NONG LAM UNIVERSITY

HO CHI MINH CITY

Faculty of Food Science and Technology

Practical Report
REAL-TIME PCR

Instructor : Dr. Vu Thi Lam An

Name Student ID Class

Hoàng Nguyễn Trúc Linh 15125351 DH15TP
Phạm Ngọc Giao Uyên 15125387 DH15TP
Nguyễn Mỹ Ha ̣nh 15125339 DH15TP
Nguyễn Thi ̣Nam 15125128 DH15TP
Nguyễn Thi ̣Ngo ̣c Thon 15125373 DH15TP

Ho Chi Minh City / 2019

 Table of Contents

I. INTRODUCTION ................................................................................................ 1

II. MATERIALS AND METHODS ......................................................................... 2

III. RESULTS .............................................................................................................. 4

IV. DISCUSSION ........................................................................................................ 6

V. CONCLUSIONS ................................................................................................... 8

VI. RECOMMENDATIONS...................................................................................... 8

VII. REFERENCES...................................................................................................... 9
 I. INTRODUCTION

The genus Salmonella belongs to the family Enterobacteriaceae. Salmonella

are facultative anaerobic Gram-negative rods. They are non-spore forming, usually
motile with peritrichous flagella, capable of growing on ordinary media. The genus
Salmonella is the leading cause of foodborne outbreaks and remains a major public
health concern around the world. Salmonella organisms penetrate from the gut lumen
into the epithelium of the small intestine, causing acute gastrointestinal illness such as
gastroenteritis, organ focal infection, and systemic febrile infection. Food products
such as meat, eggs, poultry, seafood, produces, and pepper have been recognized as
major vehicles of Salmonella infection in humans.

The expeditious detection of Salmonella species in food and environmental

samples requires rapid, efficient, and validated methods and one of significant
methods is Real-Time PCR technique.

A real-time polymerase chain reaction (Real-Time PCR), also known as

quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular
biology based on the polymerase chain reaction (PCR). It monitors the amplification of
a targeted DNA molecule during the PCR. Real time PCR facilitates the automation and
direct detection of PCR products in less than half the time of conventional PCR, with
no post processing steps required. Real-time PCR employs the 5 exonuclease activity
of Taq DNA polymerase to cleave the reporter from the hybridized specific TaqMan
gene probe, resulting in a fluorescence signal, which is then recorded in real time. The
reduction in time and labor makes this highly sensitive and specific real-time PCR assay
an excellent alternative to conventional culture methods for surveillance and research
studies to improve food safety.

Objectives: Application of the real-time qPCR method for detection of Salmonella spp.
in pork meat.

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 II. MATERIALS & METHODS

1. Materials

The following materials were used in the experiment. Shoulder pork and a scissor
were obtained for this experiment. Furthermore, a tweezer, a micropippet, and one
micro centrifuge tube were used. In preparing peptone water solution, 1.35 g peptone
was mixed with 90mL distilled water. One 100- mL beaker were used to contain
peptone water. In preparing NaCl 0.85% solution, 0.04g NaCl was mixed with 5 mL
distilled water. Two 50-mL beakers were used to contain 5ml NaCl 0.85% and 5ml
distilled water. All of the materials were sterilized in the autoclave.

2. Methods

Overview steps:

Sampl
Sample

 Environment
 Incubated condition,
time
Enrichment
 Containers
 Homogeneous
sample

Detection

 DNA ( extracted)
Real-time PCR  Kit

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Salmonella detection

(Classical cultural method and real time PCR)

Sample

Pre-enrichment • 18 hours
<24hrs

DNA purification • 45 min

Real time PCR • 90 min

 Procedure

Prepared sample:
To start with, 10g of pork shoulder was cut and put into a plastic (should not
touch inside and the head of plastic bag). Next, peptone water was poured and stirred in
30 – 60s (all process must under 15 minutes), then it was incubated at 370C in 24 hours
(O2 was existed).

DNA Template Preparation

First, 1ml of overnight enrichment was pipetted to a micro centrifuge tube and
centrifuged 14000 rpm for 3 minutes. Second, the supernatant was removed and
completely resuspend pellet in 1ml 0.85% NaCl solution.Third, the micro centrifuge
tube was centrifuged 14000 rpm for 3 minutes again. Next, the supernatant was removed
and completely resuspend pellet in 1ml sterile water. After that, the micro centrifuge
tube was boiled in a waterbath or heat block capable of maintaining 1000C for 15
minutes. After a while, the micro centrifuge tube was centrifuged 14000 rpm for 3
minutes. Next, the supernatant was removed and saved as DNA template. This might

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be frozen, minimum at -200C, for future PCR tests. Finally, PCR experiment was run.
In addition, a 1:10 dilution of this template was made and used 1μl for testing by real
time PCR. For pure cultures ( also included control cultures), 1ml of broth culture or
colony growth from agar plate was prepared in the same way as in these step above,
and suspended in 0.85% saline. Templates might be frozen at minimum – 200C for
future use.

III. RESULTS

Figure1: The curve of positive control sample

In the initiation phase, the fluorescence emission cannot be distinguished from

the baseline (Acquisition timeline).

During the exponential or log phase (34:00) .there is an exponential increase in

fluorescence, before the plateau phase is reached.

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 In the last phase, the reagents are exhausted, and no increase in fluorescence is
observed

Figure 2: The curve of negative control sample

The line of negative control sample does not increase significantly in all analysis
time

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 Figure 3: The curve of sample

The line of sample showed positive on the real-time PCR run. Therefore, this
sample (raw pork meat) has salmonella spp.

IV. DISCUSSION

Real-time PCR

A real-time polymerase chain reaction (Real-Time PCR), also known as

quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular
biology based on the polymerase chain reaction (PCR). It monitors the amplification of
a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in
conventional PCR.

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 Real-time PCR is carried out in a thermal cycler with the capacity to illuminate
each sample with a beam of light of at least one specified wavelength and detect the
fluorescence emitted by the excited fluorophore.

The key feature in RT-PCR is that amplification of DNA is detected in real time
as PCR is in progress by the use of fluorescent reporter. The fluorescent reporter signal
strength is directly proportional to the number of amplified DNA molecules.

Real-time PCR uses an increase in the intensity of a fluorescent signal generated

by an intercalating dye or from the breakdown of a dye-labeled probe during
amplification of a target sequence to detect nucleic acids either for their presence or
absence or for their amount.

Analyzing the results

In the figure 1, the fluorescence emission during qPCR is proportional to the

synthesized DNA, and can be can be visualized as an amplification plot. Typically, an
amplification curve presents three different phases: The first is called the initiation
phase, it occurs during the first PCR cycles where the emitted fluorescence cannot be
distinguished from the baseline. During the exponential or log phase there is an
exponential increase in fluorescence, before the plateau phase is reached. In this last
phase, the reagents are exhausted, and no increase in fluorescence is observed. Only in
the exponential phase, quantification is possible.

There is not fluorescence emission during qPCR in figure 2 because this is

negative control sample. In particular, it does not contain any of the bacterial DNA
analyzed (Salmonella spp.)

About the figure 3, fluorescence emission appears in the line of the analyzed
sample after analyzing time. Therefore, this sample (raw pork meat) contains DNA of
Salmonella or Salmonella presents in the sample.

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 V. CONCLUSIONS

In conclusion, the Polymerase Chain Reaction is a very helpful way to replicate

DNA. It is a very realizable way to test and analyze the DNA and is also very efficient
in the sense that it only takes a few hours.

The strengths of this developing technology are manifold: ease and speed of
assay execution for large batches of samples; high sensitivity; wide dynamic range of
detection and quantification with a linear relation between log target to detection
threshold cycle; and differentiation of detected nucleic acid sequences.

False-positive results may be caused by contamination during sample handling

and nucleic acid extraction. Careful sample handling, dedicated laboratories and
equipment, and well-trained personel are essential to control systematic contamination
problems. Besides, main cause of false-negative results is lack of robustness of real-
time PCR assay, resulting also in poor reproducibility.

The invention of PCR and real-time PCR has led to many major scientific
advances. Though both methods are still regularly used in laboratories, real-time PCR
is gaining popularity and quickly becoming the most cost- and time-effective method
for analyzing DNA products.

The use of real-time PCR expands to many areas of the clinical laboratory
including genetics, virology, and microbiology. The uses mentioned throughout this
course are only a small sampling of the many different applications in which this
technology is used. With more real-time PCR platforms and practices being created, the
growth and potential of this technology is just beginning.

VI. RECOMMENDATIONS

To avoid mistakes, in the process of conducting experiments, we need to

consider the following issues. Firstly, material for getting and keeping must be
biologically purified to avoid inhibiting the amplification reaction. Secondly, The DNA
must be extracted in maximum amount, pure and free of proteins which can inhibit the
amplification reaction. Finally, check the quality and sensitivity of PCR mix before
using and carefully set parameters on PCR program before starting the test.

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 VII. REFERENCES

Bustin, S. A., Benes, V., Nolan, T., & Pfaffl, M. W. (2005). Quantitative real-time RT-
PCR–a perspective. Journal of molecular endocrinology, 34(3), 597-601.

Logan, Julie, et al., eds. Real-time PCR: current technology and applications. Horizon
Scientific Press, 2009.

Van Poucke, L. S. G. 1990. Salmonella-Tek, a rapid screening method for Salmonella

species in food. Appl. Environ. Microbiol. 56: 924–927.