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DNA Amplification Technology

Extent: 5 days.   
Time: Annually in the middle of October   
Maximum numbers of students: 8   
DNA/RNA amplification and quantification in areas such as diagnostic PCR and molecular biology has been greatly improved by the introduction of real-time PCR technology. While this technology has tremendous potential for accurate and sensitive quantification, careful considerations regarding experimental design, sample preparation and reagent optimisation are required before it can be used correctly. The course will cover different aspects of polymerase chain reaction such as (i) absolute and relative quantification, (ii) conventional and real-time PCR, (iii) optimisation of PCR, (iv) primer/probe design, and (v) sample preparation and strategies to generate PCR-compatible samples.
The laboratory exercises will illustrate essential theoretical aspects of PCR and the results will be thoroughly discussed during the course. In the end of the course, we will mention various applications such as gene expression, cloning, diagnostic PCR, mutagenesis, RAPD, RNA preparation etc., in the form of oral presentations and general discussions. The course spans five days and is held at the library and laboratories of Applied Microbiology (Kemicentrum). The number of participants is limited to eight. Course manual and literature will be provided.
The course was originally developed 1997 for the Biomedical Research School, and has since then been a popular course with about 40-60 applicants per year. The real-time PCR technology was established as a laboratory exercise 1999 in the course.
Below can you find some of our publications in this field divided into relevant categories:  
  Diagnostic PCR    Gene expression/quantification   Sample preparation  
  PCR inhibitors   Amplification facilitators   RAPD   
Diagnostic PCR   
1 Löfström C, Knutsson R, Engdahl Axelsson C, Rådström P (2004) Rapid and specific detection of Salmonella spp. in animal feed samples by PCR after culture enrichment. Appl Environ Microbiol 70:69-75   
2 Hoorfar J, Ahrens P, Rådström P (2000) Automated 5´nuclease PCR assay for identification of Salmonella enterica. J Clin Microbiol 38:3429-3435  
  3 Lübeck PS, Wolffs P, On SL, Ahrens P, Rådström P, Hoorfar J (2003) Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation. Appl Environ Microbiol 69:5664-5669  
  4 Dahlenborg M, Borch E, Rådström P (2003) Prevalence of Clostridium botulinum types B, E, and F in faecal samples from Swedish cattle. Int J Food Microbiol 82:105-110  
  5 Knutsson R, Rådström P (2003) Detection of pathogenic Yersinia enterocolitica by a swab enrichment PCR procedure. Methods Mol Biol 216:311-324  
  6 Knutsson R, Fontanesi M, Grage H, Rådström P (2002) Development of a PCR-compatible enrichment medium for Yersinia entercolitica: amplification precision and dynamic detection range during cultivation. Int J Food Microbiol 72:185-201  
  7 Dahlenborg M, Borch E, Rådström P (2001) Development of a combined selection and enrichment PCR procedure for Clostridium botulinum types B, E and F and its use to determine prevalence in fecal samples from slaughtered pigs. Appl Environ Microbiol 67:4781-4788  
  8 Olcén P, Lantz PG, Bäckman A, Rådström P (1995) Rapid diagnosis of bacterial meningitis by a seminested PCR strategy. Scand J Infect Dis 27:537-539  
  9 Rådström P, Bäckman A, Qian N, Kragsbjerg P, Påhlson C, Olcén P (1994) Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus  influenzae , and streptococci using a seminested PCR strategy. J Clin Microbiol 32:2738-2744  
  10 Kristiansen B-E, Ask E, Jenkins A, Fermér C, Rådström P, Sköld O (1991) Rapid diagnosis of meningococcal meningitis by polymerase chain reaction. Lancet 337:1568-1569  
  Gene expression/quantification  
  1 Wolffs P, Norling B, Hoorfar J, Griffiths M, Rådström P (2005) Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR. Appl Environ Microbiol 71:5759-5764  
  2 Wolffs P, Norling B, Rådström P (2005) Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells. J Microbiol Methods 60:315-323   
  3 Lövenklev M, Artin I, Hagberg O, Borch E, Holst E, Rådström P (2004) Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B. Appl Environ Microbiol 70:2928-2934  
  4 Lövenklev M, Holst E, Borch E, Rådström P (2004) Relative neurotoxin gene expression in Clostridium botulinum type B, determined using quantitative reverse transcription-PCR. Appl Environ Microbiol 70:2919-2927  
  5 Wolffs P, Knutsson R, Norling B, Rådström P (2004) Rapid quantification of Yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR. J Clin Microbiol 42:1042-1047  
  6 Wolffs P, Grage H, Hagberg O, Rådström P (2004) Impact of DNA polymerases and their buffer systems on quantitative real-time PCR. J Clin Microbiol 42:408-411  
  7 Knutsson R, Löfström C, Grage H, Hoorfar J, Rådström P (2002) Modeling of 5´nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica. J Clin Microbiol 40:52-60  
  Sample preparation  
  1 Rådström P, Knutsson R, Wolffs P, Lövenklev M, Löfström C (2004) Pre-PCR processing: strategies to generate PCR-compatible samples. Mol Biotechnol. 26:133-146  
  2 Rådström P, Lövenklev M, Wolffs P, Löfström C, Knutsson R (2004) Pre-PCR processing strategies. In T. Weissensteiner, HG Griffin and A Griffin (ed.), PCR Technology: Current Innovations, Second Edition. CRC Press LLC. p. 37-47  
  3 Rådström P, Löfström C, Lövenklev M, Knutsson R, Wolffs P (2003) Strategies for overcoming PCR inhibition. In C.W. Dieffenbach and G.S. Dveksler (ed.), PCR Primer: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. p. 149-161  
  4 Rådström P, Knutsson R, Wolffs P, Dahlenborg M, Löfström C (2003) Pre-PCR processing of samples. Methods Mol Biol 216:31-50  
  5 Lantz PG, Abu Al-Soud W, Knutsson R, Hahn-Hägerdal B, Rådström P (2000) Biotechnical use of polymerase chain reaction for microbiological analysis of biological samples. Biotechnol Annu Rev 5:87-130  
  6 Lantz PG, Tjerneld F, Hahn-Hägerdal B, Rådström P (1996) Use of aqueous two-phase systems in sample preparation for polymerase chain reaction-based detection of microorganisms. J Chromatogr B Biomed Appl 680:165-170  
  7 Lantz PG, Hahn-Hägerdal B, Rådström P (1994) Sample preparation methods in PCR-based detection of food pathogens. Trends Food Sci Technol 5:384-389  
  8 Lantz PG, Tjerneld F, Borch E, Hahn-Hägerdal B, Rådström P (1994) Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method. Appl Environ Microbiol 60:3416-3418  
  PCR inhibitors  
  1 Abu Al-Soud W, Rådström P (2001) Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39:485-493  
  2 Abu Al-Soud W, Jönsson LJ, Rådström P (2000) Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 38:345-350  
  3 Lantz PG, Hahn-Hägerdal B, Rådström P (1998) PCR-inhibitory components in complex biological samples. Recent Res Develop in Microbiology 2:627-640  
  4 Abu Al-Soud W, Lantz PG, Bäckman A, Olcén P, Rådström P (1998) A sample preparation method which facilitates detection of bacteria in blood cultures by the polymerase chain reaction. J Microbiol Methods 32:217-224  
  5 Lantz PG, Matsson M, Wadström T, Rådström P (1997) Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. J Microbiol Methods 28:159-167  
  Amplification facilitators  
  1 Abu Al-Soud W, Rådström P (2000) Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces and meat. J Clin Microbiol 38:4463-4470  
  2 Abu Al-Soud W, Rådström P (1998) Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl Environ Microbiol 64:3748-3753  
  1 Eriksson J, Löfström C, Aspán A, Gunnarsson A, Karlsson I, Borch E, de Jong B, Rådström P (2005) Comparison of genotyping methods by application to Salmonella livingstone strains associated with an outbreak of human salmonellosis. Int J Food Microbiol 104:93-103  
  2 Blixt Y, Knutsson R, Borch E, Rådström P (2003) Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Y. enterocolitica-like bacteria. Int J Food Microbiol 83:15-26  

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Johannes Hedman
Tel: +46 (0)46 222 83 29

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