree to which the genetic structure and variability of the slender green-winged grasshopper (Aiolopus thalassinus (Fabricius) (Orthoptera: Acrididae)) were impacted by heavy metal pollution and distance. We used the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method to examine the genetic variability of populations in 3 heavy metal-polluted and 3 unpolluted locations across varying geographical distances in Egypt. The heavy metal concentrations of cadmium, copper, lead, and zinc were measured from the grasshopper tissue and soils. Sixty-nine unique and polymorphic bands were produced by 4 primers. Cluster and principal component analyses separated the populations inside and outside Cairo into 2 main branches, which were further divided into smaller branches corresponding to their geographical regions. We found no differences in the Shannon genetic diversity index between populations or with increasing heavy metal concentrations in either the soil or the grasshopper tissue. Our results showed a greater genetic variation among populations than between populations within the same location, indicating populations within locations were less differentiated than those between locations. The moderate correlation between genetic similarity and spatial distance suggests geographical isolation influenced grasshopper population differentiation. Based on the RAPD analysis, environmental pollutants and geographical distances impact the A. thalassinus population structure, potentially restricting gene flow between sites even at small spatial scales.

Capsular gene distribution and RAPD typing of Streptococcus agalactiae isolated from pregnant women

Streptococcus agalactiae has different virulence factors, from which the capsule has the most significant role in the pathogenesis of this organism. We aimed to investigate the distribution of more prevalent capsular genes among different Random Amplified Polymorphic DNA (RAPD) types of S. agalactiae isolated from pregnant women. A total of 106 isolates were collected from 420 vaginal and rectal swabs obtained from pregnant women. The specimens were transferred using Todd Hewitt Broth and were cultured on a blood agar containing antibiotics. The S. agalactiae isolates were identified by the standard microbiological and biochemical tests. The genomic DNAs of S. agalactiae isolates were extracted using an extraction kit. Then, the PCR method was used to detection of the capsular genes. Moreover, The RAPD PCR was used to genotyping of the isolates. The colonization rate of the pregnant women was 25.23%, and there was a statistically significant correlation between the weeks of gestation and the probability of colonization (p-value < 0.05). Also, 31 (29.24%) and 18 (16.98%) pregnant women had a history of abortion and membrane rupture, respectively. In addition, 20 (18.86%), 32 (30.18%), 4 (3.77%), and 6 (5.66%) isolates carried genes encoding capsular types Ia, Ib, III, and V, respectively. None isolates had the type II capsular gene, and other 44 isolates were non-typeable. Nine clones (clusters) of S. agalactiae were observed in the present study with 70% similarity, and 53 different types were identified among the isolates. Except for capsular types III and V that belonged to clones 3, 5, 7, and 9, other capsular types were detected in different RAPD types. We found that the capsular types Ib and Ia were predominant among pregnant women in this area, indicating their significance for vaccine designation. Also, our isolates showed a lower genotypic diversity in RAPD typing. This may be due to the same sources of most isolates.

Virulence Gene Properties and Phylogenetic Relationship of Salmonella Serotypes Isolated from Different Sources in North of Iran

Background: Salmonella species can cause various infections in humans and animals. The presence of certain genes determines the virulence of a Salmonella serotype. Objectives: The current research endeavor was undertaken to assess the virulence characteristics and genotypic traits of Salmonella serotypes extracted from various sources within the geographical boundaries of Iran. Methods: Salmonella isolates, previously retrieved and preserved in the veterinary microbiology laboratory, underwent serotyping and polymerase chain reaction (PCR) identification of nine virulence-associated genes. Genotyping was carried out using random amplified polymorphic DNA-PCR (RAPD-PCR). Results: All Salmonella isolates showed the presence of invA, sdiA, hilA, and iroB virulence genes. There were a total of 17 different virulence gene patterns among Salmonella serotypes. The presence of fliC and sefA genes and their related patterns were significant among S. typhimurium and S. enteritidis serotypes, respectively (P &lt; 0.05). In the RAPD-PCR fingerprinting, 11 distinct clusters were obtained, and 16 isolates (26.66%) were classified as untypeable strains. There was a significant association between RAPD genotypes and Salmonella serotypes (P &lt; 0.05), while the association between these RAPD patterns and the source of the isolates was not significant (P &gt; 0.05). Conclusions: According to the results, Salmonella serotypes from non-human sources carry significant virulence determinants and show similar genotypic patterns with human isolates. These findings provide valuable insights into the virulence properties and genetic diversity of Salmonella serotypes in Iran, which could inform the development of effective control and prevention strategies for salmonellosis in the region.

Assessment of protein and DNA polymorphisms in corn (Zea mays) under the effect of non-ionizing electromagnetic radiation

Many reports highlight biological responses of crop plants after non-ionizing electromagnetic radiation (EMR) exposure based on the phenotypic and physiological levels. So, this study aimed to estimate genetic alterations in proteins, isozymes, and DNA banding patterns as well as the extent of nuclear DNA damage of economic corn (Zea mays) under the stress of EMR using accurate and reliable bioassays like sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes (Leucine- aminopeptidase, Esterases, Peroxidase, and Catalases), random amplified polymorphic DNA- polymerase chain reaction (RAPD-PCR), and Comet Assay, respectively. SDS-PAGE analysis showed distinct polymorphisms (96.66%) between EMR exposed and non-exposed corn seedlings depending on the number and type of bands, their intensities as well as molecular weight which ranged from (60.27 to 192.35 kDa), gain, and loss of bands. The four isozymes generated varies isozymatic polymorphisms based on relative front, zymogram number, and optical intensities. RAPD analysis generated 85 amplified DNA products with high polymorphism values ranged from 90.91 to 100% based on primers, band type, DNA sizes which ranged from 153 to 1008-bp, lose, gain, and intensity of DNA bands. Comet Assay scored highest extent of loosed DNA from nuclei (DNA damage) reached the value of (tailed ratio 20%) at EMR exposed corn nuclei for 5 days compared to non-exposed nuclei which reached the value of (tailed ratio 3%). This study concluded that each EMR exposure time had unique interaction with proteins, isozymes, and DNA of corn cells exhibiting wide range of genotoxic stress and subsequently, adversely effect on growth and yield of this sensitive crop plants.

Isolation, characterization, and fingerprinting of some multidrug resistance clinical isolates from patients in Al-Qaseem Hospitals, Saudi Arabia

The aim of this study was to investigate the prevalence of antibiotic resistance genes in bacteria isolated from Saudi Arabian patients in the Al-Qassim region. We also examine the application of Random Amplified Polymorphic DNA- Polymerase chain reaction (RAPD-PCR) in genetic diversity of the strains in use. In this study, we have used 29 different clinical samples from Al-Qassim hospitals which consists of wound swabs, blood, sputum and urine. Clinical samples were streaked onto sterile Petri dishes containing nutrient agar media using sterile dry swabs. Additionally antibiotic sensitivity tests, micro scan walkway susceptibility tests and extraction of genomic DNA, amplification of RAPD and Inter simple sequence repeat assay were performed in this study. This study results confirmed from the clinical samples were streaked onto sterile Petri dishes containing nutrient agar media using sterile dry swabs. The PCR analysis in ISSR was confirmed that 30–70% of E. coli in Asia and Africa carried ESBLs in the GLASS 2020 study, which is on par with the 59% in VINARES 2016–2017. Comparing VINARES 2016–2017 to other nations, the prevalence of ESBL carriage among K. pneumoniae was 35%. However, RAPD results showed that all employed strains and primers could successfully fingerprint DNA. Between isolated bacterial strains, similarity dendrograms were produced. The technique generated bands with the same intensity as the typical PCR carried out using pure DNA, and it worked for all 29 bacterial strains examined. In conclusion, the results show that all of the isolates have at least one antibiotic-resistance gene, and that the PCR approach is a quick, easy, and reliable way to identify genes.

High Frequency of blaOXA-48like Producing Klebsiella pneumoniae Isolated from Nosocomial Infection in Azerbaijan, Iran.

Klebsiella pneumoniae is one of the significant agents of hospital-acquired infections. In recent years, carbapenem-resistant K. pneumoniae (CRKP) isolates have been found in numerous epidemics of nosocomial infections. This study aimed to determine carbapenem resistance mechanisms and molecular epidemiological of CRKP infections in Azerbaijan, Iran. A total of 50 non-duplicated CRKP from January 2020 to December 2020 were isolated form Sina and Imam Reza Hospitals in Tabriz, Iran. Antimicrobial susceptibility testing was performed by the disk-diffusion method. The carbapenem resistance mechanisms were determined by the phenotypic and PCR procedures. CRKP isolates were typed by the Random Amplified Polymorphic DNA PCR (RAPD-PCR) technique. Amikacin was the most effective antibiotics against CRKP isolates. AmpC overproduction was observed in five CRKP isolates. Efflux pump activity was found in one isolate by the phenotypic method. Carba NP test could find carbapenemases genes in 96% of isolates. The most common carbapenemases gene in CRKP isolates were blaOXA-48-like (76%) followed by blaNDM (50%), blaIMP (22%), blaVIM (10%), and blaKPC (10%). The outer membrane protein genes (OmpK36 and OmpK35) were identified in 76% and 82% of CRKP isolates, respectively. RAPD-PCR analysis yielded 37 distinct RAPD-types. Most blaOXA-48-like positive CRKP isolates were obtained from patients hospitalized in intensive care unit (ICU) wards with urinary tract infections. The blaOXA-48-like is the main carbapenemase among CRKP isolates in this area. Most blaOXA-48-like producer CRKP strains were collected from the ICU ward and urine samples. To control infections due to CRKP, a strict control program in hospital settings is required.

Genetic Diversity of Candida spp. Isolates Colonizing Twins and Their Family Members.

A wide range of options for studying Candida species are available through genetic methods. Twins, particularly monozygotic ones and their families may be fitting subjects for studying those microorganisms. The question is: How specific can yeast flora be in an individual? The study aimed to analyze the strain relatedness among commensal yeasts isolated from various parts of the bodies of healthy people and to compare correlations between the genotypes of the isolates. Yeasts were isolated from 63 twins and their family members (n = 25) from the oral cavity, anus, interdigital space and navel. After species identification, Candida albicans (n = 139), C. parapsilosis (n = 39), C. guilliermondii (n = 25), C. dubliniensis (n = 11) and C. krusei (n = 9) isolates were analyzed using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) optimization method. The similarities between the strains were calculated based on the Dice (Sab) coefficient and are displayed graphically as dendrograms. Using cluster analysis, the following relatedness was distinguished: 13 genotypes and three unique (Un) patterns among C. albicans; 10 genotypes and four Un patterns among C. parapsilosis; three genotypes and one Un pattern among C. guilliermondii and C. dubliniensis; and three genotypes among C. krusei isolates. The presence of identical, similar or both genotypes among the strains isolated from family members shows the transmission of yeasts between ontocenoses in the same person and between individuals. The similarity between the genotypes of C. albicans, C. guilliermondii, C. dubliniensis and C. krusei was more remarkable than between the genotypes of C. parapsilosis in the strains isolated from ontocenoses of the same individual and their family members. The degrees of genetic similarity between Candida spp. strains isolated from monozygotic twins and those obtained from their relatives did not differ.

Analysis of genetic diversity of E.coli bacteria isolates from UTI after exposure to some biological effects by using RAPD technique

Background and Objective: Escherichia coli bacteria are one of the most important bacterial races causing urinary tract infection, which is responsible for 90% of the causes of urinary tract infection and originated in the human intestine, this is Gram- Negative bacteria and facultative aerobic that moves by peripheral flagella and belongs to the bacteria of the Enterobacteriaceae.&#x0D; Materials and methods: The bacteria were isolated from UTI, after they were diagnosed using light microscope and conducting biochemical tests, then exposed to the antibiotic Ciprofloxacin, trimethoprim, and alcoholic extract of Punica granatum and Trigonella foenum graicum. genomic DNA was extracted for all samples and Random amplified polymorphic DNA - Polymerase chain reaction (RAPD-PCR) marker was carried out using five random primers. &#x0D; Results: The results of RAPD-PCR profiles shown that exposed to antibiotics (Ciprofloxacin and Trimethoprim) and alcoholic extracts (Punica granatum and Trigonella foenum graicum) lead to the disappearance or appearance new bands compared with non-exposed samples, and the highest rate of polymorphism for all each treatment and primers in sample 5 was 102.27% where the ratio % GTS for all treatment and primers is 6.04% in the same sample.&#x0D; Conclusion: All treatments caused genetic changes in the DNA of E.coli bacteria cells especially the Punica granatum which gave the highest effect than the rest of the treatments, this indicates its efficiency in treating bacterial infections.

Agarose Gel Electrophoresis-Based RAPD-PCR-An Optimization of the Conditions to Rapidly Detect Similarity of the Alert Pathogens for the Purpose of Epidemiological Studies.

Agarose gel electrophoresis is a well-known tool to detect DNA fragments amplified in polymerase chain reaction (PCR). Its usefulness has also been confirmed for epidemiological studies based on restriction fragments length polymorphism (RFLP), usually performed using pulsed-field gel electrophoresis (PFGE). Little is known on the effectiveness for alert-pathogen epidemiological studies of another less time-consuming and costly technique called randomly amplified polymorphic DNA-PCR (RAPD-PCR). Meanwhile, its usefulness is believed to be comparable to RFLP-PFGE. Therefore, the aim of the study was to establish and optimize the conditions of agarose gel electrophoresis following RAPD-PCR for 19 Enterococcus faecium strains derived from epidemic outbreaks at intensive care units. An application of different PCR primers, primer combinations, and, in particular, agarose gel concentrations and electrophoresis conditions revealed the usefulness of this relatively fast and inexpensive method based on RAPD-PCR for epidemiological studies without a compulsion to use the specialized equipment necessary for RFLP-PFGE.

The Human Milk Microbiota Produces Potential Therapeutic Biomolecules and Shapes the Intestinal Microbiota of Infants.

Human milk not only provides a perfect balance of nutrients to meet all the needs of the infant in the first months of life but also contains a variety of bacteria that play a key role in tailoring the neonatal faecal microbiome. Microbiome analysis of human milk and infant faeces from mother-breastfed infant pairs was performed by sequencing the V1-V3 region of the 16S rRNA gene using the Illumina MiSeq platform. According to the results, there is a connection in the composition of the microbiome in each mother-breastfed infant pair, supporting the hypothesis that the infant's gut is colonised with bacteria from human milk. MiSeq sequencing also revealed high biodiversity of the human milk microbiome and the infant faecal microbiome, whose composition changes during lactation and infant development, respectively. A total of 28 genetically distinct strains were selected by hierarchical cluster analysis of RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) electrophoresis profiles of 100 strains isolated from human milk and identified by 16S RNA sequencing. Since certain cellular molecules may support their use as probiotics, the next focus was to detect (S)-layer proteins, bacteriocins and exopolysaccharides (EPSs) that have potential as therapeutic biomolecules. SDS-PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis) coupled with LC-MS (liquid chromatography-mass spectrometry) analysis revealed that four Levilactobacillus brevis strains expressed S-layer proteins, which were identified for the first time in strains isolated from human milk. The potential biosynthesis of plantaricin was detected in six Lactiplantibacillus plantarum strains by PCR analysis and in vitro antibacterial studies. 1H NMR (Proton Nuclear Magnetic Resonance) analysis confirmed EPS production in only one strain, Limosilactobacillus fermentum MC1. The overall microbiome analysis suggests that human milk contributes to the establishment of the intestinal microbiota of infants. In addition, it is a promising source of novel Lactobacillus strains expressing specific functional biomolecules.

Evaluating the Quality of Cheese Slices Packaged with Na-Alginate Edible Films Supplemented with Functional Lactic Acid Bacteria Cultures after High-Pressure Processing.

The aim of the current study was to assess the efficacy of Na-alginate edible films as vehicles for delivering lactic acid bacteria (LAB) with functional properties to sliced cheeses, with or without high-pressure processing (HPP). A three-strain LAB cocktail (Lactococcus lactis Τ4, Leuconostoc mesenteroides Τ25 and Lacticaseibacillus paracasei Τ26) was incorporated into Na-alginate solution in a final population of 9 log CFU/mL. The cheese slices (without or with HPP treatment at 500 MPa for 2 min) were packaged in contact with the LAB edible films (LEFs), and subsequently vacuum packed and stored at 4 °C. Cheese slices without the addition of films, with or without HPP treatment, were used as controls. In all cases, microbiological, pH and sensory analyses were performed, while the presence and the relative abundance of each strain during storage was evaluated using Random Amplified Polymorphic DNA-PCR (RAPD-PCR). In addition, organic acid determination and peptide analysis were performed using high-performance liquid chromatography. The results showed that in cheeses without HPP treatment, the microbiota consisted mostly of mesophilic LAB and lactococci (>7.0 log CFU/g), while HPP caused a reduction in the indigenous microbiota population of approximately 1–1.5 log CFU/g. In the LEF samples, the populations of mesophilic LAB and lactococci were maintained at levels of >6.35 log CFU/g during storage, regardless of the HPP treatment. Sensory evaluation revealed that the LEF samples without HPP had a slightly more acidic taste compared to the control, whereas the HPP-LEF samples exhibited the best organoleptic characteristics. RAPD-PCR confirmed that the recovered strains were attributed to the three strains that had been entrapped in the films, while the strain distribution during storage was random. Overall, the results of the study are promising since the functional LAB strains were successfully delivered to the products by the edible films until the end of storage.

ANALISIS KERAGAMAN GENETIK VARIETAS JAGUNG (Zea mays L.) HIBRIDA MENGGUNAKAN MARKA RAPD (RANDOM AMPLIFIED POLYMORPHIC DNA)

Analysis of genetic diversity is essential in developing hybrid maize (Zea mays L) varieties. The purpose of this study was to analyze the genetic diversity of six maize varieties using the PCR-RAPD (Polymerase Chain Reaction-Random Amplified Polymorphic DNA) technique. Hybrid maize varieties used in this study were Srikandi, Arumba, Mira, Magenta, and Simba. A local maize variety originating from Rote Island, East Nusa Tenggara Province was used as a comparison. Seeds were planted for the observation of morphological characteristics such as roots, leaves, and stems, as well as DNA extraction samples. DNA was extracted using the CTAB method and PCR-RAPD was performed using 3 primers. The size of the PCR product was determined using semilog paper. Genetic diversity between varieties were analyzed using the MVSP (Multi-Variate Statistical Package) program with the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method. The results of PCR-RAPD gave 28 DNA bands with sizes ranging from 310 bp to 1550 bp and produced 23 polymorphic bands. Based on the dendrogram resulting from PCR-RAPD data, there was one cluster consisting of Rote, Srikandi, Arumba, Mira, and Magenta varieties, as well as another cluster consisting of only the Simba variety. The local variety of Rote and the hybrid variety of Srikandi have the closest genetic distance, while the Simba variety has the farthest genetic distance from the other five varieties.&#x0D;

Genetic analysis of micro propagated plants of Tinospora cordifolia (Willd.) Miers by using RAPD–PCR markers

Random Amplified Polymorphic DNA (RAPD) is the simplest and cost-effective tool used for the analysis of the genetic fidelity of in vitro propagated plants of Tinospora cordifolia. A high-quality genomic DNA free from any contaminants, was isolated from five genotypes by the modified CTAB method. Out of 30 random decamer primers, OPA-02, OPA-03, OPA-09, OPA-10, OPB-02, OPB- 04, OPB-09 and OPK-06 showed fingerprints. Out of total 27 scored bands, 26 produced were monomorphic bands (96.2%) and one was polymorphic band (3.8%). The highest number of bands (6) were produced by OPA -03 and OPK-06 and minimum number of bands (1) was produced by OPB -04. The amplicon size varied from 200 bp to 1500 bp. A coefficient similarity value ranging from 0.87–0.93 was obtained. Maximum similarity value 0.93 was exhibited by both the genotypes namely, SB1 and SB3. From the present study, it has been concluded that the RAPD analysis can be used to check the genetic fidelity in the micro propagated plants of Tinospora cordifolia.

Genetic Diversity of Exobasidium vexans, the Causal Agent of Blister Blight on Tea in Pagilaran, Central Java, Indonesia Using PCR-RAPD

Indonesia is one of the ten largest tea-producing countries in the world, with a plantation area of 104,420 hectares and a production of 139,285 thousand tons in 2018. Blister blight can cause massive crop losses across tea-growing regions of Asia, particularly in India, Sri Lanka, Indonesia, and Japan. The infection causes a 40% yield loss. The study aimed to determine the genetic diversity in Exobasidium vexans that cause blister blight based on polymerase chain reaction-random amplified polymorphic DNA (PCR-RAPD). Sampling was conducted at Pagilaran, a tea plantation located in Central Java, Indonesia, with sampling based on altitude, Andongsili (&gt;1,000 meters above sea level [masl]), Kayulandak (±1,000 masl), and Pagilaran (&lt;1,000 masl) with clones TRI 2024, TRI 2025, Gambung 3, Gambung 7, Gambung 9, and Pagilaran 15. This study used the PCR method using internal transcribed spacers (ITS) 1F and ITS 4 primers. Four primers used in PCR-RAPD were OPA-02, OPA-03, OPA-05, and OPB-17. The characteristics of E. vexans observed were ellipse-shaped basidiospore, hyaline, unicellular with one septate, formed at the tip of the sterigma with hyaline and elliptical shapes, with a range size of 7–15.5 μm x 2.3–4.5 μm. PCR-RAPD method was able to show the diversity of E. vexans samples between clones, in which three clusters were formed at a coefficient of 0.63. Cluster I consisted of TRI 2024 Andongsili and PGL 15 Pagilaran; Cluster II consisted of TRI 2025 Andongsili and Gambung 3 Andongsili; Cluster III consisted of Gambung 7 Andongsili, Gambung 7 Kayulandak, and Gambung 9 Andongsili.

RAPD-PCR-Based Fingerprinting Method as a Tool for Epidemiological Analysis of Trueperella pyogenes Infections.

In this study, a Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) method for genetic typing of Trueperella pyogenes, an opportunistic bacterial pathogen, was designed. The method optimization was performed for 37 clinical T. pyogenes strains isolated from various infections in different animal species. Optimal conditions for reliable and reproducible DNA fingerprinting were determined according to the modified Taguchi method. The developed method was assessed regarding its typeability, reproducibility, and discriminatory power using the Hunter’s and Gatsons’ index of discrimination. A high degree of genetic diversity was shown between the studied strains, which represented 31 genotypes. The generated RAPD profiles were relatively complex and simultaneously easy to interpret due to the wide size range of amplicons. The discriminatory index of the designed method was sufficiently high; thus, only strains epidemiologically related displayed identical RAPD genotypes. In conclusion, the DNA fingerprinting of T. pyogenes by the developed RAPD-PCR method is a reliable typing tool that may allow a better understanding of the epidemiology as well as pathogenesis of infections caused by this pathogen.

Assessment of larvicidal and genotoxic potentials of extracts of Hyptis suaveolens against Culex quinquefasciatus based on enzyme profile and RAPD-PCR assay

Vector control strategies have focused on the development of effective and ecofriendly alternatives. In the present study, investigation of larvicidal and genotoxic effects of leaves of Hyptis suaveolens from four different extraction solvents (aqueous, hexane, methanol and acetone) on fourth instar larvae of Culex quinquefasciatus was carried out. Extraction was done using soxhlet apparatus and the characteristics functional group of active constituents were identified using Fourier Transform Infrared spectrophotometer. Larvicidal activities were screened using three different concentrations (50, 150 and 250 mg/mL) following WHO standard protocol and mortality was recorded after 24, 48 and 72 hr. Hexane extract showed the highest mortality (27.92, 38.75, 90.42 %; LC50: 272.5, 191.3, 114.8 mg/mL), followed by aqueous extract (20.83, 34.58, 59.58 %; LC50: 496.6, 392.9, 208.1 mg/mL) and acetone extract (20.83, 32.08, 59.58 %; LC50: 1111.2, 393.6, 266.1 mg/mL) and methanol extract (17.92, 29.17, 52.92 %; LC50: 466.0, 400.1, 272.3 mg/mL). Enzyme profile such as superoxide dismutase (SOD), glutathione-S-transferase (GST), catalase (CAT), glutathione (GSH) and alkaline phosphatase (ALP) were significantly altered in the larvae exposed to the four extracts. Phytochemical screening of all solvents extract revealed the presence of saponins, flavonoids, terpenoids and alkaloids as common constituents. Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) profile implied hexane and aqueous extracts altered the DNA of larvae. Furthermore, FTIR Spectroscopic analysis revealed phenols, alcohols, aliphatic primary amines and saponins as the major groups in the extracts. Conclusively, this study established the lethal potential of extracts of H. suaveolens as alternative plant-based and eco-friendly larvicide against Cx. quinquefasciatus.