The document summarizes a study on characterizing the 16S rRNA gene of Flavobacterium columnare strains isolated from different fish species. The objectives were to characterize the 16S rRNA gene and construct a phylogenetic relationship among F. columnare strains. Samples were collected from fish in CIFA ponds and village ponds, and the 16S rRNA gene was amplified and sequenced. BLAST analysis showed similarity to F. columnare sequences. ClustalW alignment and phylogenetic analysis grouped the isolates. The study characterized the 16S rRNA gene to identify and establish relationships among F. columnare strains affecting freshwater aquaculture.
ExpressMAX™ Expression-ready Human GPCR ORF Clones are a ready-to-use solution that offers highest levels of GPCR expression with a wide spectrum of strong constitutive Promoters.
Designed to Accelerate your Research, Drug Discovery labs devote time and energy into developing practical methods for the discovery, isolation and characterization in the process of put on surface a heterologous G protein-coupled receptors (GPCRs) of its interest.
Trials are made in a Canvax™ Proprietary and Patented Platform, FRIDA GPCR, a robust cell-based homogeneous assay validated for GPCRs (Patent Info: (US 9347942 B2 (WO/2012/013204) and WO 2013113369 A1) that offfers Highest levels of GPCR expression.
The expression vector included in each ExpressMAX™ GPCR ORF Clones has been selected using 7TMbRN Surface GPCR Expression Vector System. The 7TMbRN Surface system comprises a group of ten for GPCR membrane proteins. Each GPCR has been cloned in this vector set, which incorporates vectors with different promoters, tags and glycosylation signal (GS) sequences.
Available now from Accuscience Ireland. Many topical and geographically important tests have been developed to allow producers and companion animal owners/veterinarians to screen animals quickly and with the high sensitivity of qPCR. Within the veterinary and agricultural range of pathogens detection kits are diseases and infections which can have significanteconomic impact. If your test is not available contact us to enquire about getting a custom kit made for you.
The Delta Seek detection kits utilise the sensitivity and speed of qPCR to get the most accurate data as quickly as possible. Each kit is specifically designed by our bioinformatics team to ensure the broadest possible detection profile and detection of all clinically relevant strains and subtypes. All test kits are validated in house on multiple qPCR platforms to ensure cross platform functionality.
The slide set describes the staining patterns for the VE1 antibody. The validation and verification process is also described. Additionally external quality assurance and image analysis processes for the VE1 antibody are presented.
Verification of an Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel, workf...Thermo Fisher Scientific
Fusion transcripts resulting from translocation events in the oncogenic driver
genes ALK, RET, ROS1, and NTRK1 play an important role in lung
adenocarcinoma. There is a need to detect these fusion transcripts with up to
date technologies as they may serve as viable therapeutic targets. We have
utilized a targeted sequencing approach and developed an Ion AmpliSeq™ RNA
Lung Fusion panel, a workflow, and an Ion Reporter™ analysis solution to detect
these known fusion events. The panel detects transcripts from 37 ALK, 9 RET, 15
ROS1, and 11 NTRK fusion variants along with 5 housekeeping genes to serve
as internal controls. The workflow is FFPE compatible requiring an input of only
10 ng of total RNA with the capacity to multiplex up to 16 libraries on a single Ion
318™ chip. The panel was initially validated using 10ng of total RNA from a
cocktail of 3 cell lines containing known lung cancer fusions (H2228 – EML4-ALK
variant 3a and 3b, HCC78 – SLC34A2-ROS1 and LC-2/ad – CCDC6-RET). The
library was sequenced using the Ion PGM™ system and analyzed with the
AmpliSeq™ RNA Lung Fusion workflow in Ion Reporter™. Analysis showed that
the positive control sample contained all expected fusions and control genes and
reported zero false positives fusions. This multiplexed fusion transcript targeted
sequencing solution is currently being validated by all members of the
OncoNetwork Consortium who will test lung cancer tissue samples that have
been well characterized by FISH, real-time PCR, IHC, and/or massarray. Initial
results from OncoNetwork Consortia members reveal 100% concordance
between the AmpliSeq™ RNA Lung Fusion panel and FISH in 25 lung tissue
samples.
ExpressMAX™ Expression-ready Human GPCR ORF Clones are a ready-to-use solution that offers highest levels of GPCR expression with a wide spectrum of strong constitutive Promoters.
Designed to Accelerate your Research, Drug Discovery labs devote time and energy into developing practical methods for the discovery, isolation and characterization in the process of put on surface a heterologous G protein-coupled receptors (GPCRs) of its interest.
Trials are made in a Canvax™ Proprietary and Patented Platform, FRIDA GPCR, a robust cell-based homogeneous assay validated for GPCRs (Patent Info: (US 9347942 B2 (WO/2012/013204) and WO 2013113369 A1) that offfers Highest levels of GPCR expression.
The expression vector included in each ExpressMAX™ GPCR ORF Clones has been selected using 7TMbRN Surface GPCR Expression Vector System. The 7TMbRN Surface system comprises a group of ten for GPCR membrane proteins. Each GPCR has been cloned in this vector set, which incorporates vectors with different promoters, tags and glycosylation signal (GS) sequences.
Available now from Accuscience Ireland. Many topical and geographically important tests have been developed to allow producers and companion animal owners/veterinarians to screen animals quickly and with the high sensitivity of qPCR. Within the veterinary and agricultural range of pathogens detection kits are diseases and infections which can have significanteconomic impact. If your test is not available contact us to enquire about getting a custom kit made for you.
The Delta Seek detection kits utilise the sensitivity and speed of qPCR to get the most accurate data as quickly as possible. Each kit is specifically designed by our bioinformatics team to ensure the broadest possible detection profile and detection of all clinically relevant strains and subtypes. All test kits are validated in house on multiple qPCR platforms to ensure cross platform functionality.
The slide set describes the staining patterns for the VE1 antibody. The validation and verification process is also described. Additionally external quality assurance and image analysis processes for the VE1 antibody are presented.
Verification of an Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel, workf...Thermo Fisher Scientific
Fusion transcripts resulting from translocation events in the oncogenic driver
genes ALK, RET, ROS1, and NTRK1 play an important role in lung
adenocarcinoma. There is a need to detect these fusion transcripts with up to
date technologies as they may serve as viable therapeutic targets. We have
utilized a targeted sequencing approach and developed an Ion AmpliSeq™ RNA
Lung Fusion panel, a workflow, and an Ion Reporter™ analysis solution to detect
these known fusion events. The panel detects transcripts from 37 ALK, 9 RET, 15
ROS1, and 11 NTRK fusion variants along with 5 housekeeping genes to serve
as internal controls. The workflow is FFPE compatible requiring an input of only
10 ng of total RNA with the capacity to multiplex up to 16 libraries on a single Ion
318™ chip. The panel was initially validated using 10ng of total RNA from a
cocktail of 3 cell lines containing known lung cancer fusions (H2228 – EML4-ALK
variant 3a and 3b, HCC78 – SLC34A2-ROS1 and LC-2/ad – CCDC6-RET). The
library was sequenced using the Ion PGM™ system and analyzed with the
AmpliSeq™ RNA Lung Fusion workflow in Ion Reporter™. Analysis showed that
the positive control sample contained all expected fusions and control genes and
reported zero false positives fusions. This multiplexed fusion transcript targeted
sequencing solution is currently being validated by all members of the
OncoNetwork Consortium who will test lung cancer tissue samples that have
been well characterized by FISH, real-time PCR, IHC, and/or massarray. Initial
results from OncoNetwork Consortia members reveal 100% concordance
between the AmpliSeq™ RNA Lung Fusion panel and FISH in 25 lung tissue
samples.
In this research paper from the Spring 2015 semester, I described my analysis of certain genome scaffolds, or gaps within the Malaclemys terrapin genome. I examined seven of these scaffolds and determined their approximate sizes through Polymerase Chain Reaction (PCR) and Gel Electrophoresis. The DNA was then prepped to be sent for sequencing by an external source. The resulting chromatograms gave inconclusive results on the exact sequences of these scaffolds.
CAP Trapper Technologies and Applications, CAP Analysis of Gene Expression (C...Laura Berry
Presented at the NGS Tech and Applications Congress: USA. To find out more, visit:
www.global-engage.com
Masayoshi Itoh is a Senior Scientist at the RIKEN Center for Life Science Technologies (CLST) and Coordinator of the RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI). In this presentation Masayoshi introduces CAP trapper technologies and presents the findings of the FANTOM5 project.
Fusion Gene Detection and Gene Expression Analysis of Circulating RNA in Plas...Thermo Fisher Scientific
The presence of circulating (cell-free) nucleic acids in the bloodstream offers a potential non-invasive approach to monitor disease status and guide treatment options. In past years, increasing interest has been shown for circulating RNA; especially circulating small RNAs for their potential application as biomarkers that may lead toward more effective diagnosis and prognosis in the future. However, widespread inconsistencies have been observed among the studies due to biases generated during sample collection, handling, RNA extraction and analysis. We have developed a complete workflow that includes blood collection, plasma preparation, circulating RNA extraction, followed by expression analysis and gene fusion detection on Ion TorrentTM Next-Generation Sequencing platforms. Blood plasma research samples from normal research samples were utilized for circulating RNA isolation following a TRIzolTM LS Reagent and mirVanaTM miRNA Isolation Kit-based method to maximize circulating RNA recovery. Ion AmpliSeqTM library preparation was performed on purified circulating RNA using either Ion AmpliSeq Transcriptome panel for expression profiling of 21K coding and non-coding genes, or an Ion AmpliSeq panel targeting fusion transcript detection from RNA. Ion AmpliSeq Transcriptome data was analyzed using ampliSeqRNA plugin in Torrent Suite™ Software. ~3000 genes were detected in cfDNA from plasma research samples with high correlation (r>0.8) observed between normal research samples. Ion Reporter™ Software was used to analyze fusion transcript panel data. Detection of fusion gene transcripts was demonstrated by spiking trace amounts of RNA from a fusion positive cell line into circulating RNA from normal research samples, indicating high sensitivity of the detection system. In summary, this study demonstrated the feasibility of gene expression profiling and gene fusion detection from circulating RNA in plasma research samples on Ion Torrent NGS platforms.
Development of a Multi-Variant Frequency Ladder™ for Next Generation Sequenci...Thermo Fisher Scientific
Increasing adoption of NGS has shed light on the need for more
standardized controls to evaluate and optimize system performance.
Samples containing mutations of interest are difficult to source and cell
line pooling experiments to determine limit of detection require significant
investments of time and money. To simultaneously evaluate variant
calling performance in >200 unique amplicons across 50 genes targeted
by NGS tests, AcroMetrix® has developed a proprietary
genomic/synthetic DNA material containing over 550 mutations as a
mixture of SNV’s indels and MNP’s. The limit of detection was then
determined for >400 variants using multiple platforms. Tumor samples
were diluted with matched normal samples to mimic a range of
frequencies. Linearity between the material and diluted tumor tissue
samples were compared. Overall, highly multiplex controls with tunable
frequencies allow for much more extensive, yet streamlined, assay
evaluation and facilitate implementation and impart confidence to NGS
testing.
In this research paper from the Spring 2015 semester, I described my analysis of certain genome scaffolds, or gaps within the Malaclemys terrapin genome. I examined seven of these scaffolds and determined their approximate sizes through Polymerase Chain Reaction (PCR) and Gel Electrophoresis. The DNA was then prepped to be sent for sequencing by an external source. The resulting chromatograms gave inconclusive results on the exact sequences of these scaffolds.
CAP Trapper Technologies and Applications, CAP Analysis of Gene Expression (C...Laura Berry
Presented at the NGS Tech and Applications Congress: USA. To find out more, visit:
www.global-engage.com
Masayoshi Itoh is a Senior Scientist at the RIKEN Center for Life Science Technologies (CLST) and Coordinator of the RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI). In this presentation Masayoshi introduces CAP trapper technologies and presents the findings of the FANTOM5 project.
Fusion Gene Detection and Gene Expression Analysis of Circulating RNA in Plas...Thermo Fisher Scientific
The presence of circulating (cell-free) nucleic acids in the bloodstream offers a potential non-invasive approach to monitor disease status and guide treatment options. In past years, increasing interest has been shown for circulating RNA; especially circulating small RNAs for their potential application as biomarkers that may lead toward more effective diagnosis and prognosis in the future. However, widespread inconsistencies have been observed among the studies due to biases generated during sample collection, handling, RNA extraction and analysis. We have developed a complete workflow that includes blood collection, plasma preparation, circulating RNA extraction, followed by expression analysis and gene fusion detection on Ion TorrentTM Next-Generation Sequencing platforms. Blood plasma research samples from normal research samples were utilized for circulating RNA isolation following a TRIzolTM LS Reagent and mirVanaTM miRNA Isolation Kit-based method to maximize circulating RNA recovery. Ion AmpliSeqTM library preparation was performed on purified circulating RNA using either Ion AmpliSeq Transcriptome panel for expression profiling of 21K coding and non-coding genes, or an Ion AmpliSeq panel targeting fusion transcript detection from RNA. Ion AmpliSeq Transcriptome data was analyzed using ampliSeqRNA plugin in Torrent Suite™ Software. ~3000 genes were detected in cfDNA from plasma research samples with high correlation (r>0.8) observed between normal research samples. Ion Reporter™ Software was used to analyze fusion transcript panel data. Detection of fusion gene transcripts was demonstrated by spiking trace amounts of RNA from a fusion positive cell line into circulating RNA from normal research samples, indicating high sensitivity of the detection system. In summary, this study demonstrated the feasibility of gene expression profiling and gene fusion detection from circulating RNA in plasma research samples on Ion Torrent NGS platforms.
Development of a Multi-Variant Frequency Ladder™ for Next Generation Sequenci...Thermo Fisher Scientific
Increasing adoption of NGS has shed light on the need for more
standardized controls to evaluate and optimize system performance.
Samples containing mutations of interest are difficult to source and cell
line pooling experiments to determine limit of detection require significant
investments of time and money. To simultaneously evaluate variant
calling performance in >200 unique amplicons across 50 genes targeted
by NGS tests, AcroMetrix® has developed a proprietary
genomic/synthetic DNA material containing over 550 mutations as a
mixture of SNV’s indels and MNP’s. The limit of detection was then
determined for >400 variants using multiple platforms. Tumor samples
were diluted with matched normal samples to mimic a range of
frequencies. Linearity between the material and diluted tumor tissue
samples were compared. Overall, highly multiplex controls with tunable
frequencies allow for much more extensive, yet streamlined, assay
evaluation and facilitate implementation and impart confidence to NGS
testing.
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
Presentation of Fridtof Lund-Johansen in 1st International Antibody Validatio...St John's Laboratory Ltd
Fridtjof Lund-Johansen is an MDPhD who has worked for 30 years with antibodies and flow cytometry. He took his degree at the University of Bergen in Norway. He then did post-docs in California, first at Becton Dickinson in San Jose and then at DNAX instiute of Immunology in Palo Alto. He is now a PI at the Institute of Immunology at Oslo University Hospital where he leads an effort to develop antibody-based proteomics. His technology is called MAP for microsphere affinity proteomics. He will talk about how MAP can be used to test thousands of antibodies in parallel.
For more details about 1st international antibody validation forum please check on http://www.stjohnslabs.com/ac_cms/blog
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
High-throughput processing to maximize genomic analysis through simultaneous ...Thermo Fisher Scientific
As personalized cancer care evolves, the patient’s nucleic acid becomes ever so important to provide valuable information regarding their genetic makeup and disease state. Common sample types for these analyses include biopsies, which can be very limited in material making the downstream measurement of more than one analyte rather difficult. Obtaining another biopsy, using a different section or splitting the sample can be problematic because of tumor heterogeneity. Even adjacent areas of the same tumor tissue can result in different RNA/DNA profiles so the ability to isolate multiple analytes from the same sample offer a number of benefits, which include preserving samples and data consistency eliminating any sample to sample variation. As more tests are developed to simultaneously monitor genetic alterations, there is a strong need to efficiently isolate both DNA and RNA from the same starting sample in a format compatible with high-throughput processing.
HIV Vaccines Process Development & Manufacturing - Pitfalls & PossibilitiesKBI Biopharma
Originally presented at the HIV Vaccine Manufacturing Workshop –July 19th& 20th, 2017 by Abhinav A. Shukla, Ph.D.Senior Vice PresidentDevelopment & ManufacturingKBI Biopharma, Durham NC
Multiplex Real-Time RT-PCR for Detection of FMDV, Rift Valley Fever Virus and...EuFMD
The European Commission for the Control of Foot-and-Mouth Disease (EuFMD), one of FAO’s oldest Commissions, came into being on the 12th June 1954, with the pledge of the sixth founding member state to the principles of a coordinated and common action against Foot-and-mouth Disease.
I presented my ongoing research from Massachusetts General Hospital at the American Society of Transplantation Annual Scientific Exchange meeting in Orlando, FL.
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
The Gram stain is a fundamental technique in microbiology used to classify bacteria based on their cell wall structure. It provides a quick and simple method to distinguish between Gram-positive and Gram-negative bacteria, which have different susceptibilities to antibiotics
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Adv. biopharm. APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMSAkankshaAshtankar
MIP 201T & MPH 202T
ADVANCED BIOPHARMACEUTICS & PHARMACOKINETICS : UNIT 5
APPLICATION OF PHARMACOKINETICS : TARGETED DRUG DELIVERY SYSTEMS By - AKANKSHA ASHTANKAR
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
1. STUDY ON 16S rRNA gene of
Flavobacterium columnare
Guided by: Dr. B. K. Das
Principal Scientist
Fish Health Management Division
Central Institute of Freshwater Aquaculture
Kausalyaganga,Bhubaneswar
Presented by: Soumya Sankar Rath
(Roll No-14716V114013)
2. •Aquaculture is one of the fastest animal food producing sector in the world.
•Fish is a rich source of protein.
•Worldwide people obtained 25% protein from fish, but due to pathogenic bacteria, the
production rate is reducing day by day.
•The most frequently encountered bacterial agent associated with fish disease is
Flavobacterium columnare.
•It is an aerobic, gliding, Gram negative, long rod-shaped bacterium.
INTRODUCTION
4. OBJECTIVES
• To characterize the 16S rRNA gene of F. columnare strain isolated
from different organs of freshwater fishes.
• To construct the phylogenetic relationship among the different
strains of F. columnare.
5. MATERIALS AND METHODS
• Sample collection: Samples were collected from the Freshwater fishes from CIFA ponds and village ponds
of Bhubaneswar.
• Colony Morphology: Colony morphology was studied by inoculating bacteria on TSA plates followed by
incubation for 24 hrs at 370C.
• Cell Morphology: Gram staining was done.
• Biochemical Tests: The isolates were processed for different biochemical test
• Oxidase Test
• Catalase Test
• Gelatin Test
6. • DNA Extraction: The DNA was extracted by using both CTAB and HipuraTM Kit
method)
• Quantity Analysis of isolated DNA: The concentration and quantity of the genomic
DNA obtained was determined by spectrophotometric analysis and agarose gel
electrophoresis.
• Quality checking of the Isolated DNA: The qualities of isolated DNA samples were
checked by 0.8% agarose gel electrophoresis.
• Primer design: primer design was done by using Primer 3 Plus Software.
MOLECULAR CHARACTERIZATION
7. – Amplification of 16S rRNA was done by taking (Forward primer- 5’
AAGAGTTTGATCCTGGCTCAG 3’, Reverse Primer-5’
GGTTACCTTGTTACGACTT 3’) obtained from Bangalore Genei.
– Both the amplification was carried out along with dNTP, MgCl2, 10X Assay
Buffer, Taq DNA Polymerase and genomic DNA.
AMPLIFICATION 16S RIBOSOMAL RNA
8. • Purification of PCR Products
– The purification of PCR products were done by MinElute Gel Extraction
Kit.
• Analysis of PCR-amplified products
– The PCR-amplified products were analyzed through 1.2% agarose gel
electrophoresis
• Sequencing of amplified PCR Products
– The amplified PCR products were sent to Eurofin Laboratory Pvt. Ltd. for
sequencing.
9. – BLAST Analysis of F. columnare : After sequencing, database searches were conducted
with the BLAST (Basic Local Alignment Search Tool) algorithm provided by NCBI
(hhttp://blast.ncbi.nlm.nih.gov/ ).
– ClustalW Alignment of F. columnare :
ClustalW ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ) is a tool for aligning multiple
protein or nucleotide sequences.
– Phylogenetic Analysis of F.columnare : MEGA 4.0 software software was used to do
phylogenetic analysis.
BIOINFORMATICS TOOLS
12. RESULTS
• Isolation of bacteria: (Table 1)
• Morphology of F. columnare : (Fig. 1)
• Identification
– Biochemical Test (Table 2)
• Catalase Test
• Oxidase Test
• Gelatin test
• Genome analysis
– Isolation of DNA
– Quantitative Analysis of isolated DNA
– Quality checking of the Isolated DNA (Fig. 2)
– Amplification of 16S rRNA gene and Analysis of PCR products: (Fig. 3)
13. – BLAST analysis of 16S rRNA (Table 3)
– Clustal analysis of 16S rRNA of F. columnare :(Table 5, 6, 7, 8, 9 and
10)(Fig. 4, 5, 6, 7, 8 and 9).
– Phylogenetic Analysis of F. columnare (Fig. 10, 11, 12, 13, 14 and 15)
14. Table 1: Isolation of F. columnare from different organs of catla, rohu, goldfish and anabas
Sl. No. Bacteria Code Species Organ Area Of Isolation
1 CFCCO41 Catla Operculum CIFA Research pond
2 CFCRVB43 Rohu Ventral belly CIFA Research pond
3 CFCGFG50 Gold fish Gill CIFA Research pond
4 CFCRG55 Rohu Gill CIFA Research pond
5 CFCCG62 Catla Gill Village pond BBSR
6 CFCCSL66 Catla Skin lesions CIFA Research pond
7 CFCACR72 Anabas Caudal region CIFA Research pond
15. Table 2: Biochemical test result of F. columnare from catla, rohu, goldfish and anabas
Sl. No. Organism Gram Staining Catalase Test Oxidase Test Gelatin
1 CFCCO41 - + - +
2 CFCRVB43 - + - +
3 CFCGFG50 - + - +
4 CFCRG55 - + - +
5 CFCCG62 - + - +
6 CFCCSL66 - + - +
7 CFCACR72 - + - +
19. Code Organism After
alignment
New Code Id No. Submitted base pair
MS 41 FC 41 CFCCO41 1629257 1128
MS 43 FC 43 CFCRVB43 1629259 1155
MS 50 FC 50 CFCGFG50 1629262 1157
MS 55 FC 55 CFCRG55 1629263 1216
MS 62 FC 62 CFCCG62 1629265 1151
MS 66 FC 66 CFCCSL66 1629267 1164
MS 72 FC 72 CFCACR72 1629269 1167
Table 4: Sequences submitted to NCBI GenBank.
20. Seq A Name Length Seq B Name Length Score
1 CFCCO41 1128 2 CFCCG62 1151 96.37
1 CFCCO41 1128 3 CFCCSL66 1164 98.76
1 CFCCO41 1128 4 ATCC 1477 76.95
2 CFCCG62 1151 3 CFCCSL66 1164 97.48
2 CFCCG62 1151 4 ATCC 1477 75.76
3 CFCCSL66 1164 4 ATCC 1477 77.32
Table 5: Clustal Analysis of 16S rRNA gene of F. columnare from gill, operculum and skin of catla.
Table 6: Clustal Analysis of 16S rRNA of F. columnare from gill and ventral belly of rohu.
Seq A Name Length Seq B Name Length Score
1 CFCRVB43 1155 2 CFCRG55 1216 81.47
1 CFCRVB43 1155 3 ATCC 1477 74.98
2 CFCRG55 1216 3 ATCC 1477 73.6
Note: 1: CFCCO41; 2: CFCCG62; 3: CFCCSL66; 4: F. columnare 16S ribosomal RNA, partial sequence, strain: ATCC 49513
21. Table 7: Clustal Analysis of 16S rRNA of F. columnare from gill of goldfish and caudal region of anabas
Seq A Name Length Seq B Name Length Score
1 CFCGFG50 1157 2 CFCACR72 1167 93.09
1 CFCGFG50 1157 3 ATCC 1477 77.27
2 CFCACR72 1167 3 ATCC 1477 76.52
Table 8: Clustal Analysis of 16S rRNA of F. columnare from gill of goldfish, rohu and catla
Seq A Name Length Seq B Name Length Score
1 CFCGFG50 1157 2 CFCRG55 1216 93.26
1 CFCGFG50 1157 3 CFCCG62 1151 92.79
1 CFCGFG50 1157 4 ATCC 1477 77.27
2 CFCRG55 1216 3 CFCCG62 1151 97.05
2 CFCRG55 1216 4 ATCC 1477 73.6
3 CFCCG62 1151 4 ATCC 1477 75.76
22. Seq A Name Length Seq B Name Length Score
1 CFCCO41 1128 2 CFCRVB43 1155 81.91
1 CFCCO41 1128 3 CFCCSL66 1164 98.76
1 CFCCO41 1128 4 CFCACR72 1167 98.14
1 CFCCO41 1128 5 ATCC 1477 76.95
2 CFCCO41 1155 3 CFCCSL66 1164 82.08
2 CFCRVB43 1155 4 CFCACR72 1167 81.82
2 CFCRVB43 1155 5 ATCC 1477 74.98
3 CFCCSL66 1164 4 CFCACR72 1167 98.8
3 CFCCSL66 1164 5 ATCC 1477 77.32
4 CFCACR72 1167 5 ATCC 1477 76.52
Table 9: Clustal Analysis of 16S rRNA of F. columnare from operculum and ventral belly of rohu, skin lesion of catla and
caudal region of anabas.
24. Fig. 4: Clustal Analysis of 16S rRNA of F. columnare from gill, operculum, and skin lesion of catla.
25. Fig.5 : Clustal Analysis of 16S rRNA of F. columnare from gill and ventral belly of rohu.
26. Fig. 6: Clustal Analysis of 16S rRNA of F. columnare from gill of goldfish and caudal region of anabas.
27. Fig. 7: Clustal Analysis of 16S rRNA of F. columnare from gill of goldfish and caudal region of anabas
28. Fig. 8: Clustal Analysis of 16S rRNA of F. columnare from operculum of rohu, ventral belly of rohu, skin lesion of catla and caudal region of anabas
29. Fig.9 : Clustal Analysis of 16S rRNA of F. columnare from gill, operculum, skin lesions of catla (C. catla), gill, and ventral belly of rohu (L. rohita), gill of
goldfish (C. auratus) and caudal region of anabas (A. testudineus)
30. Fig.10: Phylogenetic tree of F. columnare from gill, operculum, and skin of catla.
Fig.11: Phylogenetic tree of F. columnare from gill and ventral belly of rohu.
31. Fig.13: Phylogenetic tree of F. columnare from operculum and ventral belly of rohu, skin lesion of catla and caudal region of anabas.
Fig.12: Phylogenetic tree of F. columnare from gill of goldfish and caudal region of anabas.
32. Fig.22: Phylogenetic tree of F. columnare from gill, operculum, skin lesions of
catla (C. catla), gill, and ventral belly of rohu (L. rohita), gill of goldfish (C. auratus) and
caudal region of anabas (A.s testudineus)
Fig.21: Phylogenetic tree of F. columnar from operculum of rohu,
ventral belly of rohu skin lesion of catla and caudal region of anabas
33. CONCLUSION
•Amplification of 16S rRNA is done to identify the Flavobacterium columnare strains which
were isolated from different organs of different diseased fish.
•From the BLAST analysis of 16S rRNA, we found that all the strains showed 72% to 98%
similarity with Flavobacterium columnare strains in GenBank.
•From the ClustalW alignment it was conclude that, the strains isolated from same species and
different species showed 86% to 95% similarity.
•After analyzing the phylogenetic tree it could be stated that there may exist a tissue preference
for different strains of F. columnare even in the same species.
34. • Sequencing the 16S rRNA gene enables querying a database such
as GenBank to yield an alternative identification without the need
for any additional laboratory work.