Development and Validation of a Direct qPCR Method for the Detection of Pork Adulteration in Processed Meat Products
Applied Food Biotechnology,
Vol. 10 No. 3 (2023),
17 May 2023
,
Page 165-176
https://doi.org/10.22037/afb.v10i3.41897
Abstract
Background and Objective: The number of Muslims in Indonesia is increasing; hence, consumption of halal foods is increasing. This urges halal detection of foods as well. Direct qPCR is a method for DNA-based halal detection using crude DNA samples. One factor that affects the success of direct qPCR is the crude DNA. In this study, DNA extraction process was optimized by modifying the lysis step to achieve high-quality crude DNA, optimizing the qPCR conditions and validating the method.
Material and Methods: Materials used for DNA extraction from meats and meat products included lysis buffer containing EDTA, NaCl, SDS, NaOH and tween-20. Materials used in qPCR assay included crude DNA, ddH20, primers and master mix. Genomes from meats and processed products were extracted using lysis buffer by optimizing lysis conditions (temperature and incubation time). The crude DNA was assayed for concentration and purity; then, results were compared to chloroform: isoamyl and the commercial methods. The DNA extract from lysis buffer was amplified using qPCR machine. Operational conditions such as DNA concentration, annealing temperature and primer concentration were optimized. Results were validated using specificity, repeatability, reproducibility, applicability, robustness and limit of detection assays.
Results and Conclusion: Temperature for optimum lysis in extraction lysis buffer method included 75 °C for 25 min. Concentrations of DNA from the lysis buffer, chloroform-isoamyl and commercial methods did not significantly vary (p-value=0.094). The optimum conditions for qPCR method were set at DNA concentration of 50 ng µl-1, annealing temperature of 53 °C and primer concentration of 10 pmol. For the validation result, specificity assay showed that the method could be used to detect pork and wild boar in meats using ND4 primers. In addition, repeatability assay included 1.06% and reproducibility assay included 1.57%. Direct qPCR method can be used for various processed meat products and is resistant to inhibitors (alginate, calcium ions, EDTA and cellulose), except for polysaccharides. The LOD value achieved from the PCR sensitivity, linearity and efficiency assay were 0.001 ng µl-1, 0.996 and 110%, respectively. Results verify that the direct qPCR method is easy, fast, applicable, accurate and precise for the pork detection.
Conflict of interest: The authors declare no conflict of interest.
- direct qPCR ▪ halal food ▪ lysis buffer ▪ pork ▪ wild boar
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References
Pew Research Center Religion and Public Life. The future of world religions: population growth projections, 2010-2050. 2015. 2010-2050 p.
Kim YS, Yu HK, Lee BZ, Hong KW. Effect of DNA extraction methods on the detection of porcine ingredients in halal cosmetics using real-time PCR. Appl Biol Chem. 2018; 61(5): 549-55.
https://doi.org/10.1007/s13765-018-0389-x
Elisa Andriyani, Nur Lutfi Fais, Siti Muatifah. Perkembangan penelitian metode deteksi kandungan babi untuk menjamin kehalalan porduk olahan pangan olahan. J Islam Stud Humanit. 2019; 4(1):104-126.
https://doi.org/10.21580/jish.41.4888
Guan F, Jin Y, Zhao J, Ai J, Luo Y. A novel direct PCR lysis buffer can improve PCR from meat matrices. Food Anal Methods. 2019; 12(1): 100-7.
https://doi.org/10.1007/s12161-018-1342-7
Ayu D, Nlpi D. Perkembangan Teknologi Reverse Transcriptase-Polymerase Chain Reaction Dalam Mengidentifikasi Genom Avian Influenza dan Newcastle Diseases. 2014; 24(1): 16-29.
Rohman A, Orbayinah S, Hermawan A, Sudjadi S, Windarsih A, Handayani S. The development of real-time polymerase chain reaction for identification of beef meatball. Appl Food Res. 2022; 2(2): 100148.
https://doi.org/10.1016/j.afres.2022.100148
Septiani T. Detection of porcine DNA in processed beef products using real time- polymerase chain reaction. Indones J Halal Res. 2019;1(2):31-34.
https://doi.org/10.15575/ijhar.v1i2.5601
Sudjadi, Wardani HS, Sepminarti T, Rohman A. Analysis of porcine gelatin DNA in a commercial capsule shell using real-time polymerase chain reaction for halal authentication. Int J Food Prop. 2016; 19(9): 2127-34.
https://doi.org/10.1080/10942912.2015.1110164
Farrokhi R, Jafari Joozani R. Identification of pork genome in commercial meat extracts for Halal authentication by SYBR green I real-time PCR. Int J Food Sci Technol. 2011; 46(5): 951-955.
https://doi.org/10.1111/j.1365-2621.2011.02577.x
Rachmadhani, Warisman MA, Suryani, Desriani D. In-house validation and calibration of pork detection using duplex SYBR-Green I real-time PCR approach. Int Food Res J. 2019; 26(2): 509-516.
Chiş LM, Vodnar DC. Detection of the species of origin for pork, chicken and beef in meat food products by real-time PCR. Safety. 2019; 5(4): 1-8.
https://doi.org/10.3390/safety5040083
Sajali N, Ting SML, Koh CC, Desa MNM, Wong SC, Abu BS. Meatball model of porcine DNA detection by TaqMan probe real-time PCR. Food Res. 2022; 6(3): 136-144.
https://doi.org/10.26656/fr.2017.6(3).384
Lubis H, Salihah NT, Norizan NA, Hossain MM, Ahmed MU. Fast and sensitive real-time PCR-based detection of porcine DNA in food samples by using evaGreen dye. Food Sci Technol Res. 2018; 24(5): 803-810.
https://doi.org/10.3136/fstr.24.803
Rohman A, Pebriyanti NW, Sismindari, Windarsih A, Ramadhani D, Larasati R, Yulisa H. Real-time polymerase chain reaction for identification of dog meat in adulterated beef meatball using specific primer targeting on cytochrome-b for halal authentication. Int J Food Prop. 2020; 23(1): 2231-2241.
https://doi.org/10.1080/10942912.2020.1844748
Yalçınkaya B, Yumbul E, Mozioglu E, Akgoz M. Comparison of DNA extraction methods for meat analysis. Food Chem. 2017; 221: 1253-1257.
https://doi.org/10.1016/j.foodchem.2016.11.032
Girish PS, Haunshi S, Vaithiyanathan S, Rajitha R, Ramakrishna C. A rapid method for authentication of buffalo (Bubalus bubalis) meat by alkaline lysis method of DNA extraction and species specific polymerase chain reaction. J Food Sci Technol. 2013; 50(1): 141-146.
https://doi.org/10.1007/s13197-011-0230-6
Zhao G, Wang J, Yao C, Xie P, Li X, Xu Z, Xian Y, Lei H, Shen X. Alkaline lysis-recombinase polymerase amplification combined with CRISPR/Cas12a assay for the ultrafast visual identification of pork in meat products. Food Chem. 2022;383:132318.
https://doi.org/10.1016/j.foodchem.2022.132318
Suadi Z, Bilung LM, Apun K, Azmi AA. Efficiency of traditional DNA extraction method in PCR detection of porcine DNA in meat mixtures. J Teknol. 2020; 82(5): 85-90.
https://doi.org/10.11113/jt.v82.14636
Gargouri H, Kacem HH. Evaluation of alternative dna extraction protocols for the species determination in turkey salami authentication assays. Int J Food Prop. 2018; 21(1): 733-745.
https://doi.org/10.1080/10942912.2017.1422263
Piskata Z, Servusova E, Babak V, Nesvadbova M, Borilova G. The quality of DNA isolated from processed food and feed via different extraction procedures. Molecules. 2019; 24(6): 1-10.
https://doi.org/10.3390/molecules24061188
Al-Kahtani HA, Ismail EA, Asif Ahmed M. Pork detection in binary meat mixtures and some commercial food products using conventional and real-time PCR techniques. Food Chem. 2017; 219: 54-60.
http://doi.org/10.1016/j.foodchem.2016.09.108
Hoorzook KB, Barnard TG. Comparison of DNA extraction methods for the direct quantification of bacteria from water using quantitative Real-Time PCR. Water (Switzerland). 2022; 14(22).
https://doi.org/10.3390/w14223736
Zhao N, Cai J, Zhang C, Guo Z, Lu W, Yang B, Tian FW, Liu XM, Zhang H, Chen W. Suitability of various DNA extraction methods for a traditional Chinese paocai system. Bioengineered. 2017; 8(5): 642-650.
https://doi.org/10.1080/21655979.2017.1300736
Yahya A, Firmansyah M, Arlisyah A, Risandiansyah R. Comparison of DNA extraction methods between conventional, kit, alkali and buffer-Only for PCR amplification on raw and boiled Bovine and porcine meat. J Exp Life Sci. 2017; 7(2): 110-114.
https://doi.org/10.21776/ub.jels.2017.007.02.09
Mano J, Hatano S, Futo S, Minegishi Y, Ninomiya K, Nakamura K, Kondo K, Teshima R, Takabatake R, Kitta K. Development of direct real-time PCR system applicable to a wide range of foods and agricultural products. J Food Hyg Soc Japan. 2014; 55(1): 25-33.
https://doi.org/10.3358/shokueishi.55.25
Khairil Mokhtar NF, El Sheikha AF, Azmi NI, Mustafa S. Potential authentication of various meat-based products using simple and efficient DNA extraction method. J Sci Food Agric. 2020; 100(4): 1687-1693.
https://doi.org/10.1002/jsfa.10183
Kusnadi J, Mahardita P, Al-Awwaly KU, Arumingtyas EL. Novel primer targeting the mitochondrial NADH dehydrogenase subunit 4 (ND4) and NADH dehydrogenase subunit 5 (ND5) for detection of porcine (Sus scrofa) DNA fragments in food products for halal authentication. IOP Conf Ser Earth Environ Sci. 2021; 924(1): 1-7.
https://doi.org/10.1088/1755-1315/924/1/012004
Kusnadi J, Hernandi KH, Al-Awwaly KU, Arumingtyas EL, Hakiki HM, Istianah N. Design and performance assay of specific primers to detect bovine DNA fragments using multiplex PCR technique for halal authentification. Indones J Halal Res. 2022; 4(2): 45-52.
https://journal.uinsgd.ac.id/index.php/ijhar/article/view/15573
Broeders S, Huber I, Grohmann L, Berben G, Taverniers I, Mazzara M, Roosens N, Morisset D. Guidelines for validation of qualitative real-time PCR methods. Trends Food Sci Technol. 2014; 37(2): 115-126.
https://doi.org/10.1016/j.tifs.2014.03.008
Islam MS, Aryasomayajula A, Selvaganapathy PR. A review on macroscale and microscale cell lysis methods. Micromachines. 2017; 8(3): 1-27.
https://doi.org/10.3390/mi8030083
Ribeiro JC, Tamanini R, Soares BF, De Oliveira AM, De Godoi Silva F, Da Silva FF, Augusto NA, Beloti V. Efficiency of boiling and four other methods for genomic DNA extraction of deteriorating spore-forming bacteria from milk. Semin Agrar. 2016; 37(5): 3069-3078.
https://doi.org/10.5433/1679-0359.2016v37n5p3069
Shen C-H. Detection and Analysis of Nucleic Acids. Diagnostic Molecular Biology. 2019. 167-185.
https://doi.org/10.1016/b978-0-12-802823-0.00007-9
Rao SN, Manissero D, Steele VR, Pareja J. A narrative systematic review of the clinical utility of cycle threshold values in the context of COVID-19. Infect Dis Ther. 2020; 9(3): 573-586.
https://doi.org/10.1007/s40121-020-00324-3
Pertiwi NPD, Mahardika IGN, Watiniasaih NL. Optimasi amplifikasi DNA menggunakan metode PCR (Polymerase Chain Reaction) pada ikan karang anggota famili Pseudochromidae (Dottyback). J Biol. 2015; 19(2): 1-5.
Orbayinah S, Hermawan A, Sismindari S, Rohman A. Detection of pork in meatballs using probe TaqMan Real-time Polymerase. 2020;4: 1563-1568.
https://doi.org/10.26656/fr.2017.4(5).227
Kaltenbrunner M, Mayer W, Kerkhoff K, Epp R, Ruggeberg H, Hochegger R, Markl MC. Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCR. Sci Rep. 2019; 9(1): 1-12.
https://doi.org/10.1038/s41598-019-45564-7
Eugster A, Ruf J, Rentsch J, Koppel R. Guidelines for the validation of analytical methods for nucleic acid sequence‐based analysis. Eur Food Res Technol. 2009; 230(1): 55-61.
Ahuja A. Modified protocol for plant genomic DNA isolation modified protocol for plant genomic DNA isolation. Indian Res J Genet Biotech. 2017; 9(4): 478- 485.
Hariyadi S, Narulita E, Rais MA. Perbandingan metode lisis jaringan hewan dalam proses isolasi DNA genom pada organ liver tikus putih (Rattus norvegicus). Biol Educ Conf. 2018; 15(1): 689-692.
Simamora A. Struktur dan fungsi membran sel. Available from:
https://www.academia.edu/32877855/STUKTUR_DAN_FUNGSI_MEMBRAN_SEL_doc [Accessed 2 January 2023].
Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012; 113(5): 1014-1026.
https://doi.org/10.1111/j.1365-2672.2012.05384.x
Sakalar E, Abasiyanik MF, Bektik E, Tayyrov A. Effect of heat processing on DNA quantification of meat species. J Food Sci. 2012; 77(9): 1-5.
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