Diagnosis of Infectious Diseases

DIAGNOSIS OF INFECTIOUS DISEASES

Each pathogenic microorganism contains genetic material that distinguishes it from its host and from other microorganisms. This specific material constitutes a signature that allows the identification of a particular microorganism from a complex mixed population. In the diagnosis of infectious diseases, identification of specific sequences from microbial pathogens will allow appropriate treatment at an early stage as well as prevention of the spread of disease.

The two main techniques used for the diagnosis of infectious disease targeting nucleic acids are hybridization and PCR-based amplification. There are currently specific primers and probes for the detection of more than 100 infectious disease. Table 25.6 shows just a few examples.

A)    DNA Hybridization Techniques

Nucleic acid hybridization is based on the precise nucleotide base pairing and hydrogen bonding between one strand of nucleotides and a complementary nucleotide sequence. Any diagnostic nucleic acid hybridization consists of three essential elements: a DNA probe, the target DNA and the signal detection system. Recent developments in detection systems and improvements in safety have enabled the use of highly sensitive nonradioactive methods. Figure 25.13 illustrates the general steps required for DNA hybridization using chemiluminescent-based detection. This non-radioactive system achieves signal amplification by enzymatic conversion of a chemiluminescent substrate. First of all, DNA from the biological sample including the target DNA from the pathogen to be identified needs to be extracted. A diagnostic biotin-labelled probe is then mixed with the target DNA bound to a membrane support. The hybridized probe is then incubated with streptavidin, which has multiple sites that avidly bind biotin. Subsequent incubation with biotin conjugated to alkaline phosphatase results in the enzymatic labelling of the bound streptavidin. Finally, addition of a special substrate for the alkaline phosphatase results in the conversion of this substrate into a product which emits light and which can be detected after exposure to X-ray film or by using sensitive imaging cameras.

The procedure described above can also be scaled down, automated and redesigned to use thousands of sequence-specific probes at once in what are called microarrays. In this case non-labelled DNA probes are synthesized and minute droplets are spotted at high density on to glass slides where they will remain bound. The DNA from the biological sample is then labelled at the extremities with a fluorescent dye and after denaturation into single strands it is allowed to anneal with the probes bound on the slide. After washing, labelled DNA fragments which annealed to specific probes are visualized by illuminating the slide with light of the appropriate wavelength to cause the dyes to fluoresce. As microarrays can carry thousands of probes per square centimetre, this is performed by automated scanning devices and dedicated software which identifies the probes producing positive signals. This technique allows the screening at once of a very large number of pathogens or defined genetic markers.

B)   PCR Amplification Using Fluorescent Primers

As in many other fields, PCR has brought a revolutionary change in nucleic acid-based diagnosis. In a clinical setting, PCR has many desirable features such as the sensitivity to work with tiny amounts of DNA samples from blood or tissue to achieve a specific and significant amplification of target sequences. Furthermore, the rapidity of this process, as explained previously in this chapter, provides a significant advantage in the early diagnosis and treatment of infectious diseases.

A PCR fluorescence-based technique has been used successfully in the diagnosis of infectious diseases. In this case the PCR primers are labelled with a fluorescent dye that is bound to the 5′ end of each primer. Two main types of fluorescent dyes are normally used: one is fluorescein, which appears green under certain light wavelengths, and the other is rhodamine, which appears red. After PCR amplification of the target sequence with the fluorescent-labelled primers, the primers are removed by chromatographic separation, and the presence of the labelled PCR product is detected. The absence of labelled PCR product is interpreted as the absence of the target DNA sequence.

The procedure can also be applied to detect specific RNAs, for example to detect RNA viruses. In this case the extracted RNA has to be converted first to ds cDNA with reverse transcriptase before the PCR amplification. The procedure is then called reverse transcriptase PCR or RT-PCR.