Group members

Phùng Thị Bích Mận BTBTIU11160

Ngô Nguyễn Tiến Đạt BTBTIU13042
Phạm Hoàng Đăng BTBTIU13038
International University-Vietnam National University
Lê Ngọc Mai BTBTIU13104

School of Cẩm
Nguyễn Hữu Biotechnology
Tú BTBTIU13224

Molecular
Diagnostics
PROPOSAL
Instructor: Dr. Nguyễn Thị Huệ and Ms. Nguyen Dien Thanh Giang

April 13th, 2016

Table of Contents

Introduction………………………………………………………………………………2
DNA extraction…………………………………………………………………………..3
DNA quantification and qualification……………………………………………………8
Detection by Multiplex PCR……………………………………………………………..12
Detection HBV by Real-time PCR……………………………………………………….17
Work plan…………………………………………………………………………………23
References ……………………………………………………………………………….25

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 I. INTRODUCTION
1.GENERAL INFORMATION
1.1 Name of study
Detection of Aspergillus flavus and Aspergillus parasiticus in corn by multiplex PCR.
1.2 Field of study
Molecular diagnosis
1.3 Instructor
Asosociate Professor Nguyễn Thị Huệ Asosociate Professor and Ms Nguyễn Diên Thanh
Giang
1.4 Host institute
Lab A1 702, International University
1.5 Time
From April 2016 to May 2016
2. PROJECT CONTENTS
2.1 Background
2.1.1. Literature review
Aspergillus flavus and Aspergillus paracitucus are multicellular fungi species that infect corn
and peanut kernels respectively. A. flavus and A. paracitucus infections occur to kernels
during the development of the kernels but often show symptoms after the kernels are
harvested. In addition to causing preharvest and postharvest infections, many strains produce
significant quantities of toxic compounds known as mycotoxins, which, when consumed, are
toxic to mammals. Specifically A. paracitucus which can produce a potent carcinogen
known as aflatoxin.
Aflatoxin need to be detected by the most flexible and reliable technique .Modified PCR is
power tools for detecting pathogens and have various advantages over morphology
method. In this study, multiplex PCR was applied and conclude of 3 main steps: DNA
extraction, DNA quality and quantity determination and detection of 2 pathogens by
multiplex PCR.
2.1.2 Why this study need to be carried out
About 25% of world food crops are affected and countries located in tropical and subtropical
area suffer from Aspergillus flavus and Aspergillus parasiticus (the United Nations Food and

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 Agriculture Organization). This study provide a method with more benefit comparing
to morphology method for detecting and diagnosing of aflatoxins contamination for
food safety checking and further study.
2.2 Experimental purpose
In order to design and manipulate an molecular diagnostics test for the present of either
Aspergillus flavus or Aspergillus parasitucus in infected corn kernels through DNA
extraction and multiplex PCR as an amplification tools.
2.3 Hypothesis
There is the presence of either Aspergillus flavus or Aspergillus parasitucus in infected corn
kernels. The infection can be detected by using multiplex PCR because multiplex PCR can
detect mutiple target gene in single reaction

2.4 PROCESS OF PROJECT

DNA quality
Detection by
DNA extraction and quantity
multiplex DNA
determination
II. DETAIL CONTENT
1. PRACTICAL 3: DNA EXTRACTION
1.1 Purpose
DNA of Aspergillus flavus and Aspergillus paracitucus is extracted from infected corn and
peanut kernels using isopropanol extraction method. The extraction of DNA from these two
fungi allow us to perform many important molecular biology techniques such as PCR,
restriction fragment, cloning etc. which can help us identify the species infecting the kernels.
The first step in any DNA extraction technique is the cell lysis to disrupt the cell. Addition of
sand powder in the grinding step aids in the breakage of corn and peanut kernels’ cell wall.
Protease K and 2-mercaptoethanol are used to denature the protein components of the cells
which aid in their separation from DNA.

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 b. Materials and method

Sample Quantity
Aspergillus flavus and Aspergillus
10 corn kernels
paracitucus Infected corn kernels
Aspergillus flavus and Aspergillus
10 peanut kernels
paracitucus Infected peanut kernels
Sand powder 1 pellet : 3 sand

Chemicals Quantity Function

Neutralize the negative
charges and allowing the
NaCl 0.85 % 25mL (15mL for back up)
DNA molecules to come
together

Cells lysis buffer 3.925mg (0.025 mL 1000mM)

TrisHCl 18.75mg (0.05 mL 1000mM)
EDTA 125mg (1.25mL 10%) Destroy cell membrane
SDS 98.875mg (0.625 mL 2800mM)
NaCl  Volume: 2.5mL (1.5mL for
back up)
Protease K 25 µL (10 µL for back up) Remove the protein
Denature proteins by
breaking disulphide bonds.
2-mercapto ethanol
10µL (5 µL for back up) Remove tannins and other
polyphenols often present
in the crude plant extract. 
Solubilize lipids and a lot
Chloroform chloroform : supernatant ratio 1:1 of proteins to remove them
from the DNA.
Ice-cold 0.6 volume DNA precipitation (ethanol
isopropanol is less polar than water to
solution disrupts the

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 screening of charges by
water. If enough ethanol is
added, the electrical
attraction between
phosphate groups and any
positive ions present in
solution becomes strong
enough to form stable ionic
bonds and DNA
precipitation)
Ammonium acetate 0.08 volume Raise the salt
7.5M concentration, cause the
DNA to precipitate out of
solution.
Ethanol 70% 500 µL Wash DNA
TE buffer Protect DNA from
TrisHCL 3.925mg (0.025 mL 1000mM) degradation
EDTA 0.0025 mL (1000mM) Storage of DNA
 Volume: 200 µL (100 µL for
back up)

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 Equipments Quantity
50ml falcon 2
15ml falcon 2
Disposable transfer pipette 1
2 ml centrifuge tubes 1
1.5 ml centrifuge tubes 2
10 – 1000 µl micropipettes and 1
tips
Pestles 1
Water bath set up at 650C 1
Centrifuge – 15 ml falcon tube 1
Centrifuge – 2 ml centrifuge 1
tube
Fume hood 1

1.2 Procedure
1. Pour 10ml NaCl 0.85% into 50ml falcon containing 10 corn kernels and shake for 5
min at 1500rpm.
2. Collect the cells by centrifugation at 10000 rpm for 10mins at 25oC, discard the
supernatant.
Note: The pellet may not attach tightly onto the falcon bottom. Thus, you are advised
to use the transfer pipette to remove the supernatant. When the remaining amount of
supernatant in the falcon is about 1 – 2ml, transfer it into a new 2ml Eppendorf
(weigh the eppendorf before use). Centrifuge at 13000rpm for 5 min at 25oC to
collect pellet. Determine the quantity of the pellet.
3. Grind pellet with sterilized sand powder with the ratio (1 pellet: 3 sterilized sand
powder) for 3 min.
4. Add 1 ml of lysis buffer, continue grinding for 2 min.
5. Cap the tube, and vortex your tube for 1 min.
6. Add 10 μl of proteinase K, and 5 μl of 2-mercapto-ethanol in to your eppendorf.
7. Note: this step needs to be carried out in the fume hood because of the toxicity and
8. the smell of 2-mercapto-ethanol.
9. Heatshock

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 10. Incubate the tube in the water bath at 65oC for 30 min. Transfer it into -80oC fridge
for 5 min. After that, incubate in the water bath at 65oC for 10min.
Note: the time needs to be managed carefully.
11. Centrifuge the tube at 8000 rpm for 5 min at 25oC.
12. Transfer supernatant to new 2ml tubes.
13. Add chloroform into the tube (with the chloroform: supernatant ratio 1:1),
immediately invert the tube for 1 min.
14. Centrifuge the tube at 13000 rpm for 5 min at 25oC. Transfer aqueous layer to new
2ml tubes. Being careful not to disturb the interphase and organic phase.
15. Repeat adding chloroform step. Transfer aqueous layer to new 2ml tubes.
16. Add 0.6 volume of ice-cold isopropanol and 0.08 volume of ammonium acetate
7.5M,and gently invert several times.
17. Incubate the tube in -20oC fridge for 30 min.
18. Centrifuge at 13000rpm for 15 min at 4oC, collect the pellet.
19. Wash the pellet with 200ul cold ethanol 70%, centrifuge at 13000rpm for 5 min at
4oC.
20. Discard the supernatant, do not disturb the pellet.
21. Repeat the washing step, centrifuge at 13000rpm for 5 min at 4oC. Discard the
supernatant, do not disturb the pellet. Air dry overnight.
22. Resuspend the pellet in 100μl of TE buffer. Store at -200C.
1.3 Expected Result
The above DNA extraction method employ the phase separation between the organic
chloroform phase and the aqueous phase. Cell debris is partitioned in the organic phase while
DNA is partitioned in the aqueous phase along with other contaminants such as salts and
sugars. DNA is then precipitated by addition of isopropanol, after which, it is washed with
ethanol to remove any remaining unwanted substances. Resuspension of precipitated DNA in
TE buffer yields a solution containing purified DNA.

2. Practical 4: DNA QUANTIFICATION AND QUALIFICATION

2.1 Purpose
Determine how much DNA is present in DNA’s extract. Determine how pure the DNA is.

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 Gel electrophoresis is employed for evaluate the quality and quantity of extracted DNA for
further analysis. The method provides quick, simple, and inexpensive quantification and
qualification. Gel electrophoresis separates DNA according to molecular size. DNA sample,
which is negatively charged, is pulled by electric current from negative end to positive end of
agarose gel with pores. The smaller the molecule, the faster it moves through the pores,
which results in series of DNA bands containing DNA with larger size near the positive end
and DNA with smaller size closer to the positive end. The brightness of the band, in addition,
allows estimation of DNA concentration by comparing with DNA ladder.
2.2 Material and equipment
Gel electrophoresis Function
DNA solution
Agarose gel system
Form polysaccharide matrix which allows separation of
Agarose powder
DNA at different sizes
A dye that inserts between base pairs in DNA double
Edithium bromide helix. It can absorb UV light and re-emit a lower energy
light, which allows visualization of DNA bands.
Make DNA sample more dense and settle in the well and
Orange G loading dye
allow tracking of DNA migration during gel run
DNA ladder 1kb
Conduct electricity
1X SB buffer (NaOH, boric acid,
Prevent gel melting during gel run and maintain pH
dH2O)

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 Equipment
1 UV light system with camera
1 pH meter
1 UV Spectrophotometry
1 Electronic balance
4 Micro pipette
10 Tip
10 Eppendoft
4 Glove
4 Mask
1 Volumetric flask
1 Graduated Cylinder
1 Beaker 1000ml
Aluminum foil 1m
1 Seal tape
2.3 Procedure
1. Since the target is fungus genomic DNA, the gel is prepared with 0.7% agarose in
1X SB buffer and 1mM edithium bromide. Components of SB buffer include
10mM sodium hydroxide NaOH and 0.0364M boric acid dissolved in distilled
water.
2. The pH of the buffer is adjusted to 8.0.
3. Mixture of 0.175g agarose powder dissolved in 25ml SB buffer is boiled in
microwave for 1 minute.
4. The boiled mixture should be left cool for 3 minutes before adding 2µl edithium
bromide.
5. The mixture is then poured into prepared gel tank and comb is inserted.
6. After the mixture has solidified at room temperature for 30 minutes, comb should be
removed with care.
7. Submerge the gel in SB buffer.
8. 5g of DNA 1kb ladder is loaded in the first well.

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 9. 10 µl of DNA solution is mixed well with 3 µl of orange G loading dye, and the
mixture is then loaded in the second well.
10. Connect the electric current to the gel so that the wells are close to the negative
electrode and the bottom the gel is close to the positive electrode. The gel is run in
85 voltage for 30 minutes.
11. The gel is then visualized under UV light.
12. Analyze the gel result: Analyze the presence of the band and the smear to evaluate
the quality of extracted DNA. Estimate the concentration of extracted DNA base on
the comparison with the ladder, due to the bright level and the molecular weight.
2.4 Expected result
Our DNA solution is expected to contain mixture genomic DNA of Aspergillus flavus,
Aspergillus parasiticus and either of corn or peanut. However, the genomic DNA of A.
flavus and A. parasiticus are large. They possess 8 chromosomes with total lengths of about
37 Mb and 40 Mb respectively according to NCBI report [1], [2]. The result we should obtain
is a band near the top of the gel (close to the well) as the DNA sequences are too large to
travel far in 30 minutes of gel run. For qualification and quantification of the extracted DNA,
the band should be sharp and bright so that the extracted DNA is not smeared and in large
quantity.

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 The figure is obtained from A. flavus — primary causative agent of aflatoxins in dried figs by
Oktay & al. [5]. The bands showed are the genomic DNA extracted from A.flavus, A.
parasiticus, and another Aspergillus fungus. The genomic DNA are single bands at each
well, which demonstrates our expected result.

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 3. PRACTICAL 5: DETECTION OF A. FLAVUS AND A. PARASITICUS IN CORN
BY MULTIPLEX PCR
3.1 Purpose
Detect the presence of either Aspergillus flavus or Aspergillus parasiticus in infected
corn/peanut kernels.
Multiplex PCR
Multiplex polymerase chain reaction (Multiplex PCR), a revolution of PCR technique, has
been developed to overcome the inherent disadvantage of cost, time and to improve the
diagnostic capacity of the test. The principle of multiplex PCR based on a variant of the test
in which more than one target sequence is amplified using more than one pair of primers
which mean amplification of multiple targets in a single PCR experiment. Design of specific
primer sets is essential for a successful multiplex reaction. The important primer design
considerations described below are a key to specific amplification with high yield.
 Primer length
 Melting temperature
 Specificity
 Avoid primer dimer formation
Multiplexing reactions can be broadly divided in two categories:
Single Template PCR Reaction
This technique uses a single template which can be a genomic DNA along with several pairs
of forward and reverse primers to amplify specific regions within a template.
Multiple Template PCR Reaction
It uses multiple templates and several primer sets in the same reaction tube. Presence of
multiple primers may lead to cross hybridization with each other and the possibility of mis-
priming with other templates.
The main application of multiplex PCR is to detect viral, bacterial, and/or other infectious
agents. Particularly, multiplex PCR has several applications listed below
 Pathogen Identification
 High Throughput SNP Genotyping
 Mutation Analysis
 Gene Deletion Analysis

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  Template Quantitation
 Linkage Analysis
 RNA Detection
 Forensic Studies
3.2 Material and equipment
Material Function
Form polysaccharide matrix which allows
Agarose powder
separation of DNA at different sizes
DNA ladder 100bp DNA size marker
Gel is able to run at high voltage when
1x SB buffer
separating DNA at short time
It can absorb UV light and re-emit a
Ethidium Bromide lower energy light, which allows
visualization of DNA bands.
Taq DNA polymerase master
DNA synthesis
mix(qiagen)
Need for the target sequence and DNA
Primer AFLA-F 10uM
polymerase to synthesize new strand
Need for the target sequence and DNA
Primer AFLA-R 10uM
polymerase to synthesize new strand
Need for the target sequence and DNA
Primer APLA-F 10uM
polymerase to synthesize new strand
Need for the target sequence and DNA
Primer APLA-R 10uM
polymerase to synthesize new strand

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 Equipment
1 PCR machine
1 Electrophoresis system
1 Microwave
1 Gel tray and comb
1 UV light and camera
10 µl, 100 µl, 1000 µl Micropipettes and tips
4 PCR tube 0.2 ml
5 Eppendorf tube 1.5 ml
1 100 ml beaker
3.3 Procedure
PCR master mix for multiplex PCR
Master mix For 1 reaction For 10 reactions
Taq DNA
polymerase master 12.5 µl 125 µl
mix
Primer ALFA-F
0.5 µl 5 µl
10uM
Primer ALFA-R
0.5 µl 5 µl
10uM
Primer ALPA-F
0.75 µl 7.5 µl
10uM
Primer ALPA-R
0.75 µl 7.5 µl
10uM

PCR reaction (total volume: 25 µl)

Reagents For 1 reaction
Master mix 15 µl
DNA 100ng/reaction
dH20

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 Set up temperature program in PCR machine
 Incubation step (activate DNA polymerase): 95oC for 5 mins
 Denaturation step: 94oC for 30 seconds
 Annealing step: 60oC for 30 seconds
 Extension step: 72oC for 1 min
 Final extension step: 72oC for 10 min
 Repeat step 2 to step 4 for 35 cycles
 Analyze PCR products on a 2% agarose gel
Running PCR assay
Place PCR tubes into PCR machine in the right position. All tubes are in the holes of plate.
Close the cap of machine tightly. Run program.
Prepare agarose gel
Measure 0.5g agarose powder
Prepare a bottle with 25ml 1X SB buffer
Mix well 0.5g agarose with 25ml 1X SB buffer
Boil the mixture in microwave for 1 minute
Leave gel cool for 3 minutes Add 2 µl of ethidium bromide into the gel
Prepare the tank
Pour gel into gel tank. Insert gel comb.
Leave the gel cool at room temperature for 30 min.
Analysis of PCR products
Mix 10 µl of PCR product and 3 µl of loading dye. Load the mixture into gel. The first well
contains 6 µl of 100bp DNA ladder.
Run the gel with 85 voltage power for 30 min
Visualize the gel under UV light. Take a picture of the gel.
Analyze the gel result
Analyze the presence of the band and the smear to evaluate the quality of extracted DNA.
Estimate the concentration of extracted DNA base on the comparison with the ladder, due to
the bright level and the molecular weight
3.4 Expected result
The PCR results were analyzed by gel electrophoresis should be like the following image

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 Based on the image, the expected results of our practical are:
If the samples were not infected by both A. flavus and A. parisiticus, there would be no band
being seen in the loaded wells.
If the loaded wells had only one band with molecular weight just over 400bp being observed,
it indicated the presence of A. flavus in the samples.
If the loaded wells had two separate bands at positions around 300bp and 400bp, implied the
presences of both A. flavus and A. parasiticus in the samples.
The positive control should be like lane 6 in the image.
The negative control should be like lane 7 in the image.

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 PRACTICAL 6: DETECTION HBV BY REAL-TIME PCR

1. INTRODUCTION
a) General information of HBV
HBV (Hepatitis B virus) which belongs to genus Orthohepadnavirus (part of Hepadnaviridae
family) is the cause of Hepatitis B infectious disease [1]. The common symptoms after infection
include vomiting, yellowish skin, tiredness, dark urine and abdominal pain which can last a few
weeks and rarely does this disease result in death. The virus is transmitted by exposure to
infectious blood or body fluids [2].
HBV is divided into four major serotypes (adr, adw, ayr and ayw) based on antigenic epitopes
and into eight genotypes (A–H) according to overall nucleotide sequence variation of the
genome. The genome of HBV is a circular DNA, but not fully double-stranded with one end of
the full length strand is linked to the viral DNA polymerase. The genome is 3020–3320
nucleotides long (for the full length strand) and 1700–2800 nucleotides long (for the short length
strand) [3]. There are four known genes encoded by the genome, called C (code for pre-core
protein), S (code for surface antigen), P (DNA polymerase gene), and X [4]. The function of the
protein coded for by gene X is not fully understood but it is associated with the development of
liver cancer [5].

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b) Detect HBV by Real-time PCR
Hybrid capture and branched DNA (bDNA) signal amplification [6-9], assays based on real-time
PCR technology [10] are the common method for to detection and quantification of HBV DNA.
In this case, Real-time PCR will be used as the main technique. In the PCR mix, a probe labeled
with a fluorescent reporter dye and a quenching dye, as well as two primers are added to initiate
DNA replication. During amplification, DNA polymerase elongates new synthesized strand and
at the same time, break the close proximity of the reporter and the quencher which get the
reporter cleaved from the probe. Fluorescent light emissions from released reporter dye can be
captured and detected by machine. In real-time PCR, there is a direct relationship between the
starting template copy number and the number of cycles needed to measure a positive signal
from the reporter dye which we can base on and give conclusion about the presence of HBV
inside the samples.
However, due to the great variation in HBV genomic sequences, it is difficult to design proper
primer-probe sets to detect and quantify all HBV genotypes by real-time PCR. In this
experiment, we utilize a highly sensitive and accurate real-time quantitative PCR assay capable
of equally detecting and quantifying HBV genotypes A through G using a single degenerate
primer-probe set within the X gene [11].
2. MATERIAL & METHOD
a) Material & Equipment
• NaOH 0.4 mol/l (for pH adjustment)
• Tris-HCl (pH7.5) mol/l (for maintaining pH)
• Forward primer HBV-F3, 5’-GGCCATCAGCGCATGC-3’
• Reverse primer HBV-R3M3,5’-C[5-NitIdl]GCTGCGAGCAAAACA-3’
• Probe HBV-P3, 5’-FAM-CTCTGCCGATCCATACTGCGG AACTC-TAMRA-3’
• Control template
• Taq DNA polymerase (enzyme add nucleotides to new synthesized strands)
• dNTP (source of free nucleotides)
• 0.2ml PCR tubes (contain PCR mix)
• Microcentrifuge
• 2ml microcentrifuge tube (contain extracted DNA)
• 0.5ml microcentrifuge tube ( contain standard samples)

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• Micropipette
• Realtime PCR machine
• Spectrophotometer
b) Method
Plasmid control template and standard curve preparation.
The plasmid pAM6 which contains the full-length HBV genomic sequence (nucleotide position
from 1 to 3020) inserted into vector pBR322 was used as the template to prepare the standard
curve for the real-time PCR assay. The number of DNA copies is estimated by
spectrophotometry method for nucleic acid quantification at the wavelength 260 nm (Spectramax
384Plus). Five different concentrations of HBV DNA from 5 × 10^1 IU/ml to 5 × 10^5 IU/ml for
five separated PCR reaction are obtained from serial dilution (by Tris-HCl) of plasmid stock
solution [15].
Positive and negative controls preparation.
One of the five concentrations of HBV DNA samples constructing the standard curve can be also
taken as the positive control in this real-time PCR. A PCR reaction mixture that contains all the
necessary reagents with dH2O instead of HBV DNA is used as the negative control.
DNA extraction.
HBV DNA is extracted from specimens using the QIAamp MinElute Virus Spin kit (QIAGEN)
following the manufacturer's protocol. Proteinase K is added in this extraction procedure to
remove the viral DNA polymerase which covalently linked to the HBV DNA.
PCR primers and probe design.
HBV genome can be obtained by selection on GenBank nucleotide database using the search
terms “human hepatitis B virus”. After a process of alignment, those most conserved regions of
HBV genome are identified. For divergent positions, the alignment program replaced the
corresponding base in the consensus sequence with a universal base (such as P and K nucleoside
phosphoramitides to mimic a C/T and an A/G mix, respectively) (12, 13). The primers and probe
are selected from the candidates after investigation of X gene region and consideration about
sequence, HBV genome variation, common mutations, melting temperature, self-
complementarity and secondary structure using Northwestern University's Online
Oligonucleotide Properties Calculator
(http://www.basic.northwestern.edu/biotools/oligocalc.html). BLAST (Basic Local Alignment

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Search Tool) is performed on all sets of primer and probe oligonucleotides to assess their
potential for amplifying non-HBV sequences. No matches with any human or microbial
nucleotide sequences were found for any of the selected oligonucleotides.
Each potential set of primers-probe is finally tested to amplify the plasmid pAM6 as template.
Primer-probe sets that successfully amplified pAM6 were subsequently used to construct the
standard curve with dilution series of the plasmid. The HBV X gene primer and probe sequences
that is selected after many evaluation steps are:
Forward primer HBV-F3, 5′-GGCCATCAGCGCATGC-3′
Reverse primer HBV-R3M3, 5′-C [5-NitIdl] GCTGCGAGCAAAACA-3′
Probe HBV-P3, 5′-R-CTCTGCCGATCCATACTGCGGAACTC-Q-3′
To detect many HBV strains, a-5-nitroindole residue is added at heterogeneous position near 5’
end of reverse primer [13]. The probe is labeled with the reporter dye (R) FAM (6-
carboxyfluorescein) at 5’ end and with the quencher dye (Q) TAMRA (6-
carboxytetramethylrhodamine at 3’ end. These primers amplify an 86-bp fragment of the HBV
genome X gene [11].
Real-time PCR.
The prepared PCR mix include 5 standard tubes, sample tube and control tubes are put in the
microcentrifuge prior to Real-time PCR for all reagents to set down to bottom of the tubes /wells.
Real-time PCR is performed using an ABI Prism 7900HT or 7700 Sequence Detection System
(Applied Biosystems). The component of a PCR mix:
 Buffer (1.5mM MgCl2) 2.5 μl (maintain pH and act as solvent)
 dNTP (25mM each) 0.5 μl (source of free nucleotides)
 MgCl2 (25mM) 1.5 μl (provide Mg2+ as cofactor)
 HBV-F3 (20mM) 0.5 μl (forward primer)
 HBV-R3M3(20mM) 0.5 μl (reverse primer)
 Probe HBV-P3 (10mM) 0.5 μl
 HotstartTaq (5u/μl) 0.25 μl (enzyme add nucleotide to new synthesized strands)
 dH2O 13.75 μl (to obtain a specific PCR volume)
 DNA 5 μl (template)

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- Set up temperature program
 Step 1 95oC for 10 mins
 Step 2 95 oC for 30 seconds
 Step 3 60 oC for 1 min
 Repeat step 2 to step 3 for 45 cycles
 Read data at step 3
 Save the temperature program.
ABI Prism 7700 or 7900HT software will plot C T as with respect to the input copy number. The
copy number at start for each reaction is calculated using the C T value of each PCR interpolated
against the linear regression of the standard curve.
Data analysis.
Results will be converted to copies/ml prior to analysis, and all statistical analyses are performed
using SAS Enterprise Guide software, version 9.0. Statistics are performed to determine the
sensitivity of this real-time PCR method in HBV detection.
3. EXPECTED RESULT
a) Standard sample

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During the PCR reaction, the accumulation of a fluorescent signal after every cycle will be
captured for plotting the graph which presents the fluorescent signal with respect to the cycle
number in the sample mix. ∆Rn is the fluorescent intensity that subtracts background
fluorescence (baseline). The horizontal line (threshold line) indicates the statistical significant
increase in detected signal which should be set before running PCR. CT value is the cycle number
PCR mix need to run to reach the threshold limit. This value is inverse to the number of initial
copy and depends on many other factors: instruments, reagents, preparation… [14]so all the
samples should run in the same conditions and time.

Each sample gives a different CT value correlating with that sample’s number of initial copy.
This relationship constructs the second graph which helps us quantify the amount of target DNA
in the unknown sample from its C T value. Since we run 45 cycles in real-time PCR, according to
the second graph, we can detect even only 11 PCR copies/well (95% confidence level) [11].
b) Positive result
Depend on the limit point (number of HBV DNA copies/mL) we chose, a specific sample will
give positive or negative result. From the second graph, if we set the limit point is 10^1 IU/mL
(100 copies/mL), any sample showed a CT of <35 is scored as positive for this test.
c) Negative result
From the second graph, if we set the limit point is 10^1 IU/mL (100 copies/mL), any sample
showed a CT of >35 is scored as negative for this test.

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IV. WORK PLAN:
1) Estimated time

CONTENTS TIME
Practical 3: 3 hours
DNA EXTRACTION
Practical 4: 2 hours
DNA QUALITY AND QUANTITY
Practical 5: 2 hours
DETECTION OF A. FLAVUS AND A.PARASICICUS
BY MULTIPLEX PCR.

Practical 6: The whole project is estimated to spend through a period of 36 days (from April 10 th
to May 16th).
10-Apr 15-Apr 20-Apr 25-Apr 30-Apr 5-May 10-May 15-May

Prepare chemicals, kits, specimens

Select Genome

Design Primer-Probe

Arrange Laboratory

Extract DNA from sample

Perform Real-time PCR

Analyze Data

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2) Estimated cost
Content Cost
Practical 3: DNA extraction 700 USD
Practical 4: DNA quantity and quality 569 USD
Practical 5: Detection using multiplex PCR 990 USD
Practical 6: HBV detection 1699 USD
Total 3958 USD

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REFERENCES
1. Hunt, Richard (2007-11-21). "Hepatitis viruses". University of Southern California,
Department of Pathology and Microbiology. Retrieved 2008-03-13.
2. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, et al.
Global and regional mortality from 235 causes of death for 20 age groups in 1990 and
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