Laboratory Tests for Diagnosis

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Last modified 18/02/2015

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Chapter 5

Laboratory Tests for Diagnosis

Laboratory Assays for Detecting Nucleic Acids (Table 5-1)

• Detection of RNA or DNA is rapid and sensitive and can detect microbes that are too virulent or not readily grown in the laboratory.

• Methods depend on hybridization of a probe (primer) sequence with a complementary sequence in the sample.

• Probes are either radioactive or chemically labeled to allow detection on hybridization with sample.

Southern blotting for DNA and Northern blotting for RNA detect electrophoretically separated genome sequences.

In situ hybridization detects viral DNA or RNA within infected cells.

Polymerase chain reaction (PCR) (DNA), reverse transcriptase (RT)-PCR (RNA), and related technologies permit detection, identification, and amplification of specific nucleic acid sequences.

1. PCR: heat-stabile DNA polymerase amplifies DNA between sequence specific primers (Fig. 5-1)

2. RT-PCR: RT makes a complementary DNA copy of RNA, which is then amplified by PCR using sequence specific primers.

3. Quantitative PCR (qPCR; real-time PCR): rate of production of DNA during PCR reaction determines concentration of DNA in sample.

4. Other DNA and RNA detection methods include branched-chain DNA assay and antibody capture solution hybridization assay.

II Immunologic Assays (seeTable 5-1)

• Assays can be used to detect immune responses to infection, or specific antibody can be used to detect soluble and cell-associated antigen.

Antibody-antigen binding (see alsoChapter 3, section IV)

1. Precipitin reactions: Ouchterlony immunodiffusion distinguishes identical, similar, and different antigens based on the precipitin line formed by antigen-antibody precipitation (Fig. 5-2)

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