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A. Overview of diagnostic methods of virus infection

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.

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A. Overview of diagnostic methods of virus infection

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  1. 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.

  2. 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

  3. 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  

  4. 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.

  5. Serology Classical TechniquesNewer 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.

  6. 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.

  7. Rapid Diagnosis of Virus Infections Viral Genome Detection by PCR

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

  9. 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.

  10. 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

  11. Schematic of  Polymerase Chain Reaction

  12. 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. 

  13. Advantages of PCR • Extremely high sensitivity, may detect down to one viral genome per sample volume • Easy to set up • Fast turnaround time

  14. 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.

  15. 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.

  16. Detection of HPV DNA from Cervical cancer tissue by polymerase chain reaction (PCR)

  17. 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 .

  18. 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 oligonucleo-tide primers flanking this segment. • There is a protein component (a DNA polymerase), appropriate dNTPs, a buffer, and salts.

  19. Human papillomavirus (HPV) • The cervical cancer and Condylomaacuminate 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.

  20. Genome of HPV • Double-stranded circular DNA 7.9kb: • Early region (E) • late region (L) • Up regulateregion (URR)

  21. 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 .

  22. 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 are seen exclusively in the nuclei of the cells .

  23. Procedure Cervical cancer tissue nucleic acid DNA With type specific primer AmplifyE6 gene by PCR electrophoresis on an agarose gel stain the DNA with ethidium bromide

  24. 1. Preparation of HPV DNA extract from Cervical cancer tissue

  25. 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

  26. 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%) shake gently proteinase K (200 µ g/ml) incubate 30 min at 60°C in a waterbath. shake gently incubate 12-24 h at 37°C in a waterbath.

  27. incubate 30 min at 60°C in a waterbath. centrifuge at 5000g for 10min Add 500 ul of t phenol shake Remove the supernatant into microtube Add 500 ulchloroform:Isopropanol(24:1) centrifuge at 5000g for 10min Removethe 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

  28. 2. Quantitation DNA Determination of DNA concentration using the UV spectrophotometer

  29. MATERIALS • Apparatus: • the UV spectrophotometer • Pipettes • Reagents: • Extracted DNA • ddH2O

  30. 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.

  31. 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 phenol and should be re-precipitated with ethanol.

  32. OD260 x 100 (dilution factor) x 50 µg/ml DNA concentration (µg/µl) = 10 1 OD260=ds DNA 50µg/ml

  33. 3. Amplification by PCR

  34. 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

  35. PCR system • P1 (10um/L)2.5ul • P2 (10um/L) 2.5ul • dNTP(2.5mmol/L) 4ul • 5ul 10Xbuffer 5ul • H2O 34ul • Sample extracted DNA (200ng/ul) 1 ul • Taq DNA polymerase (10u/ ul) 1 ul

  36. 95℃ 5min • 95℃ 1min • 55℃ 1min • 72℃ 2min 40 sec • 72℃ 7 min • 4 ℃ preservation 35 cycles

  37. 4. Identification of PCR products Agarose gel electrophoresis analysis

  38. Materials • Apparatus: • electrophoresis apparatus • UV light box • Pipettes:10 ul • Reagents: • Agarose • TAE buffer, pH8 • 0.5 ug/ml ethidium bromide

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

  40. 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.

  41. 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.

  42. 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 .

  43. 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.

  44. Result analysis • IF specific bandvisualize 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

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