Rapid Diagnostic Technique of Virus

RAPID DIAGNOSTIC TECHNIQUES

These include:

1. Electron microscopy

2. Viral nucleic acid detection

3. Serology/detection of viral antigen and antibody

1. Electron microscopy

This is used to demonstrate the presence of virus in clinical specimens and to study the

morphology or symmetry of the virus. With electron microscopy, it is possible to

recognized mixed viral infection. It is also used for detection of non-viable viruses and

those that cannot be grown in vitro. However, equipment required for the procedure is

rather expensive. Besides, large number of viral particles in excess of 106/ml must be

present in the sample before they can be detected. It is difficult to differentiate viruses

with similar morphology especially those from the same family with electron

microscopy.

Method:

 Homogenize sample.

 Centrifuge at low speed to remove large particulate debris.

 Ultracentrifugation to sediment available virus particles.

 Negative staining with heavy metal compound such as phosphotungstic acid or uranyl

acetate. (negative staining stains the background to increase contrast so that bright

virion stand out against a dark background).

 Addition of immune serum (immunoelectron microscopy) to increase sensitivity by

clumping/agglutinating virus particles and enhance recovery following centrifugation).

 Observe at X13,000-100,000 magnification.

2. Detection of viral nucleic acid:

Oligonucleotide probes are used for the detection of viral DNA or RNA. Insufficient viral

nucleic acid in sample is increased by amplification using polymerase chain reaction.

Probes anneal to the targeted viral nucleic acid sequence which can be the whole genome,

specific gene or nucleic acid segment. Variable or conserved sequence can be targeted.

Double stranded genomes are first separated by heating. Oligonucleotide probes are

labeled with radioactive isotopes such as 32P or 35S to allow viewing. Non-radioactive

labels such as alkaline phosphatase fluorescein and horse radish peroxidase can be used

for direct viewing whilebiotin and digoxigenin are used for indirect viewing.

I. Dot-blot hybridization

 Nucleic acid, usually DNA is extracted from sample

 Extracted nucleic acid is spotted directly onto charged nylon or nitrocellulose membrane

 Nucleic acid binds firmly onto membrane after baking

 Fluorescent dye- or radioisotope- labeled probe is added

 The membrane is washed to remove unbound materials

 Binding of probe to targeted nucleic acid is detected by autoradiography or by colour

precipitation

II. In-situ hybridization

Viral nucleic as is detected in frozen section of infected cells with the aid of labeled

oligonucleotide probes. Intracellular location of viral nucleic acid is revealed by

autoradiography or immunoperoxidase cytochemistry

III. Southern blot hybridization

 Restriction enzymes are used to cleave DNA into short oligonucleotides

 Oligonucleotides are separated by agarose electrophoresis or acrylamide gel

electrophoresis

 Separated oligonucleotides are transferred by blotting onto nitrocellulose

membrane or nylon

 Probes are added and reaction detected by autoradiography or by colour

development

IV. Northern blot hybridization

RNA hybridization similar to southern blot

V. Western blot

Application to protein identification

VI. Polymerase chain reaction

 Extraction of DNA or RNA nucleic acid from sample

 Amplification of the extracted nucleic acid in a series of repeated cycles of denaturation,

primer annealing and polymerization using heat-resistant Taq (Thermus aquaticus)

polymerase. Specific primers recognize and bind to the targeted gene to initiate the

amplification reaction.

 Amplified sequences are stained with ethidium bromide and separated by

electrophoresis

 The separated sequence is viewed under ultraviolet transillumination.

 This procedure is very specific and highly sensitive