Tetraplex PCR AllMeat 9 pdf

Title Multiplex real-time PCR for the detection and quantification of DNA from beef, pork, chicken and turkey René Köppel2*, Jüg Ruf1, Franziska Zimmerli2 and Alda Breitenmoser2 1

Official Food Control Authority of the Canton Thurgau, 2Official Food Control Authority of the Kanton Zürich, Zürich Switzerland Abstract Many meat products are composed of two or more meat species. To determine the proportion of these meat fractions a quantitative multiplex PCR was developed for the quantification of beef, pork chicken and turkey. This system proved its applicability precision and accuracy examining different meat products from the market. Thus it allows the efficient control of composed meat products in official food control and production control laboratories. Keywords multiplex, real-time quantitative PCR, beef, pork, chicken, turkey Footnotes *Author for correspondence: phone: +41 43 244 72 24; e-mail: [email protected] Introduction Composed meat products enclose raw or boiled sausages or cold cut products. Due to ethnological or legal reasons, some of these products are produced without pork (e.g. "halal") or they contain a certain proportion of veal (e.g. Kalbsbratwurst). The prices for different sorts of meat may differ significantly. As for example veal is much more expensive than pork. Fraud may be attractive for producers in a highly competitive market. Usually, fraud encloses diminished contents of expensive meat, only seldom the products lack the declared meat at all. The determination of meat fractions in processed food is therefore an important issue for official food control laboratories [1] or production control. To detect fraudulent production, analytical methods must be able to quantify all potential components within these complex matrices. Pork, beef, chicken and turkey are the main ingredients for meat products in Europe. Precision and accuracy of any analytical method are crucial performance criteria to ensure reliable results. PCR-techniques proved their applicability for the analysis of mixed processed food already in the past [2,3,4,5,6,7,8,9,10,11,13,14]. However, as soon as several meat species have to be determined these PCR-systems become laborious, expensive and time consuming. Furthermore, applying numerous single PCR-systems may be a source of higher error rates. The individual systems require many pipetting steps and an extensive compilation of all single results. Therefore they are prone to confusions and mistakes. In contrast, multiplex systems require minimal pipetting steps and often compensate pipetting errors. The results don’t have to be compiled; they can be collected at once. Here, we present a multiplex PCRsystem (fourMeat) for the quantitative determination of beef, pork, chicken and turkey in one PCR-run and its performance.

Materials and methods Meat and sausage samples Tetraplex PCR AllMeat 9

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Boiled and raw sausages as well as fresh meat used as reference material was also obtained from local suppliers. The samples A-D were derived from an interlaboratory trial which took place 2006 [12]. . DNA extraction DNA extraction from all sample matrices was performed using the Wizard Plus Miniprep DNA purification system (Promega, Madison, USA). Usually 200 mg of ground sample material was extracted and DNA was eluted in 50 µl elution buffer according to the supplier's manual. The concentration was determined photometrically and adjusted by dilution to 20 ng/µl. Primers and probes Primers and probes were established "in house" or in case of the turkey-system from the Official Food control Authority of the Canton Thurgau" (see table 1). All primers and probes were synthesized by Microsynth AG, Balgach, Switzerland. Labelling of probes with fluorescent markers was done according to the recommendations of the Rotorgene 6000 manual and are listed in table 1. Real-time PCR procedure 5 µl DNA extracts were added to 20 µl of reaction mix containing QuantiTect multiplex PCR no ROX Master Mix (Qiagen AG, Germany), and all primers and probes listed in table 1. For final concentrations see table 1. PCR was performed on a Rotorgene 6000 real-time system (Corbett, Australia) according to the following cycling protocol: Initial step of 15 min at 95 °C; followed by 45 cycles of 10 sec at 95 °C and 1 min at 60 °C (according to the recommendations for the QuantiTect multiplex PCR no ROX MasterMix).

Results Design of the multiplex real-time PCR System For all applied PCR systems specificity is prerequisite for multiplex assays. After designing primers by the Beacon-designer 5.1 software (Premier Biosoft, Palo Alto, USA), they were first tested without probes in the single SYBR-green format. This approach is useful to detect amplification of unspecific products. After this, a first check of the specificity was performed. Therefore, DNA of several related meat species was isolated and used as template for the PCR-reaction. In addition a dilution row of each target revealed the amplification efficiency which is crucial to reach maximal sensitivity for each target. If these first results were satisfactory, Taqman-probes were designed according to the channels of the Rotorgene 6000. We choose the fluorophores FAM, Joe, ROX and Cy5 for labelling (for details see table 1). Specificity During the design, all primers and probes were successfully checked for relevant homologies by BLAST nr search within GenBank databases. To test the specificity extensively, DNA from a wide range of animals and other food ingredients was isolated. DNA of the following organisms was isolated and tested in this multiplex PCR-system: sheep, goat, horse, chamois, duck, ostrich, quail, red-legged partridge, pheasant, guinea fowl, springbok, wild boar, roe, deer, fallow deer, elk, hare, bunny, kangaroo, trout, pangasius, paprika, caraway, cumin, white pepper, onion, garlic, nutmeg, almond and yeast. Tetraplex PCR AllMeat 9

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The only cross-reactivity above 1% emerged with the pork system when using wild boar as a template. This species produces similar signals like pork. The pork-system therefore does not discriminate between pork and wild boar. This cross-reactivity has to be considered examining meat products enclosing meat from wild animals. Sensitivity, precision and uncertainty To evaluate the sensitivity, DNA-extracts of each target species (DNA concentration 20 ng/µl) were diluted to 0.1% in half-logarithmic steps. Herring sperm DNA-solution (20 ng/µl) was used as diluent. These single analyte dilutions were mixed according to table 2 in order to produce a multiplex standard, simulating samples containing all four analytes. Each data-point was analysed 6 times (N=6) in 4 different runs. These results were used for the calculation of the amplification efficiency, relative standard deviation (RSD) in order to assign the precision. To estimate the accuracy, the mean deviation to the true value was calculated (see compilation tables 3). All samples with DNA contents down to 0.32 % (0.32 ng) of the analytes were reproducible detectable and therefore quantifiable, 0.1% was not detected in all cases. This results give a detection and quantification range of 0.32 to 32 % (0.32 ng to 32 ng total template DNA). None of the 10 negative controls containing herring sperm DNA (20ng/µl) gave a positive result. Quantification performance data is shown in table 3. It has to be considered that these data only count for the quantification of DNA contents and not the weight composition of meat products. Analysis of interlaboratory trial samples Quantification of meat fractions in real products of the market by PCR is difficult. Quantitative results may not correlate to the recipe of the product. Reasons are: degradation of the DNA during production (heat, acids), storage conditions as well as the fact that such products are composed of different meat tissues, that contain different contents of DNA (e.g. muscle fibres or fatty meat) [14]. To make the transformation form measurements of DNA-contents towards determination of Weight-composition a interlaboratory trial [12] was performed. In this interlaboratory trial reference sausages containing 30 % to 60 % veal and complementary amounts of pork. No chicken or turkey was enclosed. For the composition of the reference sausages see table 4. These reference sausages were applied as calibrators in place of the DNA calibrators. "Unknown" samples were also composed of pork and veal only. For the true composition and measured values of the "unknown" sausages see table 5. Except for sample C, the values measured by fourMeat-system correlate well with the true values and the values determined in an earlier proficiency trial (see table 5). The use of sausages with defined contents of meat as calibrators is crucial to generate reliable results [12]. Analysis of commercial samples 13 commercially available products were analysed by fourMeat and a qualitative PCR system for pork, beef and poultry which was used earlier as screening tool for the determination of the composition of meat products. The qualitative system [13] has a lower detection limit than the fourMeat-system. The qualitative results between the two PCR-systems correlate well (for the results see table 6). Conclusion The here applied primers showed almost ideal properties. They are highly specific, have an equal optimum of annealing temperature and requirement of MgCl2concentration. Additionally the amplification-kit used here, showed a high tolerance in conjunction with high amplification capacity. All these findings reveal that quantitative Tetraplex PCR AllMeat 9

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multiplex PCR gives sensitive and accurate results providing that amplification of one target does not interfere with another one in the same tube. We assume that the simultaneous quantitative determination of DNA of pork, beef, turkey and chicken by the fourMeat-multiplex PCR-system is feasible and gives reproducible results in a very efficient manner. However, the total procedure of determination the net weight of meat products may require the consideration of many important determinants like, DNA-content of different tissues, ploidy of genomes and determination of total animal DNA [14]. To circumvent these considerations, matrix specific calibrators can be selected. They must be composed similar to the unknown samples. Using such matrix specific calibrators, meat composition, which reflect the meat proportions of production recipe, may be determined in a fast and accurate manner using the fourMeatmultiplex PCR-system. Determination of the content of total amplifiable animal DNA as proposed earlier [14] sees not to be required.

Acknowledgements We thank the cantonal laboratory of Zürich for providing the resources for this work.

References [1] Verordnung über Lebensmittel tierischer Herkunft 23.November 2005 Art. 8 Abs. 5 [2] Binke R., Spiegel K., Schwägele F. (2005) Vergleichende Untersuchung von mitochondrialen und nukleären Gensequenzen zur Identifizierung von tierischen Bestandteilen in Fleischerzeugnissen mittels PCR Mitteilungsblatt der Fleischforschung Kulmbach 44 169: 201-210 [3] Lopez-Andreo M., Lugo L., Garrido-Pertierra A., Prieto I. , Puyet A. (2005) Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction Analytical Biochemistry 339:73-82 [4] Hird H., James Chisholm J., Brown J. (2005) The detection of commercial duck species in food using a single probe-multiple species-specific primer real time PCR assay European Food research Technology 221:559-563 Tetraplex PCR AllMeat 9

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[5] Rodriguez M. A., Garcia T., Gonzales I., Asensio L., Pablo E. Hernandez P.E, Rosario Martin R. (2004) Quantiation of Mule Duck in Goose Foie Gras using TaqMan Real-Time Polymerase chain Reaction J. Agric. Food Chem. 52: 1478-1483 [6] Laube I., Spiegelberg A., Butschke A., Zagon J., Schauzu M., Kroh L., Broll H. (2003) Methods for the detection of beef and pork in foods using real-time polymerase chain reaction International Journal of Food Science and Technology 38:111-118 [7] Calvo J. H. , Osta R., Zaragoza P. (2002) Quantitative PCR Detection of Pork in Raw and heated Ground Beef and Pâté J. Agric Food Chem. 50: 5265-5267 [8] Meyer R., Candrian U., Lüthy J. (1994) Detection of Pork in Heated Meat Products by the Polymerase Chain Reaction Journal of AOAC international 77(3): 617-622

[9] Laube I., Butschke A., Zagon J., Spiegelberg A., Schauzu M., Bögl K.-W., Kroh L.W. Broll H. (2001) Nachweisverfahren für Rindfleisch in Lebensmitteln unter Anwendung der TaqManTM-Technologie Bundesgesundheitsbl. 44: 326-330 [10] Brodmann P. and Moor D. (2003) Sensitive and semi-quantitative TaqManTM real-time polymerase chain reaction systems for the detection of beef (Bos taurus) and the detection of the family Mammalia in food and feed Meat Science 65: 599-607 [11] Sawyer J., Wood C., Shanahan D., Gout S., McDowell D(2003) Real-time PCR for quantitative meat species testing Food Control 14: 579-583 [12] Alber Eugster A., Jürg Ruf J., Jürg Rentsch J., Philipp Hübner P.,Köppel R.

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Quantification of beef and pork fraction in sausages by real-time PCR analysis: results of an interlaboratory trial Eur Food Res Technol In press (DOI 10.1007/s00217-007-0686-9) [13] Matsunaga T. , Chikuni K., Tanabe R., Muroya S., Shibata K., Yamada J., Shimura Y. (1999) A quick and simple method for the identification of meat species and meat products by PCR assays Meat Science 51: 143-148 [14] Ines Laube I., Jutta Zagon J., Hermann Broll H. (2007) Quantitative determination of commercially relevant species in foods by real-time PCR International Journal of Food Science and Technology 42: 336-341

Illustrations and tables tetraplex qPCR-system fourMeat beef Primer/Probe final conc. µM Rd 1 F 0.2

sequence

amplicon GenBank acc.no. /source/ labelling

GTA GGT GCA CAG TAC GTT CTG AAG GGC CAG ACT GGG CAC ATG GAA CCT CAT TCT GGG GCC CCG

96bp

Primer/Probe final conc. µM Sus1 F 0.2

sequence


CGA GAG GCT GCC GTA AAG G

80bp

Sus1 R Sus1 TMP

TGC AAG GAA CAC GGC TAA GTG TCT GAC GTG ACT CCC CGA CCT GG

Rd 1 R Rd 1 TMP

0.2 0.08

beta-actin-gen EH170825 1) KLZH ROX-BHQ2

pork

0.2 0.08

beta-actin-gen DQ452569 1) KLZH JOE-BHQ1

chicken Primer/Probe final conc. µM

sequence



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Gallus1 F

0.2

CAG CTG GCC TGC CGG

76bp

Gallus1 R Gallus1 TMP

0.2 0.08

CCC AGT GGA ATG TGG TAT TCA TCT GCC ACT CCT CTG CAC CCA GT

TF-GB3 X6009 1)KLZH FAM-BHQ1

turkey Primer/Probe final conc. µM MG-ProlR-F 0.2 MG-ProlR-R

0.2 0.08

MG-ProlR-Cy5

sequence


CAA AGA AAG CAG GGA AAA GGA

83bp

TGC ACT CTC GTT GTT AAA AAG GA CTG GGA AAG TTA CTG TGT AGC CTC AGA ACG

prolactin receptor, L76587 2)KLTG Cy5-BHQ2

Table 1. Multiplex qPCR-system fourMeat for the simultaneously determination of beef (Rd), pork (Sus), chicken (Gallus) and turkey (MG-ProlR). 1) 2) Official Food Control Authority of the Kanton Zürich. Official Food Control Authority of the Canton Thurgau

fourMeat No. 1 2 3 4 5 6

turkey 32 10 3.2 1 0.32 0.1

chicken 0.32 0.1 32 10 3.2 1

pork 3.2 1 0.32 0.1 32 10

beef 0.1 32 10 3.2 1 0.32

Table 2. Multiplex serial dilution of standards for fourMeat in the range from 0.1% to 32% (0.1 to 32ng of total temlate DNA). The DNA was isolated from pure fresh low fat meat of the appropriate species.

fourMeat Amplification efficiencies precision +/-% accuracy +/-%

turkey

chicken

pork

beef

1.03 9.1 14.1

1.24 12.6 15.7

1.27 10.3 15.5

1.05 8.5 15.5

Table 3. Performance of the fourMeat. The Multiplex serial dilution were taken as calibrator to evaluate these properties. The amplification efficiencies were taken from the Rotorgen algorithm directly. The here shown numbers represent mean values from 6 single experiments.

Reference sausage composition portion of veal in total meat ingredient veal pork bacon


60% 60 g/100 g 38.5 2.9 24.4

50% 50 g/100 g 31.7 9.8 24.4

40% 40 g/100g 24.9 16.6 24.4

30% 30 g/100 g 18 23.4 24.4

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calf's head incl. 50% ice ice/water Total

4.9 29.3 100

4.9 29.3 100

4.9 29.3 100

4.9 29.3 100

Table 4. Composition of reference fried sausages ranging from 60% to 30% veal (beef) proportion in total meat ingredient.

Reference sausage samples sample A sample B sample C sample D

measured proportion of beef 33 48 43 50

true value 34 48 50* 55

measured proportion of pork 67 52 57 50

True value 66 52 50 45

Table 5. Values measured by fourMeat of the unknown samples A-D. Values are composed by the average value of 3 single determinations. *The sample C is a commercial available sample of unknown receipe. The value of 50% veal was estimated. The measured proportion of beef using single PCR-Systems yielded 42-44%

fourMeat pork