scholarly journals Bacillus sp. As a Decomposition Agent in The Maintenance of Brachionus rotundiformis Which Uses Raw Fish As a Source of Nutrition

2019 ◽  
Vol 7 (1) ◽  
pp. 158
Author(s):  
Hatopan G. Napitupulu ◽  
Inneke F. M. Rumengan ◽  
Stenlly Wullur ◽  
Elvy L. Ginting ◽  
Joice R. T. S. L. Rimper ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Culture Media ◽  
Morphological Characterization ◽  
Nutrient Broth ◽  
Bacillus Sp ◽  
Pcr Product ◽  
16S Rna ◽  
Pcr Products ◽  
Bacteria Culture ◽  
Reaction Chain

The research was conducted at the Laboratory of Marine Molecular Biology and Pharmacy, Faculty of Fisheries and Marine Sciences, Sam Ratulangi University. This research aims to isolate and analyze the morphology and molecular types of bacteria associated in rotifer’s culture media that use fisheries waste.The research was begin by culturing bacteria in rotifer maintenance media using Nutrient Broth media. After bacterial isolates were obtained, morphological characterization and DNA extraction was carried out. extraction was done using DNeasy Blood and Tissue Kit (Qiagen). After DNA was obtained, DNA was amplified through the Polymerase Reaction Chain (PCR) machine using a 16S RNA primer, followed by the separation of PCR products through electrophoresis and detection through UV Transluminator. The target PCR product was determined by comparing the 100 bp ladder DNA, with a yield of around 1400 bp, which was measured using ladder DNA available in the laboratory. The DNA that was successfully amplified was sent to be sequenced to determine the species of each microbe obtained.Based on the results of the research conducted, obtained Bacillus sp. bacteria associated with rotifer maintenance media.Keywords: Bacteria, Culture Media, DNA Extraction, PCR, Sequencing ABSTRAKPenelitian ini dilakukan di Laboratorium Biologi Molekuler dan Farmasetika Laut, Fakultas Perikanan dan Ilmu Kelautan, Universitas Sam Ratulangi.  Penelitian ini bertujuan untuk mengisolasi dan menganalisis morfologi dan molekuler jenis-jenis bakteri yang berasosiasi dalam media pemeliharaan rotifer yang menggunakan limbah perikanan.Penelitian dilakukan dengan cara mengkultur bakteri yang ada pada media pemeliharaan rotifer menggunakan media Nutrient Broth. Setelah isolat bakteri didapatkan, dilakukan karakterisasi morfologi dan dilakukan ekstraksi DNA.  ekstraksi dilakukan menggunakan DNeasy Blood and Tissue Kit (Qiagen).  Setelah DNA didapatkan, DNA diamplifikasi melalui mesin Polymerase Reaction Chain (PCR) menggunakan primer 16S RNA, diikuti dengan pemisahan produk PCR melalui electrophorisis dan deteksi melalui UV Transluminator. Produk PCR target ditentukan dengan membandingkan ladder DNA 100 bp, dengan hasil sekitar 1400 bp, yang diukur menggunakan ladder DNA yang tersedia di laboratorium.  DNA yang berhasil diamplifikasi, dikirim untuk dilakukan sekuensing untuk mengetahui spesies dari setiap mikroba yang didapatkan.  Berdasarkan hasil penelitian yang dilakukan, diperoleh bakteri Bacillus sp. yang berasosiasi pada media pemeliharaan rotifer. Kata Kunci: Bakteri,  Media Pemeliharaan, Ekstraksi DNA, PCR, Sekuensing

scholarly journals Microsatellite markers-based characterisation of elephant grass (Pennisetum purpureum) harvested from selected locations in South-West Nigeria

2020 ◽  
Vol 37 (1) ◽  
pp. 38-45
Author(s):  
O.A. Okukenu ◽  
A.A. Olajide ◽  
P.A. Dele ◽  
M. Wheto ◽  
B.T. Akinyemi ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Microsatellite Markers ◽  
Dna Extraction ◽  
Morphological Characterization ◽  
Ethanol Solution ◽  
Pennisetum Purpureum ◽  
Elephant Grass ◽  
South West ◽  
Ssr Primers ◽  
Genetic Index

This study was carried out to characterise Pennisetum purpureum harvested from some selected locations in S outh-W estern Nigeria using microsatellite markers. Leaf parts of growing young elephant grass (Pennisetum purpureum) were harvested and immediately preserved in ethanol solution before DNA extraction. Two (2) SSR primers (CTM59 and Xtxp278) were used to assess genetic diversity in Pennisetum purpureum. The result shows that 72% of the molecular variations in the elephant grass exists within the population with 28% among the population; there were no unique characteristics among the Nine (9) populations. Nei genetic index ranged from 0.067 (lowest) observed between Isokan and Odeda populations to 0.158 (highest), between Ifedore and Ikoyi Populations. Morphological characterization showed moderate diversity with two major clusters and one minor cluster. Keyword: Elephant grass; cultivars; locations; markers

scholarly journals A PCR-Based Method of Detection and Differentiation of K88+ Adhesive Escherichia Coli

1996 ◽  
Vol 8 (4) ◽  
pp. 460-463 ◽  
Author(s):  
Mark A. Franklin ◽  
David H. Francis ◽  
Diane Baker ◽  
Alan G. Mathew
Keyword(s):  
Escherichia Coli ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antigenic Variant ◽  
Variable Regions ◽  
E Coli ◽  
Clinical Assay ◽  
Pcr Product ◽  
Structural Subunit ◽  
Pcr Products ◽  
Polymerase Chain

The objective of this study was to develop a polymerase chain reaction (PCR)-based method to detect and differentiate among Escherichia coli strains containing genes for the expression of 3 antigenic variants of the fimbrial adhesin K88 (K88ab, K88ac, and K88ad). Five primers were designed that allowed detection of K88+ E. coli, regardless of antigenic variant, and the separate detection of the ab, ac, and ad variants. Primers AM005 and AM006 are 21 base pair (bp) oligomers that correspond to a region of the K88 operon that is common to all 3 antigenic variants. Primers MF007, MF008, and MF009 are 24-bp oligomers that matched variable regions specific to ab, ac, and ad, respectively. Using primers AM005 and AM006, a PCR product was obtained that corresponds to a 764-bp region within the large structural subunit of the K88 operon common to all 3 antigenic variants. Primer AM005 used with MF007, MF008, or MF009 produced PCR products approximately 500-bp in length from within the large structural subunit of the K88 operon of the 3 respective antigenic variants. Fragments were identified by rates of migration on a 1% agarose gel relative to each other as well as to BstEII-digested lambda fragments. This PCR-based method was comparable to the enzyme-linked immunosorbent assay and western blot test in the ability to differentiate between the antigenic variants. K88+ E. coli were differentiated from among laboratory strains and detected in ileal samples taken from cannulated pigs challenged with a known K88+ variant. K88+ E. coli were also detected from fecal swabs taken from newly weaned pigs, thus confirming that this PCR-based test could provide a convenient clinical assay for the detection of K88+ E. coli. Detection and differentiation of K88+ E. coli using general and specific primers was successful. PCR methods of detection should permit identification of K88+ antigenic variants regardless of the level of expression of the antigen.

scholarly journals Characterization and Lipolytic Activity of Bacteria Isolates from Freshwater Clam (Mercenaria Mercenaria) in Bayelsa State, Nigeria

2020 ◽  
pp. 1-4
Author(s):  
Opara C N ◽  
◽  
Anumudu C K ◽  
Keyword(s):  
Protein Content ◽  
Culture Media ◽  
Lipolytic Activity ◽  
Cell Mass ◽  
Mercenaria Mercenaria ◽  
Total Protein Content ◽  
Growth Media ◽  
Pseudomonas Sp ◽  
Bacillus Sp ◽  
Freshwater Clam

Lipases form an important group of relevant enzymes which have applications in various fields including; food, pharmaceutical, detergent, textile and cosmetic industries. Lipases can be produced from diverse sources including microorganisms. This study evaluated the potential of bacteria isolates from fresh-water clam Mercenaria Mercenaria to produce lipolytic enzymes. Ten samples of Clam (Mercenaria Mercenaria) were screened for the presence of lipase producing bacteria using classical culture methods. Eleven bacteria species were obtained, of which six (Actinomyces sp., E. coli, Bacillus sp., Pseudomonas sp., Clostridium sp. and Klebsiella sp.) produced lipases that had lipolytic activity in breaking down olive oil used in media supplementation. The best culture media and conditions for optimal production of lipases was studied and it was shown that supplementation of growth media with 2% dextrose at neutral pH gave the greatest yield of lipases when lipase producing isolates were grown in shake flasks. Measurement of biomass by culture and turbidimetric methods indicates that the highest cell mass was recorded by Pseudomonas sp at 7.8 x 105 CFU/ml, closely followed by Actinomyces sp. and Bacillus sp., at 6.2 x 105 CFU/ml and 5.3 x 105 respectively. The produced lipases were partially purified by precipitating with ammonium sulphate followed by dialysis. The total protein content of produced lipases was evaluated by the Lowry’s method, showing that estimated protein content followed the same trend as cell biomass with the highest recorded by Pseudomonas sp. at 1.53mg/ml, followed by Actinomyces sp. and Bacillus sp. at 1.47mg/ml and 1.32mg/ml respectively. The results obtained in this study shows that isolates obtained from freshwater clam can produce potent lipases which can be employed for industrial, food and other diverse uses

scholarly journals HEAVY METAL-RESISTANT RHIZOBACTERIA FROM GOLD MINE IN PONGKOR INDONESIA AND COPPER MINE IN MARINDUQUE PHILIPPINES

Agric ◽  
2019 ◽  
Vol 31 (1) ◽  
pp. 75-88 ◽  
Author(s):  
Erny Yuniarti ◽  
Ida F Dalmacio ◽  
Erlinda S Paterno
Keyword(s):  
Resistance Mechanism ◽  
Morphological Characterization ◽  
Gold Mine ◽  
Isolation Medium ◽  
Mine Site ◽  
Copper Mine ◽  
Gram Staining ◽  
16S Rna ◽  
Agar Media

The purposes of the study was to isolate, to characterize, and to identify rhizobacteria from plant rhizosphere growing in gold and copper mine. The isolation of rhizobacteria used N-free semisolid agar media, TSA, and SLP plus heavy metals (HMs), namely Pb, Cd, and or Cu. Isolated rhizobacteria were subsequently characterized for resistance to higher level of Pb, Cd, Cu in SLP media. Cultural and morphological characterization of rhizobacteria were conducted for cell morphology, motility, Gram staining, and biofilm formation. The rhizobacteria identification used sequence analysis of the 16S RNA gene fragments. The results showed that the majority of rhizobacterial from Cu mine site (66.7% of 21 isolates) were resistant to Cu (72150 ppm) while the majority of rhizobacteria from gold mine site (77.8% of 18 isolates) were sensitive to 72 ppm Cu. Majority of Cu in the soil was insoluble as granules attaching to gravel so that rhizobacteria of Cu mine site have been exposed and adapted to available Cu. This fact, explaining that the rhizobacteria’s MIC value was lower than the total Cu level in the soil. Three HMs-resistant rhizobacter (PbSM 2.1, MGR 334, and CuNFbM 4.1) formed biofilms, which was as one of the resistance mechanism to HMs. This research informed that HM contaminated-soil is better source for obtaining HM resistant rhizobacteria than HM uncontaminated-soil. The use four isolation media produce rhizobacteria which was more diverse than rhizobacteria from each isolation medium. Further characterization needs to be done to obtain HM resistant-rhizobacteria which can be used as biofertilizers and phytoremediation agent.

scholarly journals First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 360-360 ◽  
Author(s):  
A. M. Al-Subhi ◽  
N. A. Al-Saady ◽  
A. J. Khan ◽  
M. L. Deadman
Keyword(s):  
Nested Pcr ◽  
Solanum Melongena ◽  
Positive Control ◽  
Rrna Gene ◽  
Direct Pcr ◽  
First Report ◽  
Pcr Product ◽  
Pcr Products ◽  
Broom Disease ◽  
Phytoplasma Infection

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

scholarly journals Genotyping Single-Nucleotide Polymorphisms by the Invader Assay with Dual-Color Fluorescence Polarization Detection

2001 ◽  
Vol 47 (8) ◽  
pp. 1373-1377 ◽  
Author(s):  
Tony M Hsu ◽  
Scott M Law ◽  
Shenghui Duan ◽  
Bruce P Neri ◽  
Pui-Yan Kwok
Keyword(s):  
Fluorescence Polarization ◽  
Cost Effective ◽  
Snp Markers ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Clear Cut ◽  
Pcr Product ◽  
Invader Assay ◽  
Pcr Products ◽  
Incorporation Assay

Abstract Background: The PCR-Invader® assay is a robust, homogeneous assay that has been shown to be highly sensitive and specific in genotyping single-nucleotide polymorphism (SNP) markers. In this study, we introduce two changes to improve the assay: (a) we streamline the PCR-Invader method by assaying both alleles for each SNP in one reaction; and (b) we reduce the cost of the method by adopting fluorescence polarization (FP) as the detection method. Methods: PCR product was incubated with Invader oligonucleotide and two primary probes at 93 °C for 5 min. Signal probes corresponding to the cleaved flaps of the primary probes [labeled with fluorescein and 6-carboxytetramethylrhodamine (TAMRA) dye] and Cleavase® VIII enzyme (a flap endonuclease) were then added to the mixture. This reaction mixture was incubated at 63 °C for 5 min. FP measurements were made with a fluorescence plate reader. Results: Eighty-eight individuals were genotyped across a panel of 10 SNPs, using PCR product as template, for a total of 880 genotypes. An average “no call” rate of 3.2% was observed after first round of experiments. PCR products were remade in those samples that failed to produce any genotype in the first round, and all gave clear-cut genotypes. When the genotypes determined by the PCR-Invader assay and template-directed dye-terminator incorporation assay with FP were compared, they were in 100% concordance for all SNP markers and experiments. Conclusions: The improvements introduced in this study make PCR-Invader assay simpler and more cost-effective, and therefore more suitable for high-throughput genotyping.

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction

1993 ◽  
Vol 39 (9) ◽  
pp. 1927-1933 ◽  
Author(s):  
J B Findlay ◽  
S M Atwood ◽  
L Bergmeyer ◽  
J Chemelli ◽  
K Christy ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Automated System ◽  
Closed Vessel ◽  
Chain Reaction ◽  
Panel Testing ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

Abstract An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

scholarly journals Rapid sequencing of MRSA direct from clinical plates in a routine microbiology laboratory

2019 ◽  
Vol 74 (8) ◽  
pp. 2153-2156 ◽  
Author(s):  
Beth Blane ◽  
Kathy E Raven ◽  
Danielle Leek ◽  
Nicholas Brown ◽  
Julian Parkhill ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Clinical Laboratory ◽  
Culture Media ◽  
Direct Sequencing ◽  
Laboratory Culture ◽  
Outbreak Detection ◽  
Snp Analysis ◽  
University Hospitals ◽  
Quality Control Metrics ◽  
Clinical Culture

Abstract Background Routine sequencing of MRSA could bring about significant improvements to outbreak detection and investigation. Sequencing is commonly performed using DNA extracted from a pure culture, but overcoming the delay associated with this step could reduce the time to infection control interventions. Objectives To develop and evaluate rapid sequencing of MRSA using primary clinical cultures. Methods Patients with samples submitted to the clinical laboratory at the Cambridge University Hospitals NHS Foundation Trust from which MRSA was isolated were identified, the routine laboratory culture plates obtained and DNA extraction and sequencing performed. Results An evaluation of routine MRSA cultures from 30 patients demonstrated that direct sequencing from bacterial colonies picked from four different culture media was feasible. The 30 clinical MRSA isolates were sequenced on the day of plate retrieval over five runs and passed quality control metrics for sequencing depth and coverage. The maximum contamination detected using Kraken was 1.09% fragments, which were identified as Prevotella dentalis. The most common contaminants were other staphylococcal species (25 isolate sequences) and Burkholderia dolosa (11 isolate sequences). Core genome pairwise SNP analysis to identify clusters based on isolates that were ≤50 SNPs different was used to triage cases for further investigation. This identified three clusters, but more detailed genomic and epidemiological evaluation excluded an acute outbreak. Conclusions Rapid sequencing of MRSA from clinical culture plates is feasible and reduces the delay associated with purity culture prior to DNA extraction.

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