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mô tả cách thực hành xét nghiệm Realtime PCR. Sử dụng thịt heo thịt gà để xét nghiệm. báo cáo được viết bằng tiếng anh. mô tả cách thực hành xét nghiệm Realtime PCR. Sử dụng thịt heo thịt gà để xét nghiệm. báo cáo được viết bằng tiếng anh.
MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY FACULTY OF CHEMICAL ENGINEERING AND FOOD TECHNOLOGY LAB REPORT POLYMERASE CHAIN REACTION SALMONELLA REALTIME PCR Dr Nguyễn Minh Xuân Hồng Instructor : Name : Nguyễn Thị Ngọc Huyền ID : 19125516 Class : DH19TP School year : 2019 – 2024 Ph.D Nguyễn Hoàng Thảo Ly Ho Chi Minh City, May, 2023 Contents List of Table List of figure I INTRODUCTION Overview of Salmonella 2 Overview of PCR testing .3 Salmonella Realtime PCR Kit II Materials, equipments, and methods Materials .6 Equipment Methods 3.1 Prepare sample 3.2 Sample proliferation 3.3 DNA extraction procedure IV Results .12 V Discussion 14 V Conclusion 14 References .15 List of Table Table TQPCR Salmonella (SM) results 14 List of figure Figure The step of PCR Figure The pattern of exponential growth Figure Preparation Figure Chicken Figure Minced pork Figure Sample homogenization Figure Incubated the sample Figure Aspirate the enrichment solution Figure Centrifuge Figure 10 Step 10 Figure 11 Added distilled water 10 Figure 12 Wisetherm 10 Figure 13 Running Realtime PCR 11 Figure 14 HEX channel of Minced Pork 12 Figure 15 FAM channel of minced pork 12 Figure 16 Temperature data 13 Figure 17 FAM channel of Chicken 13 Figure 18 HEX channel of Chicken 13 1|Page I INTRODUCTION Lifetime is developing more and more, many modern devices have been invented to help the food industry advance, to meet people's needs for food Additionally, food processing methods have also been improved, many methods to improve product quality have been introduced, helping to shorten processing time, increase texture, color, and taste of food product (Hein, Flekna, Krassnig, & Wagner, 2006) However, the most concerned issue in the food industry is the issue of food hygiene and safety One of the microorganisms with high potential to cause poisoning and the most concern is Salmonella Salmonella is the common name for many types of bacteria They can be found in poultry and eggs, or in fruits and vegetables as well Most types of Salmonella affect the stomach directly, causing stomach pain, diarrhea and vomiting for a few days There are also types that enter the intestinal tract, causing typhoid, causing the patient to die Therefore, the detection of Salmonella in food is a top priority The more recent modern method that is widely used is PCR (Polymerase Chain Reaction) which is a specialized method of amplifying DNA using specialized primers In this report, the PCR method used to detect Salmonella in food is presented Overview of Salmonella Salmonella is classified into the kingdom Bacteria, the phylum Proteobacteria, the family Enterobacteriaceae, and the genus Salmonella The genus Salmonella is currently divided into two species: Salmonella bongori (non-pathogenic) and Salmonella enterica (pathogenic) (Guthrie, 2018) S enterica is further divided into different species: S enterica (S entericaI), S salamae (S enterica II), S arizonae (S enterica IIIa), S diarizonae (S enterica IIIb), S.houtenae (S enterica IV) and S indica (S enterica VI) For the most part, S.enterica I serotypes are responsible for more than 99.9% of infections in humans and animals Salmonella infection (salmonellosis) is a common bacterial disease that affects the intestinal tract Salmonella bacteria typically live in animal and human intestines and are shed through stool (feces) Humans become infected most frequently through contaminated water or food 2|Page Some people with salmonella infection have no symptoms Most people develop diarrhea, fever, and stomach (abdominal) cramps within to 72 hours after exposure Most healthy people recover within a few days to a week without specific treatment In some cases, diarrhea can cause severe dehydration and requires prompt medical attention Life-threatening complications also may develop if the infection spreads beyond the intestines The risk of getting Salmonella infection is higher with travel to countries without clean drinking water and proper sewage disposal Gram-negative, flagellated, facultatively anaerobic Salmonella species are bacilli with O, H, and Vi antigens Average size from - x 0.5 - µm, migrating by flagella except S gallimarum and S pullorum, non-spore forming, they grow well at 6oC – 42oC, most suitable at 350C – 370C, pH – and most suitable at pH = 7.2 At temperatures from 180C to 400C bacteria can live up to 15 days Salmonella is a facultative anaerobic bacterium that can be grown on conventional culture media On the right medium, bacteria will grow after 24 hours Can grow on media with selective inhibitors such as DCA (deoxycholate citrate agar) and XLD (xylose lysine deoxycholate), in which XLD medium is less inhibitory so it is often used for isolation of Salmonella Typical colonies of Salmonella on this medium are round, convex, transparent, with a black center, sometimes a large black center covering the colony, the surrounding medium turns red Overview of PCR testing Polymerase chain reaction (abbreviated PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks) The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized 3|Page The basic steps are: - Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands This provides single-stranded template for the next step - Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA - Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA Figure The step of PCR This cycle repeats 25- 35 times in a typical PCR reaction, which generally takes - hours, depending on the length of the DNA region being copied If the reaction is efficient (works well), the target region can go from just one or a few copies to billions That’s because it’s not just the original DNA that’s used as a template each time Instead, the new DNA that’s made in one round can serve as a template in the next round of DNA synthesis There are many copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the number of DNA molecules can 4|Page roughly double in each round of cycling This pattern of exponential growth is shown in the image below Figure The pattern of exponential growth Salmonella Realtime PCR Kit Kit for real-time detection Real-time PCR is used to quickly and accurately detect Salmonella spp in a straightforward manner The assay uses real-time PCR reactions based on 5' nuclease to amplify a specific genomic sequence in the target microorganism The maximum sensitivity and specificity are ensured by the precisely crafted primers and probe we use A pathogen detection assay mix, a positive control, and an internal control (IC) are all included in the kit (Hein et al., 2006) - PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus) - Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis In a PCR reaction, the experimenter determines the region of DNA that will be copied, or amplified, by the primers she or he chooses PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length 5|Page - The FAM + HEX Calibrator Set contains everything necessary to calibrate the Open qPCR dual channel instrument (Boiştean, Chirsanova, Zgardan, Mitina, & Găină, 2020) II Materials, equipments, and methods Materials Chicken Distiled water Ground pork Master Mix Peptone Equipment Spindown Homogenizer Realtime PCR Wisetherm Pipette tips Pipette Autoclave 6|Page Centrifuge Methods 3.1 Prepare sample - Prepare laboratory bottle 250mL - Weigh 3.375g of BPW into each bottle, then add 225 mL to each bottle, obtaining peptone solution medium - Bring scissors, clamps, pipette tips, BPW bottles into the Autoclave sterilizer at 120oC Figure Preparation 7|Page 3.2 Sample proliferation - Take 25g of sample and put it in sterile peptone buffered water (BPW) medium Figure Ground pork Figure Chicken - Insert the sample into the homogenizer Ground pork Chicken Figure Sample homogenization 8|Page - Incubated at 37oC ± for 24h Figure Incubated the sample 3.3 DNA extraction procedure - Step 1: Aspirate ml of the enrichment solution into an Eppendorf tube, and close the tube tightly Figure Aspirate the enrichment solution - Step 2: Centrifuge 13000RPM for minutes Figure Centrifuge 9|Page - Step 3: Remove the floating water, keep the residue Figure 10 Step - Step 4: Add 100 𝜇𝑙 distilled water, shake gently Figure 11 Added distilled water - Step 5: Dry heat at 90°C for 10 minutes Figure 12 Wisetherm - Step 6: Centrifuge 13000RPM for minutes Filtrate after centrifugation is used to run PCR 10 | P a g e - Step 7: Transfer supernatant to new eppendorf - Step 8: Using the micropipette dispense 15 𝜇𝑙 master mix into the appropriate PCR tubes Use dry ice to keep it cold - Step 9: Put 5𝜇𝑙 DNA extracts of the test sample into each PCR tube containing the prepared real-time PCR master mix, and close the PCR tubes Then use spindown centrifuge to settle the entire solution in the PCR tube to the bottom of the test tube - Step 10: Run a one-step Realtime PCR program + cycle of 95oC for 15 minutes + 45 cycles with temperature steps ✓ 95oC for 15 seconds ✓ 60oC for minutes Note: select FAM and HEX channels when running PCR Figure 13 Running Realtime PCR 11 | P a g e IV Results Figure 15 FAM channel of Ground pork Figure 14 HEX channel of Ground Pork 12 | P a g e Figure 18 HEX channel of Chicken Figure 17 FAM channel of Chicken Figure 16 Temperature data 13 | P a g e V Discussion Base on the resuls, as below: Table TQPCR Salmonella (SM) results Ingredients Ground pork (A1) Chicken (B1) Dye Channel FAM HEX FAM HEX Signal [-] [+] [-] [-] Result Salmonella (SM) negative Inhibited Sample - Chicken (B1): Both dye channels in the samples were inhibited based on the amplification curve, as shown in Table The main reason the sample was inhibited was because it took longer than the allotted time (less than minute) to transfer the DNA extraction from the Eppendorf tube to the PCR tube As a result, neither the threshold cycle values for the two dye channels nor the threshold cycle period for the chicken sample could be determined - Ground Pork (A1): the HEX channel give out a positive results which indicate the Cq value or the threshold cycle of around 18.86 The FAM channel give out a negative results Because the eppendorf tube was the wrong one during implementation, it required an additional operation to transfer the sample to a tube that was appropriate for the device, which caused noisy samples to produce inaccurate results - Temperature data: This chart shows that there are 45 cycles After 16 minutes, they start to replicate; after 1.5 hours, they stop V Conclusion As discussed in this study, real-time PCR is a cutting-edge approach that is quickly taking over as the accepted way to measure cytokine mRNA levels in organs, cells, or cell cultures The method is extremely quick, accurate, and sensitive, and it has a lower risk of PCR contamination than earlier endpoint PCR assays It is clear that the test developed here for measuring the expression of cytokine genes can be easily applied to other groups of mRNA Overall, the method has made it possible for researchers to quickly and largely automatically get a deeper understanding of many immunological systems and disorders 14 | P a g e References Boiştean, A., Chirsanova, A., Zgardan, D., Mitina, I., & Găină, B (2020) Methodological aspects of real-time PCR usage in acetobacter detection Journal of Engineering Sciences(3), 232-238 Guthrie, R K (2018) Salmonella: CRC press 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 Journal of microbiological methods, 66(3), 538547 15 | P a g e