Zeynep Ceren Karahan
School of Medicine Department of Microbiology and Clinical Microbiology
Ankara University Turkey
The most important duty of a microbiology laboratory is to provide a rapid and reliable diagnosis of infectious agents. Traditional diagnostic tools (ie, culture, microscopy and serological tests) have some limitations. Culture is time-consuming, and results are affected by specimen quality and transportation conditions. The organism isolation rate is significantly reduced in patients receiving antibiotics. For fastidious or uncultivable organisms, culture may even fail. Microscopic diagnosis can be useful for specimens obtained from sterile body sites, but it usually requires a relatively large number of organisms to become positive, and identification of the organism by microscopy is not possible. Serologic tests are indirect proofs of disease; they often provide retrospective information only, and they are affected by the patient’s immune status.(1-4)
Molecular methods overcome many of these disadvantages. Polymerase chain reaction (PCR), an enzyme-driven process for in-vitro amplification of short nucleic acid fragments, is the most frequently used molecular method in microbiology. The main advantages of PCR over traditional diagnostics include its speed, simplicity, increased sensitivity and the ability to determine the agents in inadequately stored specimens. Databases and phylogenetic trees can be made by PCR, and automation is possible. PCR applications in microbiology are summarised in Box 1.(2-6)
PCR applications in microbiology
The most frequently used PCR approach in microbiology is broad-range PCR that uses primers complementary to 16S rDNA sequences. The amplicon obtained is sequenced, and the result is compared with the ones in the databases. Some available databases include: GenBank (www. ncbi. nlm.nih.gov/Genbank); Genomes OnLine Database (www. genomesonline.org); the Institute for Genomic Research (www.tigr.org); and the Ribosomal Database Project (http://rdp.cme.msu.edu).
Other PCR-based diagnostic approaches include multiplex PCR, nested PCR and reverse transcriptase PCR (RT-PCR). Multiplex PCR enables the simultaneous detection of several target sequences by incorporating multiple sets of primers. It saves time and effort in the laboratory and identifies bacteria more accurately. Nested PCR uses two PCR rounds with different primer sets designed internally to each other, and is used to increase sensitivity and specificity. RT-PCR is performed to detect RNA viruses and viable organisms. In this assay, RNA is converted to complementary DNA (cDNA) by the enzyme reverse transcriptase, and then cDNA is amplified by PCR.(3,6)
Genotyping the agent is important for epidemiological studies, for determining virulence factors and antimicrobial susceptibilities, and for predicting the response to therapy. PCR-based approaches for genotyping include: arbitrarily primed PCR, randomly amplified polymorphic DNA, 16-23S rDNA spacer PCR, amplified ribosomal DNA restriction analysis, amplified fragment-length polymorphism, terminal-restriction fragment-length polymorphism, multilocus sequence typing, multispacer typing, variable number tandem repeat, genome level-informed PCR, repetitive-sequence -based PCR and ligation-dependent PCR.(3,5,6)
Although PCR is widely used in the infectious disease arena, conventional PCR technology has some disadvantages. It is more expensive than traditional methods, infectious agents cannot be differentiated according to their viability and isolated for further studies, specimen adequacy cannot be determined, there is a risk of contamination, confirmatory tests are often necessary and it is not yet standardised or widely accepted.(2) Resistance detection by PCR does not always show true resistance and is not quantitative. (2,3) Other limitations of conventional PCR assays include false-positive results from background DNA contaminations, potential false-negative test results (due to small sample volume, poor DNA recovery, inadequate removal of PCR inhibitors in the sample), test sensitivity exceeding clinical significance, limited detection space of the assay platform for simultaneous identification of multiple species, virulence factors or drug resistance genes.(3) Newer technologies (eg, real-time PCR and microarrays) and methodologies (eg, loop-mediated amplification [LAMP] and helicase-dependent amplification [HAD]) are being developed to overcome these limitations.
Real-time PCR is a significant advance in PCR technology. It combines amplification and detection steps in a closed system, and quantification is possible. It is faster than conventional PCR assays due to a reduced cycle time, reduced amplicon size and the elimination of additional steps for product detection. Other advantages include its simplicity, reproducibility, reduced contamination risk and quantitative capacity.(3,7) By linking real-time PCR technology to automated nucleic acid extractions, assays may take as little as two hours to perform.(7) In clinical microbiology, real-time assays are used for detecting bacterial, viral, parasitic and fungal targets; analysing genes that confer drug resistance and genetic determinants of pathogenesis; detecting bioterrorism agents; and for determining microbial load by quantitative analysis. The main limitation of this technology is its inadequate multiplexing capabilities.(7)
DNA microarrays are constructed by spatially isolating specific genome sequences to prearranged areas on a microchip. This technology can be used for microbial identification at the species level, genotyping and resistance determination. Complex multiplex PCR assays, which are often difficult to optimise, can be done by these systems. The main advantage of using microarrays for pathogen detection is the potentially large number of target sequences the system can discriminate simultaneously.(3) The combination of broad-range PCR with DNA microarray technology offers interesting possibilities for identifying microbial pathogens and determining host genes conferring susceptibility or resistance to infectious diseases. Efforts to improve sensitivity, reproducibility and user-friendly approaches to complex data analysis are still needed before they can be used clinically.(3) Other promising strategies include emerging biochip technologies; for example, putting nucleic acid amplification directly onto the oligonucleotide biochip or using arrays coupled with ultrasensitive detection methods that don’t require previous amplification of the nucleic acid target.(8,9)
The LAMP method amplifies nucleic acids under isothermal conditions in the range of 65ºC. As a result, it allows the use of simple and cost-effective reaction equipment. Another characteristic of this method is that it has both high specificity and high-amplification efficiency.(10) Its detection limit is a few copies, comparable to that of PCR. LAMP is simple and easy to perform, requiring only four primers, a DNA polymerase and a regular laboratory water bath or heat block for reaction. By combination with reverse transcription, LAMP can amplify RNA sequences with high efficiency.(11)
HDA uses a DNA helicase to separate double-stranded templates for primer hybridisation and subsequent extension. As the DNA helicase enzymatically unwinds dsDNA, the initial heat denaturation and subsequent thermocycling steps required by PCR can be omitted. Therefore, HDA provides a simple DNA amplification scheme – one temperature from the beginning to the end of the reaction. The simplicity and true isothermal nature of the HDA platform offers a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point of care. Future experiments will be directed towards improving the efficiency of HDA by testing different helicases/polymerases and by optimising the existing HDA systems by varying the ratio and concentration of each component.(12)
Differentiating viable from nonviable organisms is another important issue in molecular diagnosis. As RNA is degraded in minutes after cell death, it is proposed to be an accurate indicator of viable organisms. RNA can be detected by RT-PCR or real-time RT-PCR, but it is usually difficult to extract detectable concentrations of intact RNA from a small number of organisms.(3) RT-PCR cannot be used for quantitative differentiation of specific cells in mixed populations. A recently developed method, the ethidium–monoazide PCR (EMA-PCR) offers a novel, real-time PCR method for quantitative distinction between viable and dead cells. EMA penetrates only dead cells with compromised membrane/cell wall systems, and DNA covalently bound to EMA cannot be amplified. Therefore, only DNA from viable cells can be detected. EMA-PCR may promote new applications in viable/dead diagnostics.(13)
It is obvious that the infectious disease arena needs a reliable and standardised diagnostic system. Although molecular methods are increasingly used for diagnosing infectious diseases, routine use of PCR in the clinical microbiology laboratory must be balanced against cost, expertise and time associated with routine testing. The assay must be used when the benefits outweigh its cost, especially in developing countries. Institutional resources and manpower issues must be considered before decisions are made about replacing traditional diagnostic methodologies. Tests must be standardised and quality control programmes must be established before they are routinely used.
Education of practitioners who will interpret the results is another important issue. Clinicians must be trained about the principles, diagnostic values and limitations of the assays used in the laboratory. Because PCR can detect pathogens at concentrations below those previously established gold-standard reference methods, interpretive guidelines based on the correlation of results with clinical presentation and existing standards will be required before these assays can be used for definitive diagnosis and treatment decisions.
Congress of Molecular Medicine
24–26 March 2007 Istanbul, Turkey.
25th International Congress of Chemotherapy & 17th European Congress of Clinical Microbiology and Infectious Diseases (joint conference)
31 March–3 April 2007 Munich, Germany
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- Yang S, Rothman RE. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis 2004;4:337-48.
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- Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophy Res Communi 2001;289:150-4.
- Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28:i-vii, e63.
- Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification. EMBO Reports 2004;5:795-800.
- Rudi K, Moen B, Dromtorp SM, Holck AL. Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. Appl Environ Microbiol 2005;71:1018-24.