scholarly journals KARAKTERISASI FITOPLASMA PENYEBAB PENYAKIT LAYU KELAPA DI PULAU DERAWAN MENGGUNAKAN RFLP IN SILICO

2018 ◽  
Vol 17 (2) ◽  
pp. 105
Author(s):  
Agus Eko Prasetyo ◽  
Kikin Hamzah Mutaqin ◽  
Giyanto .
Keyword(s):  
Nested Pcr ◽  
In Silico ◽  
Bacterial Species ◽  
Rflp Analysis ◽  
Wilt Disease ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Gram Positive Bacteria ◽  
Pcr Method

Characterization of phytoplasmas associated with coconut wilt disease in Derawan Island using in silico RFLP. Coconutwilt disease has been reported in Derawan Island that resulted in eradication up to 10% of the total cultivated palms. Theobjective of this study was to detect and characterize phytoplasmas associated with coconut wilt disease in Derawan islandusing nested PCR technique and in silico RFLP based on 16S rRNA gene sequences. Detection of phytoplasmas was performedusing nested PCR method, cloning of nPCR products, sequencing, and analysis of sequencing results using in silico RFLP.The results revealed that phytoplasmas could not be detected by PCR using P1/P7 primer pairs however it could be amplifiedby nested PCR using R16F2n/R16R2 primer pairs resulting amplicon at about 1.25 kb. In silico RFLP analysis indicated thatphytoplasmas associated with coconut wilt disease in Derawan Island belong to 16SrII (witches broom phytoplasma). PCRproduct of the nPCR need to be sequenced because the R16F2n/R16R2 primer will also amplify the other bacterial species, mainly from Gram positive bacteria.

Isolation and Characterization of Levoglucosan Metabolizing Bacteria

2021 ◽  
Author(s):  
Ajay S. Arya ◽  
Minh T. H. Hang ◽  
Mark A. Eiteman
Keyword(s):  
Recombinant Expression ◽  
Bacterial Species ◽  
Soluble Carbohydrate ◽  
Rrna Gene ◽  
Gene Products ◽  
16S Rrna Gene Sequences ◽  
E Coli ◽  
Isolation And Characterization ◽  
Charred Wood

Bacteria were isolated from wastewater and soil containing charred wood remnants based on their ability to use levoglucosan as a sole carbon source and on their levoglucosan dehydrogenase (LGDH) activity. On the basis of their 16S rRNA gene sequences, these bacteria represented diverse genera of Microbacterium, Paenibacillus , Shinella , and Klebsiella . Genomic sequencing of the isolates verified that two isolates represented novel species, Paenibacillus athensensis MEC069 T and Shinella sumterensis MEC087 T , while the remaining isolates were closely related to either Microbacterium lacusdiani or Klebsiella pneumoniae . The genetic sequence of LGDH, lgdA , was found in the genomes of these four isolates as well as Pseudarthrobacter phenanthrenivorans Sphe3. The identity of the P. phenanthrenivorans LGDH was experimentally verified following recombinant expression in E. coli . Comparison of the putative genes surrounding lgdA in the isolate genomes indicated that several other gene products facilitate the bacterial catabolism of levoglucosan, including a putative sugar isomerase and several transport proteins. Importance Levoglucosan is the most prevalent soluble carbohydrate remaining after high temperature pyrolysis of lignocellulosic biomass, but it is not fermented by typical production microbes such as Escherichia coli and Saccharomyces cerevisiae . A few fungi metabolize levoglucosan via the enzyme levoglucosan kinase, while several bacteria metabolize levoglucosan via levoglucosan dehydrogenase. This study describes the isolation and characterization of four bacterial species which degrade levoglucosan. Each isolate is shown to contain several genes within an operon involved in levoglucosan degradation, furthering our understanding of bacteria which metabolize levoglucosan.

scholarly journals Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 396
Author(s):  
Ewa Sajnaga ◽  
Marcin Skowronek ◽  
Agnieszka Kalwasińska ◽  
Waldemar Kazimierczak ◽  
Karolina Ferenc ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Entomopathogenic Nematodes ◽  
Bacterial Species ◽  
Antagonistic Activity ◽  
Rrna Gene ◽  
Nanopore Sequencing ◽  
Bacterial Phyla ◽  
Melolontha Melolontha

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

2006 ◽  
Vol 106 (3) ◽  
pp. 297-306 ◽  
Author(s):  
A. Llorens ◽  
M.J. Hinojo ◽  
R. Mateo ◽  
M.T. González-Jaén ◽  
F.M. Valle-Algarra ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Rrna Gene ◽  
Fusarium Spp ◽  
Intergenic Spacer Region ◽  
Pcr Rflp

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

scholarly journals Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tsui-Kang Hsu ◽  
Jung-Sheng Chen ◽  
Hsin-Chi Tsai ◽  
Chi-Wei Tao ◽  
Yu-Yin Yang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
In Silico ◽  
Environmental Samples ◽  
Method Development ◽  
Small Volume ◽  
Detection Efficiency ◽  
Genotyping Method ◽  
Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

scholarly journals Molecular Characterization of anEndozoicomonas-Like Organism Causing Infection in the King Scallop (Pecten maximusL.)

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Irene Cano ◽  
Ronny van Aerle ◽  
Stuart Ross ◽  
David W. Verner-Jeffreys ◽  
Richard K. Paley ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Mass Mortality ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Content Type ◽  
The 16S Rrna Gene

ABSTRACTOne of the fastest growing fisheries in the UK is the king scallop (Pecten maximusL.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resemblingRickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from “CandidatusEndonucleobacter bathymodioli” and 95% withEndozoicomonasspecies.In situhybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences fromEndozoicomonasspp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.IMPORTANCEMolluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of anEndozoicomonas-like organism (ELO) associated with an important commercial scallop species.

scholarly journals Characterization of the GenusBifidobacteriumby Automated Ribotyping and 16S rRNA Gene Sequences

2006 ◽  
Vol 50 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Shinji Sakata ◽  
Chun Sun Ryu ◽  
Maki Kitahara ◽  
Mitsuo Sakamoto ◽  
Hidenori Hayashi ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Automated Ribotyping

Current state of knowledge: the canine gastrointestinal microbiome

2012 ◽  
Vol 13 (1) ◽  
pp. 78-88 ◽  
Author(s):  
Seema Hooda ◽  
Yasushi Minamoto ◽  
Jan S. Suchodolski ◽  
Kelly S. Swanson
Keyword(s):  
Bacterial Species ◽  
Rrna Gene ◽  
Dietary Interventions ◽  
Health Indices ◽  
Recent Emergence ◽  
Spatial Differences ◽  
Bowel Diseases ◽  
Health And Disease ◽  
Cultivation Techniques

AbstractGastrointestinal (GI) microbes have important roles in the nutritional, immunological, and physiologic processes of the host. Traditional cultivation techniques have revealed bacterial density ranges from 104to 105colony forming units (CFU)/g in the stomach, from 105to 107CFU/g in the small intestine, and from 109to 1011CFU/g in the colon of healthy dogs. As a small number of bacterial species can be grown and studied in culture, however, progress was limited until the recent emergence of DNA-based techniques. In recent years, DNA sequencing technology and bioinformatics have allowed for better phylogenetic and functional/metabolic characterization of the canine gut microbiome. Predominant phyla include Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria. Studies using 16S ribosomal RNA (rRNA) gene pyrosequencing have demonstrated spatial differences along the GI tract and among microbes adhered to the GI mucosa compared to those in intestinal contents or feces. Similar to humans, GI microbiome dysbiosis is common in canine GI diseases such as chronic diarrhea and inflammatory bowel diseases. DNA-based assays have also identified key pathogens contributing to such conditions, including variousClostridium,Campylobacter,Salmonella, andEscherichiaspp. Moreover, nutritionists have applied DNA-based techniques to study the effects of dietary interventions such as dietary fiber, prebiotics, and probiotics on the canine GI microbiome and associated health indices. Despite recent advances in the field, the canine GI microbiome is far from being fully characterized and a deeper characterization of the phylogenetic and functional/metabolic capacity of the GI microbiome in health and disease is needed. This paper provides an overview of recent studies performed to characterize the canine GI microbiome.

scholarly journals Characterization of Tambjamines Pigment from Marine Bacterium Pseudoalteromonas sp. PM2 Indigenous from Alor Island, Indonesia

2021 ◽  
Vol 3 (1) ◽  
pp. 16-23
Author(s):  
Edi Setiyono ◽  
Marcelinus Alfasisurya Setya Adhiwibawa ◽  
Matheus Randy Prabowo ◽  
Tatas H.P. Brotosudarmo
Keyword(s):  
16S Rrna ◽  
Marine Bacteria ◽  
Marine Bacterium ◽  
Rrna Gene ◽  
Accession Number ◽  
16S Rrna Gene Sequences ◽  
Different Types ◽  
New Finding ◽  
Yellow Pigments

Pigments from marine bacteria have attracted the attention for scientists because of their extensive applications and currently exploration of new pigment sources from marine bacteria is still ongoing. Recently, we have successfully isolated six new yellow-pigmented marine bacteria, strain PS2, PM2, SB11, SB13, SB21, and SB23, isolated from seawater from different sampling sites on Alor Island, Indonesia. The UV−Vis and FTIR spectra of the crude pigment extracts of the six strains showed the characteristics of tambjamines, a group of yellow pigments commonly found in nudibranchs and bryozoans. Moreover, separation and characterization of crude tambjamines extract resulted in five different types of tambjamine with maximum absorbance at the wavelength of 374−392 nm. Based on the analysis of 16S rRNA gene sequences, strain PM2 was closely related to several species in genus Pseudoalteromonas with a similarity of more than 99%. Strain PM2 was designed as Pseudoalteromonas sp. PM2 with accession number LC505058. So far, only two marine bacteria have been known to produce tambjamine and they are from genus Pseudoalteromonas. Our new finding indicated that in the group of marine bacteria, tambjamine might be only synthesized by members from genus Pseudoalteromonas. 

scholarly journals Molecular Characterization of Zinc (Zn) Resistant Bacteria in Banger River, Pekalongan, Indonesia

2018 ◽  
Vol 10 (3) ◽  
pp. 622-628
Author(s):  
Fitri Arum Sasi ◽  
Hermin Pancasakti Kusumaningrum ◽  
Anto Budiharjo
Keyword(s):  
Bacterial Species ◽  
Selective Medium ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Indigenous Bacteria ◽  
Gram Positive Bacteria ◽  
Sequences Analysis

Indigenous bacteria are able to remove the metals contamination in environment. This study aimed to assess the resistance of bacterial species to Zinc (Zn) in Banger River, Pekalongan City. The bacteria from three different parts of Banger River were isolated and inoculated in Zn-selective medium. Then, molecular identification to determine the bacteria species was conducted using polymerase chain reaction (PCR) by applying forward-reverse 16SrRNA gene primers. The sequences analysis was conducted using MUSCLE and MEGA6. There were seven dominant species that possibly resistant to Zn. Approximately, every isolate could reach more than 95 % from 2000 ppm of Zn in the medium. The higher absorption of Zn was found in Z5 isolate. The seven bacteria species were clustered into nine genera i.e. Klebsiela, Xenorhabdus, Cronobacter, Enterobacter, Escherichia, Shigella and Sporomusa known as Gram Negative bacteria and Clostridium and Bacillus as Gram Positive bacteria. In Gram Positive bacteria, especially Bacillus sp, carboxyl group in peptidoglycan play a role as metal binder. In Gram-negative bacteria, lipopolysaccharide (LPS) which is highly anionic component on the outer membrane, able to catch the Zn. Besides that, Enterobacter activates endogen antioxidants such as glutathione peroxidase (GSHPx), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD). The research found there was possible seven novel indigenous bacteria species in Banger that able to remove Zn from the sediment extremely. This finding can be developed as an eco-friendly approach to reduce metals pollution using local microorganisms.

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