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 :
Ph.D Nguyễn Hoàng Thảo Ly
Name : Nguyễn Thị Ngọc Huyền
ID : 19125516
Class : DH19TP
School year : 2019 – 2024

Ho Chi Minh City, May, 2023

Contents
List of Table ....................................................................................................................1

List of figure ...................................................................................................................1

I. INTRODUCTION ......................................................................................................2

1. Overview of Salmonella ......................................................................................2

2. Overview of PCR testing .....................................................................................3

3. Salmonella Realtime PCR Kit ............................................................................5

II. Materials, equipments, and methods ......................................................................6

1. Materials...............................................................................................................6

2. Equipment ............................................................................................................6

3. Methods ................................................................................................................7

3.1. Prepare sample ................................................................................................7

3.2. Sample proliferation ........................................................................................8

3.3. DNA extraction procedure ..............................................................................9

IV. Results .....................................................................................................................12

V. Discussion .................................................................................................................14

V. Conclusion ................................................................................................................14

References .....................................................................................................................15
 List of Table
Table 1 TQPCR Salmonella (SM) results .................................................................. 14

List of figure
Figure 1 The step of PCR .......................................................................................................... 4
Figure 2 The pattern of exponential growth ................................................................................. 5
Figure 3 Preparation ................................................................................................................. 7
Figure 4 Chicken ...................................................................................................................... 8
Figure 5 Minced pork ............................................................................................................... 8
Figure 6 Sample homogenization ............................................................................................... 8
Figure 7 Incubated the sample.................................................................................................... 9
Figure 8 Aspirate the enrichment solution.................................................................................... 9
Figure 9 Centrifuge .................................................................................................................. 9
Figure 10 Step 3 ..................................................................................................................... 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

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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.

1. 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 6 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.

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Some people with salmonella infection have no symptoms. Most people develop
diarrhea, fever, and stomach (abdominal) cramps within 8 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 2 - 3 x 0.5 - 1 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 6 – 9 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.

2. 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.

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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 1 The step of PCR

This cycle repeats 25- 35 times in a typical PCR reaction, which generally takes 2 - 4
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
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roughly double in each round of cycling. This pattern of exponential growth is shown
in the image below.

Figure 2 The pattern of exponential growth

3. 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.

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- 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

1. Materials

Chicken Ground pork

Distiled water Master Mix

Peptone
2. Equipment

Spindown Homogenizer
Realtime PCR Wisetherm

Pipette tips

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Pipette Autoclave Centrifuge
 3. Methods
3.1. Prepare sample
- Prepare 2 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, 2 BPW bottles into the Autoclave sterilizer at
120oC

Figure 3 Preparation

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3.2. Sample proliferation
- Take 25g of sample and put it in sterile peptone buffered water (BPW) medium

Figure 5 Ground pork Figure 4 Chicken

- Insert the sample into the homogenizer

Ground pork Chicken

Figure 6 Sample homogenization

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- Incubated at 37oC ± 1 for 24h

Figure 7 Incubated the sample

3.3. DNA extraction procedure

- Step 1: Aspirate 1 ml of the enrichment solution into an Eppendorf tube, and close
the tube tightly.

Figure 8 Aspirate the enrichment solution

- Step 2: Centrifuge 13000RPM for 5 minutes

Figure 9 Centrifuge 9|Page

- Step 3: Remove the floating water, keep the residue

Figure 10 Step 3
- 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 5 minutes. Filtrate after centrifugation is used to
run PCR

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- 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

+ 1 cycle of 95oC for 15 minutes
+ 45 cycles with 2 temperature steps
✓ 95oC for 15 seconds
✓ 60oC for 1 minutes
Note: select FAM and HEX channels when running PCR

Figure 13 Running Realtime PCR

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IV. Results

Figure 15 FAM channel of Ground pork

Figure 14 HEX channel of Ground Pork

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Figure 18 HEX channel of Chicken

Figure 17 FAM channel of Chicken

Figure 16 Temperature data

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V. Discussion
Base on the resuls, as below:
Table 1 TQPCR Salmonella (SM) results
Ingredients Dye Channel Signal Result
FAM [-] Salmonella (SM)
Ground pork (A1)
HEX [+] negative
FAM [-]
Chicken (B1) Inhibited Sample
HEX [-]

- Chicken (B1): Both dye channels in the samples were inhibited based on the
amplification curve, as shown in Table 1. The main reason the sample was inhibited
was because it took longer than the allotted time (less than 1 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.

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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), 538-
547.

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