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Salmonella Gallinarum ve Salmonella Pullorum için qPCR Tanı Kitinin Geliştirilmesi

Yıl 2019, Cilt 30, Sayı 2, 137 - 142, 27.12.2019
https://doi.org/10.35864/evmd.631225

Öz

Konak spesifik ve biovar olan Salmonella Gallinarum ve Salmonella Pullorum başta tavuk olmak üzere hindi, bıldırcın, güvercin, serçe ve papağanlarda önemli hastalıklara sebep olmaktadır. Bu nedenle kanatlı hayvanlarda bu serotipleri saptamak için hızlı ve güvenilir tanı yöntemleri hastalığın etkin kontrolü için gerekli olmaktadır. Bu amaçla biovarları saptamak ve birbirinden ayrımını sağlamak için eş zamanlı olarak S. Gallinarum/Pullorum ve internal kontrol hedefli reaksiyonları içeren hızlı qPCR tanı kiti geliştirildi. S. Gallinarum/Pullorum referans ve VKMAE Kanatlı Hastalıkları Laboratuvarı'ndan temin edilen S. Gallinarum suşlarına qPCR metodu uygulandı. Çalışmada qPCR analizinin duyarlılığı ve özgüllüğü değerlendirildi. Sonuçlar geliştirilen qPCR yönteminin S. Gallinarum/Pullorum'u  kesin olarak saptayabildiğini gösterdi. qPCR yönteminin kullanım kolaylığı, güvenirliliği, teknik olarak basit, tam otomasyon, spesifik, hassas, hızlı olmasından dolayı bu biovarların teşhisi ve ayrımında daha fazla avantaj sağlayacağı sonucuna varıldı.

Kaynakça

  • 1. Akan M, (2008). Kanatlılarda Salmonella infeksiyonları ve kontrolünde temel prensipler. Mektup Ankara. 6, 3-4.2. Barrow PA, Freitas Neto OC, (2011). Pullorum disease and fowl typhoid–new thoughts on old diseases: a review. Avian Pathol. 40: 1-13.3. Berchieri JRA, Murphy CK, Marston K, Barrow PA, (2001). Observations on the persistence and vertical transmission of Salmonella enterica serovars Pullorum and Gallinarum in chickens: effect of bacterial and host genetic background. Avian Pathol. 30, 229-239. 4. Bohaychuk VM, Gensler GE, McFall ME, King RK, Render DG, (2007). A real-time PCR assay for the detection of Salmonella in a wide variety of food-animal matrices. J Food Prot. 70, 1080-1087.5. Christensen JP, Olsen JE, Hansen HC, Bisgaard M, (1992). Characterisation of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum by plasmid profiling and biochemical analysis. Avian Pathol. 21,461-470. 6. Christensen JP, Olsen JE, Bisgaard M,(1993). Ribotypes of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum. Avian Pathol. 22,725-738. 7. Çarlı KT, Ünal CB, Caner V, Eyigör A, (2001). Detection of Salmonella chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis. J Clin Microbiol. 39, 1871-1876.8. Dorak TM, (2007). Real-Time PCR. New York, NY, USA: Taylor&Francis.9. Eyigor A, Carli KT,(2003). Rapid detection of Salmonella from poultry by real-time polymerase chain reaction with fluorescent hybridization probes. Avian Dis. 47, 380-386. 
10. Eyigor A, Carli KT, (2007). A PCR-ELISA for the detection of Salmonella from chicken intestine. J Biol Environ Sci. 1, 45-49. 11. Ellingson JL, Anderson JL, Carlson Sa, Sharma VK, (2004). Twelve hour real-time PCR technique for the sensitive and specific detection of Salmonella in raw and ready-to-eat meat products. Mol Cell Prob. 18, 51-57. 12. Hein I, Flekna G, Krassnig M, Wagner M, (2006). Real-time PCR for the detection of Salmonella spp. in food: an alternative approach to a conventional PCR system suggested by the FOOD-PCR project. J Microbiol Methods. 66, 538–547.13. Hoorfar J, Cook N, Malorny B, Rådström P, De Medici D, Abdulmawjood A, Fach P, (2003). Diagnostic PCR: making internal amplification control mandatory. J Clin Microbiol. 41, 5835. 14. Feng Y, Johnston RN, Liu G, Liu S, (2013). Genomic comparison between Salmonella Gallinarum and Pullorum: differential pseudogene formation under common host restriction. PLoS ONE. 8, 1-6. 15. ISO, (2007). Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. Amendment 1: Annex D: Detection of Salmonella spp. in animal faeces and in environmental samples from the primary production stage (ISO 6579:2002/A1:2007). International Organization for Standardization, Geneva, Switzerland.16. ISO, (2012). Microbiology of food and animal feed - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 2: Enumeration by a miniaturized most probable number technique (EN ISO/TS 6579-2). International Organization for Standardization, Geneva, Switzerland. 17. Jones MA, Wigley P, Page KL, Hulme SD, Barrow PA, (2001). Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens. Infect Immun. 69, 5471-5476.18. Josefsen MH, Krause M, Hansen F, Hoorfar J, (2007). Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat. Appl Environ Microbiol. 73, 3040-3048.19. Kang MS, Kwon YK, Jung BY, Kim A, Lee KM, An BK, Song EA, Kwon JH, Chung, GS, (2011). Differential identification of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum based on polymorphic regions of glgC and speC genes. Vet Microbiol. 147, 181–185.20. Kang MS, Kwon YK, Kım HR, Oh JY, Kım MJ, An BK, Shın EG, Kwon JH, Park CK, (2012). Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R. Vet Microbial. 160, 491-495.21. Kisiela D, Kuczkowski M, Kiczak L, Wieliczko A, Ugorski M, (2005). Differentiation of Salmonella Gallinarum biovar Gallinarum from Salmonella Gallinarum biovar Pullorum by PCR-RFLP of the fimH gene. J Vet Med B. 52 (2005), 214-218.22. Le Minör L, (1992). The Genus Salmonella. In: A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Application, Ed.: Balows A, Truper, H.G., Dworkin, M., Harder, W., Schleifer, K.H. 2nd Ed., Springer-Verlag, New York, Berlin, Heidelberg, 2760-2774.23. Löfström C, Hansen F, Hoorfar J, (2010). Validation of a 20-h real-time PCR method for screening of Salmonella in poultry faecal samples. Vet Microbiol. 144, 511-514. 24. Malorny B, Paccasoni E, Fach P, Bunge C, Martin A, Helmuth R (2004). Diagnostic real-time PCR for detection of Salmonella in food. Appl Environ Microbiol. 70, 7046-7052. 25. OIE, (2018). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: Fowl Typhoid and Pullorum Disease, Bölüm 2.3.11.26. Paiva JB, Cavallini JS, Silva MD, Almeida MA, Angela HL, Berchieri JA, (2009) Molecular differentiation of Salmonella Gallinarum and Salmonella Pullorum by RFLP of fliC gene from Brazilian isolates. Brazilian J Poult Sci. 11, 271-276.27. Rijpens N, Herman L, Vereecken F, Jannes G, Smedt JD, Zutter LD, (1999). Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic seperation and PCR. Int J Food Microbiol. 46, 37-44.28. Rubio S, Antonio R, Penha, C, Maria A, Almeida D, Junior AB, (2017). Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum. Avian Pathol. 46, 644-651. 29. Sareyyüpoğlu B, Cantekin Z, (2009). Use of a multiplex-polymerase chain reaction for detection of Salmonella and Chlamydophila psittaci from caged birds. Ankara Üniv Vet Fak Derg. 56, 269-273.30. Shivaprasad HL, (2008). Pullorum disease and fowl typhoid Y.M. Siaf (Ed.), Diseases of poultry., Iowa State University Press, Ames, Iowa, pp. 568-582.31. Schoder D, Schmalwieser A, Schauberger G, Kuhn M, Hoorfar J, Wagner M, (2003). Physical characteristics of six new thermocyclers. Clin Chem. 49, 960-963. 32. Soria MC, Soria MA, Bueno DJ, (2012). Comparison of 2 culture methods and PCR assays for Salmonella detection in poultry feces. Poult Sci. 91, 616-626. 33. Trabulsi LR, Edwards PR, (1962). The differentiation of Salmonella Pullorum and Salmonella Gallinarum by biochemical methods. Cornell Vet. 52, 563-569.34. Xiong D, Song L, Tao J, Zheng H, Zhou Z, Geng S, Pan Z, Jiao X, (2017). An efficient multiplex PCR-based assay as a novel tool for accurate inter-serovar discrimination of Salmonella Enteritidis, S. Pullorum/Gallinarum and S. Dublin. Front Microbiol. 8,420-429. 35. Xu LJ, Liu ZJ, Li Y, Yin C, Hu YC, Xie XL, Li QC, Jiao XA, (2018). A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ. Avian Pathol. 47(3):238–44.

Development of qPCR Diagnostic Kit for Salmonella Gallinarum and Salmonella Pullorum

Yıl 2019, Cilt 30, Sayı 2, 137 - 142, 27.12.2019
https://doi.org/10.35864/evmd.631225

Öz

Salmonella Gallinarum and Salmonella Pullorum cause important disease of chicken, the others are turkey, quail, pigeon, sparrow and parrot, which are host specific and biovars of each other. Therefore, rapid and reliable meth-ods to detect these poultry-associated Salmonella serotypes are necessary for efficient control of Salmonella in poultry. For this purpose, a rapid qPCR diagnostic kit including S. Gallinarum / Pullorum and internal control targeted reactions was developed simultaneously to detect and differentiate biovars. S. Gallinarum/Pullorum reference and S. Gallinarum strains that were developed from VKMAE Poultry Laboratory were performed using qPCR method. In this study, sensitivity and specificity of qPCR analysis were evaluated. The results showed that the developed qPCR method was able to 

detect Salmonella Gallinarum/Pullorum correctly. Since the ease of use, reliability, technically simple, full automation, specific, sensitive and fast, the qPCR method is thought to provide more advantages in the diagnosis and differentiation of these biovars.

Kaynakça

  • 1. Akan M, (2008). Kanatlılarda Salmonella infeksiyonları ve kontrolünde temel prensipler. Mektup Ankara. 6, 3-4.2. Barrow PA, Freitas Neto OC, (2011). Pullorum disease and fowl typhoid–new thoughts on old diseases: a review. Avian Pathol. 40: 1-13.3. Berchieri JRA, Murphy CK, Marston K, Barrow PA, (2001). Observations on the persistence and vertical transmission of Salmonella enterica serovars Pullorum and Gallinarum in chickens: effect of bacterial and host genetic background. Avian Pathol. 30, 229-239. 4. Bohaychuk VM, Gensler GE, McFall ME, King RK, Render DG, (2007). A real-time PCR assay for the detection of Salmonella in a wide variety of food-animal matrices. J Food Prot. 70, 1080-1087.5. Christensen JP, Olsen JE, Hansen HC, Bisgaard M, (1992). Characterisation of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum by plasmid profiling and biochemical analysis. Avian Pathol. 21,461-470. 6. Christensen JP, Olsen JE, Bisgaard M,(1993). Ribotypes of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum. Avian Pathol. 22,725-738. 7. Çarlı KT, Ünal CB, Caner V, Eyigör A, (2001). Detection of Salmonella chicken feces by a combination of tetrathionate broth enrichment, capillary PCR, and capillary gel electrophoresis. J Clin Microbiol. 39, 1871-1876.8. Dorak TM, (2007). Real-Time PCR. New York, NY, USA: Taylor&Francis.9. Eyigor A, Carli KT,(2003). Rapid detection of Salmonella from poultry by real-time polymerase chain reaction with fluorescent hybridization probes. Avian Dis. 47, 380-386. 
10. Eyigor A, Carli KT, (2007). A PCR-ELISA for the detection of Salmonella from chicken intestine. J Biol Environ Sci. 1, 45-49. 11. Ellingson JL, Anderson JL, Carlson Sa, Sharma VK, (2004). Twelve hour real-time PCR technique for the sensitive and specific detection of Salmonella in raw and ready-to-eat meat products. Mol Cell Prob. 18, 51-57. 12. Hein I, Flekna G, Krassnig M, Wagner M, (2006). Real-time PCR for the detection of Salmonella spp. in food: an alternative approach to a conventional PCR system suggested by the FOOD-PCR project. J Microbiol Methods. 66, 538–547.13. Hoorfar J, Cook N, Malorny B, Rådström P, De Medici D, Abdulmawjood A, Fach P, (2003). Diagnostic PCR: making internal amplification control mandatory. J Clin Microbiol. 41, 5835. 14. Feng Y, Johnston RN, Liu G, Liu S, (2013). Genomic comparison between Salmonella Gallinarum and Pullorum: differential pseudogene formation under common host restriction. PLoS ONE. 8, 1-6. 15. ISO, (2007). Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. Amendment 1: Annex D: Detection of Salmonella spp. in animal faeces and in environmental samples from the primary production stage (ISO 6579:2002/A1:2007). International Organization for Standardization, Geneva, Switzerland.16. ISO, (2012). Microbiology of food and animal feed - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 2: Enumeration by a miniaturized most probable number technique (EN ISO/TS 6579-2). International Organization for Standardization, Geneva, Switzerland. 17. Jones MA, Wigley P, Page KL, Hulme SD, Barrow PA, (2001). Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens. Infect Immun. 69, 5471-5476.18. Josefsen MH, Krause M, Hansen F, Hoorfar J, (2007). Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat. Appl Environ Microbiol. 73, 3040-3048.19. Kang MS, Kwon YK, Jung BY, Kim A, Lee KM, An BK, Song EA, Kwon JH, Chung, GS, (2011). Differential identification of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum based on polymorphic regions of glgC and speC genes. Vet Microbiol. 147, 181–185.20. Kang MS, Kwon YK, Kım HR, Oh JY, Kım MJ, An BK, Shın EG, Kwon JH, Park CK, (2012). Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R. Vet Microbial. 160, 491-495.21. Kisiela D, Kuczkowski M, Kiczak L, Wieliczko A, Ugorski M, (2005). Differentiation of Salmonella Gallinarum biovar Gallinarum from Salmonella Gallinarum biovar Pullorum by PCR-RFLP of the fimH gene. J Vet Med B. 52 (2005), 214-218.22. Le Minör L, (1992). The Genus Salmonella. In: A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Application, Ed.: Balows A, Truper, H.G., Dworkin, M., Harder, W., Schleifer, K.H. 2nd Ed., Springer-Verlag, New York, Berlin, Heidelberg, 2760-2774.23. Löfström C, Hansen F, Hoorfar J, (2010). Validation of a 20-h real-time PCR method for screening of Salmonella in poultry faecal samples. Vet Microbiol. 144, 511-514. 24. Malorny B, Paccasoni E, Fach P, Bunge C, Martin A, Helmuth R (2004). Diagnostic real-time PCR for detection of Salmonella in food. Appl Environ Microbiol. 70, 7046-7052. 25. OIE, (2018). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: Fowl Typhoid and Pullorum Disease, Bölüm 2.3.11.26. Paiva JB, Cavallini JS, Silva MD, Almeida MA, Angela HL, Berchieri JA, (2009) Molecular differentiation of Salmonella Gallinarum and Salmonella Pullorum by RFLP of fliC gene from Brazilian isolates. Brazilian J Poult Sci. 11, 271-276.27. Rijpens N, Herman L, Vereecken F, Jannes G, Smedt JD, Zutter LD, (1999). Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic seperation and PCR. Int J Food Microbiol. 46, 37-44.28. Rubio S, Antonio R, Penha, C, Maria A, Almeida D, Junior AB, (2017). Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum. Avian Pathol. 46, 644-651. 29. Sareyyüpoğlu B, Cantekin Z, (2009). Use of a multiplex-polymerase chain reaction for detection of Salmonella and Chlamydophila psittaci from caged birds. Ankara Üniv Vet Fak Derg. 56, 269-273.30. Shivaprasad HL, (2008). Pullorum disease and fowl typhoid Y.M. Siaf (Ed.), Diseases of poultry., Iowa State University Press, Ames, Iowa, pp. 568-582.31. Schoder D, Schmalwieser A, Schauberger G, Kuhn M, Hoorfar J, Wagner M, (2003). Physical characteristics of six new thermocyclers. Clin Chem. 49, 960-963. 32. Soria MC, Soria MA, Bueno DJ, (2012). Comparison of 2 culture methods and PCR assays for Salmonella detection in poultry feces. Poult Sci. 91, 616-626. 33. Trabulsi LR, Edwards PR, (1962). The differentiation of Salmonella Pullorum and Salmonella Gallinarum by biochemical methods. Cornell Vet. 52, 563-569.34. Xiong D, Song L, Tao J, Zheng H, Zhou Z, Geng S, Pan Z, Jiao X, (2017). An efficient multiplex PCR-based assay as a novel tool for accurate inter-serovar discrimination of Salmonella Enteritidis, S. Pullorum/Gallinarum and S. Dublin. Front Microbiol. 8,420-429. 35. Xu LJ, Liu ZJ, Li Y, Yin C, Hu YC, Xie XL, Li QC, Jiao XA, (2018). A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ. Avian Pathol. 47(3):238–44.

Ayrıntılar

Birincil Dil Türkçe
Konular Veteriner Hekimlik
Bölüm Araştırma Makaleleri
Yazarlar

Özlem ŞAHAN YAPICIER>

0000-0003-3579-9425
Türkiye

Yayımlanma Tarihi 27 Aralık 2019
Gönderilme Tarihi 9 Ekim 2019
Kabul Tarihi 6 Aralık 2019
Yayınlandığı Sayı Yıl 2019, Cilt 30, Sayı 2

Kaynak Göster

APA Şahan Yapıcıer, Ö. (2019). Salmonella Gallinarum ve Salmonella Pullorum için qPCR Tanı Kitinin Geliştirilmesi . Etlik Veteriner Mikrobiyoloji Dergisi , 30 (2) , 137-142 . DOI: 10.35864/evmd.631225


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