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Experiment nine
A. Overview of diagnostic methods
of virus infection
• In general, diagnostic tests can be
grouped into 3 categories.:
– direct detection
– indirect examination (virus
isolation)
– serological methods.
Direct detection
• In direct examination, the clinical specimen is
examined directly for the presence of virus
particles, virus antigen or viral nucleic acids.
– Electron
Microscopy
morphology
/
immune electron microscopy
– Light microscopy for histological
appearance - e.g. inclusion bodies
– Antigen detection with
immunofluorescence, ELISA etc.
– Molecular techniques for the direct
detection of viral genomes
Indirect examination
• Inoculating the specimen in cell culture,
eggs or animals to cultivate virus: this is
called virus isolation.
– Cell
Culture
cytopathic
effect,
haemadsorption,
confirmation
by
neutralization,
interference
immunofluorescence etc.
– Eggs pocks on CAM - haemagglutination,
inclusion bodies
– Animals disease or death confirmation by
neutralization
Serological diagnosis
• A serological diagnosis can be made by
the detection of rising titres of antibody
between acute and convalescent stages of
infection, or the detection of IgM.
• In general, the majority of common viral
infections can be diagnosed by serological
methods.
Serology
Classical Techniques
1. Complement fixation tests (CFT)
Newer Techniques
1. Radioimmunoassay (RIA)
2. Haemagglutination inhibition tests 2. Enzyme linked
immunosorbent assay (EIA)
3. Immunofluorescence techniques (IF) 3. Particle agglutination
4. Neutralization tests
4. Western Blot (WB)
5. Single Radial Haemolysis
5. Recombinant immunoblot
assay (RIBA), line
immunoassay (Liatek) etc.
Direct examination- rapid diagnostic methods
• Direct examination methods are often also
called rapid diagnostic methods because
they can usually give a result either within
the same or the next day.
• With the advent of effective antiviral
chemotherapy, rapid diagnostic methods
are expected to play an increasingly
important role in the diagnosis of viral
infections.
Rapid Diagnosis of Virus
Infections
Viral Genome Detection by
PCR
Detection of viral nucleic acid
• Methods based on the detection of viral
genome are also commonly known as
molecular methods.
Polymerase Chain Reaction
(PCR)
• PCR allows the in vitro amplification of
specific target DNA sequences by a
factor of 106 and is thus an extremely
sensitive technique.
• It is based on an enzymatic reaction
involving the use of synthetic
oligonucleotides flanking the target nucleic
sequence of interest.
PCR
• These oligonucleotides act as primers for
the thermostable Taq polymerase.
• Repeated the below cycles (usually 25 to
40)
– denaturation of the template DNA (at 94oC).
– Annealing of primers
to their complementary sequences (50oC)
– Primer extension (70oC) resulting in the
exponential production of the specific target
fragment
Schematic of Polymerase Chain Reaction
Detection of DNA sequence
product of the PCR
• Detection of DNA sequence product of the PCR
assay may be performed in several ways.
– The least sensitive and specific method is to
size fractionate the reaction product on an
agarose or acrylamide gel and stain the
DNA with ethidium bromide.
– A more sensitive technique involves the
attachment of DNA to a membrane through
dot or slot-blot techniques followed by
hybridization with a labelled homologous
oligonucleotide probe.
Advantages of PCR
• Extremely high sensitivity, may detect
down to one viral genome per sample
volume
• Easy to set up
• Fast turnaround time
Disadvantages of PCR
•
•
•
•
Extremely liable to contamination
High degree of operator skill required
Not easy to quantitate results
A positive result may be difficult to interpret,
especially with latent viruses such as CMV,
where any seropositive person will have virus
present in their blood irrespective whether
they have disease or not.
Detection of viral nucleic acid
• Highly sensitive and specific.
• The viral nucleic acid can be either DNA or RNA,
double-stranded or single-stranded.
– Nucleic acid hybridization (include dot
hybridization, norther or southern blot
hybridization, hybridization in situ, etc.),
– Polymerase chain reaction (PCR) plus gel
electrophoresis techniques are increasingly
being used to detect viral DNA or RNA in
tissue samples.
Detection of HPV DNA from
Cervical cancer tissue by
polymerase chain reaction
(PCR)
objective
• To master basic principle and method
of PCR diagnosis of virus infection.
• PCR diagnostic method only apply to
detection of virus known sequences,
for examples: HBV、HIV、HPV、HCV、
CMV .
outline of PCR
• The polymerase chain reaction (PCR) is a
rapid procedure for in vitro enzymatic
amplification of a specific segment of
DNA.
• There are three nucleic acid segments : the
segment of double-stranded DNA to be
amplified and two single-stranded oligonucleotide primers flanking this segment.
• There is a protein component (a DNA
polymerase), appropriate dNTPs, a buffer, and
Human papillomavirus (HPV)
• The cervical cancer and Condyloma acuminate
•
occur are close related to HPV infection.
Epidemiology indicates HPV 16,18,33,52 and 58
cause cervical cancer ,they are higher-perilous
types; Cervical cancer tissue are infected
with HPV higher- perilous types,in which
HPV16 and 18types are frequently checked.
Genome of HPV
• Double-stranded circular DNA 7.9kb:
– Early region (E)
– late region (L)
– Up regulate region (URR)
Virion structure of HPV
• Papillomaviruses are a family of nonenveloped,
doublestranded DNA viruses which infect
many species, and at this time more than 100
different types of human papillomaviruses
(HPVs) have been identified.
• Image analysis of cryoelectron micrographs of
HPV has demonstrated a structure comprised
of 72 pentameric capsomeres arranged on a
T57 icosahedral capsid lattice .
• The papillomavirus genome encodes two
structural proteins, L1 and L2, synthesized late
in infection. In the virion, the ratio of L1 (55 kDa)
to L2 (74 kDa) has been estimated over a range
from 5:1 to 30:1 .
• HPV have been expressed in mammalian cells
with vaccinia virus and insect cells with
baculovirus. These expression systems all
generate virus-like particles (VLPs) similar in
appearance to empty capsids, and these VLPs
have immunologic characteristics suggesting a
native conformation. When cells expressing L1
are examined by electron microscopy, the VLPs
Procedure
Cervical cancer tissue
nucleic acid
DNA
With type specific primer
Amplify E6 gene by PCR
electrophoresis on an agarose gel
stain the DNA with ethidium bromide
1. Preparation of HPV DNA
extract from Cervical cancer
tissue
MATERIALS
• Apparatus:
– centrifuge
– Bath water
• Reagents:
– Lysis buffer (0.1M EDTA pH8.0, 0.1% SDS,
20%Rnase, 10mM Tris-HCl pH8.0, TE buffer
pH8.0)
– Saturated phenol
– Chloroform:Isopropanol(24:1)
– 3M sodium acetate, pH5.2
– 100% and 70% ethanol
– Proteinase K
• Condyloma acuminate tissue
Procedure
Put 25 mg of tissue in a petri dish
and divide the tissue into little
pieces.
Add
40 ul lysis
buffer without SDS
tissue homogenate
Add 100 ul lysis buffer
Remove into microtube
Add SDS (1%)
proteinase K (200 µ g/ml) shake gently
incubate 30 min at 60°C in a waterbath.
shake gently
incubate 12-24 h at 37°C in a waterbath.
incubate 30 min at 60°C in a waterbath.
Add 500 ul of t phenol
centrifuge at 5000g for 10min
shake
Remove the supernatant into microtube
Add 500 ul
chloroform:Isopropanol(24:1)
centrifuge at 5000g for 10min
Remove the supernatant into microtube
Add 0.2 fold volume 3M sodium
acetate and 2.5fold volume cool
ethanol
centrifuge at 10000g for 15min at 4 °C
Discard the supernatant, 70%
ethanol wash the pillet two times
Dry ,dissolve the DNA in TE or
sterile water ,store at -20°C
2. Quantitation DNA
Determination of DNA
concentration using the UV
spectrophotometer
MATERIALS
• Apparatus:
– the UV spectrophotometer
– Pipettes
• Reagents:
– Extracted DNA
– ddH2O
procedure
• Turn on machine and wait for it to warm up .
• Wash glass cuvettes thoroughly before use and if
•
•
two cuvettes are used then make sure they are a
‘matching’ pair.
Insert 1ml of ddH2O into machine and press
‘autozero’ .
Add 10 µl of each sample to 990 µl dd, into
cuvette mix well, and read OD260 and OD280
to determine purity.
procedure
• The ratio OD260/OD280 should be determined
in order to assess the purity of the sample. If
this ratio is 1.8 -2.0, the absorption is probably
due to nucleic acids.
• A ratio less than 1.8 in-dicates that there may
be proteins and/or other UV absorbers in the
sample, in which case it is advisable to
reprecipitate the DNA.
• A ratio higher than 2.0 indicates the samples
may be con-taminated with chloroform or
OD260 x 100 (dilution factor) x 50 µg/ml
DNA concentration (µg/µl) =
1 OD260=ds DNA 50µg/ml
10
3. Amplification by PCR
MATERIALS
• Apparatus:
– PCR instrument
– Pipettes:10,200 ul
• Reagents:
– HPV16 E6 specific primer
• Upstream P1: 5’CTGACCAAGCTCCTTCAT
• Downstream P2: 5’ AACTATTGTGTCATGCAAC
– Taq DNA polymerase
– dNTP(5-10mmol/L)
– 10xbuffer
– Deionized water
PCR system
• P1 (10um/L)
2.5ul
• P2 (10um/L)
2.5ul
• dNTP(2.5mmol/L)
ul
• 5 ul 10Xbuffer
ul
• H2O
ul
4
5
34
• 95℃
• 95℃
• 55℃
• 72℃
• 72℃
•4℃
5min
1min
1min
2min 40 sec
7 min
preservation
35 cycles
4. Identification of PCR
products
Agarose gel electrophoresis
analysis
Materials
• Apparatus:
– electrophoresis apparatus
– UV light box
– Pipettes:10 ul
• Reagents:
– Agarose
– TAE buffer, pH8
– 0.5 ug/ml ethidium bromide
Procedure
Dried agarose 1 g is dissolved in 1XTAE buffer 100ml by
heating and the warm gel solution then is poured into a
mold), which is fitted with a well-forming comb.
Agarose gels are submerged in electrophoresis buffer
in a horizontal electrophoresis apparatus.
The DNA samples(200bp maker 5 ul and PCR products 10ul) are
mixed with gel tracking dye and loaded into the sample wells
Electrophoresis usually is at 150 - 200 mA for 0.5-1 hour at
room temperature, depending on the desired separation.
After electrophoresis, stain with EB 15 min,then the gel is placed on
a UV light box and a picture of the fluorescent ethidium bromidestained DNA separation pattern is taken with a camera.
procedure
• Prepare an 1% agarose gel
– Dried agarose 1 g is dissolved in 1XTAE
buffer 100ml by heating in a 300 ml
Ehrlenmeyer flask, and heating in a
microwave for 2-4 minutes until the
agarose is dissolved.
– Pour the gel onto a taped plate with
casting combs in place. Allow 20-30
minutes for solidification.
Procedure
• Electrophoresis
– Carefully remove the tape and the gel
casting combs and place the gel in a
horizontal electrophoresis apparatus.
Add 1X TAE electrophoresis buffer to the
reservoirs until the buffer just covers
the agarose gel.
Procedure
• Electrophoresis
– Add at least one-sixth volume of 6X
agarose gel loading dye to each DNA
sample, mix, and load into the wells.
Electrophorese the gel at 150-200 mA
until the required separation has been
achieved, usually 0.5-1 hour .
Procedure
• Observation
– After electrophoresis, stain with EB 15
min.
– Visualize the DNA fragments on a long
wave UV light box and photograph with
a Polaroid camera.
Result analysis
• IF specific band visualize at 1500 bp, then
may identify this Condyloma acuminate
tissue was infected by corresponding type HPV.
Fig.1 Amplification of HPV 11L1gene by PCR
1. marker:λDNA/HindIII and EcoRI
2.3.4. L1 gene PCR products(1.5kb)
5. negative control