Role of the sample weight in norovirus detection in oysters by using molecular techniques Student: Noor Meersseman Work placement employer: Leena Maunula.

Slides:

Advertisements

Similar presentations

PCR, Gel Electrophoresis, and Southern Blotting

Advertisements

REAL TIME PCR ………A step forward in medicine

Amplification and Detection of Nucleic Acid by the Real-Time RT-PCR Procedure Janice C. Pedersen, Microbiologist Avian Section Diagnostic Virology Laboratory.

TACKLING OSTEOARTHRITIS -Research Tools At Our Disposal Mahita Kadmiel July 21, 2005.

Analysis of gene expression by real-time PCR RBCS3 and Cab-1b transcript quantitation by real time PCR.

RT-PCR lab You have a cell…is a certain gene on (by “on,” we mean active and producing mRNA?)? If a certain gene is on when the cell divides, the gene.

Real-Time PCR mRNA quantification. What do mRNA levels tell us? DNA  mRNA  protein Reflect level of gene expression Information about cell response.

PCR quantitative en temps réel Lydie Pradel. PCR.

PCR quantitativo.

Carolyn Dunn, Annabel Whibley, Lionel Willatt and Ingrid Simonic

Analysis of Gene Expression of Arabidopsis using RT-PCR and DNA Cloning Presented by Neha Jain ABE Workshop 2006 June 30, 2006.

Applied Biosystems 7900HT Fast Real-Time PCR System I. Real-time RT-PCR analysis of siRNA-induced knockdown in mammalian cells (Amit Berson, Mor Hanan.

An introduction to qPCR BCH452 Spring So far in BCH452 you have studied Carbohydrates: Glucose Proteins: ADH and LDH DNA: Plant DNA and viral DNA.

Analysis of Gene Expression - Overview -

Genomic DNA purification

Principles and Important Considerations

Real Time PCR = Quantitative PCR.

Measuring RNA transcript levels. Research problem – A small RNA, RteR, inhibits transfer of the 65 kb conjugative transposon CTnDOT attR attL ermF tetQ.

IGEM 101: Session 6 4/9/15Jarrod Shilts 4/11/15Ophir Ospovat.

Lecture 19, Chapter 11 Analysis of transgenic plants part II Neal Stewart.

Laboratory Training for Field Epidemiologists Polymerase Chain Reaction Investigation strategies and methods May 2007.

Variants of PCR Lecture 4

Last class Overall goal for labs 7-9 How to pick candidate miRNA targets Overview: “Validate” target prediction.

Abstract Automated, High-Throughput Total and Viral RNA Purification Byung-in Lee 1, Seachol Oak 1, Sharon Khietala 2, Beate Schikora 2, and Karl H Hecker.

COBAS AmpliPrep/Cobas TaqMan HIV-1 Test

APPLICATIONS OF MOLECULAR BIOLOGY TECHNIQUES TO MEDICAL MICROBIOLOGY.

Analysis of Transgenic Plants. 1.Regeneration on Selective Medium Selectable Marker Gene.

REAL-TIME RT-PCR BASED ASSAY ON BLOOD CLOT SPECIMENS FOR DIAGNOSIS OF HIV-1 INFECTION IN CHILDREN, MALAWI Hua Yang 1, Rita Helfand 2, Desiree Witte 3,

Analyzing your clone 1) FISH 2) “Restriction mapping” 3) Southern analysis : DNA 4) Northern analysis: RNA tells size tells which tissues or conditions.

PCR POLYMERASE CHAIN REACTION Dauphin Island Graduate Neurobiology.

Dr. Sumbul Fatma Department of Medical Biochemistry.

Real time RT-PCR Quantitating Gene Expression.

Quantification of RNA by real-time PCR

Development and Validation of a Novel Real-Time RT-PCR Assay to Distinguish Porcine Reproductive and Respiratory Syndrome (PRRS) Virus Subtypes with High.

Results Alien Reference RNA QRT-PCR Detection Kit for Monitoring the Overall Performance of QRT-PCR Assays Bahram Arezi, Melissa McCarthy & Holly Hogrefe.

PCR reaction to be visualized “in real time” as the reaction progresses to quantify the amount of DNA in the sample at the start of the reaction REAL TIME.

How do you identify and clone a gene of interest? Shotgun approach? Is there a better way?

Genetics Techniques: RFLP & PCR AP Biology Unit 3.

POLYMERASE CHAIN REACTION (PCR)

Thitiporn Miiksombat 1, Narumol Bhummaphan 1, Teerakul Arpornsuwan 2 1 Student in Medical Technology Program, Faculty of Allied Health Sciences, Thammasat.

Polymerase Chain Reaction PCR. PCR allows for amplification of a small piece of DNA. Some applications of PCR are in: –forensics (paternity testing, crimes)

HCV PCR By Henrietta Orji July 31 st, 2010 Hepatitis C Virus by Polymerase Chain Reaction.

Molecular Techniques in Microbiology These include 9 techniques (1) Standard polymerase chain reaction Kary Mullis invented the PCR in 1983 (USA)Kary.

 DNA (gene mutations, paternity, organs compatibility for transplantations)  RNA  Proteins (gene expression)

Molecular Testing and Clinical Diagnosis

INTRODUCTION. INTRODUCTION Introduction In the past, amplifying (replication) of DNA was done in bacteria and took weeks. In 1971, paper in the.

Northern blotting & mRNA detection by qPCR - part 2.

Polymerase Chain Reaction (PCR)

Supplementary Figures Fig. 1 Transcript levels of Tissue Factor, PAR-1 and PAR-2 Expression if TF, PAR-1 and PAR-2 was analyzed in unstimulated HUVECs,

6.3 Advanced Molecular Biological Techniques 1. Polymerase chain reaction (PCR) 2. Restriction fragment length polymorphism (RFLP) 3. DNA sequencing.

Polymerase Chain Reaction (PCR) Nahla Bakhamis. Multiple copies of specific DNA sequences; ‘Molecular Photocopying’

Nada M. Melhem, PhD American University of Beirut

Introduction to PCR Polymerase Chain Reaction

Topic Cloning and analyzing oxalate degrading enzymes to see if they dissolve kidney stones with Dr. VanWert.

RNA isolation from monolayer cell Vascular Genomics Laboratory

Genome Regulation Center

CATEGORY: EXPERIMENTAL TECHNIQUES Polymerase Chain Reaction (PCR) Tarnjit Khera, University of Bristol, UK Background The polymerase chain reaction (PCR)

Rajan sharma.  Polymerase chain reaction Is a in vitro method of enzymatic synthesis of specific DNA sequences.  This method was first time developed.

RT-PCR analysis 생화학 실험 2 조교 : 이 선 민 내선 7699, 첨단과학기술관 201-B 호 신과학원 S438 호.

Aim: To develop a new one-step RT-PCR assay to detect H1N1 by designing new primer to target NP gene Experimental approach: -nasopharyngeal swabs from.

Last class Overall goal for labs 7-9 How to pick candidate miRNA targets Overview: “Validate” target prediction.

The stroke size should be 0.25

PCR Basics A review.

Last class Overall goal for labs 7-9

Gel electrophoresis analysis Automated DNA analyzer.

PCR Polymerase Chain Reaction

Testing for viruses – the way forward with PCR

Molecular diagnosis of viral hepatitis

Principles of Quantitative PCR

Presentation transcript:

Role of the sample weight in norovirus detection in oysters by using molecular techniques Student: Noor Meersseman Work placement employer: Leena Maunula Work placement mentor: Kirsi Söderberg Work placement supervisor: Griet Vanbillemont

Overview presentation 1.Introduction: background and aims 2.Methods 2.1 Sample collection and processing of the oysters 2.2 RNA extraction 2.3 RT-qPCR 3.Results 3.1 RT-PCR inhibition 3.2 Extraction efficiency 3.3 Sample quantification 4.Conclusion 5.Future 17/06/2015 2

1.Background and aims Risks of raw oysters: norovirus Cause of non-bacterial gastroenteritis Several outbreaks each year Analyze raw oysters for the presence of noroviruses Calculate amount of genome copies in positive samples Determine importance of weight/ sample size 17/06/2015 3

2.Methods 17/06/ batches, each 6 oysters 5 oysters1 oyster Virus extraction from digestive gland RNA extraction using NucliSENS miniMAG RT-qPCR (NoV GI + NoV GII)

2.Methods 2.1 Sample collection and processing of the oysters Crassostrea gigas or Pacific oyster 10 weeks  10 batches  60 oysters in total Virus extraction: 17/06/ Digestive gland Mengo virus control Proteinase K solution Incubation Centrifugation Process control

2.Methods 2.2 RNA extraction Supernatant + lysisbuffer NucliSENS miniMAG extraction machine + reagents Silica-containing magnetic beads 17/06/2015 6

2.Methods 17/06/2015 7

2.Methods 2.3 RT-qPCR Principle: One-step PCR  RT step + PCR reaction in one single tube RNA  cDNA (Reverse transcription enzyme) cDNA  exponential amplification Real-time PCR  amplification is followed during run (probes) 17/06/2015 8

2.Methods 2.3 RT-qPCR Mengo virus, NoV GI and NoV GII Specific primers and probes Rotor Gene - ILS15  Initial activation: 55°C – 60 min  Second activation: 95°C – 15 min  45 cycles: 95°C – 15 sec, 60°C – 60 sec, 65°C – 60 sec Also included in run:  EC RNA (RT-PCR inhibition)  dsDNA (quantification) 17/06/2015 9

3.Results 3.1 RT-PCR inhibition Sample + EC RNA C t values Above 75% = Fail 17/06/

3.Results 17/06/ GIGII

3.Results 3.2 Extraction efficiency Sample C t values from Mengo virus PCR runs Below 1% = Fail 2/ 19 failed Extraction repeated with batch 3, 7 and 9 17/06/

3.Results 3.3 Sample quantification: all results GI and GII 14/19 positive for GI (74%) 3 also positive for GII 17/06/ GI GII

3.Results 3.3 Sample quantification: results according to batch number 17/06/

4.Conclusion Detection of noroviruses successful in both sample types Quantification: amount of detected genome copies/g slightly higher in 1-oyster samples Inhibition: better results using 5-oyster method Extration efficiency: better mean result for 5-oyster samples 17/06/

5.Future Further research is needed Bigger amount of samples Winter  summer 17/06/

Role of the sample weight in norovirus detection in oysters by using molecular techniques 17/06/ Thank you for your attention! Any questions?