Journal of Food and Nutrition Research (ISSN 1336-8672) Vol. 55, 2016, No. 4, pp. 325–329
Detection method based on real-time polymerase chain reaction for celery (Apium graveolens) in beverages and dehydrated soups
Ľubica Piknová – Veronika Janská – Peter Siekel
Summary According to European legislation, food ingredients with potential allergenic properties, including celery (Apium gra- veolens), have to be declared. In order to provide an appropriate detection method for specific matrices, a method based on real-time polymerase chain reaction (PCR) for celery in beverages and dehydrated soups was developed and validated. Three varieties of celery frequently processed in food production were used for artificial contamination of vegetable and fruit beverages as well as instant soups in order to prepare model samples. Detection limits of 2.8–9.2 pg celery DNA per sample (200 mg of food sample) for all three varieties were reached. Inclusivity and exclusivity of the real-time PCR system was 100 %. A practical detection limit of 10 mg·kg-1 was determined for artificially contaminated beverages and soups. Real samples of fresh pressed vegetable and fruit juices with and without the celery component were analysed with identical results stated by the manufacturer.
Keywords allergen; detection; Apium graveolens; celery; real-time polymerase chain reaction
Celery (Apium graveolens) belongs to the profilin allergen families. The cross-reactive car- Apiaceae family (or Umbelliferae), three varieties bohydrate determinats also seem to be allergenic being mostly used in food production: Apium gra- [1]. Allergic symptoms occurred after ingestion veolens L. var. rapaceum (Mill.) Gaud. (celeriac, of celery within 30 min to 1 h in 46 % and as little celery root), Apium graveolens L. var. secalinum as 700 mg of celery induced allergic symptoms in Alef. (Chinese, leaf or cutting celery) and Apium 48 % of patients [2]. graveolens L. var. dulce (Mill.) Pers. (celery). As the only way of allergic reaction preven- Celery is consumed raw, cooked or dried as a spice tion, for sensitive persons, is avoiding of con- [1]. However, IgE-mediated reactions to celery sumption, all allergenic food components have are common in food allergic adults in Europe to be declared. Celery and products thereof must and it can induce allergic reactions of immediate always be declared according to the European type, from oral contact, skin contact (urticaria), legislation presented in Directive 2003/89/EC and to anaphylactic shock, if consumed. Sensitization Regulation (EU) No 1169/2011 [3, 4]. Control of to celery is frequently associated with birch and/ declared food components requires appropriate or mugwort pollinosis, hence the term „birch- diagnostic methods. Besides direct immuno- mugwort-celery-syndrome“ was established. Birch chemical methods, which detect the allergenic pollen and celery allergy are highly incidential in component(s), most of the detection methods are Central Europe, while celery allergy is most fre- based on polymerase chain reaction (PCR) [5–9] quently related to mugwort pollen in Southern Eu- or multiplex PCR [10–16]. rope [2]. Production and consumption of fresh fruit and The major celery antigen Api g 1 belongs to vegetable juices and beverages became very popu- Bet v 1 family. Other identified allergens are lar in European countries, including Slovakia. Pro- Api g 2, Api g 4 and Api g 6 from prolamin and duction lines should be cleaned and sanitized to be
Ľubica Piknová, Veronika Janská, Peter Siekel, National Agricultural and Food Centre, Food Research Institute, P. O. Box 25, Priemyselná 4, 824 75 Bratislava, Slovakia. Correspondence author: Ľubica Piknová, e-mail: [email protected]
© 2016 National Agricultural and Food Centre (Slovakia) 325 Piknová, Ľ. – Janská, V. – Siekel, P. J. Food Nutr. Res., Vol. 55, 2016, pp. 325–329 free from allergens if several products of different were homogenized in HGB2WTS3 mixer with composition are produced. Sanitation procedures a stainless steel container (Waring, Torrington, of production facilities needs to be safe also from Connecticut, USA) fro approx. 5 min. Aliquots of the allergen point of view as in the same manu- the celery powder from all three varieties, alterna- facture several products of different composition tively, were added to homogenized instant soups are produced. Effectiveness of cleaning has to be to gain identical celery component contents as it checked by appropriate methods. was in the case of artificially contaminated drinks In this study, the method of celery detection (100 000, 10 000, 1 000, 100, 10, 1 and 0.1 mg·kg-1). in beverages, starting from the preparation of raw material to Taqman real-time PCR detection, Real food samples was provided. The model samples of artificially Real samples of green smoothie pure vegeta- contaminated non-celery beverages were pre- ble and fruit (containing celery), 100% juice from pared, as well as real samples were used. Target- pressed vegetable mixture with celery, 100% juice ing development of comprehensive method also from pressed vegetable mixture without celery, another matrix, instant vegetable soup, was used, 100% fruit and vegetable juice, and 100% juice as it is one of mostly produced celery-containing from pressed fruit mixture were kindly provided by products in food industry. The sequences for real- the Slovak producer of fresh juices. time PCR primers and probe oriented to the man- nitol dehydrogenase gene were utilized [17]. The DNA extraction concentration of extracted DNA was determined DNA from leaf celery for determination of fluorimetrically and amplifiability was checked by the detection limit as well as DNA for determi- the 18S rRNA-oriented real-time PCR method nation of selectivity (leaves from parsley, parsnip, [18]. The comprehensive detection method en- dill, coriander; all from the market in Bratislava, compassed the sample preparation, DNA extrac- Slovakia) were extracted by chaotropic solid- tion, optimization and intralaboratory validation phase extraction (SPE) using DNeasy Plant Mini of real-time PCR analysis. Kit (Qiagen, Hilden, Germany). DNA for detec- tion limit experiments from celeriac and celery and DNA from food for selectivity testing (po- Materials and methods tato, carrot, kohlrabi, leek, onion, garlic, paprika, pumpkin, soya, maize, wheat, chives, cumin, beef, Materials for artificial contamination chicken, pork; all from the market in Bratislava, Celery (Apium graveolens L. var. dulce), Slovakia) were extracted by chaotropic SPE using celeriac (Apium graveolens L. var. rapaceum) and Nucleospin Food Kit (Macherey Nagel, Düren, leaf celery (Apium graveolens L. var. secalinum Germany). The latter method was used also for Alef.) from the market in Bratislava (Slovakia) extraction of DNA from real food samples. Both were used for artificial contamination. Matrices kits were used according to manufacturers’ manu- from all three celery varieties for artificial con- als. Blank for the control of reagents and possible tamination of beverages and instant soups were contamination was included in every extraction manually grated using stainless steel cutter and experiment. Amplifiability of the extracted DNA dried as follows: celery and celeriac over 72 h, leaf was checked by universal eukaryotic real-time celery overnight. Matrices were dried at labora- PCR [18]. DNA concentration was determined tory temperature, being covered with filtration pa- fluorimetrically using Quant-iT PicoGreen Kit per to avoid contamination. Dried materials were (Invitrogen Molecular Probes, Eugene, Oregon, homogenized for approx. 10 min in a mortar with USA) with fluoresence measured by Tecan Safire2 a pestle, till the powder was obtained. fluorimeter (Tecan, Grödig bei Salzburg, Austria).
Preparation of model samples Apium graveolens L. specific real-time PCR Beverages were obtained from fruit and Real-time PCR assays were performed in mi- vegetable beverages producer in Slovakia and con- croplates with individual reaction volumes of taminated using celery root, celeriac or leaf celery 25 µl containing 5 µl of DNA, 1× reaction buffer powder. Samples of beverages with 100 000, 10 000, supplied with DNA polymerase, 2.5 mmol·l-1 -1 1 000, 100, 10, 1 and 0.1 mg·kg of a celery com- MgCl2, 200 µmol·l-1 dNTP mixture (Applied Bio- ponent were prepared. Artificially contaminated systems, Foster City, California, USA) and 1.5 U samples of beverages were incubated overnight of Cheetah Hot Start Taq DNA polymerase at laboratory temperature and DNA was then ex- (Biotium, Hayward, California, USA). Primers tracted. Instant soups (Tiha, Thuan An, Vietnam) Ag637F (AGC CTG TTT CCC GTA CGA
326 Detection of celery in beverages and dehydrated soups
GAT) and Ag749R (CTC ATC ACA CCG TAA terms of the lowest concentration reliably de- TCC AAA CAT) were used in concentrations of tectable in all 4 replicate analyses. Standard 500 nmol l-1, and the probe Ag690P (5’-FAM-TAC curves for celery (slope k = –3.39 ± 0.08, coef- ACG CTC ATC GTG ACT CAG CA-BHQ1) was ficient of determination R2 = 0.99) and celeri- used at a concentration of 200 nmol l-1 (oligonu- ac (k = –2.61 ± 0.1, R2 = 0.97) were linear and cleotides synthesized by Eurofins MWG Operon nearly identical. However, the calibration curve (Ebersberg, Germany). The reaction was carried for leaf celery was also linear (k = –3.29 ± 0.08, out in a real-time PCR thermal cycler ABI 7900 R2 = 0.99). The absolute LOD was 2.8 pg (1.6 hap- (Applied Biosystems) using a temperature pro- loid genome copy) for celeriac, 4.2 pg (2.4 haploid gramme as follows: initial denaturation at 95 °C genome copy) for celery and 9.2 pg (5.3 haploid ge- for 2 min, 45 cycles of denaturation at 95 °C for nome copy) for leaf celery, as the haploid genome 15 s and annealing at 60 °C for 60 s. Fluorescence size for Apium graveolens is 1.73 pg (1.69 × 109 bp) data were collected and processed using internal [19]. The determined values of LOD were found to software of the instrument. be comparable with other real-time PCR methods reported previously [7, 9, 12]. Parameters of practical analytical applicabil- Results and discussion ity were determined using model samples of ar- tificially contaminated vegetable and fruit juices The aim of this work was development of sam- and a dehydrated soup. The commodities used ple preparation, optimization and intralaboratory as model samples were found to be free of celery validation of the real-time PCR method for celery before the samples were used in experiments. The detection and its adaptation to the beverages and vegetable and fruit juices represent a complicated dehydrated soup analyses. The specific real-time PCR system was oriented to the target gene en- coding for mannitol dehydrogenase of celery. The Tab. 1. Selectivity of real-time PCR method amplification product had a length of 113 bp, the for detection of celery. real-time PCR exhibited 100% selectivity when Species Latin nomenclature Result tested all three variants of celery (celery, celeriac and leaf celery) and 100% selectivity when Celery Apium graveolens var. dulce + 20 different plant and animal samples were tested Celeriac Apium graveolens var. rapaceum + (Tab. 1). Selectivity test was implemented using Leaf celery Apium graveolens var. secalinum + DNA extracted in two replicates and each PCR Carrot Daucus carota – was run on in 6 replicate measurements. Species Parsley Petroselinum crispum – from Apiaceae family and food, which could be used along with celery, were chosen for selectivity Parsnip Pastinaca sativa – testing. Potato Solanum tuberosum – The procedure of grating, pressing and filtra- Onion Allium cepa – tion the juice from celery root was found to be Leek Allium porrum – laborious and time-consuming. The procedure Garlic Allium sativum – of grating, drying and powdering the celery root Chives Allium schoenoprasum – was found to have identical yield in DNA extrac- tion (data not shown) and was easier implement- Pumpkin Cucurbita pepo – ed. Thus, the latter was chosen for preparation Kohlrabi Brassica oleracea – of samples artificially contaminated by celery, Paprika Capsicum annuum – beverages as well as dehydrated soups. Amplifia- Maize Zea mays – bility of DNA was confirmed by universal eukaryo- Soy Glycine max – tic real-time PCR oriented to 18S rRNA gene. DNA-based detection limit was determined Wheat Triticum aestivum – by analysis of series of four times diluted samples Coriander Coriandrum sativum – of DNA extracted from celery, leaf celery and Cumin Carum carvi – celeriac, measured in 4 replicates. Standard curves Dill Anethum graveolens – were generated by plotting the average cycle Beef Bos taurus – threshold values against the logarithm of corre- Pork Sus scrofa domesticus – sponding DNA concentration in nanograms per microlitre (Fig. 1). Chicken Gallus gallus domesticus – Limit of detection (LOD) was expressed in (+) – positive PCR result; (–) – negative PCR result.
327 Piknová, Ľ. – Janská, V. – Siekel, P. J. Food Nutr. Res., Vol. 55, 2016, pp. 325–329
50 matrix for DNA extraction, which are however of Celery interest due to the increasing trend in their con- 45 Celeriac sumption. Developing a method for juice analysis
e Leaf celery l reflects the problems with these products being c 40 y produced in the same equipment as celery-free d c 35 juices and the necessity to monitor the efficiency h o l of sanitary procedures. The model sample of de- 30
T h r e s hydrated soup was used as it is the most often con- sumed celery-containing product available on the 25 market in Slovakia. 20 Results of beverages samples in Fig. 2 10-3 10-2 10-1 1 10 102 (celery k = –2.74 ± 0.09, R2 = 0.99; celeriac DNA concentration [ng·µl-1 ] k = –2.58 ± 0.21, R2 = 0.92; leaf celery Fig. 1. Calibration lines k = –2.54 ± 0.15, R2 = 0.96) and instant soup sam- for the celery-specific real-time PCR. ples in Fig. 3 (celery k = –3.23 ± 0.22, R2 = 0.94; 2 Three celery varieties were analysed. celeriac k = –3.05 ± 0.14, R = 0.97; leaf celery k = –2.83 ± 0.12, R2 = 0.97) present the deter- mined calibration lines. The practical detec- tion limit of 10 mg·kg-1 was achieved for all used celery varieties in beverages and in soups. This is 40 in accordance with other celery-specific real-time Celery PCR-based methods published previously [7, 9, Celeriac 12]. 35
e Leaf celery
l Real samples of beverages were also analysed,
c y with the aim to verify the developed method for d 30 celery detection. All samples had been pasteur- ized. Celery was declared on the package of two e s h o l samples of beverages. The developed method for T h r 25 celery detection was able to detect celery in both samples declared to contain it. We assume that 20 the method is suitable for celery detection in fruit 1 10 102 103 104 105 106 and vegetable freshly pressed beverages and also Content [mg·kg-1 ] in other food products, such as dehydrated soups. Fig. 2. Practical detection limit of the celery-specific real-time PCR using an artificially contaminated fruit Acknowledgements and vegetable juice. This research was carried out in frames of the project 471/2014-310 „Authentication of products and cross-contamination characterization of plant aller- gens in production processes applying molecular-bio- logical methods“ of the Ministry of Agriculture and Regional Development of the Slovak Republic. It is 40 a result of implementation of the project of ITMS Celery 26240120013 “Establishment of a HiTech Centre for Celeriac Research on Formation, Elimination and Assessment 35
e Leaf celery l of Contaminants in Food” supported by the Research
c y & Development Operational Programme funded by the d 30 ERDF. e s h o l T h r 25 References
1. Scientific Opinion on the evaluation of allergenic 20 1 10 102 103 104 105 106 foods and food ingredients for labelling purpos- Content [mg·kg-1 ] es. EFSA Journal, 12, 2014, pp. 158–163. DOI: 10.2903/j.efsa.2014.3894. Fig. 3. Practical detection limit of the celery-specific 2. Ballmer-Weber, B. – Besler, M. – Hoffmann- real-time PCR using artificially contaminated dehy- Sommergruber, K. – Vieths S. – Wuthrich, B.: drated soup. Allergen data collection: Celery (Apium graveolens).
328 Detection of celery in beverages and dehydrated soups
Internet Symposium on Food Allergens, 2 (3), 2000, 2009, pp. 367–374. DOI: 10.1007/s00217-009-1164-3. pp. 145–167. ISSN: 1437-0573.
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