Issue 4 Summer 2002 - Applied Biosystems

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34 new product review In MRM mode the signal gain, when combined with a reduction in background noise, translates into between 5 to 20 times better signal-to-noise ratios. The response was linear with an upper limit of determination of 2000 pg on column for Testosterone, and 4000 pg on column for Ethynyl Estradiol. Relative response for Testosterone and Ethynyl Estradiol with PhotoSpray (PS) and APCI, as recorded in MRM mode. The same respective amount of compounds were injected on column using Normal phase chromatographic conditions. Estimated LODs and improvement Factor of S/N versus APCI Normal Reversed Phase Phase Steroid LODs PS Ratio PS/APCI Steroid LODs PS Ratio PI/APCI Testosterone 0.16 pg 7.5 Testosterone 0.08 pg 22.2 Ethynyl Estradiol 1.0 pg 5.0 Ethynyl Estradiol 1.1 pg 8.1 Polycyclic Aromatic Hydrocarbons (PAHs) The PhotoSpray source was used to develop an analytical method for polycyclic aromatic hydrocarbons (PAHs), comparing the difference under reverse and normal phase chromatographic conditions. A total of 16 PAHs commonly found in atmospheric samples from both rural and urban areas were analysed. Airborne PAHs, usually found in aerosol particles, are implicated in heterogeneous atmospheric processing and as important indicators of gas-phase atmospheric processes. Certain carcinogenic PAHs may be carried by ultra fine particles in ambient air into the lungs and are often the targets of rigorous environmental monitoring. A 20ppm stock standard solution containing 16 common PAHs was used for this experiment 5 . Reverse Phase PAH separation showing ten MRM transistions for 16 common PAHs Conclusion The PhotoSpray source provides a better detection limit to the API 150EX , API 3000 and QSTAR Pulsar systems for low polar class of compounds. The API 3000 and QSTAR Pulsar LC/MS/MS systems are the only tandem MS systems with three different ionisation sources, which allows researchers to have a high ionisation range flexibility. References 1. Bruins, A. P., Robb, D.B.: A New Ionisation Technique for LC/MS, 16th Montreux LC/MS Symposium, Nov. 3rd, 1999 (WO3) 2. Robb, D.B., Covey, T.R., Bruins, A.P., Anal. Chem. 2000, 72, 3653-3659. 3. Robb, D.B., Bruins, A.P., Peters, H.A.M., Jacobs, P.L.; Atmospheric Pressure Photoionisation (APPI) for High Sensitivity LC/MS in Bioanalysis, ASMS 2000 poster MPA-015 4. Alary, J.F., Comparative Study: LC-MS/MS Analysis of Four Steroid Compounds Using a New Photoionisation Source and a Conventional APCI, ASMS 2001 poster TPA-009 5. Impey, G., Kieser, B., Alary, J.F., The Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) by LC/MS/MS using a New Atmospheric Pressure Photoionisation Source, ASMS 2001 poster TPH-187 For more information on: PhotoSpray Ionisation Source enter: No. 423 new product review TaqMan ® Assays for Food Testing and Food Pathogen Detection F ood quality and food safety is a subject of growing public concern. Public food control institutions, private service laboratories and commercial food manufacturers see a growing need to test for genetically modified organisms (GMO) and pathogens in food. In July 1999, Switzerland implemented legal regulations which require food containing more than 1% GMO to be labelled. The European Community and many other countries around the world followed this example. Assays for detecting GMO must be quantitative, sensitive and reproducible. Immunoassays are fast and inexpensive, but not quantitative. Common PCR and its gel-based analysis is only semi-quantitative and bares a high risk of cross-contamination. Real-time quantitative PCR produces quantitative results in a closed tube assay, which avoids crossover contamination. The use of fluorescent TaqMan probes adds an extra level of specificity to the PCR. An internal positive control labelled with a different fluorescent dye controls for false-negative results. TaqMan GMO Detection Kits for soy and maize from Applied Biosystems use a universal target to detect the presence of the transcriptional regulatory elements common to most GMOs, which express the desired traits such as resistance to insects and herbicides. This transcriptional regulator is the 35S promoter of the cauliflower mosaic virus. Figure 1. Amplification plot of processed foods purchased from a local supermarket and tested with the TaqMan GMO Soy 35S Detection Kit run on an ABI PRISM ® 7700 Sequence Detection System. A) Soy-based infant formula, 50% GMO content (yellow) B) Energy bar, 8% GMO content (green) C) Soy-based drink, GMO-negative (red) A plant-specific endogenous control has two advantages: it allows a relative quantitation of GMO soy or GMO maize in the sample, and no amplification of the control indicates the presence of PCR inhibitors. The probe for the endogenous control is labelled with VIC ® dye, so the 35S target and the control can be detected simultaneously in a multiplex reaction. Figure 2. Amplification plot of processed foods purchased from a local supermarket and tested with the TaqMan GMO Maize 35S Detection Kit run on an ABI PRISM ® 7000 Sequence Detection System. A) Puppy food, 50% GMO content (green) B) Corn snack, 5% GMO content (red) C) Blue corn chips, GMO-negative (blue) A different source of public concern is contamination by food-borne pathogens. Salmonella is the second most frequent cause of food-borne illness in Europe and the United States. According to the World Health Organisation (WHO), diseases caused by the major pathogens alone are estimated to cost up to US $35 billion annually (1997) in medical costs and lost productivity in the USA alone. Applied Biosystems recently released the new TaqMan ® Salmonella Gold Detection Kit. Now a complete line of assays for food testing is available, including kits for detection of Salmonella, E. coli O157:H7 and E. coli STX-1/2 (Shiga-like toxin; VT1 and VT2). The TaqMan Salmonella Gold Kit detects all serotypes of Salmonella while closely related non-Salmonella bacteria test negatively. Hot-start with AmpliTaq Gold ® DNA Polymerase and the TaqMan probe contribute to the high level of specificity. After pre-enrichment, 10-100 cfu per sample can be detected reproducibly. Salmonella-specific and internal positive control assays use FAM ® and VIC reporter dyes. They are run as duplex 5' nuclease assays, which can be analysed and detected on the ABI PRISM ® 7000, 7700, and 7900HT Sequence Detection Systems. Like the GMO Kits, the Salmonella Detection Kit provides positive and negative controls. In addition, the GMO and Food Pathogen Kits include a label licence, which conveys certain PCR service rights when used with an authorised thermal cycler. For more information on: TaqMan assays for food testing enter: No. 424 TaqMan assays for food pathogen detection enter: No. 425 35
36 promotions Free Subscription! To the Award-Winning Online Magazine from Applied Biosystems B ioBeat ® Online Magazine (www.biobeat.com) covers life science research around the world that is enabled by technology from Applied Biosystems. This award-winning magazine currently offers over 120 articles on advances such as: ➜ Identification of a dog narcolepsy gene ➜ Identification of the human baldness gene ➜ Identification of the total colour blindness gene ➜ The complete genome sequencing of the plague and typhus organisms ➜ The discovery of influenza virus in seals ➜ The effective use of dog DNA evidence in a murder trial A wide variety of other topics are also covered, including proteomics, PNA, anthrax resistance, cancer research, suicide PCR, real-time quantitative PCR, and the origin of the global AIDS epidemic. The BioBeat home page BioBeat articles include numerous links to related articles and web sites, as well as illustrative graphics and photos of researchers. BioBeat has over 5,500 subscribers in 109 countries around the world. The stimulating content attracts subscribers ranging from biotech CEOs and CSOs, to lab directors, to physicians and bench scientists, to journalists from the New York Times and Wall Street Journal, to university students and teachers, to the general public. The BioBeat subscription page BioBeat’s Journal Watch feature provides an extensive list of recent peer-reviewed journal articles with links to article and Medline abstracts. BioBeat’s Conference Calendar offers a comprehensive list of upcoming science conferences, with links to conference web sites. BioBeat’s Journal Watch and Conference Calendar are e-mailed periodically to BioBeat subscribers. Subscribers are also notified by email whenever a new article is posted in BioBeat Online Magazine. You may obtain a free subscription to BioBeat Online Magazine by going to the BioBeat home page at www.biobeat.com and first clicking on the Subscribe link at the upper left. Then, simply fill out the brief subscription form that appears, click submit, and you will have your free ticket to cutting-edge content. For additional information on BioBeat Online Magazine, please contact BioBeat editor Mike O’Neill at: oneillmd@appliedbiosystems.com T he Gene Expression package from Applied Biosystems takes your experiments from RNA sample preparation to successful real-time quantitative PCR with TaqMan ® MGB probes. The Gene Expression package includes: ABI PRISM 6100 Nucleic Acid PrepStation ➜ For highly reproducible preparation of high yield, high purity RNA from cultured cells, plant and animal tissue and whole blood. ABI PRISM ® 7000 Sequence Detection System ➜ Accurate and highly reproducible results with multicolour detection for multiplex quantitation Set of Reagents and Disposables ➜ All you need to get started with your Gene Expression studies, including • TaqMan ® Universal PCR Master Mix Simplified assay set up with standardised components ensures robust real-time PCR reactions • TaqMan ® MGB Probes Easy and efficient design of shorter probes with very high specificity due to Minor Groove Binder Special Package Pricing* promotions Start off your Gene Expression Studies! Reagents and disposables Switzerland Denmark UK Norway Sweden E CHF DKK GBP NOK SEK $** Your Package Price 71.779,50 110.937,50 535.690,00 44,654.40 591.855,00 621.190,00 64.217,50 You save 9.629,50 14.871,50 71.790,00 5,984.40 79.355,00 83.090,00 9.317,50 % discount 12% 12% 12% 12% 12% 12% 12% *Terms & conditions for this offer: Offer available until 30 June 2003. No other discounts apply. All prices exclude delivery and local tax. E&OE. **For pricing in other non EU countries. ABI PRISM 6100 Nucleic Acid PrepStation ABI PRISM 7000 Sequence Detection System For more information on: ABI PRISM 6100 Nucleic Acid PrepStation enter: No. 426 Gene Expression Package enter: No. 428 ABI PRISM 7000 Sequence Detection System enter: No. 427 Real-Time PCR Information Pack enter: No. 429 37
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Issue 4 Summer 2002 - Applied Biosystems

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