practical virology laboratory

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY
INSTITUTE OF BIOTECHNOLOGY RESEARCH & DEVELOPMENT

LAB REPORT
PRACTICAL VIROLOGY
CODE MM414C
GROUP 3

INSTRUCTOR

STUDENTS

TRƯƠNG THỊ BÍCH VÂN, PhD. Trần Thị Thảo My – B1904532

Trần Yến Nhi – B1904693
Trần Nguyễn Nguyệt Thanh – B1904697

12/2021
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PART I: EXTRACTION OF WHITE SPOT SYNDROM VIRUS DNA
I. Introduction
a. Questioned
The Mekong Delta is one of the largest spearhead industries in Vietnam such as aquaculture,
agriculture, etc. Talking about the aquaculture industry, mention shrimp farms in Vietnam, with a
large area that has brought economic benefits in processing and exporting. Nowadays, to serve the
domestic and foreign, leading to the development of scientific and technologically applied shrimp
farming models. However, shrimp farms always have the potential to cause disease, causing

damage to shrimp farmers and the aquaculture industry. As the result, the emergence of a drugresistant mutant strain of viruses is one of the biggest obstacles to the economic development of
our country. Specifically, white spot syndrome virus (WSSV) with the ability to spread quickly
causing mass death of shrimp is one of them. WSSV does not have an effective cure, so it is very
important to do well in the prevention and early diagnosis. Polymerase chain reaction (PCR)
method has shown detecting viral diseases and it is essential to understand how the mechanism
works practically.
Identification of white spot syndrome virus (WSSV): The virus belongs to genus Whispovirus
in the family Nimaviridae with a rod-shaped, double-stranded DNA (group I according to
Baltimore classification of viruses). Enveloped ovoidal particles of about 275 nm in length and 120
nm in width, with a tail-like appendage at one end. The genome of WSSV is circular dsDNA about
300 kb in size, encoding for at least 531 ORF. The modes of transmission of WSSV in the natural
environment are mainly by vertical and/or horizontal ways. WSSV can spread quickly among the
farms and sites. WSSV has a wide host range that contains all cultured, wild marine shrimps, crabs,
lobsters, crayfishes, Squilla, copepods, and freshwater cultures species and this caused to be the
control of disease more difficult.

White Spot Syndrome Virus

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Disease symptoms: The most clinical signs are redness of body coloration and appendages,
reduction of feeding, lethargy, and characteristic of white spots on the carapace white spot on the
shell of infected shrimp under scanning electron, dome-shaped spots on the carapace measuring
0.5 to 2.0mm in diameter. Chemical composition of the spots is similar to the carapace, calcium
forming 80-90% of the total material, and may have derived from abnormalities of the cuticular
epidermis.

WSSV infection in shrimp
b. Objectives

− Know how to use Laboratory equipment (Micropipette, Centrifuge, PCR machine, Gel
electrophoresis equipment).
− Know the procedure of PCR techniques (Polymerase chain reaction or PCR, is a technique
to make many copies of a specific DNA region in vitro (in a test tube rather than an organism).
PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers
designed specifically for the DNA region of interest.
II. Method
a. Preparation & equipment
− Prepare
+
Lysis buffer.
+
TE 0.1X.
+
Absolutely alcohol.
+
Shrimp sample.
+
Mytag mix.
+
Agarose gel: 0.48g agarose, 70mL TAX 1X, SafeView.
− Centrifuge: Using in DNA extraction.
− PCR machine: Carrying out reactions in PCR.

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− Gel electrophoresis equipment: Used to separate mixtures of DNA, RNA, or proteins
according to molecular size.
− Bio-rad machine.

− Micropipette.
− Eppendorf.
b. Extraction WWSSV DNA from shrimp process: There are many different methods of DNA
extraction, so it is necessary to choose a suitable method depending on the study subjects. The
quick DNA extraction method is a simple and efficient technique that allows us to have DNA
samples to test quickly in molecular biology.
WSSV belongs to the group of gram-positive viruses, so the method that is supposed to have
the most stable performance is shown below.

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100 – 150 gram of infected shrimp gill
or epidermis under the head is crashed
thoroughly

Cells-breaking by physical methods

Sample is mixed with 1mL of lysis
buffer and leave for 10 minutes

Break up membrane structure

Centrifuging (14000rpm) for 5 minutes,
transfer 400 µL solution to a new
eppendorf

Add 600 µL of Ethanol 95%, undergo
centrifugation (12000rpm) for 5
minutes

Remove the liquid. 1mL of Ethanol
70% is added, continue to centrifuge
(12000rpm) for 2 minutes.
This process can be done twice.

Separate DNA from orthe particle

Elimiate the solvation shell that surrounds
the DNA, thus allowing the DNA to precipitate in
pellet form.

DNA washing.
Allows the salts to dissolve while minimizing DNA
solubility.

Use the absorbent paper to remove any
droplet

Help the sample dry faster

Dry the sample
(vaccum centrifuge can be used)

Drain out any ethanol particle which will prevent
any later reaaction.

Solubilize DNA while protecting it from
degradation

150 µL TE 0.1 is added to dissovle the
DNA

Electrophoresis a part and store the
rest to use in PCR

Detect the present of DNA

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III. Result
 Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or
proteins according to molecular size, charge, or conformation. In gel electrophoresis, the molecules
to be separated are pushed by an electrical field through a gel that contains small pores. Agarose is
appropriate for separating DNA fragments ranging in size from a few hundred base pairs to about
20 kb.
 SafeView products represent a new and safe class of nucleic acid stains for the visualization
of double-stranded DNA, single-stranded DNA, and RNA in agarose gels. The dyes are developed
to replace toxic Ethidium Bromide (EB, a potent mutagen), commonly used in gel electrophoresis
for visualization of nucleic acids in agarose gels. SafeView Classic is used the same way as
Ethidium Bromide in agarose gel electrophoresis. It emits green fluorescence when bound to
dsDNA and red fluorescence when bound to ssDNA or RNA. This stain has two fluorescence
excitation maxima when bound to nucleic acid, at approximately 290nm and 490nm.
DNA extraction result

1

2

3

4

5

6

7

8 9 10 11 12 13 14 15 16 17

DNA

Proteins

Large agarose gel (17 wells) after electrophoresis
Meaning
No.8: Trần Thị Thảo My
No.9: Trần Yến Nhi
No.10: Trần Nguyễn Nguyệt Thanh
− All samples contain DNA.
− DNA’s purity is low as the presence of protein.
Disscusion
Purify it to reduce the number of contaminants that can compromise the results of your research
and shorten the shelf-life of your precious samples. If the DNA samples are not purified completely
due to samples not mixed well enough during extraction or not washed thoroughly, the samples
need to be repurified to removes all the remaining cellular debris and unwanted material.

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 Polymerase chain reaction (PCR) is an in vitro or laboratory technique used to amplify
sequences of DNA of interest. It is an artificial process which mimics a natural DNA replication,
also known as molecular photocopying.
 Positive control is one that you expect to work under the conditions given. The positive
control will test your master mix, MgCl2 amounts, primer annealing temperature and extension
times. If your positive control does not work, those results indicate that something is wrong with
your annealing or extension times or temperatures, MgCl2, master mix set up.
 Negative control is one which should not give you amplicons, typically the negative control
will contain no template or have one/ the other primer. Setting up two negative controls, each
containing only the forward or reverse primer, should not provide visible amplicons. Thus, any
visible bands might be a result of contamination or multiple opposing binding sites for the designed
primers.
 Ladder consists of a set of DNA fragments of different sizes. These DNA fragments are
separated and visualised as DNA bands on agarose or SDS DNA gels. DNA ladders are used during
gel electrophoresis to determine both sizes as well as for the quantification of PCR products. In
this experiment, we used the Invitrogen 100 bp DNA Ladder. This ladder is designed for sizing and
approximate quantification of double-stranded DNA in the range of 100 bp to 2,000 bp. It is
prepared from a plasmid containing repeats of a 100 bp DNA fragment. The ladder consists of 15
blunt-ended fragments ranging in length from 100 to 1,500 bp, at 100 bp increments, and an
additional fragment at 2,072 bp. 100 bp DNA Ladder is ideal for separation on 1–2% agarose gels.


PCR mechanism

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PCR tube components

Chemical/ Solution
H2 O
My Taq Mix 2X
DNA
Primer Vp26 F
Primer Vp26 R
Total

Volume (L)
14
8
2
0.5
0.5
25

Primer Sequence
5’-3’ sequence
F: GTCTTCTGACGCAATCGTTG
R: ATACGAGTGGTTGCTGTCATG

Vibrio Primer
Gen ToxR
Kim et al. (1999)

Amplifier Length (bp)
368

Processing
Step

Initialization

Process
Denaturation
Annealing
Elongation
Final elongation

Amplifier

Final hold

Temperature
94
94
54
72
72
10

Time
5 min
50 sec
30 sec
1 min 20 second
5 min
Forever

Cycle

35

PCR result
Positive control

Ladder

1

2 3

4 5

6 7

8 9 10 11 12 13 14 15

1500bp

500bp

Large agarose gel electrophoresis result from white spot syndrome virus

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Meaning
No.8: Trần Thị Thảo My
No.9: Trần Yến Nhi
No.10: Trần Nguyễn Nguyệt Thanh

− All samples were tested negative for WSSV.
− The result has proven that the shrimps we used for this experiment were not infected by
WSSV.
Disscusion
Although the result has clearly proven that WSSV did not infects the shrimps, there are still
some theories that may cause errors in PCR result and lead to false-negative test result:
− When we used the micropipette to take the sample from eppendorf, we did not shake the
tube carefully. Thus, we just took the water on the surface which is not contain the DNA.
− An incorrect PCR primer can lead to a failed reaction - one in which the wrong gene
fragment or no fragment is synthesized.
− The DNA samples were not stored in negative temperature. Thus, the DNA was damaged
and inactivated.
Questions
1. Why is a DNA standard ladder important?
The DNA ladder is a composition of standard-size fragments that runs according to their
fragment size. It contains a series of DNA fragments of known molecular weight that we compare
to our experimental samples and helps to determine the size of DNA fragments and to be popular
markers.
2. What kind of chemical is used to dye the gel to see the bands in the gel? Why?
The chemical uses to dye the gel is Safeview because the sample is dyed with better quality,
safe and minimizes the possibility of toxicity to users. Besides, Safeview can be decomposed in
sunlight, we can rest assured about the number of toxins left on the skin.
3. When to use large comb and small comb in electrophoresis?
We use the large comb when there are few samples with large volumes because the large comb
has few wells and the gap between each slit is large, so the sample with a large volume will be
easier to pass.
Small comb is used when there are many samples with a small volumes because the small comb
has many wells and the distance between each slit is small, then it is easier for sample with a small
volume to pass through the slit.

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PART II: INVESTIGATE THE EFFECTS OF BACTERIOPHAGES ON
BACTERIA
I.

Introduction
Bacteriophages (phages) are viruses of bacteria that can kill and lyse the bacteria they infect.
After their discovery early in the 20th century, phages were widely used to treat various bacterial
diseases in people and animals. Phages get into a bacterium, where they multiply, and finally they
break the bacterial cell open to release the new viruses.
Theoretically, there are no bacteria that cannot be lysed by at least one bacteriophage. In this
regard, bacteriophages are significantly more effective than antibiotics, as, although some
antimicrobial drugs have a very large spectrum of activity, an antibiotic able to kill all the bacterial
species does not exist. However, the most attractive characteristic of bacteriophages is their
specificity of action, i.e., their ability to kill only the pathogen that they can recognize.
Purpose: The goal of this method is to investigate the effects of bacteriophages on bacteria,
some factors are used in this experiment such as the concentration of bacteriophage population
solution, time factor, etc. After the experiment, we can discover bacteriophages’ factors which have
effects on the number of colonies of bacteria.
II.

Calculate

104 plaques

𝑝𝑙𝑎𝑞𝑢𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

𝑃𝑓𝑢 =

𝑣𝑜𝑙𝑢𝑚𝑒 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛

=

104 𝑥 8
1000 𝑥 500 𝑥 10−3

= 1.664 (Pfu/ml)

19 plaques

𝑃𝑓𝑢 =

𝑝𝑙𝑎𝑞𝑢𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
𝑣𝑜𝑙𝑢𝑚𝑒 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛

10

=

19
1000 𝑥 500 𝑥 10−3

= 0.038 (Pfu/ml)


25 plaques

𝑃𝑓𝑢 =

𝑝𝑙𝑎𝑞𝑢𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
𝑣𝑜𝑙𝑢𝑚𝑒 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛

=

25
1000 𝑥 500 𝑥 10−3

= 0.05 (Pfu/ml)

IMOJEV vaccine contains 4,0 – 5,8 log
PFU. It indicates that in 0.5mL of the
solution contains enough virus particles to
produce 4,0 – 5,8 log infectious plaques.

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PART III: INDIVIDUAL POINTS
After this virology laboratory course, our group have been provided basic and specialised
knowledge of viruses and methods used in virology, for examples, DNA extraction and
purification, PCR (Polymerase chain reaction). Besides, we have been taught about principle of
basic tool and equipments in molecular biology lab for virus research such as manipulation of using
micropipette and operating PCR, etc. We also know how to calculate CFU, PFU, MOI and how to
operate the laboratory equipments. This thing is extremely meaningful because the majority of the
group’s members have never practiced with the PCR experiments before. Moreover, we were
trained about self-study ability and problem-solving skills in the learning and working process. We
really appreciate the enthusiastic guidance of Ms. Trương Thị Bích Vân, Mr. Trần Văn Bé Năm
and teachers in Biotechnology Institute, supporting and giving us the opportunities to work in the

laboratory in the most effective way while ensuring the safety.
Nevertheless, our group has some suggestions for improvement for virology laboratory course:
− Althought the lecturers have provided related textbooks and taught us about the lessons
before doing the experiments, it would be better if we were given printed books. During
experiments, we can note important information directly into the textbooks and we can prepare the
lessons by reading step by step before doing experiments.
− The laboratory is full of equipment but the space is limited. In the COVID pandemic
situation, it was difficult to keep distance from others and very hard to observe clearly while
lecturers were operating the equipment. If we have a chace to work in this laboratory in next
semesters, we request a larger space for the safety and convenience.
− In this semester, our class does not have much time left in the laboratory due to the
pandemic, thus, the practical time is limited. We hope that we will have more time to do the
experiments carefully and repeat experiments many times, so we can reduce the risk of errors in
the results and also practice some crucial techniques while operating or using equipment.

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