SIMPLEKS DAN MULTIPLEKS PRE-ENRICHMENT-PCR UNTUK DETEKSI Salmonella Enteritidis DAN Typhimurium PADA KARKAS AYAM
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Accepted
17 January 2022
Published
7 February 2022
Keywords:
-
chicken carcass, PCR, pre-enrichment, Salmonella Enteritidis, Salmonella Typhimurium
Abstract
A PCR assay has been developed and applied to detect Salmonella contamination in chicken carcasses. However, a concentration fewer than 3 cells per gram lead to false-negative results due to difficulties in the DNA extraction. The objective of this study was to evaluate of the influence of pre-enrichment on the sensitivity of simplex and multiplex PCR methods the detection of for Salmonella spp., S. Enteritidis and S. Typhimurium in chicken carcasses. Artificial contamination was done using very low number of Salmonella Hadar, S. Enteritidis dan S. Typhimurium and pre-enrichment was carried out by 8 hours incubation in non-selective (BPW) medium. The results showed that simplex PCR could detect Salmonella spp., S. Enteritidis and S. Typhimurium at initial numbers of 2.3, 0.9 and 2.3 MPN/mL of cells in broth medium, respectively. A multiplex PCR could detect mixed culture of the three Salmonella serovars at an initial number of 0.73 MPN/mL of cells. When compared to non-enrichment treatment, simplex pre-enrichment-PCR gave an increase in the percentage of positive results in chicken carcasses (n= 12), from 75 to 100% for Salmonella spp., from 8 to 58% for S. Typhimurium, and from 58 to 75% for S. Enteritidis. Increasing in the positive percentage was also occurred at multiplex pre-enrichment-PCR, however the concentration of S. Enteritidis primer was not optimum for detection. Pre-enrichment step significantly increases the sensitivity of PCR-based assay for detection Salmonella.
References
Aerita AN, Pawenang ET, Mardiana. 2014. Hubu-ngan higiene pedagang dan sanitasi dengan kontaminasi Salmonella pada daging ayam potong. Unnes J Public Health 3: 9-16.
Afendy ATM, Son R. 2015. Pre-enrichment effect on PCR detection of Salmonella Enteritidis in artifi-cally-contaminated raw chicken meat. Int Food Res J 22: 2571-2576.
Asthiti NGAM. 2019. Peningkatan Sensitivitas De-teksi Cemaran Salmonella spp., S. Typhimu-rium, dan S. Enteritidis menggunakan Multiplex PCR dengan Tahap Pre-enrichment. [skripsi]. Bogor: Fakultas Teknologi Pertanian, Institut Pertanian Bogor.
Bakara VFS, Tafsin M, Hasnudi. 2014. Analisis bakteri Salmonella spp. pada daging ayam po-tong yang dipasarkan pada pasar tradisional dan pasar modern di Kota Medan. J Peter-nakan Integratif 3: 71-83. DOI: 10.32734/jpi. v3i1.2746.
Chiang Y-C, Wang H-H, Ramireddy L, Chen H-Y, Shih, C-M, Lin C-K, Tsen H-Y. 2018. Designing a biochip following multiplex polymerase chain reaction for the detection of Salmonella sero-vars Typhimurium, Enteritidis, Infantis, Hadar, and Virchow in poultry products. J Food Drug Anal 26: 58-66. DOI: 10.1016/j.jfda.2016.11.0 19.
Crump JA, Wain J. 2017. Salmonella. International Encyclopedia of Public Health 2: 425-433. DOI: 10.1016/B978-0-12-803678-5.00394-5.
El-Sharkawy H, Tahoun A, El-Gohary AEGA, El-Abasy M, El-Khayat F, Gillespie T, Kitade Y, Hafez HM, Neubar H, El-Adawy H. 2017. Epi-demiological, molecular characterization and antibiotic resistance of Salmonella enterica serovars isolated from chicken farms in Egypt. Gut Pathog 9: 1-12. DOI: 10.1186/s13099-017-0157-1.
Eng S-K, Pusparajah P, Ab-Mutalib N-S, Ser H-L, Chan K-G, Lee L-H. 2015. Salmonella: a review on pathogenesis, epidemiology and antibiotic resistance. Front Life Sci 8: 284-293. DOI: 10.1 080/21553769.2015.1051243.
Gandra EA, Fernandez MA, Silva JA, da Silva WP. 2016. Detection by multiplex PCR of Staphylo-coccus aureus, S. intermedius and S. hyicus in artificially contaminated milk. Ciênc Rural 46: 1418-1423. DOI: 10.1590/0103-8478cr201513 91.
Gorski L. 2012. Selective enrichment media bias the types of Salmonella enterica strains isolated from mixed strain cultures and complex enrich-ment broths. PLoS ONE 7: 1-8. DOI: 10.1371/ journal.pone.0034722.
Hadjinicolaou A, Dmetriou VL, Emmanuel MA, Kako-yiannis CK, Kostrikis LG. 2009. Molecular beacon-based real-time PCR detection of pri-mary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and cli-nical samples. BMC Microbiol 9: 1-14. DOI: 10. 1186/1471-2180-9-97.
Hoelzer K, Switt AIM, Wiedmann M. 2011. Animal contact as a source of human non-typhoidal salmonellosis. Vet Res 42: 1-28. DOI: 10.1186/1297-9716-42-34.
[ISO] International Organization for Standardization. 2017. ISO 6579:1 Detection of Salmonella spp.. Switzerland (CH): ISO.
[Kementan RI] Kementerian Pertanian Republik Indonesia. 2020. Statistik Peternakan dan Kesehatan Hewan 2020. Direktorat Jenderal Peternakan dan Kesehatan Hewan, Jakarta.
Krämer N, Löfström C, Vigre H, Hoorfar J, Bunge C, Malorny B. 2011. A novel strategy to obtain quantitative data for modelling: Combined en-richment and real-time PCR for enumeration of salmonellae from pig carcasses. Int J Food Microbiol 145: 86-96. DOI: 10.1016/j.ijfoodmicro. 2010.08.026.
Liu B, Zhou X, Zhang L, Liu W, Dan X, Shi C, Shi X. 2012. Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis. Food Control 27: 87-93. DOI: 10.1016/j.foodcont.201 2.01.062.
Maciel BM, Dias JCT, Romano CC, Sriranganathan N, Brendel M, Rezende RP. 2011. Detection of Salmonella Enteritidis in asymptomatic carrier animals: comparison of quantitative real-time PCR and becteriological cultue methods. Genet Mol Res 10: 2578-2588.
Maksum IP, Suhaili, Amalia R, Kamara DS, Rach-man SD, Rachman RW. 2018. PCR Multiplex untuk identifikasi Mycobacterium tuberculosis resisten terhadap isoniazid dan rifampisin pada galur lokal balai laboratorium kesehatan Provinsi Jawa Barat. J Kimia Valensi 4: 107-118. DOI: 10.15408/jkv.v4i2.7226.
Matias BG, Pinto PSA, Cossi MVC, Silva JA, Vanetti MCD, Nero LA. 2010. Evaluation of a PCR protocol for the detection of Salmonella species directly from superficial samples of chicken carcasses and pre-enrichment broth. Poultry Sci 89: 1524-1529. DOI: 10.3382/ps.2009-003 68.
Myint MS, Johnson YJ, Tablante NL, Heckert RA. 2006. The effect of pre-enrichment protocol on the sensitivity and specificity of PCR for detec-tion of naturally contaminated Salmonella in raw poultry compared to conventional culture. Food Microbiol 23: 599-604. DOI: 10.1016/j.fm.2005. 09.002
Nurjanah S, Rahayu WP, Komalasari E. 2017. Sensitivity of multiplex real-time PCR assay for detection of pathogenic E. coli on ice sample. November 14-17 2017. Ho Chi Minh City, Vietnam. Ho Chi Minh City (VI): Proceedings of the 15th ASEAN Conference on Food Science and Technology.
Nurjanah S, Rahayu WP. Mutaqin LA. 2018. Detection method for Salmonella Typhimurium and Salmonella Enteritidis using real-time polymerase chain reaction. Int J Eng Technol 7: 302-306. DOI: 10.14419/ijet.v7i4.14.27661.
Park SH, Kim HJ, Cho WH, Kim JH, Oh MH, Kim SH, Lee BK, Ricke SC, Kim HY. 2009. Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR. FEMS Microbiol Lett 30: 137-146. DOI: 10.1111/j.1574-6968.20 09.01809.x.
Radji M, Puspaningrum A, Sumiati A. 2010. Deteksi cepat bakteri Escherichia coli dalam sampel air dengan metode polymerase chain reaction menggunakan primer 16e1 dan 16e2. Makara J Sci 14: 39-43. DOI: 10.7454/mss.v14i1.474.
Reyes-Escogido L, Balam-Chi M, Rodriguez-Buenfil I, Valdés J, Kameyama L, Martinez-Pérez F. 2010. Purification of bacterial genomic DNA in less than 20 mins using chelex-100 microwave: examples from stains of lactic acid bacteria isolated from soil samples. Anton Leeuw 98: 465-474. DOI: 10.1007/s10482-010-9462-0.
Saeki EK, Alves J, Bonfante RC, Hirooka EY, de Oliveira TCRM. 2012. Multiplex PCR (mPCR) for detection of Salmonella spp. and the diffe-rentiation of Typhimurium and Enteritidis sero-vars in chicken meat. J Food Safety 33: 25-29. DOI: 10.1111/jfs.12019.
Safitri E, Hidayati NA, Hertati R. 2019. Prevalensi bakteri Salmonella pada ayam potong yang dijual di pasar tradisional pangkalpinang. Ekotomia: J Penelitian Biologi, Botani, Zoologi, dan Mikrobiologi 4: 25-30. DOI: 10.33019/ ekotonia.v4i1.1012.
Soumet C, Ermel G, Rose N, Rose V, Drouin P, Salvat G, Colin P. 1999. Evaluation of multiplex PCR assay for simultaneous identification of Salmonella spp., Salmonella Enteritidis and Salmonella Typhimurium from environmental swabs of poultry houses. Lett Appl Microbiol 28: 113-117. DOI: 10.1046/j.1365-2672.1999.0048 8.x.
Taskila S, Tuomola M, Ojamo H. 2012. Enrichment cultivation in detection of food-borne Salmo-nella. Food Control 26: 369-377. DOI: 10.1016/ j.foodcont.2012.01.043.
Tortajada-Genaro LA, Rodrigo A, Hevia E, Mena S, Niñoles R, Maquieira Á. 2015. Microarray on digital versatile disc for identification and geno-typing of Salmonella and Campylobacter in meat products. Anal Bioanal Chem 407: 7285-7294. DOI: 10.1007/s00216-015-8890-0
Tran HH, Trinh KTL, Lee NY. 2013. Pressure-driven one-step solid phase-based on-chip sample preparation on a microfabricated plastic device and integration with flow-through polymerase chain reaction (PCR). J Chromatogr B 936: 88-94. DOI: 10.1016/j.jchromb.2013.06.037.
Triyani Y, Nafsi N, Yuniarti L, Sekarwana N, Sutedja E, Gurnida DA, Parwati I, Alisjahbana B. 2016. Rancangan primer spesifik gen macrophage mannose receptor (MMR) untuk Polymerase Chain Reaction (PCR) dan sekuensing Deoxyri-bose Nucleic Acid (DNA). Indonesian J Clin Pathol Med Lab 22: 158-162. DOI: 10.24293/ ijcpml.v22i2.1120.
Utari LK, Riyanti, Santosa PE. 2016. Status mikro-biologis daging broiler di pasar tradisional kabu-paten Pringsewu. J Ilmiah Peternakan Terpadu 4: 63-66.
Wulan HA, Nurjanah S, Rahayu, WP. 2021. Sensi-tivity of enrichment-PCR method for Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis analysis in chicken carcasses. Food Res 5: 54-61. DOI: 10.26656/ fr.2017.5(2).429.
Yosua A. 2018. Deteksi Salmonella Hadar, Salmonella Typhimurium, dan Salmonella Ente-ritidis menggunakan Polymerase Chain Reac-tion (PCR). [Skripsi]. Bogor: Fakultas Teknologi Pertanian, Institut Pertanian Bogor.
[WHO] World Health Organization. 2014. WHO Global Salmonella survey: Progress Report 2008-2013. World Health Organization.
Afendy ATM, Son R. 2015. Pre-enrichment effect on PCR detection of Salmonella Enteritidis in artifi-cally-contaminated raw chicken meat. Int Food Res J 22: 2571-2576.
Asthiti NGAM. 2019. Peningkatan Sensitivitas De-teksi Cemaran Salmonella spp., S. Typhimu-rium, dan S. Enteritidis menggunakan Multiplex PCR dengan Tahap Pre-enrichment. [skripsi]. Bogor: Fakultas Teknologi Pertanian, Institut Pertanian Bogor.
Bakara VFS, Tafsin M, Hasnudi. 2014. Analisis bakteri Salmonella spp. pada daging ayam po-tong yang dipasarkan pada pasar tradisional dan pasar modern di Kota Medan. J Peter-nakan Integratif 3: 71-83. DOI: 10.32734/jpi. v3i1.2746.
Chiang Y-C, Wang H-H, Ramireddy L, Chen H-Y, Shih, C-M, Lin C-K, Tsen H-Y. 2018. Designing a biochip following multiplex polymerase chain reaction for the detection of Salmonella sero-vars Typhimurium, Enteritidis, Infantis, Hadar, and Virchow in poultry products. J Food Drug Anal 26: 58-66. DOI: 10.1016/j.jfda.2016.11.0 19.
Crump JA, Wain J. 2017. Salmonella. International Encyclopedia of Public Health 2: 425-433. DOI: 10.1016/B978-0-12-803678-5.00394-5.
El-Sharkawy H, Tahoun A, El-Gohary AEGA, El-Abasy M, El-Khayat F, Gillespie T, Kitade Y, Hafez HM, Neubar H, El-Adawy H. 2017. Epi-demiological, molecular characterization and antibiotic resistance of Salmonella enterica serovars isolated from chicken farms in Egypt. Gut Pathog 9: 1-12. DOI: 10.1186/s13099-017-0157-1.
Eng S-K, Pusparajah P, Ab-Mutalib N-S, Ser H-L, Chan K-G, Lee L-H. 2015. Salmonella: a review on pathogenesis, epidemiology and antibiotic resistance. Front Life Sci 8: 284-293. DOI: 10.1 080/21553769.2015.1051243.
Gandra EA, Fernandez MA, Silva JA, da Silva WP. 2016. Detection by multiplex PCR of Staphylo-coccus aureus, S. intermedius and S. hyicus in artificially contaminated milk. Ciênc Rural 46: 1418-1423. DOI: 10.1590/0103-8478cr201513 91.
Gorski L. 2012. Selective enrichment media bias the types of Salmonella enterica strains isolated from mixed strain cultures and complex enrich-ment broths. PLoS ONE 7: 1-8. DOI: 10.1371/ journal.pone.0034722.
Hadjinicolaou A, Dmetriou VL, Emmanuel MA, Kako-yiannis CK, Kostrikis LG. 2009. Molecular beacon-based real-time PCR detection of pri-mary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and cli-nical samples. BMC Microbiol 9: 1-14. DOI: 10. 1186/1471-2180-9-97.
Hoelzer K, Switt AIM, Wiedmann M. 2011. Animal contact as a source of human non-typhoidal salmonellosis. Vet Res 42: 1-28. DOI: 10.1186/1297-9716-42-34.
[ISO] International Organization for Standardization. 2017. ISO 6579:1 Detection of Salmonella spp.. Switzerland (CH): ISO.
[Kementan RI] Kementerian Pertanian Republik Indonesia. 2020. Statistik Peternakan dan Kesehatan Hewan 2020. Direktorat Jenderal Peternakan dan Kesehatan Hewan, Jakarta.
Krämer N, Löfström C, Vigre H, Hoorfar J, Bunge C, Malorny B. 2011. A novel strategy to obtain quantitative data for modelling: Combined en-richment and real-time PCR for enumeration of salmonellae from pig carcasses. Int J Food Microbiol 145: 86-96. DOI: 10.1016/j.ijfoodmicro. 2010.08.026.
Liu B, Zhou X, Zhang L, Liu W, Dan X, Shi C, Shi X. 2012. Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis. Food Control 27: 87-93. DOI: 10.1016/j.foodcont.201 2.01.062.
Maciel BM, Dias JCT, Romano CC, Sriranganathan N, Brendel M, Rezende RP. 2011. Detection of Salmonella Enteritidis in asymptomatic carrier animals: comparison of quantitative real-time PCR and becteriological cultue methods. Genet Mol Res 10: 2578-2588.
Maksum IP, Suhaili, Amalia R, Kamara DS, Rach-man SD, Rachman RW. 2018. PCR Multiplex untuk identifikasi Mycobacterium tuberculosis resisten terhadap isoniazid dan rifampisin pada galur lokal balai laboratorium kesehatan Provinsi Jawa Barat. J Kimia Valensi 4: 107-118. DOI: 10.15408/jkv.v4i2.7226.
Matias BG, Pinto PSA, Cossi MVC, Silva JA, Vanetti MCD, Nero LA. 2010. Evaluation of a PCR protocol for the detection of Salmonella species directly from superficial samples of chicken carcasses and pre-enrichment broth. Poultry Sci 89: 1524-1529. DOI: 10.3382/ps.2009-003 68.
Myint MS, Johnson YJ, Tablante NL, Heckert RA. 2006. The effect of pre-enrichment protocol on the sensitivity and specificity of PCR for detec-tion of naturally contaminated Salmonella in raw poultry compared to conventional culture. Food Microbiol 23: 599-604. DOI: 10.1016/j.fm.2005. 09.002
Nurjanah S, Rahayu WP, Komalasari E. 2017. Sensitivity of multiplex real-time PCR assay for detection of pathogenic E. coli on ice sample. November 14-17 2017. Ho Chi Minh City, Vietnam. Ho Chi Minh City (VI): Proceedings of the 15th ASEAN Conference on Food Science and Technology.
Nurjanah S, Rahayu WP. Mutaqin LA. 2018. Detection method for Salmonella Typhimurium and Salmonella Enteritidis using real-time polymerase chain reaction. Int J Eng Technol 7: 302-306. DOI: 10.14419/ijet.v7i4.14.27661.
Park SH, Kim HJ, Cho WH, Kim JH, Oh MH, Kim SH, Lee BK, Ricke SC, Kim HY. 2009. Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR. FEMS Microbiol Lett 30: 137-146. DOI: 10.1111/j.1574-6968.20 09.01809.x.
Radji M, Puspaningrum A, Sumiati A. 2010. Deteksi cepat bakteri Escherichia coli dalam sampel air dengan metode polymerase chain reaction menggunakan primer 16e1 dan 16e2. Makara J Sci 14: 39-43. DOI: 10.7454/mss.v14i1.474.
Reyes-Escogido L, Balam-Chi M, Rodriguez-Buenfil I, Valdés J, Kameyama L, Martinez-Pérez F. 2010. Purification of bacterial genomic DNA in less than 20 mins using chelex-100 microwave: examples from stains of lactic acid bacteria isolated from soil samples. Anton Leeuw 98: 465-474. DOI: 10.1007/s10482-010-9462-0.
Saeki EK, Alves J, Bonfante RC, Hirooka EY, de Oliveira TCRM. 2012. Multiplex PCR (mPCR) for detection of Salmonella spp. and the diffe-rentiation of Typhimurium and Enteritidis sero-vars in chicken meat. J Food Safety 33: 25-29. DOI: 10.1111/jfs.12019.
Safitri E, Hidayati NA, Hertati R. 2019. Prevalensi bakteri Salmonella pada ayam potong yang dijual di pasar tradisional pangkalpinang. Ekotomia: J Penelitian Biologi, Botani, Zoologi, dan Mikrobiologi 4: 25-30. DOI: 10.33019/ ekotonia.v4i1.1012.
Soumet C, Ermel G, Rose N, Rose V, Drouin P, Salvat G, Colin P. 1999. Evaluation of multiplex PCR assay for simultaneous identification of Salmonella spp., Salmonella Enteritidis and Salmonella Typhimurium from environmental swabs of poultry houses. Lett Appl Microbiol 28: 113-117. DOI: 10.1046/j.1365-2672.1999.0048 8.x.
Taskila S, Tuomola M, Ojamo H. 2012. Enrichment cultivation in detection of food-borne Salmo-nella. Food Control 26: 369-377. DOI: 10.1016/ j.foodcont.2012.01.043.
Tortajada-Genaro LA, Rodrigo A, Hevia E, Mena S, Niñoles R, Maquieira Á. 2015. Microarray on digital versatile disc for identification and geno-typing of Salmonella and Campylobacter in meat products. Anal Bioanal Chem 407: 7285-7294. DOI: 10.1007/s00216-015-8890-0
Tran HH, Trinh KTL, Lee NY. 2013. Pressure-driven one-step solid phase-based on-chip sample preparation on a microfabricated plastic device and integration with flow-through polymerase chain reaction (PCR). J Chromatogr B 936: 88-94. DOI: 10.1016/j.jchromb.2013.06.037.
Triyani Y, Nafsi N, Yuniarti L, Sekarwana N, Sutedja E, Gurnida DA, Parwati I, Alisjahbana B. 2016. Rancangan primer spesifik gen macrophage mannose receptor (MMR) untuk Polymerase Chain Reaction (PCR) dan sekuensing Deoxyri-bose Nucleic Acid (DNA). Indonesian J Clin Pathol Med Lab 22: 158-162. DOI: 10.24293/ ijcpml.v22i2.1120.
Utari LK, Riyanti, Santosa PE. 2016. Status mikro-biologis daging broiler di pasar tradisional kabu-paten Pringsewu. J Ilmiah Peternakan Terpadu 4: 63-66.
Wulan HA, Nurjanah S, Rahayu, WP. 2021. Sensi-tivity of enrichment-PCR method for Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis analysis in chicken carcasses. Food Res 5: 54-61. DOI: 10.26656/ fr.2017.5(2).429.
Yosua A. 2018. Deteksi Salmonella Hadar, Salmonella Typhimurium, dan Salmonella Ente-ritidis menggunakan Polymerase Chain Reac-tion (PCR). [Skripsi]. Bogor: Fakultas Teknologi Pertanian, Institut Pertanian Bogor.
[WHO] World Health Organization. 2014. WHO Global Salmonella survey: Progress Report 2008-2013. World Health Organization.
Authors
NurjanahS., RahayuW. P., Dewanti-Hariyadi R., AsthitiN. G. A. M. W., & MelatiR. P. (2022). SIMPLEKS DAN MULTIPLEKS PRE-ENRICHMENT-PCR UNTUK DETEKSI Salmonella Enteritidis DAN Typhimurium PADA KARKAS AYAM . Jurnal Teknologi Dan Industri Pangan, 32(2), 148-156. https://doi.org/10.6066/jtip.2021.32.2.148
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