Development of a Multiplex Real-Time PCR for Determination of

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Development of a multiplex real-time PCR for determination of apricot in marzipan using the PLEXOR® System Stefanie Schelm, Ilka Haase, Christin Fischer, and Markus Fischer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04457 • Publication Date (Web): 12 Dec 2016 Downloaded from http://pubs.acs.org on December 19, 2016

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Journal of Agricultural and Food Chemistry

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Development of a multiplex real-time PCR for determination of apricot in marzipan using the PLEXOR® System

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Stefanie Schelm1, Ilka Haase1, Christin Fischer1 and Markus Fischer1*

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Grindelallee 117, 20146 Hamburg, Germany, *Corresponding author: Tel.: +49-40-

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428384357; Fax: +49-40-428384342; E-Mail: [email protected]

HAMBURG SCHOOL OF FOOD SCIENCE; Institute of Food Chemistry, University of Hamburg,

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1 ACS Paragon Plus Environment


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ABSTRACT

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Marzipan is a confectionary which is mostly offered in form of filled chocolate, pralines or

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pure. According to the German guidelines for oil seeds only almonds, sugar and water are

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admitted ingredients of marzipan. A product very similar in taste is persipan which is used in

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the confectionary industry because of its stronger flavor. For persipan production almonds

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are replaced by debittered apricot or peach kernels. To guarantee high quality products for

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consumers, German raw paste producers have agreed a limit of apricot kernels in marzipan

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raw paste of 0.5 %.

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Different DNA based methods for quantitation of persipan contaminations in marzipan are

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already published. In order to increase the detection specificity compared to published

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intercalation dye based assays, the present work demonstrate the utilization of a multiplex

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real-time PCR based on the Plexor® technology. Thus the present work enables the detection

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of at least 0.1% apricot DNA in almond DNA or less. By analyzing DNA-mixtures, the

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theoretical limit of quantification of the duplex PCR for the quantitation of persipan raw

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paste DNA in marzipan raw paste DNA was determined as 0.05%.

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KEYWORDS

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marzipan, persipan, multiplex real-time PCR, Plexor® technology, Prunus

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INTRODUCTION

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Marzipan raw paste and sugar are the main ingredients for marzipan. Marzipan raw paste

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mainly consists of blanched, peeled, ground almonds (Prunus dulcis), water, and sugar. The

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ingredients were mixed and heated up during manufacturing. The confectionary marzipan is

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used in chocolate, pralines or offered pure as marzipan-potatoes or –figures.

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Persipan, which exhibits quite similar organoleptic properties to marzipan, shows a more

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intense flavor compared to marzipan. Thus it is used nowadays in confectionery instead of

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marzipan.

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The main difference between persipan and marzipan is the replacement of almonds by

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blanched peeled and if necessary debittered bitter almonds (P. dulcis phenotype bitter),

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apricot (P. armeniaca) or peach (P. persica) kernels during the manufacturing.

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In Germany production parameters for marzipan raw paste were defined in the guidelines

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for oil seeds.1 According to these guidelines the usage of apricot kernels in marzipan raw

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paste is not intended. The presence of apricot kernels in marzipan raw paste may be caused

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unintentional by contaminations during manufacturing by the use of the same production

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lines for marzipan and persipan raw paste. Otherwise, a deliberate addition of apricot

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kernels is also conceivable to reduce raw material costs.2 To guarantee high quality products

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for consumers, German raw paste producers have defined a limit of apricot kernels in

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marzipan raw paste of 0.5 %. Compliance with legal requirements protects the raw paste

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manufacturing industry from a loss of image and prevents for cost-intensive recall

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campaigns. Quantitative methods for the analysis of apricot kernel contents in marzipan raw

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paste are also an important requirement for quality control of incoming raw material for

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marzipan producing companies.



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Different methods for detecting apricot kernels in almond/marzipan are based on the

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tocopherol or protein pattern. These methods allow a distinction between almond and

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apricot, but the required detection limit of 0.5% is not reached.3, 4

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To quantify the amount of apricot kernels in marzipan, quantitative molecular biological

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DNA-based methods were developed. Based on sequence differences between the gene for

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the lipid transfer protein I in almond and apricot a semi-quantitative real-time PCR method

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and a ligation dependent probe amplification assay were published.5, 6 To achieve a lower

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limit of detection, Haase et al. (2013) developed a multiplex real-time PCR method based on

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the multi copy gene for ribosomal DNA (rDNA) using the intercalating dye SYBR Green I.7

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DNA-binding dyes like SYBR Green I intercalate nonspecifically into any double-stranded DNA

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generated in the PCR reaction. Hence, the specificity of such approaches is only defined by

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the specificity of the primers. This leads to an increased susceptibility for the co-detection of

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non-specific fragments and subsequently to false positive results. .8-11

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To avoid the detection of unspecific products, fluorescence-labeled primers ore probes (e.g.

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TaqMan®) can be used. Both systems have the advantage that they generate a sequence

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specific fluorescence so that nonspecific amplicons are not detected. In addition, such

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systems enable the design of multiplex assays for a simultaneous detection of multiple

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targets and an adjustment of results by normalization in a single PCR assay resulting in a

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higher accuracy of quantitative assays.12, 13 Furthermore, multiplex PCR assays using two or

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three primer pairs simultaneously are less time consuming and require less laboratory

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materials.

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For the design and use of a specific TaqMan® probe, sequence differences between the

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amplified sequence targets of almond and apricots would be necessary. However, as


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described in Haase et al., a design of a specific probe is not possible due to the close

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relationship and high sequence similarity of the two species.7

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Therefore, the aim of this study was the development of a fluorescence-labeled primer-

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based multiplex real-time PCR based on the Plexor® technology for detection and

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quantitation of persipan contaminations in marzipan. To reach a lower detection limit, the

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PCR is based on the rDNA.7, 14, 15

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The Plexor® technology is used for a wide range of applications.16-19 It works with a

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fluorescently-labeled primer synthesized with a modified nucleotide 5´-methylisocytosine

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(iso-dCTP). The iso-dCTP interacts with another modified nucleotide bound to the quencher

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4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl). During PCR, dabcyl quenches the

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fluorescence dye signal (a simplified scheme is represented in Figure S1). The decrease of

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fluorescence is direct proportional to the amount of amplified DNA, which enables

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quantitative assays.20-22

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It’s the first time, that a method is published, which uses the PLEXOR® technology for the

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authenticity control of marzipan. Furthermore it’s the first approach to develop a multiplex

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real-time PCR assays for a simultaneous detection of apricot in marzipan and an adjustment

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of results by normalization in a single PCR assay enabled by the use of fluorescently-labeled

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primers.

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MATERIALS AND METHODS

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Sample material

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Different types of almonds (purchased from the USA) as well as apricot kernels (purchased

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from China) and raw paste (made from Californian and Mediterranean almonds, or Chinese,

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Iranian, Turkish or Syrian apricot kernels, respectively) as well as marzipan and persipan

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were analyzed. The samples were taken from a previous project which is described in

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Brüning et al (see there for further sample details).2 For specificity tests, fresh frozen leaves

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and kernels from different Prunus species (almond, apricot, peach, cherry = P. cerasus) were

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used.

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DNA-Isolation

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Prior to DNA isolation from kernels, kernels were ground to a fine powder with a moulinette

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(La Moulinette, 800 W, Tefal Groupe SEB Deutschland GmbH, Germany). DNA-isolation from

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this powder or from marzipan/persipan was performed according to the protocol

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“Precipitation with isopropyl alcohol and subsequent silica adsorption” published by Brüning

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et al. (2011).2 Afterwards, DNA purity was determined photometrically (ratio at 260/280 nm)

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using a NanoDrop (Thermo Fisher Scientific Oy, Finland). The DNA concentration was

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determined fluorimetrically (SpectraMax M2, Molecular Devices, Sunnyvale, USA) using an

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external calibration with plasmid DNA.

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Preparation of spiked sample material

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To evaluate the calibration parameters for the developed real-time PCR, different spiked

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samples were prepared. By performing first experiments with mixtures of DNA from


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almonds and apricot kernels and DNA from marzipan raw paste and persipan raw paste,

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possible matrix effects were minimized.

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Isolated DNA was adjusted to a concentration of 5 ng/µL and blended in different ratios

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(series 1: 0.1 %, 1%, 5 % and 10% apricot DNA in almond DNA; series 2: 0.05%, 0.5%, 5%,

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12.5%, 25% apricot DNA in almond DNA, series 3: 0.5%, 5%, 25%, 50%, 75%, 95%, 99.5%

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apricot DNA in almond DNA).

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Spiked samples of almonds with apricot kernels were produced by mixing ground almond

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kernels with ground apricot kernels in different ratios (0.5%, 2.5%, 5%, 25%, 50% apricot

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kernel powder in almond powder). Subsequently, the DNA was isolated and adjusted to

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5 ng/µL.

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To prepare spiked samples of raw pastes, marzipan and persipan raw pastes were mixed in

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various proportions (40 g in total) using an unguator (Cito UNGUATOR® e, GAKO® Konietzko

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GmbH, Germany) for two minutes by 1260 rpm (persipan raw paste in marzipan raw paste:

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0.5%, 2.5%, 5%, 25%, 50%). In addition, a “blind study” was performed using four marzipan

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raw paste samples that were spiked with different amounts of persipan raw paste by a third

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person and provided to the authors. Spiking amounts (0.4%, 0.7%, 1.9%, 18.0%) were

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handed over to the authors after finishing the experiments.

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Isolated DNA from spiked raw paste samples was also adjusted to a concentration of

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5 ng/µL.

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Primer-Design

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For the development of a multiplex real-time PCR for the detection and quantitation of a

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possible apricot kernel content in almond/raw paste, different primer pairs were used. The

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apricot specific primer pair’s sequences targeting the ITS 1 region on Prunus rDNA (PA7 ACS Paragon Plus Environment


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fw/PA-rv, Table 1) were taken from Brüning et al. (2011).2 To quantify the apricot DNA

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content in relation to the total DNA content, or the almond DNA content respectively, one

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universal and one almond specific primer pair was designed (PD-fw/PD-rv, U2-fw/U2-rv,

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Table 1). The universal primer pair targets the ITS 2 domain, whereas the almond specific

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primer pair targets the ITS 1 domain on Prunus rDNA.

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For real-time PCR assays using the Plexor® technology the forward primers were linked to

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specific fluorophores. Regarding their absorption and emission maxima the fluorophores

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were selected, taking care, that no overlapping of maxima occurs. The fluorescently labeled

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primers were obtained from Integrated DNA Technologies, Inc., Coralville, IA, USA. All used

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primer sequences including the fluorophores are listed in Table 1 (PA-Pl-fw, PD-Pl-fw, U2-Pl-

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fw).

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Polymerase chain reaction

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Possible cross reactions between used primers in multiplex PCR as well as specificity of

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specific primers were tested via endpoint PCR using DNA from different Prunus species. The

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following reaction mix in a total volume of 20 μL was set up for PCR: 1× Taq buffer (20 mM

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MgCl2·6H2O, 100 mM Tris-HCl, 500 mM KCl, 1% Triton X-100 (m/v), pH 8.8), 0.8 mM dNTPs

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(each 2.5 mM, Bioline GmbH, Luckenwalde, Germany), 1 unit Taq polymerase (in-house

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generated, isolated from Escherichia coli), 1 μM of each primer (Thermo Fisher Scientific,

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Waltham, MA, USA). The following thermal cycle profile was used: initial denaturation at

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95 °C for 180 s, 35 cycles with denaturation at 95 °C for 20 s, annealing at 59 °C for 20 s,

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elongation at 72 °C for 20 s and final elongation for 180 s at 72 °C. Resulting PCR products

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were visualized on 3% agarose gels (170 V, 20 minutes) stained with 0.001% ethidium


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bromide. Stained gels were documented under UV light (254 nm, Biostep, Felix 1040,

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Biostep GmbH, Jahnsdorf, Germany).

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Real-time PCR using Plexor® technology

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For real-time PCR an iQ5 real-time PCR thermocycler (Bio-Rad Laboratories GmbH, Munich,

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Germany) or LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany) was used. Real-time

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PCR was performed with the following temperature program: initial denaturation for 180 s

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at 95 °C followed by 40 cycles with 20 s denaturation at 95 °C, 20 s annealing for 59 °C, 20 s

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elongation at 72 °C finished by final elongation at 72 °C for 600 s.7 Afterwards a melting

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curve analysis (55 − 95 °C, 1 °C/s) was performed. After optimization the time of

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measurement, the fluorescence was detected at 72 °C, during elongation.

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After optimization the PCR conditions for multiplex real-time PCR with the Plexor®

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technology, the PCR reaction was carried out in a total volume of 20 μL, including 20 ng of

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DNA and 10 μL of Plexor® Master Mix (qPCR system, Promega Corporation, Madison, USA).

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The primer concentrations used for multiplex real-time PCR with the Plexor® technology are

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listed in Table 2.

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Method validation

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To verify the robustness of developed real-time PCR method, the relative repeatability

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standard deviation (RSDr), the limit of detection (LoD) and the limit of quantitation (LoQ)

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were determined using the Plexor® technology, regarding the standards of European

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Network of GMO (Genetically Modified Organisms) Laboratories (ENGL).23

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The within-laboratory repeatability represented by the RSDr is defined as the standard

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deviation of measured data under repeatability conditions, using the same method and 9 ACS Paragon Plus Environment


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equipment on identical test items in the same laboratory by the same operator within short

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intervals of time.23 To determine the RSDr, a matrix standard of marzipan raw paste spiked

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with 0.51% persipan raw paste was analyzed thirty times by the same operator on the same

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day with the same equipment. Measurement was performed via duplex PCR with apricot

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specific (PA-Pl-fw/PA-rv) as well as universal (U2-Pl-fw/U2-rv) primers. The RSDr was

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calculated using the standard deviations (SD) and the average (xത) of CT values:

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RSDr [%] = 100 %/SD · xത.

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Limit of detection was calculated via real time PCR with apricot specific primers (PA-Pl-

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fw/PA-rv). Different samples of almond DNA spiked with 10%, 5%, 1% and 0.1% apricot DNA

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were analyzed six-times on LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany). The

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lowest concentration of apricot DNA which can be reliably detected was defined as LoD.

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Furthermore, DNA from marzipan raw paste spiked with 0.05% up to 25% DNA from

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persipan raw paste was analyzed in duplicate via duplex PCR using apricot specific primers

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(PA-Pl-fw/PA-rv) in combination with universal (U2-Pl-fw/U2-rv) primers to determine the

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LoQ. The sample with the lowest amount of persipan raw paste DNA showing a correlation

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between normalized CT values and persipan raw paste DNA content represents the

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theoretical LoQ of the duplex PCR.

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Results and Discussion

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DNA isolation

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In the present work DNA of unprocessed and processed apricot and almond kernels

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(marzipan, persipan and raw paste) was isolated. Therefore the method “precipitation with

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isopropyl alcohol and subsequent silica adsorption” published by Brüning et al. (2011)

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optimized for highly processed foods like raw pastes.2 In average, DNA isolation yields to a

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DNA concentration of 29.44 ng/µL for unprocessed kernels and 37.36 ng/µL for processed

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kernels, respectively. The ratio of absorbance at 260 and 280 nm was used to assess the

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purity of isolated DNA, in which values of 1.8 predict pure DNA.24 As the box and whiskers

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plot in Figure S2 implies, unprocessed and processed kernels show an average ratio at

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260 nm/280 nm of 2.0, indicating that isolated DNA is mainly free from proteins.

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Primer specificity

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For the quantitation of apricot content in marzipan raw paste an apricot specific primer pair

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(PA-fw/PA-rv) was adapted from Brüning et al. (2011) and the forward primer was

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fluorescently labelled (PA-Pl-fw).2 To enable a calculation of apricot content relative to

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almond content, almond specific primer pairs (PD-fw/PF-rv, PD-Pl-fw/PD-rv) were designed.

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After alignment-analysis, the 5.8S rDNA was chosen as target for primer design. Due to the

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fact that the rDNA is a multicopy gene a lower detection limit is expected.7, 14, 15 Primers

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were designed to amplify short PCR products in order to reliably detect also fragmented

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target DNA. Fragmentation of target DNA can be expected due to the thermal and mechanic

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steps during the manufacturing process.2

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Specificity of the created almond specific primer pair PD-fw/PD-rv was tested with DNA

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isolated from different Prunus spp. leaves via endpoint PCR. This almond specific primer pair 11 ACS Paragon Plus Environment


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shows positive results with almond (P. dulcis phenotype sweet and bitter) and peach (see

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Figure S3). Nevertheless, using this primer a reliable distinction between apricot and almond

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is possible. Results obtained with endpoint PCR were verified via real-time PCR analysis

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which was followed by a melting point analysis. Only with almond DNA as target a formation

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of almond specific PCR products was detected (see Figure 1). The characteristic almond

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specific products can be clearly identified by a higher melting point (85.6-85.9 °C) and lower

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CT values (CT values: 31.3 – 35.0) compared to the melting point of unspecific products

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generated with apricot DNA and the blank (melting point: 83.4 °C to 84.4 °C, CT values: 37.0-

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39.4).

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Primer suitability for multiplex PCR

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To detect potential cross reactions between the different primers during multiplex PCR, a

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duplex endpoint PCR was performed.

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First, the apricot specific primer pair PA-fw/PA-rv was tested in combination with the

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universal 1 primer pair U1-fw/U1-rv.2 As depicted in Figure 2 these primer combinations are

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unsuitable for duplex PCR due to the formation of an approximately 280 bp long unspecific

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amplicon. An alignment analysis indicates that the unspecific amplicon is a product of the

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apricot specific forward (PA-fw) and the universal 1 reversed (U1-rv) primer. To avoid

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undesirable cross amplifications a new primer pair (U2-fw/U2-rv, specifications see Table 1)

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based on the ITS 2 rDNA sequence from Prunus species was implemented. Compared to the

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universal 1 primer pair the universal 2 primer pair shows a larger distance to the apricot

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specific primers’ binding sides, thus cross reactions should be prevented. Figure S4 depicts

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that only primer specific PCR products with approximately 60 bp were detected in duplex

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PCR with the universal 2 and the apricot specific primer pairs, which proves the absence of 12 ACS Paragon Plus Environment

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cross amplification. Subsequently, the universal 2 primer pair was tested via endpoint PCR

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with DNA isolated from leaves of different Prunus species. Positive results with all tested

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plants were detected (see Figure S5), showing the suitability as a universal primer pair for

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the genus Prunus.

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Furthermore, the almond specific primer pair was tested in combination with the universal 2

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primer pair as well as the apricot specific primer pair. With these primer combinations only

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PCR amplicons with approximately 60 bp were detected (data not shown), which

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implements that no unspecific PCR products caused by cross amplifications are formed.

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Method validation

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To determine the RSDr, a matrix standard of marzipan raw paste spiked with 0.51% persipan

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raw paste was analyzed thirty times in duplex PCR with the Plexor® technology using the

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fluorescently-labeled apricot specific and universal 2 forward primers (PA-Pl-fw/PA-rv and

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U2-Pl-fw/U2-rv; specifications and fluorophores see Table 1). The RSDr was calculated using

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all obtained treshold cycles.

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With 0.84% (apricot specific primer pair) to 1.94% (universal 2 primer pair) the within-

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laboratory repeatability falls within the ENGL standards.23

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To determine the LoD, mixtures of almond DNA spiked with apricot DNA were analyzed in six

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fold determination with the apricot specific primer pair PA-Pl-fw/PA-rv. Because apricot DNA

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could be reliably detected in all analyzed samples, the LoD for this singleplex Plexor® system

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was determined to be at least 0.1% apricot DNA in almond DNA.

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In order to determine the linear working range and the theoretical LoQ of the developed

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duplex Plexor® PCR, mixtures of marzipan raw paste DNA with 25% - 0.05% persipan raw

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paste DNA were analyzed by real-time PCR in duplicate using both the apricot specific (PA-Pl13 ACS Paragon Plus Environment


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fw/ PA-rv) and the universal 2 (U2-Pl-fw/ U2-rv) primers. As shown in Figure 3, a correlation

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between normalized CT values and persipan raw paste DNA content is detected over the

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whole range of analyzed samples with an efficiency of E = 113 % (R2 = 0.9933). Thus, 0.05%

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persipan raw paste DNA in marzipan raw paste DNA can be defined as the theoretical LoQ of

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this duplex PCR system.

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Quality and purity of isolated DNA can be affected by co-extracted substances e.g.

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polysaccharides. The presence of such substances can result in inhibition of PCR reaction

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leading to a decrease of amplification efficiency. In further tests influences of the marzipan

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matrix on the calibration parameters were examined. Using an apricot DNA dilution series

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and different matrices samples amplification efficiency and coefficient of determination (R²)

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were determined.25

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First, an apricot DNA dilution series (75.2 ng – 0.024 ng) was prepared and analyzed by real-

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time PCR in triplicate with apricot specific primers (PA-Pl-fw/ PA-rv). A correlation between

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obtained CT values and DNA amount was observed over the whole concentration range with

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an amplification efficiency of E = 112% and a R² of 0,997 (data not shown).

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Afterwards, samples of almond kernels spiked with 0.5% up to 50% apricot kernels were

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prepared and analyzed in triplicate via duplex real-time PCR with apricot specific (PA-Pl-fw/

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PA-rv) and universal primers (U2-Pl-fw/ U2-rv). CT values obtained with both primers were

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plotted against the logarithm of the apricot content. An amplification efficiency of 77% and a

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R² from 0.990 was achieved with not normalized CT values from apricot specific primers.

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Subsequently, delta CT values (difference between the CT value of the apricot specific and

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the universal assay) were calculated. This normalization leads to an improvement of R² to

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0.998 with a 82% efficiency, indicating that normalization can compensate differences in

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total DNA amount by enhancing the coefficient of determination (data not shown). Because 14 ACS Paragon Plus Environment

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precision of calibration curves can be increased with a normalization step it should be

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performed in further quantitation assays.

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The analysis was repeated with samples of marzipan raw paste spiked with persipan raw

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paste (0.5% up to 50%), resulting in a calibration curve with an efficiency of 92% and a R² of

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0.984 from not normalized CT values (Figure 4A). After normalization an efficiency of 87%

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and a R² of 0.994 was achieved (Figure 4B).

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These results are in concordance with the results described above, demonstrating that

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normalization increases the accuracy of quantitation by enhancing the coefficient of

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determination, while the efficiency remains approximately constant. Furthermore, the

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results demonstrate that duplex real-time PCR enables quantitation of apricot respectively

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persipan raw paste content even in complex samples like marzipan raw paste.

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In addition to duplex real-time PCR a triplex real-time PCR with universal primers (U2-Pl-fw/

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U2-rv), almond (PD-Pl-fw/ PD-rv) and apricot (PA-Pl-fw/ PA-rv) specific primers was

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developed in the present work. Samples of almond DNA mixed with apricot DNA (0.5%, 5%,

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25%, 50%, 75%, 95%, 99.5% apricot DNA in almond DNA) were analyzed in triplex real-time

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PCR. Normalized CT values generated with specific primers were plotted against logarithm of

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almond/apricot content. Normalized CT values generated with the almond specific primers

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resulted in a calibration curve with a R² of 0.998 and an efficiency of 125%. Assays with

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apricot specific primers reveal 92% efficiency with a R² from 0.988 (data not shown).

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Obtained results demonstrate that the developed triplex real-time PCR opens up the

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possibility to analyze marzipan quality by calculating apricot content in complex samples

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even in presence of other Prunus spp. than almond (with the exception of peach, because of

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the cross-reaction of the almond specific primer pair with peach DNA) with almond specific

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primer and a compensation of total DNA amount using the universal 2 primer pair. 15 ACS Paragon Plus Environment


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Quantitation

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To determine a suitable quantitation method with the Plexor® technology, four matrix

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samples of marzipan raw paste spiked with a certain amount of persipan raw paste by a

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third person were analyzed in duplicate via duplex real-time PCR with apricot specific (PA-Pl-

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fw/ PA-rv) and universal (U2-Pl-fw/ U2-rv) primer pairs in a “blind study”. Calculation of the

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content of persipan raw paste was performed using (A) absolute quantitation with an

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external matrix calibration curve (marzipan raw paste spiked persipan raw paste) and (B)

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absolute quantitation with an external matrix calibration curve and normalization against

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total DNA amount using the universal primer pair. In addition to absolute quantitation, the

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calculation of the persipan raw paste amount was performed by (C) a relative quantitation

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using an external reference sample (ΔΔ CT method, matrix standard 0.5% persipan raw paste

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in marzipan raw paste) without the need of a calibration curve.26-28 The results of

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quantitation with different strategies are presented in Figure 5. Similar results were

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obtained by including the universal primer pair using the absolute quantitation strategy with

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normalization (B) as well as relative quantitation strategy via ΔΔ CT method (C). Absolute

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quantitation without normalization (strategy A) generally exhibits the most inaccurate

343

results. Furthermore, the results reveal that normalization using the universal primer pair

344

leads to an improvement of quantitative results by compensating differences in total DNA

345

amount or amplification efficiencies.29

346 347

Analysis of commercial samples

348

Sixteen commercially available samples containing marzipan (marzipan filled chocolate,

349

marzipan figures, marzipan loaves) were purchased. Samples were first analyzed in 16 ACS Paragon Plus Environment

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350

duplicates with the apricot specific primer pair PA-fw/PA-rv under the conditions published

351

elsewhere with the intercalating dye SYBR-Green I.7 One of the analyzed samples (marzipan

352

loaf) reveals positive results indicating that this sample contains apricot DNA. The positive

353

result was checked using Plexor® technology with apricot specific primers (PA-Pl-fw/ PA-rv).

354

In this experiment no apricot specific signal was observed. As the fluorescence-labeled

355

apricot specific primer generates an apricot specific fluorescence, the positive result

356

obtained with SYBR Green I appears to be false-positive. These results indicate the suitability

357

of the developed real-time PCR to control ambiguous results.

358

In conclusion, the developed multiplex real-time PCR method with the use of the Plexor®

359

technology facilitated a specific detection of apricot DNA in marzipan samples. The Plexor®

360

technology enables a sequence or species specific detection of the target of interest similar

361

to FRET probes, which reduces false-positive results and can improve the accuracy of the

362

real-time PCR results.

363

According to German food guidelines, persipan is made of apricot or peach kernels. Because

364

apricot kernels are commercially used instead of peach kernels, the developed method can

365

be used to quantify possible contaminations of persipan in marzipan. To cover also marzipan

366

contaminations with persipan made out of peach it is possible to create a peach specific

367

primer pair.

368

The use of a universal primer pair enables a normalization of real-time PCR results increasing

369

the precision of the results by compensating differences in total DNA amount of the

370

samples. The developed multiplex PCR enables a simultaneously quantitation of persipan

371

contaminations and a normalization what increases the precision of quantitative results and

372

minimizes the overall time needed.

373 17 ACS Paragon Plus Environment


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374

ABBREVIATIONS

375

dabcyl, 4-((4-(dimethylamino)phenyl)azo)benzoic acid; iso-dCTP, 5´-methylisocytosine; DNA,

376

deoxyribonucleic acid; PCR, polymerase chain reaction; rDNA, ribosomal DNA; GMO,

377

genetically modified organism; dNTP, deoxynucleotide; RSDr, repeatability standard

378

deviation; LoD, limit of detection, LoQ, limit of quantitation; ITS 2, internal transcribed

379

spacer 2; R², coefficient of determination; MRP, marzipan raw past; PRP, persipan raw paste.

380 381

ACKNOWLEDGEMENT

382

The authors gratefully thank Zentis GmbH & Co. KG, Moll Marzipan GmbH, Kessko, Kessler &

383

Comp. GmbH & Co. KG, Lubeca, Luebecker Marzipan-Fabrik v. Minden & Bruhns GmbH & Co.

384

KG, and Georg Lemke GmbH & Co. KG for providing sample material.

385 386

FUNDING

387

This research project was supported by the German Ministry of Economics and Technology

388

(via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e. V., Bonn, Germany), Project

389

AiF 15304 N.

390 391

SUPPORTING INFORMATION

392

DNA yield and DNA purity for different matrices; specificity tests for the almond specific

393

primers; agarose gel from duplex endpoint PCR with universal 2 and apricot specific primers,

394

agarose gel from endpoint PCR with universal 2 primers using isolated DNA from different

395

Prunus species.

396 397

REFERENCES 18 ACS Paragon Plus Environment

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(22) Sherrill, C. B.; Marshall, D. J.; Moser, M. J.; Larsen, C. A.; Daudé-Snow, L.; Prudent, J. R. Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence. J. Am. Chem. Soc. 2004. 126, 4550-4556. (23) Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing. http://gmo-crl.jrc.ec.europa.eu/ (24) Gallagher, S. R.; Desjardins, P. R. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Current Protocols in Human Genetics. 2007. A. 3D. 1-A. 3D. 21. (25) Cankar, K.; Štebih, D.; Dreo, T.; Žel, J.; Gruden, K. Critical points of DNA quantification by realtime PCR–effects of DNA extraction method and sample matrix on quantification of genetically modified organisms. BMC Biotechnol. 2006. 6, 1. (26) Bustin, S. A., AZ of quantitative PCR. International University Line La Jolla, CA: 2004. (27) Dorak, M. T., Real-time PCR. Taylor & Francis: 2007. (28) Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 2001. 29, e45-e45. (29) Meijerink, J.; Mandigers, C.; van de Locht, L.; Tönnissen, E.; Goodsaid, F.; Raemaekers, J. A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR. J. Mol. Diagn. 2001. 3, 55-61.

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TABLES AND FIGURES

TABLES Table 1: Primer Sequences, Accession Numbers and size of PCR products. Primers

Target

Name

Sequence 5’ – 3’

PA-fw GGGCCGTCTCGGCGT7 PA-Pl-fw 6-FAM//iMe-isodC/GGGCCGTCTCGGCGT* PA-rv TTGTACGCCCGGAAGGGTAT7 almond PD-fw GGTCCTGCGGCTCCTCGTC specific PD-Pl-fw Cy5//iMe-isodC/GGTCCTGCGGCTCCTCGTC* PD-rv CGGAAGGGTCGGCGCGAG universal 1 U1-fw GACTCTCGGCAACGGATATC7 U1-rv CGCAACTTGCGTTCAAAGACTCGA7 universal 2 U2-fw GTCGCGCATCGAGGGCTCGAA U2-Pl-fw 6TAMN//iMe-isodC/TGGGGTCGCGTTGAAAGCCGAG* U2-rv TGGGGTCGCGTTGAAAGCCGAG fw = forward; rv = reverse; *fluorescently labeled primer apricot specific

P. armeniaca AF318756 rDNA P. dulcis HE806329.1 rDNA all Prunus species rDNA all Prunus species rDNA

Product size (bp) 64

65

115 60



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Table 2: Combinations and corresponding concentrations for real-time multiplex PCR with

fluorescently labeled primers. duplex PCR I Primer pair concentration [nmol] amplificate length [bp] U2-Pl-fw/U2-rv 200 60 PD-Pl-fw/PD-rv 400 65 duplex PCR II Primer pair concentration [nmol] amplificate length [bp] U2-Pl-fw/U2-rv 200 60 PA-Pl-fw/PA-rv 200 64 triplex PCR Primer pair concentration [nmol] amplificate length [bp] U2-Pl-fw/U2-rv 200 60 PD-Pl-fw/PD-rv 200 65 PA-Pl-fw/PA-rv 200 64


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FIGURES List of Figures Figure 1: Melting point analysis with Plexor® technology. Results received with almond specific primer pair PD-Pl-fv/PD-rv. Black: apricot DNA and blank, grey: DNA isolated from marzipan raw paste and almond kernels. Almond specific amplicons are characterized through the higher melting point of 85.6 °C-85.9 °C. Figure 1: Agarose gel from duplex endpoint PCR with universal 1 U1-fv/U1-rv and apricot specific primers PA-fw/PA-rv using DNA isolated from apricot. The agarose gel shows the unspecific co-amplified PCR fragment from the apricot specific forward PA-Fw and the universal 1 reverse U1-rv primer with a length of approx. 550 bp. Figure 2: Normalized CT values of marzipan raw paste DNA spiked with persipan raw paste DNA (0.05%, 0.5%, 5%, 12.5%, 25%) from duplex real-time PCR using apricot specific and universal primers. Normalized threshold cycles (CT value) are plotted against logarithm of persipan raw paste DNA content in marzipan DNA. E = 113 %, R2 = 0.9933 Figure 3: Standard curve of duplex real-time PCR of marzipan raw paste spiked with 50%, 25%, 5.0%, 0.5% and 0.25% persipan raw paste, respectively. DNA from each sample was isolated three times and it was used to real-time PCR with the apricot specific primers and the universal 2 primers. The A) threshold cycles (CT value; black, apricot specific primer pair; grey, universal 2 primer pair) respectively, E(apricot system) = 92%, R2 (apricot system) = 0.9844%. B) normalized CT values (Delta CT value). E = 87%; R2 = 0.9942%. Figure 4: Results of quantitative analysis of spiked marzipan raw paste samples of the “blind study”. Different quantitation strategies were pursued: Absolute quantitation using a matrix calibration curve of marzipan raw paste spiked with persipan raw paste 50%, 25%, 5%, 2.5%, 0.5%, amounts were calculated A) using the calibration curve with and B) without a normalization step using the universal primers. In addition, C) relative quantitation with a matrix standard (0.5% persipan raw paste in marzipan raw paste) was used. The calculated amounts were compared to real persipan raw paste values of the samples (18.0%, 1.9%, 0.7%, 0.4%). PRP: persipan raw paste



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Figure 1

Figure 2


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Figure 3

Figure 4



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Figure 5


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TABLE OF CONTENT


Development of a Multiplex Real-Time PCR for Determination of

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