ارزیابی تشخیص سریع و بررسی حضور ژن‌های حدت اپرون spv در جدایه‌های سالمونلا با استفاده از روش‌های مولکولی simplexPCR و multiplexPCR
محورهای موضوعی :
آسیب شناسی درمانگاهی دامپزشکی
سمیه یزدی امیرخیز
1
,
یونس انزابی
2
,
ساناز مهمازی
3
1 - دانش‌آموخته کارشناسی ارشد رشته ژنتیک، دانشکده علوم پایه، واحد زنجان، دانشگاه آزاد اسلامی، زنجان، ایران.
2 - استادیار گروه پاتوبیولوژی، دانشکده دامپزشکی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.؛ مرکز تحقیقات بیوتکنولوژی، واحد تبریز، دانشگاه آزاد اسلامی، تبریز، ایران.
3 - استادیار گروه زیست‌شناسی، دانشکده علوم پایه، واحد زنجان، دانشگاه آزاد اسلامی، زنجان، ایران.
تاریخ دریافت : 1398/11/14
تاریخ پذیرش : 1399/08/06
تاریخ انتشار : 1399/08/01
کلید واژه:
PCR,
سالمونلا,
تشخیص سریع,
invA,
spv,
چکیده مقاله :
امروزه بـرای شناسـایی و تشخیص سروتیپ های سالمونلا در نمونه های بالینی و مواد غذائی از روش‌های سنتی تشخیص این باکتری استفاده مـی‌گـردد که وقت گیر و گاهی مشکل ساز می‌باشند، اما با کشف روش‌های سریع تشخیص مولکولی، این مشکلات تا حد زیادی مرتفع گردیده است. مطالعه حاضر، با هدف تشخیص سریع جدایه های مختلف سالمونلا بر اساس جسجوی ژن کروموزومی invA و نیز شناسائی جدایه های حاد حاوی ژن های حدت اپرون spv انجام گردید. بدین منظور تعداد 20 جدایه انسانی سالمونلا از بیمارستان های شهر تبریز و 20 جدایه این باکتری که از پنیرهای سنتی عرضه شده در بازار مصرف تبریز جدا شده بود، تهیه گردید. ابتدا با استفاده از پرایمر های اختصاصی ژن invA ، تائید مولکولی جدایه ها با استفاده از روشsimplex PCR ارزیابی گردید. در ادامه برای بررسی سویه های حاد باکتری مذکور براساس حضور ژن های اپرون spv، با استفاده از پرایمرهای اختصاصی مربوطه، به روشmultiplex PCR حضور ژن های R، C، B وspvA بررسی شد. یافته ها اولاً نشان دهنده تائید مولکولی همه جدایه ها بود. ثانیاً همه 40 جدایه مورد آزمایش دارای 3 ژن R، C وspvA بوده ولی هیچ کدام از آن ها دارای ژنspv B نبودند. به نظر می رسد که با توجه به محدودیت ها و مشکلات موجود در روش بررسی آزمایشگاهی سنتی سالمونلاها، می توان ازPCR به عنوان روشی سریع در تشخیص آلودگی به باکتری سالمونلا استفاده کرد. همچنین بایستی وجود 3 ژن از 4 ژن حدت اپرون spv در جدایه های مختلف سالمونلا در منطقه تبریز را یافته ای نامطلوب تلقی کرد که لزوم رعایت بیشتر اصول کنترل و پیشگیری در جوامع دامی و انسانی را تاکید می کند.
چکیده انگلیسی:
The traditional methods of diagnosing Salmonella which are time-consuming and sometimes problematic, are still used to identify Salmonella serotypes in clinical and food samples, but with the invention of rapid molecular detection methods, these problems have been largely eliminated. The present study aimed to rapidly detect different Salmonella isolates based on invA chromosomal gene search and also to identify acute isolates containing spv operon virulence genes. To this end, 20 human isolates of Salmonella were obtained from hospitals in Tabriz and 20 isolates of this bacterium were isolated from traditional cheese available on Tabriz consumer market. The molecular confirmation of isolates was first evaluated using specific primers of invA gene by simplex PCR method. Then, in order to evaluate the acute strains of the bacterium based on the presence of operon spv, the presence of spvA, B, C and R genes was examined by multiplex PCR using the relevant specific primers. The results showed that firstly, all isolates had molecular confirmation. Secondly, all 40 tested isolates had 3 spvA, C and R genes, but none of them had spvB gene. It seems that due to the limitations and problems in the traditional laboratory examination of Salmonella, PCR can be used as a rapid method to detect Salmonella infection. Also, the presence of 3 out of 4 virulence genes of opron spv in different Salmonella isolates in Tabriz region should be considered an undesirable finding, which emphasizes the need to further observe principles of control and prevention in animal and human communities.
منابع و مأخذ:
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Amini, K., Zahraei-Salehi, T., Nikbakht, G., Ranjbar, R., Amini, J. and Ashrafganjooei, S.B. (2010). Molecular detection of invA and spv virulence genes in Salmonella enteritidis isolated from human and animals in Iran. African Journal of Microbiology Research, 4(21): 2202-10.
Bailey, J. (1998). Detection of Salmonella cells within 24 to 26 hours in poultry samples with the polymerase chain reaction BAX system. Journal of Food Protection, 61(7): 792-5.
Bailey, J. and Cosby, D. (2003). Detection of Salmonella from chicken rinses and chicken hot dogs with the automated BAX PCR system. Journal of Food Protection, 66(11): 2138-40.
Borges K.A., Furian T.Q., Borsoi, A., Moraes, H.L., Salle, C.T. and Nascimento, V.P. (2003). Detection of virulence-associated genes in Salmonella Enteritidis isolates from chicken in South of Brazil. Pesquisa Veterinaria Brasileira, 33(12): 1416-1422.
D'Aoust, J.Y. (1991). Pathogenicity of food borne Salmonella. International Journal of Food Microbiology, 12(1): 17-40.
de Oliveira S.D., Rodenbusch C.R., Michael G.B., Cardoso M.I.R., Canal, C.W. and Brandelli, A. (2003). Detection of virulence genes in Salmonella Enteritidis isolated from different sources. Brazilian Journal of Microbiology, 34(1): 123-124.
Eriksson, E. and Aspan, A. (2007). Comparison of Culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry. BMC Veterinary Research, 3(1): 21.
Foley, S.L. and Lynne, A.M. (2008). Food animal-associated Salmonella challenges: pathogenicity and antimicrobial resistance. Journal of Animal Science, 86(14suppl): 173-87.
Guerra, B.S.S., Helmuth, R. and Mendoza, M.C. (2002). Characterization of a self-transferable plasmid from Salmonella enterica serotype Typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes. Antimicrobial Agents and Chemotherapy, 46(9): 2977-2981.
Guiney, D.G. and Fierer, J. (2011). The role of the spv genes in Salmonella pathogenesis. Frontiers in Microbiology, 2(1): 129.
Hochmann, H., Pust, S., von Figura, G., Aktories, K. and Barth H. (2006). Salmonella enterica spvB ADP-ribosylates actin at position Arginine-177 characterization of the catalytic domain within the spvB protein and a comparison to binary clostridial Actin-ADP-ribosylating toxins. Biochemistry, 45(4): 1271-1277.
Hoseinpour, M., Sabokbar, A., Batkhtiyari, A. and Parsa, S.H. (2013).Comparison of bacterial culture, ELISA and techniqes for detection of salmomella in poultry meat samples collected from tehran. Journal of Microbial World, 1(14): 62-72. [In persian]
Jafari, R.A., Ghorbanpoor, M., Zahraei Salehi, T., Mayahi, M. and Gholipour Azar, M. (2017). Serotyping and antibiotic resistance patterns of isolated Salmonella from broiler chickens in Ahvaz. Journal of Veterinary Clinical Pathology, 4(40): 327-336. [In persian]
Kumar, R., Surendran, P. and Thampuran N. (2008). Evaluation of Culture, ELISA and PCR assays for the detection of Salmonella in seafood. Letters in Applied Microbiology, 46(2): 221-226.
Li, H., Xu, H., Zhou, Y., Zhang, J., Long, C., Li S., et al. (2007). The phosphothreonine lyase activity of a bacterial type III effector family. Science, 315: 1000-1003.
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Lyytikäinen, O., Koort, J., Ward, L., Schildt, R., Ruutu, P., Japisson, E., et al. (2000). Molecular epidemiology of an outbreak caused by Salmonella enterica serovar Newport in Finland and the United Kingdom. Epidemiology and Infection, 124(2): 185-1892.
Mazurkiewicz, P., Thomas, J., Thompson, J.A., Liu, M., Arbibe, L., Sansonetti, P., et al. (2008). spvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen‐activated protein kinases. Molecular Microbiology, 67(6): 1371-1383.
Mayahi, M., Talazadeh, F., Jafari, R.A. and Keshavarz Zamanian, V. (2017). Isolation of Salmonella from Iranian broiler breeder farms and feed. Journal of Veterinary Clinical Pathology, 3(43): 263-276. [In persian]
McEvoy, J., Doherty, A., Sheridan, J., Blair, I. and McDowell, D.(2003). The prevalence of Salmonella spp. in bovine faecal, rumen and carcass samples at a commercial abattoir. Journal of Applied Microbiology, 94(4): 693-700.
Ngan, G.J., Ng, L.M., Lin, R.T., and Teo, J.W. (2010). Development of a novel multiplex PCR for the detection and differentiation of Salmonella enterica serovars Typhi and Paratyphi A. Research in Microbiology, 161(4): 243-248.
Nikbakht, G.H. and Tadjbakhsh, H. (2004). Study of salmonella plasmid virulence genes (spv) in salmonella enterica serovars isolated in Iran. Journal of Veterinary Research, 59(2): 137-140. [In Persian]
Nógrády, N., Kardos, G., Bistyak, A., Turcsányi, I., Mészáros, J., Galántai, Z., et al. (2008). Prevalence and characterization of Salmonella infantis isolates originating from different points of the broiler chicken–human food chain in Hungary. International Journal of Food Microbiology, 127(1-2): 162-167.
Nosrat, S., Sabokbar A., Dezfoolian, M., Tabarraie, B. and Fallah, F. (2012). Prevalence of Salmonella enteritidis, typhi and typhimurium from food products in Mofid hospital. Research in Medicine, 36(1): 43-48. [In Persian]
Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J. and Leonard, F.C. (2002). Veterinary Microbiology and Microbial Disease. Blackwell Publishing, pp: 109-118.
Ramadan, F., Unni, A., Hablas, R. and Rizk, M.(1992). Salmonella-induced enteritis. Clinical, serotypes and treatment. The Journal of the Egyptian Public Health Association, 67(3-4): 357-367.
Rastgar, H., Ghahramani, M.H., Halaj-neyshabouri, S.H., Jalali, M., Anjarani, S. and Khosrokhavar, R. (2008). Isolation and identification of salmonella typhimurium in milk by Conventional and PCR methods. Journal of Nutrition Sciences and Food Technology, 3(10): 45-52. [In Persian]
Rotger, R. and Casadesús, J. (1992). The virulence plasmids of Salmonella. International Microbiology, 2(1): 177-184.
Sabeghi, M. and Anzabi, Y. (2019). Determination of serogroup and antibiotic resistance pattern of isolated Salmonella from laying industrial poultry in Tabriz area. Journal of Veterinary Clinical Pathology, 2(50): 199-211. [In Persian]
Tabatabaei, A. and Firuzi, R. (2009). Animal diseases due to bacteria. 1st ed., Iran: Tehran University Press, pp: 206-261.
Zahraei-Salehi, T., Mahzoniae, M.R. and Ashrafi, A. (2006).To Detect invA gene in Salmonella serotypes by PCR. Journal of Veterinary Research, 61(2): 195-199.
Zahraei-Salehi, T. and Saeedzadeh, A. (2005). Detection of invA gene in isolated salmonella from broilers by PCR method. International Journal of Poultry Science, 4(8): 557-579.
_||_Ahmadi, A., Ghorbanali Zadegan, M., Najafi, A., Tavakoli, H. and Mirnejad, R. (2012). Molecular detection of Salmonella typhi in food samples by PCR using invA gene. Journal of Ilam University, 20(1): 8-17. [In Persian]
Amini, K., Zahraei-Salehi, T., Nikbakht, G., Ranjbar, R., Amini, J. and Ashrafganjooei, S.B. (2010). Molecular detection of invA and spv virulence genes in Salmonella enteritidis isolated from human and animals in Iran. African Journal of Microbiology Research, 4(21): 2202-10.
Bailey, J. (1998). Detection of Salmonella cells within 24 to 26 hours in poultry samples with the polymerase chain reaction BAX system. Journal of Food Protection, 61(7): 792-5.
Bailey, J. and Cosby, D. (2003). Detection of Salmonella from chicken rinses and chicken hot dogs with the automated BAX PCR system. Journal of Food Protection, 66(11): 2138-40.
Borges K.A., Furian T.Q., Borsoi, A., Moraes, H.L., Salle, C.T. and Nascimento, V.P. (2003). Detection of virulence-associated genes in Salmonella Enteritidis isolates from chicken in South of Brazil. Pesquisa Veterinaria Brasileira, 33(12): 1416-1422.
D'Aoust, J.Y. (1991). Pathogenicity of food borne Salmonella. International Journal of Food Microbiology, 12(1): 17-40.
de Oliveira S.D., Rodenbusch C.R., Michael G.B., Cardoso M.I.R., Canal, C.W. and Brandelli, A. (2003). Detection of virulence genes in Salmonella Enteritidis isolated from different sources. Brazilian Journal of Microbiology, 34(1): 123-124.
Eriksson, E. and Aspan, A. (2007). Comparison of Culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry. BMC Veterinary Research, 3(1): 21.
Foley, S.L. and Lynne, A.M. (2008). Food animal-associated Salmonella challenges: pathogenicity and antimicrobial resistance. Journal of Animal Science, 86(14suppl): 173-87.
Guerra, B.S.S., Helmuth, R. and Mendoza, M.C. (2002). Characterization of a self-transferable plasmid from Salmonella enterica serotype Typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes. Antimicrobial Agents and Chemotherapy, 46(9): 2977-2981.
Guiney, D.G. and Fierer, J. (2011). The role of the spv genes in Salmonella pathogenesis. Frontiers in Microbiology, 2(1): 129.
Hochmann, H., Pust, S., von Figura, G., Aktories, K. and Barth H. (2006). Salmonella enterica spvB ADP-ribosylates actin at position Arginine-177 characterization of the catalytic domain within the spvB protein and a comparison to binary clostridial Actin-ADP-ribosylating toxins. Biochemistry, 45(4): 1271-1277.
Hoseinpour, M., Sabokbar, A., Batkhtiyari, A. and Parsa, S.H. (2013).Comparison of bacterial culture, ELISA and techniqes for detection of salmomella in poultry meat samples collected from tehran. Journal of Microbial World, 1(14): 62-72. [In persian]
Jafari, R.A., Ghorbanpoor, M., Zahraei Salehi, T., Mayahi, M. and Gholipour Azar, M. (2017). Serotyping and antibiotic resistance patterns of isolated Salmonella from broiler chickens in Ahvaz. Journal of Veterinary Clinical Pathology, 4(40): 327-336. [In persian]
Kumar, R., Surendran, P. and Thampuran N. (2008). Evaluation of Culture, ELISA and PCR assays for the detection of Salmonella in seafood. Letters in Applied Microbiology, 46(2): 221-226.
Li, H., Xu, H., Zhou, Y., Zhang, J., Long, C., Li S., et al. (2007). The phosphothreonine lyase activity of a bacterial type III effector family. Science, 315: 1000-1003.
Libby, S.J., Adams, L.G., Ficht, T.A., Allen, C., Whitford, H.A., Buchmeier, N.A., et al. (1997). The spv genes on the Salmonella dublin virulence plasmid are required for severe enteritis and systemic infection in the natural host. Infection and Immunity, 65(5): 1786-1792.
Lyytikäinen, O., Koort, J., Ward, L., Schildt, R., Ruutu, P., Japisson, E., et al. (2000). Molecular epidemiology of an outbreak caused by Salmonella enterica serovar Newport in Finland and the United Kingdom. Epidemiology and Infection, 124(2): 185-1892.
Mazurkiewicz, P., Thomas, J., Thompson, J.A., Liu, M., Arbibe, L., Sansonetti, P., et al. (2008). spvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen‐activated protein kinases. Molecular Microbiology, 67(6): 1371-1383.
Mayahi, M., Talazadeh, F., Jafari, R.A. and Keshavarz Zamanian, V. (2017). Isolation of Salmonella from Iranian broiler breeder farms and feed. Journal of Veterinary Clinical Pathology, 3(43): 263-276. [In persian]
McEvoy, J., Doherty, A., Sheridan, J., Blair, I. and McDowell, D.(2003). The prevalence of Salmonella spp. in bovine faecal, rumen and carcass samples at a commercial abattoir. Journal of Applied Microbiology, 94(4): 693-700.
Ngan, G.J., Ng, L.M., Lin, R.T., and Teo, J.W. (2010). Development of a novel multiplex PCR for the detection and differentiation of Salmonella enterica serovars Typhi and Paratyphi A. Research in Microbiology, 161(4): 243-248.
Nikbakht, G.H. and Tadjbakhsh, H. (2004). Study of salmonella plasmid virulence genes (spv) in salmonella enterica serovars isolated in Iran. Journal of Veterinary Research, 59(2): 137-140. [In Persian]
Nógrády, N., Kardos, G., Bistyak, A., Turcsányi, I., Mészáros, J., Galántai, Z., et al. (2008). Prevalence and characterization of Salmonella infantis isolates originating from different points of the broiler chicken–human food chain in Hungary. International Journal of Food Microbiology, 127(1-2): 162-167.
Nosrat, S., Sabokbar A., Dezfoolian, M., Tabarraie, B. and Fallah, F. (2012). Prevalence of Salmonella enteritidis, typhi and typhimurium from food products in Mofid hospital. Research in Medicine, 36(1): 43-48. [In Persian]
Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J. and Leonard, F.C. (2002). Veterinary Microbiology and Microbial Disease. Blackwell Publishing, pp: 109-118.
Ramadan, F., Unni, A., Hablas, R. and Rizk, M.(1992). Salmonella-induced enteritis. Clinical, serotypes and treatment. The Journal of the Egyptian Public Health Association, 67(3-4): 357-367.
Rastgar, H., Ghahramani, M.H., Halaj-neyshabouri, S.H., Jalali, M., Anjarani, S. and Khosrokhavar, R. (2008). Isolation and identification of salmonella typhimurium in milk by Conventional and PCR methods. Journal of Nutrition Sciences and Food Technology, 3(10): 45-52. [In Persian]
Rotger, R. and Casadesús, J. (1992). The virulence plasmids of Salmonella. International Microbiology, 2(1): 177-184.
Sabeghi, M. and Anzabi, Y. (2019). Determination of serogroup and antibiotic resistance pattern of isolated Salmonella from laying industrial poultry in Tabriz area. Journal of Veterinary Clinical Pathology, 2(50): 199-211. [In Persian]
Tabatabaei, A. and Firuzi, R. (2009). Animal diseases due to bacteria. 1st ed., Iran: Tehran University Press, pp: 206-261.
Zahraei-Salehi, T., Mahzoniae, M.R. and Ashrafi, A. (2006).To Detect invA gene in Salmonella serotypes by PCR. Journal of Veterinary Research, 61(2): 195-199.
Zahraei-Salehi, T. and Saeedzadeh, A. (2005). Detection of invA gene in isolated salmonella from broilers by PCR method. International Journal of Poultry Science, 4(8): 557-579.
