Experiment nineExperiment nineA. Overview of diagnostic methods A. Overview of diagnostic methods of virus infection of virus infection

• In general, diagnostic tests can In general, diagnostic tests can be grouped into 3 categories.:be grouped into 3 categories.:– direct detection direct detection – indirect examination (virus indirect examination (virus

isolation) isolation) – serological methods.serological methods.

Direct detectionDirect detection• In direct examination, the clinical specimen is eIn direct examination, the clinical specimen is e

xamined directly for xamined directly for the presence of virus partithe presence of virus particlescles, , virus antigenvirus antigen or or viral nucleic acids.viral nucleic acids.– Electron Microscopy morphology / immunElectron Microscopy morphology / immun

e electron microscopy e electron microscopy – Light microscopy for histological appearaLight microscopy for histological appeara

nce - e.g. inclusion bodies nce - e.g. inclusion bodies – Antigen detection with immunofluorescenAntigen detection with immunofluorescen

ce, ELISA etc. ce, ELISA etc. – Molecular techniques for the direct detectiMolecular techniques for the direct detecti

on of viral genomes on of viral genomes

Indirect examinationIndirect examination

• Inoculating the specimen in cell culturInoculating the specimen in cell culture, eggs or animals to cultivate virus: the, eggs or animals to cultivate virus: this is called is is called virus isolationvirus isolation..– Cell Culture - cytopathic effect, haemadsorpCell Culture - cytopathic effect, haemadsorp

tion, confirmation by neutralization, interfetion, confirmation by neutralization, interference immunofluorescence etc. rence immunofluorescence etc.

– Eggs pocks on CAM - haemagglutination, incEggs pocks on CAM - haemagglutination, inclusion bodies lusion bodies

– Animals disease or death confirmation by neAnimals disease or death confirmation by neutralization utralization

Serological diagnosisSerological diagnosis

• A serological diagnosis can be made by tA serological diagnosis can be made by the detection of rising titres of antibody bhe detection of rising titres of antibody between acute and convalescent stages oetween acute and convalescent stages of infection, or the detection of IgM. f infection, or the detection of IgM.

• In general, the majority of common viral In general, the majority of common viral infections can be diagnosed by serologicinfections can be diagnosed by serological methods.al methods.

Classical Techniques Newer Techniques

1. Complement fixation tests (CFT)

2. Haemagglutination inhibition tests

3. Immunofluorescence techniques (IF)

4. Neutralization tests

5. Single Radial Haemolysis

1. Radioimmunoassay (RIA)

2. Enzyme linked immunosorbent assay (EIA)3. Particle agglutination

4. Western Blot (WB)

5. Recombinant immunoblot assay (RIBA), line immunoassay (Liatek) etc.

Serology

Direct examination- rapid diagnostic methodsDirect examination- rapid diagnostic methods

• Direct examination methods are often Direct examination methods are often also called also called rapid diagnosticrapid diagnostic methods methods because they can usually give a result because they can usually give a result either within the same or the next day. either within the same or the next day.

• With the advent of effective antiviral With the advent of effective antiviral chemotherapy, rapid diagnostic methods chemotherapy, rapid diagnostic methods are expected to play an increasingly are expected to play an increasingly important role in the diagnosis of viral important role in the diagnosis of viral infections.infections.

Rapid Diagnosis of Virus Rapid Diagnosis of Virus Infections Infections

Viral Genome Detection Viral Genome Detection by PCRby PCR

Detection of viral nucleic Detection of viral nucleic acidacid

• Methods based on the detection of Methods based on the detection of viral genome are also commonly viral genome are also commonly known as molecular methods. known as molecular methods.

Polymerase Chain ReactionPolymerase Chain Reaction (PC(PCR)R)

• PCR allows the in vitro amplification of sPCR allows the in vitro amplification of specific target DNA sequences by a factor pecific target DNA sequences by a factor of 10of 1066 and is thus an extremely sensitive and is thus an extremely sensitive technique. technique.

• It is based on an enzymatic reaction invIt is based on an enzymatic reaction involving the use of synthetic oligonucleotiolving the use of synthetic oligonucleotides flanking the target nucleic sequencedes flanking the target nucleic sequence of interest. of interest.

PCRPCR

• These oligonucleotides act as primers for These oligonucleotides act as primers for the thermostable Taq polymerase. the thermostable Taq polymerase.

• Repeated the below cycles (usually 25 to Repeated the below cycles (usually 25 to 40) 40) – denaturation of the template DNA (at 94denaturation of the template DNA (at 94ooC).C).– Annealing of primers to their complementarAnnealing of primers to their complementar

y sequences (50y sequences (50ooC)C)– Primer extension (70Primer extension (70ooC) resulting in the expoC) resulting in the expo

nential production of the specific target fragnential production of the specific target fragmentment

Schematic of  Polymerase Chain Reaction

Detection of DNA sequence Detection of DNA sequence product of the PCRproduct of the PCR

• Detection of DNA sequence product of the PCDetection of DNA sequence product of the PCR assay may be performed in several ways. R assay may be performed in several ways. – The least sensitive and specific method is to siThe least sensitive and specific method is to si

ze fractionate the reaction product ze fractionate the reaction product on an agaron an agarose or acrylamide gelose or acrylamide gel and stain the DNA with and stain the DNA with ethidium bromide. ethidium bromide.

– A more sensitive technique involves the attachA more sensitive technique involves the attachment of DNA to a membrane through dot or sloment of DNA to a membrane through dot or slot-blot techniques followed by t-blot techniques followed by hybridizationhybridization wi with a labelled homologous oligonucleotide probth a labelled homologous oligonucleotide probe. e.

Advantages of PCRAdvantages of PCR

• Extremely high sensitivity, may Extremely high sensitivity, may detect down to one viral genome per detect down to one viral genome per sample volume sample volume

• Easy to set up Easy to set up

• Fast turnaround time Fast turnaround time

Disadvantages of PCRDisadvantages of PCR

• Extremely liable to contamination Extremely liable to contamination • High degree of operator skill required High degree of operator skill required • Not easy to quantitate results Not easy to quantitate results • A positive result may be difficult to interpreA positive result may be difficult to interpre

t, especially with latent viruses such as CMV,t, especially with latent viruses such as CMV, where any seropositive person will have vir where any seropositive person will have virus present in their blood irrespective whethus present in their blood irrespective whether they have disease or not. er they have disease or not.

Detection of viral nucleic acidDetection of viral nucleic acid

• Highly sensitive and specific.Highly sensitive and specific.• The viral nucleic acid can be either DNA or RNA, The viral nucleic acid can be either DNA or RNA,

double-stranded or single-stranded. double-stranded or single-stranded. – Nucleic acid hybridization (include dot hybridizatiNucleic acid hybridization (include dot hybridizati

on, norther or southern blot hybridization, hybridion, norther or southern blot hybridization, hybridization in situ, etc.),zation in situ, etc.),

– Polymerase chain reaction (PCR) plus gel electropPolymerase chain reaction (PCR) plus gel electrophoresis techniques are increasingly being used to horesis techniques are increasingly being used to detect viral DNA or RNA in tissue samples.detect viral DNA or RNA in tissue samples.

Detection of HPV DNA from CDetection of HPV DNA from Cervical cancer tissue by polymeervical cancer tissue by polyme

rase chain reaction (PCR)rase chain reaction (PCR)

objectiveobjective

• To mTo masteraster basic basic principleprinciple and and methmethod of PCR diagnosis of virus infection.od of PCR diagnosis of virus infection.

• PCR diagnostic method only apply to PCR diagnostic method only apply to detection of virus known sequences, fdetection of virus known sequences, for examples: HBVor examples: HBV 、、 HIVHIV 、、 HPVHPV 、、 HCVHCV 、、CMV .CMV .

outline of PCRoutline of PCR

• The polymerase chain reaction (PCR) is a rapThe polymerase chain reaction (PCR) is a rapid procedure for in vitro enzymatic amplificaid procedure for in vitro enzymatic amplification of a specific segment of DNA.tion of a specific segment of DNA.

• There are three nucleic acid segments : the segThere are three nucleic acid segments : the segment of double-stranded DNA to be amplified ament of double-stranded DNA to be amplified and two single-stranded oligonucleo-tide primend two single-stranded oligonucleo-tide primers flanking this segment. rs flanking this segment.

• There is a protein component (a DNA polymeraThere is a protein component (a DNA polymerase), appropriate dNTPs, a buffer, and salts. se), appropriate dNTPs, a buffer, and salts.

Human papillomavirus (HPV)Human papillomavirus (HPV)

• The cervical cancer and CondylomaThe cervical cancer and Condyloma acuminatacuminate occur are close related to HPV infection.e occur are close related to HPV infection.

• Epidemiology indicates HPV 16,18,33,52 and 5Epidemiology indicates HPV 16,18,33,52 and 58 cause cervical cancer ,they are higher-perilo8 cause cervical cancer ,they are higher-perilous types; us types; Cervical cancer tissue are infected Cervical cancer tissue are infected with HPV higher- with HPV higher- perilous types,in which HPVperilous types,in which HPV16 and 18types are frequently checked.16 and 18types are frequently checked.

Genome of HPVGenome of HPV

•Double-stranded circular DNA Double-stranded circular DNA 7.9kb7.9kb::– Early region (E)Early region (E)– late region (L)late region (L)– Up regulateUp regulate region (URR)region (URR)

Virion structure of HPVVirion structure of HPV• Papillomaviruses are a family of nonenveloped, Papillomaviruses are a family of nonenveloped,

doublestranded DNA viruses which infect many doublestranded DNA viruses which infect many species, and at this time more than 100 different species, and at this time more than 100 different types of human papillomaviruses (HPVs) have betypes of human papillomaviruses (HPVs) have been identified. en identified.

• Image analysis of cryoelectron micrographs of HImage analysis of cryoelectron micrographs of HPV has demonstrated a structure comprised of 7PV has demonstrated a structure comprised of 72 pentameric capsomeres arranged on a T2 pentameric capsomeres arranged on a T557 ico7 icosahedral capsid lattice . sahedral capsid lattice .

• The papillomavirus genome encodes two structural proteins,The papillomavirus genome encodes two structural proteins, L1 and L2, synthesized late in infection. In the virion, the rati 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 rano of L1 (55 kDa) to L2 (74 kDa) has been estimated over a range from 5:1 to 30:1 . ge from 5:1 to 30:1 .

• HPV have been expressed in mammalian cells with vaccinia HPV have been expressed in mammalian cells with vaccinia virus and insect cells with baculovirus. These expression sysvirus and insect cells with baculovirus. These expression systems all generate virus-like particles (VLPs) similar in appeartems all generate virus-like particles (VLPs) similar in appearance to empty capsids, and these VLPs have immunologic chance to empty capsids, and these VLPs have immunologic characteristics suggesting a native conformation. When cells earacteristics suggesting a native conformation. When cells expressing L1 are examined by electron microscopy, the VLPs xpressing L1 are examined by electron microscopy, the VLPs are seen exclusively in the nuclei of the cells .are seen exclusively in the nuclei of the cells .

ProcedureProcedure

Cervical cancer tissueCervical cancer tissue

nucleic acid DNA

AmplifyAmplify E6 gene by PCRE6 gene by PCR

With type specific primerWith type specific primer

electrophoresis on an agarose gel

stain the DNA with ethidium bromide

1. 1. Preparation of Preparation of HPV HPV DNADNA ext extractract from Cervical cancer tissue from Cervical cancer tissue

MATERIALS MATERIALS • ApparatusApparatus::

– centrifuge centrifuge – Bath water Bath water

• Reagents:Reagents:– Lysis buffer (Lysis buffer (0.10.1M M EDTA EDTA pH8.0, pH8.0, 0.1% SDS, 0.1% SDS,

20%Rnase, 20%Rnase, 10mM Tris-HCl pH8.0, 10mM Tris-HCl pH8.0, TE buffer TE buffer pH8.0)pH8.0)– Saturated Saturated phenolphenol– ChloroformChloroform:: IsopropanolIsopropanol(24:1)(24:1)– 3M sodium acetate, pH5.2 3M sodium acetate, pH5.2 – 100% and 70% ethanol 100% and 70% ethanol – Proteinase KProteinase K

• Condyloma acuminate tissueCondyloma acuminate tissue

Put 25 mg of tissue in a petri dish and divide the tissue into little pieces.

ProcedureProcedure

Add 40 Add 40 ul lysis buf lysis buffer without SDSfer without SDS

tissue homogenate

Remove into microtube

Add 100 Add 100 ul lysis buffer lysis buffer

incubate 30 min at 60°C in a waterbath.

proteinase K (200 µ g/ml)Add SDS (1%)

shake gently

shake gently

incubate 12-24 h at 37°C in a waterbath.

incubate 30 min at 60°C in a waterbath.centrifuge at 5000g for 10min

Add 500 ul of t phenolshake

Remove the supernatant into microtubeRemove the supernatant into microtubeAdd Add 500 ul chloroformchloroform::IsopropaIsopropanolnol(24:1)(24:1) centrifuge at 5000g for 10min

RemoveRemove the supernatant into microtubethe supernatant into microtubeAdd 0.2 fold volume 3M sodium Add 0.2 fold volume 3M sodium acetate and 2.5fold volume cool acetate and 2.5fold volume cool ethanolethanol

centrifuge at 10000g for 15min at 4 °C

Discard the supernatant, 70% ethaDiscard the supernatant, 70% ethanol wash the pillet two timesnol wash the pillet two times

Dry ,dissolve the DNA in TE or Dry ,dissolve the DNA in TE or sterile watersterile water , ,store at -20store at -20°C

2. Quantitation DNA2. Quantitation DNA

Determination of DNA Determination of DNA concentration using the concentration using the UV UV

spectrophotometerspectrophotometer

MATERIALSMATERIALS

• Apparatus:Apparatus:– the the UV UV spectrophotometerspectrophotometer– PipettesPipettes

• Reagents:Reagents:– Extracted DNAExtracted DNA

– ddHddH22OO

procedureprocedure• Turn on machine and wait for it to warm up .Turn on machine and wait for it to warm up .• Wash glass cuvettes thoroughly before use and if tWash glass cuvettes thoroughly before use and if t

wo cuvettes are used then make sure they are a wo cuvettes are used then make sure they are a ‘matching’ pair. ‘matching’ pair.

• Insert 1ml of Insert 1ml of ddHddH22OO into machine and press ‘aut into machine and press ‘autozero’ .ozero’ .

• Add 10 µl of each sample to 990 µl dd, into Add 10 µl of each sample to 990 µl dd, into cuvcuvetteette mix well, and read OD260 and OD280 to de mix well, and read OD260 and OD280 to determine purity.termine purity.

procedureprocedure• The ratio OD260/OD280 should be determined in ordeThe ratio OD260/OD280 should be determined in orde

r to assess the purity of the sample. If this ratio is 1.8 -r to assess the purity of the sample. If this ratio is 1.8 -2.0, the absorption is probably due to nucleic acids.2.0, the absorption is probably due to nucleic acids.

• A ratio less than 1.8 in-dicates that there may be protA ratio less than 1.8 in-dicates that there may be proteins and/or other UV absorbers in the sample, in whiceins and/or other UV absorbers in the sample, in which case it is advisable to reprecipitate the DNA. h case it is advisable to reprecipitate the DNA.

• A ratio higher than 2.0 indicates the samples may be cA ratio higher than 2.0 indicates the samples may be con-taminated with chloroform or phenol and should on-taminated with chloroform or phenol and should be re-precipitated with ethanol.be re-precipitated with ethanol.

DNA concentration (µg/µl) =OD260 x 100 (dilution factor) x 50 µg/ml

10

1 OD260=ds DNA 50µg/ml

3. Amplification by PCR3. Amplification by PCR

MATERIALSMATERIALS

• Apparatus:Apparatus:– PCR instrumentPCR instrument– Pipettes:10,200 ulPipettes:10,200 ul

• Reagents:Reagents:– HPV16 E6 specific primerHPV16 E6 specific primer

• Upstream P1: 5’CTGACCAAGCTCCTTCATUpstream P1: 5’CTGACCAAGCTCCTTCAT• Downstream P2: 5’ AACTATTGTGTCATGCAACDownstream P2: 5’ AACTATTGTGTCATGCAAC

– Taq DNA polymerase Taq DNA polymerase – dNTP(5-10mmol/L)dNTP(5-10mmol/L)– 10xbuffer10xbuffer– DeionizedDeionized water water

PCR systemPCR system• P1 (10um/L)P1 (10um/L) 2.5ul2.5ul• P2 (10um/L) 2.5ulP2 (10um/L) 2.5ul• dNTP(2.5mmol/L) 4dNTP(2.5mmol/L) 4 ul ul • 55 ul ul 10Xbuffer 510Xbuffer 5 ul ul • HH22O 34O 34 ul ul • Sample Sample extracted DNA (200ng/ul) 1 uextracted DNA (200ng/ul) 1 u

l l • Taq DNA polymeraseTaq DNA polymerase (10u/ (10u/ ul) 1 ulul) 1 ul

• 9595℃ 5min℃ 5min

• 95℃ 1min95℃ 1min

• 55℃ 1min55℃ 1min

• 72℃ 2min 40 sec72℃ 2min 40 sec

• 72℃ 7 min72℃ 7 min

• 4 ℃ preservation4 ℃ preservation

35 cycles35 cycles

4. Identification of PCR 4. Identification of PCR products products

Agarose gel electrophoresis anAgarose gel electrophoresis analysisalysis

MaterialsMaterials

• Apparatus:Apparatus:– electrophoresis apparatuselectrophoresis apparatus– UV light boxUV light box – Pipettes:10 ulPipettes:10 ul

• Reagents:Reagents:– AgaroseAgarose– TAE buffer, pH8TAE buffer, pH8– 0.5 ug/ml ethidium bromide 0.5 ug/ml ethidium bromide

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.

ProcedureProcedure

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 bromide-stained DNA separation pattern is taken with a camera.

procedureprocedure

• Prepare an 1% agarose gelPrepare an 1% agarose gel– Dried agarose 1 g is dissolved in 1XTAE bDried agarose 1 g is dissolved in 1XTAE b

uffer 100ml by heating in a 300 ml Ehrlenuffer 100ml by heating in a 300 ml Ehrlenmeyer flask, and heating in a microwave fmeyer flask, and heating in a microwave for 2-4 minutes until the agarose is dissolvor 2-4 minutes until the agarose is dissolved. ed.

– Pour the gel onto a taped plate with castiPour the gel onto a taped plate with casting combs in place. Allow 20-30 minutes fong combs in place. Allow 20-30 minutes for solidification.r solidification.

ProcedureProcedure

• Electrophoresis Electrophoresis – Carefully remove the tape and the gel casCarefully remove the tape and the gel cas

ting combs and place the gel in a horizontting combs and place the gel in a horizontal electrophoresis apparatus. Add 1X TAE al electrophoresis apparatus. Add 1X TAE electrophoresis buffer to the reservoirs uelectrophoresis buffer to the reservoirs until the buffer just covers the agarose gel.ntil the buffer just covers the agarose gel.

ProcedureProcedure• Electrophoresis Electrophoresis

– Add at least one-sixth volume of 6X agaAdd at least one-sixth volume of 6X agarose gel loading dye to each DNA samplrose gel loading dye to each DNA sample, mix, and load into the wells. Electroe, mix, and load into the wells. Electrophorese the gel at 150-200 mA until the phorese the gel at 150-200 mA until the required separation has been achieved,required separation has been achieved, usually 0.5-1 hour . usually 0.5-1 hour .

ProcedureProcedure

• ObservationObservation– After electrophoresis, stain with EB After electrophoresis, stain with EB

15 min.15 min.– Visualize the DNA fragments on a Visualize the DNA fragments on a

long wave UV light box and long wave UV light box and photograph with a Polaroid cameraphotograph with a Polaroid camera. .

Result analysisResult analysis

• IF specific IF specific bandband visualize at 1500 bp, then visualize at 1500 bp, then may identify this may identify this Condyloma acuminate tissCondyloma acuminate tissue was infected by corresponding type HPV.ue was infected by corresponding type HPV.

Fig.1 Amplification of HPV 11L1gene by PCR1. marker:λDNA/HindIII and EcoRI 2.3.4. L1 gene PCR products(1.5kb) 5. negative control