The most widely used methods for detecting microbial DNA fall into three categories: 1) direct hybridization (nonamplified assay), 2) amplification methods using the polymerase chain reaction (PCR)1 or one its variations, and 3) DNA microarrays. Although not likely to completely replace culture techniques in the near future, nucleic acid–based tests for the diagnosis of infectious diseases are gaining wider acceptance as more products approved by the Food and Drug Administration become commercially available.

 A. Direct detection of pathogens without target amplification

This highly specific method of pathogen detection involves identification of the DNA of the pathogen in a patient sample or, more commonly, organisms isolated in culture. The basic strategy is to detect a relatively short sequence of nucleotide bases of DNA (target sequence) that is unique to the pathogen. This is done by hybridization with a probe, a single-stranded piece of DNA (usually labeled with a fluorescent molecule) containing a complementary sequence of bases. [Note: In bacteria, DNA sequences coding for 16S ribosomal RNA sequences (rRNA) are commonly used targets because each microorganism contains multiple copies of its specific 16S rRNA gene, thereby increasing the sensitivity of the assay.] When the probe is bound to the target, the label will give off a signal after the free probe is washed away. A limitation of standard direct probe hybridization is the requirement for a 10⁴ or greater number of copies of target nucleic acid for detection.

B. Nucleic acid amplification for diagnosis

 Nucleic acid amplification overcomes the principal limitation of direct detection with nucleic acid probes by selectively amplifying specific DNA targets present in low concentrations. The bacterial 16S rRNA gene has emerged as the most useful marker for microbial detection and identification. Ribosomal DNA genes contain highly conserved areas (that are used as targets for primers) separated by internal transcribed sequences containing variable, species-specific regions. These sequences are like fingerprints. Comparing certain locations on a 16s rRNA gene with a database of known organisms allows the identification of organisms. For virus detection, primers are con structed to target highly conserved DNA or RNA sequences unique to the pathogen. Amplification and detection of the viral genomes are highly sensitive and are especially valuable when the viral load is too low to be detected by culture or when results are needed rapidly.

 1. Conventional polymerase chain reaction: In this method, DNA polymerase repetitively amplifies targeted portions of DNA (ideally sequences that are highly conserved and unique to the pathogen). Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase in DNA with repeated cycles of amplification. The amplified DNA sequence can then be analyzed by gel electrophoresis, Southern blotting, or direct sequence determination.

2. Real-time polymerase chain reaction: This variant of PCR com bines nucleic acid amplification and fluorescent detection of the amplified product in the same closed automated system. Real time PCR limits the risk of contamination and provides a rapid (30–40 minutes) diagnosis. Real-time PCR is a quantitative method and allows the determination of the concentrtion of pathogens in various samples.

 3. Advantages of polymerase chain reaction: Methods employing nucleic acid–amplification techniques have a major advantage over direct detection with nucleic acid probes because amplification methods allow specific DNA or RNA target sequences of the pathogen to be amplified millions of times without having to culture the microorganism itself for extended periods. PCR also permits identification of noncultivatable or slow-growing microorganisms, such as mycobacteria, anaerobic bacteria, and viruses. Nucleic acid–amplification methods are sensitive, specific for the target organism, and are unaffected by the prior administration of antibiotics.

4. Applications: Nucleic acid–amplification techniques are generally quick, easy, and accurate. A major use of these techniques is for the detection of organisms that cannot be grown in vitro or for which current culture techniques are insensitive. Moreover, they are useful in the detection of organisms that require com plex media or cell cultures and/or prolonged incubation times (Figure 1).

Fig1. Commercial nucleic acid– amplification systems for diagnosis of infectious diseases. The table is not intended to be all inclusive.

5. Limitations: PCR amplification is limited by the occurrence of spurious false-positives due to cross-contamination with other microorganisms’ nucleic acid. PCR tests are often costly and require skilled personnel.

C. DNA microarrays

Although microarrays are now routinely used to measure gene expression, the technique is an emerging technology in the diagnostic microbiology laboratory. Microarrays have the unprecedented potential to simultaneously detect and identify many pathogens from the same specimen. For example, an oligonucleotide microarray targeting the 16S rRNA gene has been developed for the detection of a panel of forty predominant human intestinal bacterial pathogens in human fecal samples.

 1. Diagnostic use of microarrays: A DNA microarray consists of microscopic spots of immobilized DNA oligonucleotides, each containing specific DNA sequences, known as probes. The probes are constructed to be complementary to specific gene sequences of interest in suspected pathogens. DNA of the microorganism obtained from a clinical specimen, known as the target, is extracted and amplified using PCR and fluorescent labeling techniques. The target DNA is exposed to the probe microarray. If the labeled DNA from the microorganism and the immobilized probe have a complementary base sequence, they will hybridize, thereby increasing fluorescence intensity. After washing off of nonspecific bonding sequences, only strongly paired strands will remain hybridized and fluoresce. The intensity of fluorescence at each spot is a measure of the amount of that particular microbial DNA in the sample. Correlating fluorescence with the identity of the probe allows for the detection and quantitation of specific pathogens.

مواضيع ذات صلة

human chorionic gonadotropin (hCG, Pregnancy tests, hCG beta subunit)

homocysteine (Hcy)

HIV serologic and virologic testing

HIV RNA quantification (HIV viral load)

Molecules involved in Lipoprotein metabolism

Lipoproteins

Retinol Binding Protein

Direct Visualization Of The Organism

Direct Detection Methods for Bacillus and Similar Organisms

HIV drug resistance testing (HIV genotype)

herpes simplex (Herpesvirus types 1 and 2, Herpes simplex virus types 1 and 2 [HSV 1, HSV 2], Herpes genitalis)

hepatitis virus studies