شناسایی گونه‌های سالمونلا در فرآورده‌های خام مرغ با استفاده از کاوشگر اختصاصی به کمک روش real time pcr

هدف از انجام این پژوهش، شناسایی گونه‌های سالمونلا در فرآورده‌های مرغ با تکیه بر روش real time pcr است. بدین منظور، تعداد 45 نمونه از فرآورده‌های ماکیان که شامل سینه، کبد و سنگدان مرغ بودند از نقاط مختلف شهر مشهد و از شرکت‌های گوناگون خریداری و با رعایت موازین بهداشتی به آزمایشگاه انتقال داده شدند. طبق استانداردهای ایران و بین‌المللی، در 25 گرم از ماده غذایی، نباید هیچ سالمونلایی وجود داشته باشد. بدین منظور، 25 گرم از هر یک از نمونه‌ها جدا شده و تحت شرایط استریل به‌خوبی هموژن شدند و استخراج dna صورت گرفت. به‌منظور تایید، dna استخراج شده توسط الکتروفورز ژل آگارز مورد ارزیابی قرار گرفت. خلوص و کمیت dna استخراج شده هر یک از نمونه‌ها توسط طیف‌سنجی نوری مورد بررسی قرار گرفت. در مرحله بعد به‎منظور شناسایی جنس سالمونلا، نمونه‎ها ‎توسط روشreal time pcr  مورد بررسی قرار گرفتند. نتایج حاصل از real time pcr نشان داد که از مجموع 45 نمونه، 9 نمونه آلوده به جنس سالمونلا می‌باشد. سپس این 9 نمونه، از نظر آلودگی به گونه‌های سالمونلا تیفی موریوم و سالمونلا انتریتیدیس مورد ارزیابی قرار گرفتند که برای این منظور از روش pcr معمولی استفاده شد. نتایج نشان داد از تعداد 9 نمونه که در تست real time pcr مثبت تشخیص داده شده بود، تعداد 7 نمونه آلوده به سالمونلا تیفی موریوم بوده که از این 7 نمونه، 5 مورد مربوط به سینه مرغ و 2 مورد مربوط به کبد بود و از نظر آلودگی به سالمونلا انتریتیدیس از تعداد 9 نمونه، تنها یک نمونه آلوده بود که مربوط به سینه مرغ می‌باشد.

detection of salmonella spp. in raw chicken products using specific primer-probe set by real time-pcr method

1]introduction: poultry and meat products are the largest sources of non-typhoid salmonella infections in most countries. studies have shown that raw foods of animal origin, especially poultry and its products, are the main source of contamination of kitchens and restaurants. in terms of growth conditions, these microorganisms are resilient bacteria and easily adapt to their environmental conditions. salmonella has been known to cause intestinal disease for many years and has been reported as the most important cause of food poisoning. according to iranian and international standards, there should be no s. enteritidis or s. typhimurium in 25 grams of food. dna-based methods for the identification and differentiation of salmonella serovars have been designed and applied using specific primers at the genus and serovar levels. therefore, they can be used as useful and rapid screening tests, as well as to supplement or replace conventional biochemical and serological tests. real-time pcr, with the most accurate and reliable results using a fluorescence probe, which of course has a high cost. in this method, sequence specific fluorescence probes are used, and as a result, in the target molecule, screening and determination the presence or even the concentration of specific sequences is possible. therefore, even in the presence of other types of nucleic acid molecules, the results are obtained quickly and have a high level of specificity. under these conditions, if specific probes with different florescence dyes are used, even multiple targets can be detected in a single pcr reaction. the aim of this study was to identify s. enteritidis or s. typhimurium by pcr and salmonella spp. by real time pcr method in poultry products. material and method: in total, 45 samples of poultry products, including chicken breast, liver and gizzard (15 samples each) were purchased from different regions of mashhad and from various companies and transferred to the laboratory in accordance with hygienic standards. for each sample, 25 g of tissue was isolated and homogenized under sterile conditions and dna extraction was then performed using a dna extraction kit. the extracted dna was evaluated by agarose gel electrophoresis. the purity and quantity of dna extracted from each sample was examined by spectrophotometry method. in the next step, in order to identify the genus salmonella, the samples were examined by real time pcr. in this method we used an internal control to ensure that negative results are not false negative due to inhibitors. the results of real time pcr showed that out of 45 samples, nine samples were infected with salmonella. then, these nine samples were evaluated for salmonella typhimurium and salmonella enteritidis infection by conventional pcr method. result and discussion: the results showed that out of nine samples that were positive in real time pcr test, seven samples were contaminated with salmonella typhimurium, of which five samples were related to chicken breast and two to liver. regarding salmonella enteritidis infection, out of nine samples, only one sample was contaminated, which was related to chicken breast. conventional methods have been traditionally used to enumerate target bacteria in food. however, these methods have some limitations and require considerable time and labor. previous studies have already shown that real time pcr is more effective than conventional bacteriological methods for the detection of salmonella spp. in a study by whyte et al. (2002) the presence of salmonella was assessed by traditional culture methods and by a salmonella-specific polymerase chain reaction (pcr) test. salmonella was recovered from 16% of samples using traditional culture methods. in contrast, the pcr assay proved to be more sensitive and detected salmonella dna in 19% of the examined samples (whyte et al. 2002). results of pcr with specific primers showed that reactions in real time pcr with general primers of salmonella spp. were done correctly. despite of accuracy and speed of real time pcr to detect dna of microorganisms, further studies are developed to have more advantages. loop-mediated isothermal amplification (lamp) showed a higher sensitivity of salmonella detection in compare to qpcr (vichaibun kanchanaphum, 2020). although lamp could detect trace amount of salmonella dna but primer design for this reaction is very difficult. however, it is important to highlight that non-viable cells can be detected by real time pcr or other dna-based methods, which does not occur in traditional methods of culture and isolation that require viable cells for quantification (zeng et al., 2016).