scholarly journals Isolation and characterization of two virulent Aeromonads associated with haemorrhagic septicaemia and tail-rot disease in farmed climbing perch Anabas testudineus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Abhishek Mazumder ◽  
Hrishikesh Choudhury ◽  
Abhinit Dey ◽  
Dandadhar Sarma
Keyword(s):  
Natural Infection ◽  
Winter Season ◽  
Pcr Amplification ◽  
Body Parts ◽  
Anabas Testudineus ◽  
Climbing Perch ◽  
Isolation And Characterization ◽  
Biochemical Characterisation ◽  
Rot Disease

AbstractDiseased Anabas testudineus exhibiting signs of tail-rot and ulcerations on body were collected from a fish farm in Assam, India during the winter season (November 2018 to January 2019). Swabs from the infected body parts were streaked on sterilized nutrient agar. Two dominant bacterial colonies were obtained, which were then isolated and labelled as AM-31 and AM-05. Standard biochemical characterisation and 16S rRNA and rpoB gene sequencing identified AM-31 isolate as Aeromonas hydrophila and AM-05 as Aeromonas jandaei. Symptoms similar to that of natural infection were observed on re-infecting both bacteria to disease-free A. testudineus, which confirmed their virulence. LC50 was determined at 1.3 × 104 (A. hydrophila) and 2.5 × 104 (A. jandaei) CFU per fish in intraperitoneal injection. Further, PCR amplification of specific genes responsible for virulence (aerolysin and enterotoxin) confirmed pathogenicity of both bacteria. Histopathology of kidney and liver in the experimentally-infected fishes revealed haemorrhage, tubular degeneration and vacuolation. Antibiotic profiles were also assessed for both bacteria. To the best of our knowledge, the present work is a first report on the mortality of farmed climbing perch naturally-infected by A. hydrophila as well as A. jandaei, with no records of pathogenicity of the latter in this fish.

scholarly journals Isolation and Characterization of Pectobacterium Phage vB_PatM_CB7: New Insights into the Genus Certrevirus

Antibiotics ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 352
Author(s):  
Colin Buttimer ◽  
Caoimhe Lynch ◽  
Hanne Hendrix ◽  
Horst Neve ◽  
Jean-Paul Noben ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Burst Size ◽  
Homing Endonuclease ◽  
Soft Rot ◽  
Nucleotide Metabolism ◽  
Homing Endonucleases ◽  
Isolation And Characterization ◽  
Rot Disease ◽  
Large Terminase

To date, Certrevirus is one of two genera of bacteriophage (phage), with phages infecting Pectobacterium atrosepticum, an economically important phytopathogen that causes potato blackleg and soft rot disease. This study provides a detailed description of Pectobacterium phage CB7 (vB_PatM_CB7), which specifically infects P. atrosepticum. Host range, morphology, latent period, burst size and stability at different conditions of temperature and pH were examined. Analysis of its genome (142.8 kbp) shows that the phage forms a new species of Certrevirus, sharing sequence similarity with other members, highlighting conservation within the genus. Conserved elements include a putative early promoter like that of the Escherichia coli sigma70 promoter, which was found to be shared with other genus members. A number of dissimilarities were observed, relating to DNA methylation and nucleotide metabolism. Some members do not have homologues of a cytosine methylase and anaerobic nucleotide reductase subunits NrdD and NrdG, respectively. Furthermore, the genome of CB7 contains one of the largest numbers of homing endonucleases described in a single phage genome in the literature to date, with a total of 23 belonging to the HNH and LAGLIDADG families. Analysis by RT-PCR of the HNH homing endonuclease residing within introns of genes for the large terminase, DNA polymerase, ribonucleotide reductase subunits NrdA and NrdB show that they are splicing competent. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was also performed on the virion of CB7, allowing the identification of 26 structural proteins—20 of which were found to be shared with the type phages of the genera of Vequintavirus and Seunavirus. The results of this study provide greater insights into the phages of the Certrevirus genus as well as the subfamily Vequintavirinae.

scholarly journals Isolation and Characterization of DNA from Root Tubers

2020 ◽  
pp. 61-67
Author(s):  
O. G. Dawodu ◽  
O. R. Agbeni ◽  
V. Iwu
Keyword(s):  
Gel Electrophoresis ◽  
Manihot Esculenta ◽  
Pcr Amplification ◽  
Colocasia Esculenta ◽  
Marker Genes ◽  
Molecular Weight Determination ◽  
Isolation And Characterization ◽  
Sequencing Method

This study reports a modified SDS extraction technique for isolation of DNA from root tubers namely Dioscorea (yam), Manihot esculenta (cassava) and Colocasia esculenta (cocoyam). DNA extraction in many plants is very difficult because of secondary metabolites that interfere with DNA isolation procedure. Young fresh leaves were collected from the selected tubers and DNA extracted using SDS-extraction protocol, the DNA isolated from the extracted leaves was subjected to gel electrophoresis for determining their purity and concentration and PCR amplification was carried out on the extracted DNA, afterwards gel-electrophoresis was performed for molecular weight determination of the samples. DNA sequencing were performed on both reverse and forward using Sanger sequencing method which gave the nucleotide bases of the samples. DNA isolated by modified SDS protocol method was pure and highest level of purity was obtained from Manihot esculenta, Colocasia esculenta and Dioscorea which are 1.77, 1.72 and 1.20 with concentration of 537.7 ng/µl, 992.4 ng/µl and 149.8 ng/µl respectively. The DNA isolated from root tubers was pure and of good quality, and the isolated DNA was characterized by sequencing. The isolated DNA was successfully sequenced which coded for marker genes of the respective root tubers.

Isolation and characterization of chalcone synthase (CHS) gene from Phalaenopsis and Doritaenopsis orchids

2020 ◽  
Vol 21 (11) ◽  
Author(s):  
Dewi Sukma ◽  
Aline Sisi Handini ◽  
Sudarsono Sudarsono
Keyword(s):  
Nucleotide Sequence ◽  
Chalcone Synthase ◽  
Pcr Amplification ◽  
Nucleotide Sequence Analysis ◽  
Degenerate Primers ◽  
Multiple Sequence ◽  
Isolation And Characterization ◽  
Key Enzyme ◽  
Young Leaves

Abstract. Sukma D, Handini AS, Sudarsono. 2020. Isolation and characterization of chalcone synthase (CHS) gene from Phalaenopsis and Doritaenopsis orchids. Biodiversitas 21: 5054-5064. Chalcone synthase (CHS) is a key enzyme in flavonoid biosynthesis. The research aims to isolate and characterize the CHS gene nucleotide sequence diversity of nine Phalaenopsis and one Doritaenopsis genotypes. Genomic DNA was isolated from young leaves and used to PCR amplify the gene using CHS specific degenerate primers. Results of PCR amplification yielded DNA fragments of 700 bp. Upon sequencing and nucleotide sequence analysis, we found that the genomic fragments were partial CHS gene from Phalaenopsis and Doritaenopsis genotypes and deposited the sequences in the NCBI Genebank Database accession numbers of KR184089-KR184098. More analysis of the sequences confirmed they shared ranged from 81-99% sequence identities to known CHS genes from Phalaenopsis deposited in the NCBI Database. They also shared 84-100% amino acid residues identity to CHS polypeptides. Multiple sequence alignment (MSA) and phylogenetic analysis further revealed the determined CHS nucleotide sequences from Phalaenopsis and Doritaenopsis genotypes were closely related to CHS genes from other orchid species.

Detection, Isolation, and Characterization of Shiga Toxin–Producing Escherichia coli in Flour

2019 ◽  
Vol 82 (1) ◽  
pp. 164-167 ◽  
Author(s):  
PATRICK KINDLE ◽  
MAGDALENA NÜESCH-INDERBINEN ◽  
NICOLE CERNELA ◽  
ROGER STEPHAN
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Pcr Amplification ◽  
The United States ◽  
The European Union ◽  
Conventional Pcr ◽  
E Coli ◽  
Isolation And Characterization

ABSTRACT Wheat flour has recently been described as a novel vehicle for transmission of Shiga toxin–producing Escherichia coli (STEC). Very recently, an outbreak of STEC O121 and STEC O26 infections was linked to flour in the United States. The aim of the present study was to generate baseline data for the occurrence of STEC in flour samples from different retailers in Switzerland. In total, 70 flour samples were analyzed. After enrichment, the samples were screened for stx1 and stx2 by the Assurance GDS MPX ID assay. STEC strains were isolated and serotyped by the E. coli SeroGenoTyping AS-1 kit. The determination of stx subtypes was performed with conventional PCR amplification. Screening for eae, aggR, elt, and estIa/Ib was performed by real-time PCR. Nine (12.9%) of the flour samples tested positive for stx by PCR. STEC was recovered from eight (88.9%) of the positive samples. Two isolates were STEC O11:H48 harboring stx1c/stx1d, two were O146:H28 containing stx2b, one was O103:H2 containing stx1a and eae, and three were O nontypeable: Ont:H12 (stx2a), Ont:H14 (stx2a/stx2g), and Ont:H31 (stx1c/stx1d). STEC O103 belongs to the “top five” serogroups of human pathogenic STEC in the European Union, and STEC O146 is frequently isolated from diseased humans in Switzerland. Our results show that flour may be contaminated with a variety of STEC serogroups. Consumption of raw or undercooked flour may constitute a risk for STEC infection.

scholarly journals Generation by a Widely Applicable Approach of a Hybrid Dioxygenase Showing Improved Oxidation of Polychlorobiphenyls

2007 ◽  
Vol 73 (8) ◽  
pp. 2682-2689 ◽  
Author(s):  
Beatriz Cámara ◽  
Michael Seeger ◽  
Myriam González ◽  
Christine Standfuß-Gabisch ◽  
Silke Kahl ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
The Novel ◽  
Catabolic Pathway ◽  
Catalytic Center ◽  
Burkholderia Xenovorans ◽  
Chimeric Enzymes ◽  
The Core ◽  
Isolation And Characterization ◽  
Unknown Sequence

ABSTRACT Recently, a sequence-based approach has been developed for the fast isolation and characterization of class II aryl-hydroxylating dioxygenase activities (S. Kahl and B. Hofer, Microbiology 149:1475-1481, 2003). It comprises the PCR amplification of segments of alpha subunit genes of unknown sequence that encode the catalytic center and their fusion with sequences of the bphA gene cluster of Burkholderia xenovorans LB400. One of the resulting chimeric enzymes, harboring the core segment of a dioxygenase from Pseudomonas sp. strain B4-Magdeburg, has now been characterized with respect to the oxidation of chlorobiphenyls (CBs). Its substrate and product specificities differed favorably from those of the parental dioxygenase of strain LB400. The hybrid possessed a higher regiospecificity and yielded less unproductive dioxygenations at meta and para carbons. It attacked ortho-, meta-, and para-chlorinated rings with comparable efficiencies. It gave significantly higher yields in ortho,meta-dioxygenation of recalcitrant congeners containing a doubly ortho-chlorinated ring. While the parental enzyme yielded mainly unproductive meta, para dioxygenation of 2,5,4′-CB, the hybrid predominantly converted this congener into an ortho,meta-dioxygenated product. The subsequent enzymes of the LB400 catabolic pathway were able to transform most of the metabolites formed by the novel dioxygenase, indicating that the substrate ranges of these biocatalysts are not adapted to that of their initial pathway enzyme. Some of the catabolites, however, were identified as problematic for further degradation. Our results demonstrate that the outlined approach can successfully be applied to obtain novel dioxygenase specificities that favorably complement or supplement known ones.

scholarly journals Isolation and characterization of DNA barcodes from distinctive and rare terrestrial animals in China using universal COI and 16S primers

STEMedicine ◽  
2021 ◽  
Vol 2 (7) ◽  
pp. e95
Author(s):  
Mali Guo ◽  
Zhenzhen Peng ◽  
Xiaoting Zhang ◽  
Chaohai Yuan ◽  
Hanxi Lu ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Careful Consideration ◽  
Sequence Information ◽  
Dna Barcodes ◽  
Closely Related Species ◽  
Mitochondrial Coi ◽  
Isolation And Characterization ◽  
Ecological Resources ◽  
Case Basis

Background: Accurate taxonomic identification is the cornerstone for monitoring, conservation and management of ecological resources. China has the highest biodiversities and the richest species assemblages in the world, but is lacking in sufficient assessment to the abundant genetic variability. DNA barcoding is a proven tool employing sequence information for rapid and unambiguous species delineation. However, the ability of barcodes to distinguish species that are archaic and distinctive evolutionary lines remains largely untested.Methods: In order to investigate the resources of terrestrial animals in China, regions from mitochondrial COI and 16S are barcoded for 395 specimens belonging to 54 selected species, many of which are indigenous representatives in danger. High success rate of PCR amplification is achieved by using universal COI and 16S primers with many numts pseudogenes co-amplified from mammalian samples.Results: Application of barcodes to flag species is generally straightforward since no COI or 16S haplotypes are shared between closely related species. Barcoding gap, species resolution and phylogenetic relationships relying on our barcode libraries are further compared using distance and tree based approaches.Conclusion: Results show that the discriminatory power of the two barcode markers could differentiate on a case-by-case basis, and also suggest a careful consideration of the nuclear numts for barcoding studies as they might provide a new understanding for evolution.

scholarly journals ISOLATION AND CHARACTERIZATION OF PHENANTHRENE DEGRADATING BACTERIA FROM DAIRY WASTE SAMPLES

2020 ◽  
Vol 8 (12) ◽  
pp. 372-380
Author(s):  
Gunti Vijay Kumar ◽  
◽  
Pandu Brahmaji Rao ◽  
Keyword(s):  
Agar Medium ◽  
Micrococcus Luteus ◽  
Pcr Amplification ◽  
Molecular Techniques ◽  
Biochemical Tests ◽  
Hazardous Chemical ◽  
Isolation And Characterization ◽  
Dairy Waste ◽  
Degrading Bacteria

Phenanthrene is the commonly used hazardous chemical in various industries and the detoxification is a big problem till today. In this study, phenanthrene degrading bacteria were isolated from dairy waste samples, characterized by molecular techniques. A total of 10 samples were collected respectively from different spots of dairy industry. Minimum salt agar medium with phenanthrene composition was used to isolate the pure culture of resistant bacterium. Morphological, biochemical tests were done to identify the phenanthrene degrading bacteria. The colonies of the isolates were circular to irregular in dairy samples. Phenanthrene tolerant bacteria were isolated after the dairy waste soil samples was serially diluted and transferred onto M9 minimal agar medium amended with 10mg/l of phenanthrene. A concentration of 10 mg/l was chosen based on the prevailing concentration. Four Phenanthrene tolerant bacteria from dairy waste (HW2, HW1, HW3, HW5) were isolated. From the biochemical and 16srRNA PCR amplification, the bacterium identified was Micrococcus leuteus. The resistance to Phenanthrene by the isolate was tested with various concentrations of Phenanthrene from 0 to 10mM. The Micrococcus luteus showed a MTC value of 28Cfu/ml at 8mM and Bacillus cereus showed least.

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