Agilent 5 -Nuclease PCR Assays for Foodborne Pathogen Detection using the Agilent-Stratagene Mx3000P Q-PCR System Manual
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1. The predicted 88 bp amplicon contains sequence complimentary to the detection probe at positions 1346 to 1382 Table 2 The primerprobe sets used for the specific detection of Escherichia coli 0157 H7 Listeria and Campylobacter were extracted from previously published research 3 4 5 The TaqMan probe reporter dye for all assays was 6 carboxyfluorescein FAM and the quencher was a minor groove binding non fluorescent quencher MGBNFQ Applied Biosystems Inc Sequence 5 3 5 nuclease PCR assay The amplification reaction mixtures 25 ul contained template DNA 1000 pg 0 001 pg in 1 pl 1X Brilliant Multiplex Q PCR Master Mix Stratagene 30 nM ROX reference dye 0 3 uM each primer and 0 1 uM TaqMan probe 0 05 uM for the C jejuni assay All reactions were performed in triplicate and run on the Mx3000P thermal cycler system Stratagene The amplification profile for all assays was 1 cycle at 95 C for 10 min followed by 40 cycles at 95 C for 30 s and 60 C for 1 min Fluorescent signals were read using 516 nm FAM and 610 nm ROX filters at the end of each annealing extension step Data analysis was carried out with MxPro software Stratagene S enterica F CGT GTC CTT TGG TAT TAA TCC AAC AAT C R CCG GAG TTT CTC CCC CTC TTC E coli 0157 H7 F ATC AGC AAG GTA GCA GTC AGT ATT TCT GGG TAA CGC A TTT CAC ACT TAT TGG ATG GTC TCA R TGA GTT TAT CTG CAA GGT GAT TCC L monocytogen2. Food samples require an enrich ment step that incubates microbes if present before DNA extraction and analysis Hence absolute quantifica tion that precisely measures the extent of contamination in the initial raw sample is normally not possible for any detection method but Q PCR analysis does allow absolute quantification of enriched pathogens in unknown samples and thus gives a relative measure of contamination in the original samples Conclusions This study describes the development of sensitive and specific detection assays for four common foodborne pathogens A key component of the assays is combining gene amplifica tion detection and data analysis steps by using the Agilent Stratagene Mx3000P Q PCR platform Relative to traditional microbiological cultur ing methods for food testing our molecular detection approach offers enormous potential for reducing food analysis time from days to hours Next generation multiplexed detection assays will incorporate the integrated four color technology of the Agilent Stratagene Mx3000P Q PCR system to provide simultane ous detection of multiple pathogens in one reaction tube References 1 Mead P S et al Food related illness and death in the United States Emerg Infect Dis 5 607 25 1999 2 Galan J E Ginocchio C amp Costeas P Molecular and functional characterization of the Sa monella invasion gene invA homology of InvA to members of a new protein fa
3. resulted in a detection limit of 10 fg rxn The most sensitive Q PCR assays that use genomic DNA to detect single copy genes have detection limits between 1 10 fg rxn The quantitative aspects of each assay were evaluated by correlating log input DNA with threshold cycle signals Ct for each dilution series Figure 2 Linear regression analysis was performed on each resultant standard curve in order to obtain a regression square coefficient all of which fell within 0 996 and 1 000 Thus the regression line for each assay approximates real data points with little error The slopes of the curves were used to calculate the Q PCR reaction efficiencies which were found to be between 93 5 101 3 depending on the assay These results suggest that these assays could be used to accurately measure the load of contamination by these four infectious agents in unknown samples by interpolation of ras TOUBHEVHEABSADAND SYD Cyche number rsSTHOUNBS VT PARAM aH DHT wD PISTFTINBVGESVTVBABSASABBIHB Cycle number Cycle number Figure 1 Amplification sensitivity for the 5 nuclease PCR foodborne pathogen detection assays A range of known concentrations of genomic DNA from S enterica E coli 0157 H7 L monocytogenes all at 1000 pg 0 01 pg and C jejuni 1000 pg 0 001 pg were used in singleplex 5 nuclease PCR assays containing the primer probe set for the target gene Each trace represents the average of tripl
4. Authors Greg Richmond Lisa Gorski Dan Ryan Mary McBride Molecular Preparation Agilent Laboratories Santa Clara CA 95051 2 Produce Safety and Microbiology Research Unit USDA ARS WRRC Albany CA 94710 5 Nuclease PCR Assays for Foodborne Pathogen Detection using the Agilent Stratagene Mx3000P Q PCR System Application Food Safety Abstract Nucleic acid based assays are increasingly being applied as a rapid alterna tive to conventional culture techniques to test for pathogenic microbial contaminants in food This study details the use of real time quantitative PCR as a tool to detect four major foodborne bacteria that cause human disease Primers and probes were used to develop assays that specifically target the invA rfbE hlyA and Cj0414 genes from Salmonella enterica Escherichia coli 0157 H7 Listeria monocytogenes and Campylobacter jejuni respectively The detection limit for these assays was between 1 10 fg genomic DNA per PCR reaction quantification was linear over 6 7 log units Results can be obtained with these assays in 2 5 h following enrichment When used in tandem with an appropriate sample preparation technique this method may be suitable for food safety applications Introduction Foodborne disease is estimated to cause 76 million illnesses and 5 000 deaths annually in the United States 1 Of the known pathogens that cause foodborne illness Salmonella Escherichia coli 0157 H7 Listeria and Ca
5. es F TTC TAA CTA GGA CCG CAG AGG AAA GAG AGG AAT TA TGC AAG TCC TAA GAC GCC A R CAC TGC ATC TCC GTG GTA TAC TAA C jejuni F CGA TTT CAT CCG CGT GTT TCT TTT CG CTG AAT TTG ATA CCT TAA GTG CAG C R AGG CAC GCC TAA ACC TAT AGC T probe TCT CCT TGC TCA TCT TTA GGA TAA ATT CTT TCA CA The 5 reporter dye for all probes is FAM the 3 quencher dye for all probes is MGBNFQ Table 2 Primers and TaqMan probes used in real time quantitative PCR Target Amplicon size bp Ref invA 88 rfbE 85 3 hylA 113 4 Cj0414 86 5 Results and Discussion Individual primer probe sets were used in 5 nuclease Q PCR reactions containing purified genomic DNA to successfully amplify and detect the target gene from each of the four foodborne pathogens Figure 1 A fluorescent signal was observed for each reaction that contained DNA whereas the no template control NTC samples all gave negative results To evaluate the sensitivity of the primer probe combinations DNA from each species was serially diluted 10 fold in buffer and each dilution was subjected to PCR Seven dilutions for Q PCR amplification were created for each species utiliz ing a dynamic range that spanned seven log units between 1000 and 0 001 pg DNA reaction Only the Campylobacter assay was sensitive enough to detect a signal in the most dilute sample this assay has a detec tion limit of 1 fg rxn The Salmonella Escherichia coli 0157 H7 and Listeria assays all
6. etic Listeria monocytogenes Li2 19115D 5 nuclease PCR assays designed to Campylobacter jejuni RM3193 BAA 1234D 5 individually detect four major food borne pathogens The assays were designed by researchers at the USDA Western Regional Research Center in Albany CA and are being tested as part of an on going collaboration to develop rapid detection approaches to food borne pathogens The first gen eration assays are intended for use in research laboratories but these may also find utility as tools to aid in the USDA s response to outbreaks of foodborne illnesses The assays were conducted using an existing Agilent Stratagene Q PCR platform Brilliant Q PCR Master Mix and the Mx3000P thermal cycler system and assay performance was evaluated using the Agilent Stratagene MxPro software Materials and Methods DNA primers and probes Genomic DNA from four major food borne pathogens was purchased from ATCC Table 1 Ten fold serial dilu tions of genomic DNA were prepared in 1X TE 10mM Tris 1 mM EDTA pH Species Table 1 Bacterial isolates used as a source of genomic DNA 8 0 A primer and TagMan probe set was designed for Sa monella enterica using Primer Express v2 0 Applied Biosystems Inc to target the chromosomal invasion gene invA which mediates Salmonella entry into epithelial cells 2 PCR primers were constructed from the regions including positions 1317 to 1344 forward and 1384 to 1404 reverse
7. icate PCR reactions The cycle number is plotted as a function of the baseline subtracted fluorescent reading normalized to the reference dye dRn NTC no template control 0 998 101 3 Slope 3 290 Ct da ERGRERRUTESKEKRGERKSREBE lee SUEYCNSREKRNHUESKKE Bee Ras oot on 1 L wo ww initial quantity of genomic DNA per PCR reaction pg a a y 3 a v s u 3 d p 2 s 3 A 0 2 n on a v 5 a 2s 5 z a a A 2 2 n 2 EJ 19 v an or 1 x 00 2000 oon oo ar 2 w 10 xo initial quantity of genomic DNA per POR reaction pg initial quantity of genomic DNA per POR reaction pe Figure 2 Threshold cycle analysis of serial 10 fold dilutions of foodborne pathogen DNA A range of known concentrations of genomic DNA from S enterica E coli 0157 H7 L monocytogenes all at 1000 pg 0 01 pg and C jejuni 1000 pg 0 001 pg were used in singleplex 5 nuclease PCR assays containing the primer probe set for the target gene The starting quantity of DNA from each serial dilution is plotted as a function of threshold cycle Ct values to obtain a standard curve Each trace represents the average of triplicate PCR reactions The straight line is calculated by linear regression similar standard curves These results also demonstrate the advantage of Q PCR over conventional and real time PCR in obtaining a model with high predictive power that can be used to detect and quantify pathogen load
8. mily J Bacteriol 174 4338 49 1992 w Cooley M et al Incidence and tracking of Escherichia coli 0157 H7 in a major produce production region in California PLoS ONE 2 e1159 2007 4 Nogva H K Rudi K Naterstad K Holck A amp Lillehaug D Application of 5 nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures water skim milk and unpasteurized whole milk Appl Environ Microbiol 66 4266 71 2000 o Nogva H K Bergh A Holck A amp Rudi K Application of the 5 nuclease PCR assay in evaluation and development of methods for quantitative detection of Campylobacter jejuni App Environ Microbiol 66 4029 36 2000 TaqMan is a registered trademark of Roche Molecular Systems Inc Find an Agilent customer center in your country www agilent com chem contactus U S and Canada 1 800 424 5444 x3 agilent_inquiries agilent com Asia Pacic adinquiry_aplsca agilent com Europe info_agilent agilent com Agilent Technologies Inc September 2008 Printed in USA November 2008 5990 3161EN gt Agilent Technologies
9. mpylobacter are among the short list of micro organisms that exert the largest disease burden Bacteriological culturing has traditionally been used to detect microbial patho gens in food and this technique is still in widespread use today However the time consuming and low throughput nature of culture based detection methods warrant their replacement by more rapid methods based on biomolecular analysis like the polymerase chain reaction PCR PCR predicated on DNA synthesis and amplification is an established core technique in molecular biol ogy microbiology diagnostics genetics and environmental science applications A derivative of PCR real time quantitative PCR Q PCR kinetically monitors the reaction in real time Q PCR confers significant advantages over conventional PCR including increased reaction speed sensitivity and specificity it also eliminates the need to open the reaction tubes for post PCR analysis thus preventing cross contamination Q PCR is a fluorogenic probe based 5 nuclease assay that provides a higher level of specificity than conventional PCR thus assuring the correct DNA target is amplified and it is the preferred method to EE Agilent Technologies measure starting amounts of DNA in Species Serotype Strain ATCC number a sample Salmonella enterica Typhimurium LT2 700720D 5 In this study we describe the devel Escherichia coli 0157 H7 RIMD 0509952 BAA 460D 5 opment and composition of kin
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Agilent 5 Nuclease PCR Assays for Foodborne Pathogen Detection using the Agilent Stratagene Mx3000P