46 实验课件6.ppt

第1页：A. Overview of diagnostic

A. Overview of diagnostic

• In general, diagnostic tests can begrouped into 3 categories.:
  • direct detection
  • indirect examination (virus isolation)
  • serological methods.

第2页：Direct detection

Direct detection

• In direct examination, the clinical specimen isexamined directly forthe presence of virus particles,virus antigenorviral nucleic acids.
  • ElectronMicroscopymorphology/immuneelectronmicroscopy
  • Light microscopy for histological appearance-e.g. inclusion bodies
  • Antigen detection with immunofluorescence,ELISA etc.
  • Moleculartechniquesforthedirectdetectionofviralgenomes

第3页：Indirect examination

Indirect examination

• Inoculating the specimen in cell culture,eggs or animals to cultivate virus: this iscalledvirus isolation.
  • CellCulture-cytopathiceffect,haemadsorption,confirmationbyneutralization,interferenceimmunofluorescenceetc.
  • Eggs pocks on CAM-haemagglutination,inclusion bodies
  • Animals disease or death confirmation byneutralization

第4页：Serological diagnosis

Serological diagnosis

• A serological diagnosis can be made by thedetection of rising titres of antibodybetween acute and convalescent stages ofinfection, or the detection of IgM.
• In general, the majority of common viralinfections can be diagnosed by serologicalmethods.

第5页：Classical Techniques Newer

Classical Techniques Newer

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第6页：Direct examination-rapid

Direct examination-rapid

• Direct examination methods are often alsocalledrapid diagnosticmethods becausethey can usually give a result either withinthe same or the next day.
• With the advent of effective antiviralchemotherapy, rapid diagnostic methods areexpected to play an increasingly importantrole in the diagnosis of viral infections.

第7页：Rapid Diagnosis of Virus

Rapid Diagnosis of Virus

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第8页：Detection of viral nucleic

Detection of viral nucleic

• Methods based on the detection of viralgenome are also commonly known asmolecular methods.

第9页：Polymerase Chain Reaction(PCR)

Polymerase Chain Reaction(PCR)

• PCR allows the in vitro amplification ofspecific target DNA sequences by afactorof 106and is thus an extremelysensitive technique.
• It is based on an enzymaticreactioninvolving the use of syntheticoligonucleotides flanking the target nucleicsequenceof interest.

第10页：These oligonucleotides act as

These oligonucleotides act as

• These oligonucleotides act as primers forthethermostableTaq polymerase.
• Repeated the below cycles (usually 25 to 40)
  • denaturation ofthetemplate DNA (at 94oC).
  • Annealing of primers totheircomplementarysequences(50oC)
  • Primer extension (70oC) resulting in theexponentialproduction of the specific targetfragment

第11页：Schematic of Polymerase Chain

Schematic of Polymerase Chain

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第12页：Detection of DNA

Detection of DNA

• Detection of DNA sequence product of the PCRassay maybeperformed in several ways.
  • The least sensitive and specific methodistosizefractionatethe reaction producton an agaroseoracrylamidegeland stain the DNA with ethidiumbromide.
  • A more sensitive technique involvesthe attachment ofDNA to a membrane through dot or slot-blottechniquesfollowed byhybridizationwith a labelledhomologous oligonucleotide probe.

第13页：Advantages of PCR

Advantages of PCR

• Extremelyhighsensitivity,maydetectdowntooneviralgenomepersamplevolume
• Easy to set up
• Fast turnaround time

第14页：Disadvantages of PCR

Disadvantages of PCR

• Extremelyliabletocontamination
• 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, whereany seropositive person will have virus present intheir blood irrespective whether they have diseaseor not.

第15页：Detection of viral nucleic

Detection of viral nucleic

• 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, hybridizationin situ, etc.),
  • Polymerase chain reaction (PCR) plus gelelectrophoresis techniques are increasingly being usedto detect viral DNA or RNA in tissue samples.

第16页：Detection of HPV DNA from

Detection of HPV DNA from

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第17页：objective

objective

• To masterbasicprincipleandmethod of PCR diagnosis of virusinfection.
• PCR diagnostic method only apply todetection of virus known sequences,for examples: HBV、HIV、HPV、HCV、CMV .

第18页：outline of PCR

outline of PCR

• The polymerase chain reaction (PCR) is a rapidprocedure for in vitro enzymatic amplification ofa specific segment of DNA.
• There are three nucleic acid segments : the segmentof double-stranded DNA to be amplified and twosingle-stranded oligonucleo-tide primers flankingthis segment.
• There is a protein component (a DNA polymerase),appropriate dNTPs, a buffer, and salts.

第19页：Human papillomavirus (HPV)

Human papillomavirus (HPV)

• The cervical cancer andCondylomaacuminateoccur are closerelated toHPV infection.
• Epidemiology indicates HPV 16,18,33,52 and 58cause cervical cancer ,they are higher-periloustypes;Cervical cancer tissue are infected withHPV higher-perilous types,in which HPV16 and18types are frequently checked.

第20页：Genome of HPV

Genome of HPV

• Double-stranded circular DNA7.9kb:
  • Early region (E)
  • late region (L)
  • Up regulateregion (URR)

第21页：Virion structure of HPV

Virion structure of HPV

• Papillomaviruses are a family of nonenveloped,doublestranded DNA viruses which infect manyspecies, and at this time more than 100 differenttypes of human papillomaviruses (HPVs) have beenidentified.
• Image analysis of cryoelectron micrographs of HPVhas demonstrated a structure comprised of 72pentameric capsomeres arranged on a T57icosahedral capsid lattice .

第22页：The papillomavirus genome

The papillomavirus genome

• The papillomavirus genome encodes two structuralproteins, L1 and L2, synthesized late in infection. Inthe virion, the ratio of L1 (55 kDa) to L2 (74 kDa) hasbeen estimated over a range from 5:1 to 30:1 .
• HPV have been expressed in mammalian cells withvaccinia virus and insect cells with baculovirus.These expression systems all generate virus-likeparticles (VLPs) similar in appearance to emptycapsids, and these VLPs have immunologiccharacteristics suggesting a native conformation.When cells expressing L1 are examined by electronmicroscopy, the VLPs are seen exclusively in thenuclei of the cells .

第23页：Procedure Cervical cancer

Procedure Cervical cancer

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第24页：1.Preparation of HPVDN

1.Preparation of HPVDN

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第25页：MATERIALS

MATERIALS

• Apparatus:
  • centrifuge
  • Bath water
• Reagents:
  • Lysisbuffer(0.1MEDTApH8.0,0.1%SDS,20%Rnase,10mMTris-HClpH8.0,TEbufferpH8.0)
  • Saturatedphenol
  • Chloroform:Isopropanol(24:1)
  • 3Msodiumacetate,pH5.2
  • 100%and70%ethanol
  • ProteinaseK
• Condyloma acuminate tissue

第26页：Put 25 mg of tissue in a petri

Put 25 mg of tissue in a petri

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第27页：incubate 30 min at 60°C in a

incubate 30 min at 60°C in a

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第28页：2. Quantitation DNA

2. Quantitation DNA

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第29页：MATERIALS

MATERIALS

• Apparatus:
  • theUVspectrophotometer
  • Pipettes
• Reagents:
  • Extracted DNA
  • ddH2O

第30页：procedure

procedure

• Turn on machine and wait for it to warm up .
• Wash glass cuvettes thoroughly before use and iftwo cuvettes are used then make sure they are a‘matching’pair.
• Insert 1ml ofddH2Ointo machine and press‘autozero’.
• Add 10 µl of each sample to 990 µl dd, intocuvettemix well, and read OD260 and OD280 to determinepurity.

第31页：procedure

procedure

• The ratio OD260/OD280 should be determined inorder to assess the purity of the sample. If this ratiois 1.8-2.0, the absorption is probably due to nucleicacids.
• A ratio less than 1.8 in-dicates that there may beproteins and/or other UV absorbers in the sample, inwhich case it is advisable to reprecipitate the DNA.
• A ratio higher than 2.0 indicates the samples may becon-taminated with chloroform or phenol and shouldbe re-precipitated with ethanol.

第32页：DNA concentration (µg/µl)

DNA concentration (µg/µl)

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第33页：3. Amplification by PCR

3. Amplification by PCR

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第34页：MATERIALS

MATERIALS

• Apparatus:
  • PCR instrument
  • Pipettes:10,200ul
• Reagents:
  • HPV16 E6 specific primer
  • Upstream P1: 5’CTGACCAAGCTCCTTCAT
  • Downstream P2: 5’AACTATTGTGTCATGCAAC
  • Taq DNA polymerase
  • dNTP(5-10mmol/L)
  • 10xbuffer
  • Deionizedwater

第35页：PCR system

PCR system

• P1 (10um/L)2.5ul
• P2 (10um/L) 2.5ul
• dNTP(2.5mmol/L) 4ul
• 5ul10Xbuffer 5ul
• H2O 34ul
• Sampleextracted DNA (200ng/ul)1ul
• Taq DNA polymerase(10u/ul) 1 ul

第36页：4℃preservation35 cycles

4℃preservation35 cycles

• 95℃5min
• 95℃1min
• 55℃1min
• 72℃2min 40 sec
• 72℃7 min
• 4℃preservation35 cycles

第37页：4. Identification of PCR

4. Identification of PCR

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第38页：Materials

Materials

• Apparatus:
  • electrophoresis apparatus
  • UV light box
  • Pipettes:10ul
• Reagents:
  • Agarose
  • TAEbuffer,pH8
  • 0.5 ug/ml ethidium bromide

第39页：Dried agarose 1 g is dissolved

Dried agarose 1 g is dissolved

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第40页：procedure

procedure

• Prepare an 1% agarose gel
  • Dried agarose 1 g is dissolved in1XTAE buffer 100ml by heating in a 300ml Ehrlenmeyer flask, and heating in amicrowave for 2-4 minutes until theagarose is dissolved.
  • Pour the gel onto a taped plate withcasting combs in place. Allow 20-30minutes for solidification.

第41页：Procedure

Procedure

• Electrophoresis
  • Carefully remove the tape and the gelcasting combs and place the gel in ahorizontal electrophoresis apparatus.Add 1X TAE electrophoresis buffer tothe reservoirs until the buffer justcovers the agarose gel.

第42页：Procedure

Procedure

• Electrophoresis
  • Add at least one-sixth volume of 6Xagarose gel loading dye to each DNAsample, mix, and load into the wells.Electrophorese the gel at 150-200 mAuntil the required separation has beenachieved, usually 0.5-1 hour .

第43页：Procedure

Procedure

• Observation
  • After electrophoresis, stain with EB 15min.
  • Visualize the DNA fragments on a longwave UV light box and photograph witha Polaroid camera.

第44页：Result analysis

Result analysis

• IF specificbandvisualize at 1500 bp, thenmay identify thisCondyloma acuminate tissuewas infected by corresponding type HPV.Fig.1 Amplification of HPV 11L1gene by PCR1. marker:λDNA/HindIII and EcoRI2.3.4. L1 gene PCR products(1.5kb)5. negative control