Molecular Diagnostics: Current Technology and Applications Chapter Abstracts

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Chapter 1
Update on Molecular Tools for Detection of Plant Pathogenic Bacteria and Viruses
María M. López, Edson Bertolini, Ester Marco-Noales, Pablo Llop and Mariano Cambra

Detection of pathogenic bacteria and viruses in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The available techniques, especially those based on molecular techniques, have evolved significantly in the last few years to allow the rapid and reliable detection of pathogens,. Molecular detection is largely based on PCR or RT-PCR amplification following purification of nucleic acids from the samples, with the extraction of the target DNA a key step for optimising detection protocols. Variants of PCR, such as simple or multiplex nested-PCR in a single closed tube, co-operative-PCR, and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The lastest development is the microarray technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in genomics and proteomics represent a new source of information for the development of sensitive and specific detection techniques for these microorganisms.

Chapter 2
Molecular Diagnostic Tools for Detection of Plant Pathogenic Fungi
Rosie E. Bradshaw, Simon J. Foster and Brendon J. Monahan

Early and accurate diagnosis of fungal plant pathogens is essential to limit their impact on food supplies and other plant-based commodities. Molecular diagnostic methods are becoming established as useful additions to the plant pathologist's toolbox. Basic methods of fungal DNA extraction are discussed, followed options for PCR primer design. Recently developed techniques combining PCR with hybridization and immunological based methods for detection are outlined. Three specific applications are presented where the pathogen's DNA is often difficult to obtain in sufficient purity or quantity for testing (from woody tissue, aerial spores or herbarium samples). The crucial issues involved in the quantification of plant pathogens and the detection of mycotoxigenic fungi are also addressed. In looking towards the future of diagnostics, new methods to amplify whole genomes are presented that will enable diagnostic testing with trace amounts of starting material, and the prospects for field-based testing are discussed. Finally the impact of fungal genome sequence information on the future of diagnostic testing is evaluated.

Chapter 3
Application of Molecular Techniques for Detection of Bacterial Veterinary Pathogens
M. Murakami, T. Sekizuka and M. Matsuda

In relation to molecular techniques for the detection of bacterial veterinary pathogens, several PCR assays have been used successfully by employing genus and species specific PCR primers designed on the nucleotide sequences of several targeted genes. In addition, it is suggested that a polyphasic strategy for PCR-based-identification should be used. Nested and seminested multiplex PCR methods have also been developed. In recent years, real-time PCR methods have been developed and described for the rapid detection and identification of the several bacterial veterinary pathogens. Most recently, DNA microarrays containing species-specific oligonucleotide probes designed from specific regions of various targeted genes have been developed and applied successfully.__ DNA molecular techniques for the detection of bacterial pathogens have been clearly proven to be more useful than any traditional identification methods in veterinary medicine.__

Applications to Food Safety (Foodborne Pathogens, Pests, GMO)

Chapter 4
Molecular Diagnostics and Epidemiological Typing Methods for Common Foodborne Pathogens Campylobacter and Salmonella
Majella Maher, Sinead Lahiff and Dearbháile Morris

Campylobacter and Salmonella are significant causes of food-borne illness. Microbiological methods currently used for food testing are slow. Developments in molecular diagnostics for pathogens have increased the potential for rapid tests to compliment and in some applications replace traditional methods. The availability of rapid, reliable methods to detect the presence of pathogens in food is becoming increasingly important for food producers, legislators, for international trade, to ensure consumer safety. Molecular epidemiological typing methods have become essential tools in source identification, tracking infection and contact tracing during outbreaks. The purpose of this chapter is to inform the readership of developments over the last five years in molecular diagnostic and epidemiological typing methods for common food-borne pathogens, Campylobacter and Salmonella.

Chapter 5
DNA Biosensor Chips for GMO Detection
Maria Minunni

This chapter deals with recent advances in the detection of genetically modified organism (GMOs) based on biosensor technology. DNA-based sensing has been successfully applied - to the analysis of the promoter p35S and the TNOS terminator, which are inserted sequences in the genome, regulating the transgene expression - and present in most of the commercially available GMOs. The current biosensors applicable to GMOs are based on either optical or piezoelectric and electrochemical transduction principle. All of these systems, are coupled to the Polymerase Chain reaction (PCR) which selectively amplifies the target sequence of interest (p35S or TNOS) and this chapter describe these biosensor systems for GMO detection.

Chapter 6
Molecular Techniques for Identification of Quarantine Insects and Mites: The Potential of Microarrays
Juerg E. Frey and Monika Pfunder

Globally, many insects and mites possess an astounding potential to cause damage to agricultural production. Quarantine measures are put in place to prevent or delay introduction of such pests into areas of intensive agricultural production and correct identification of quarantine pests is among the most important factors determining success or failure of these measures. Traditionally, identification has relied primarily on external morphological characters of adult life stages. However, intercepted pest specimens often are not in the adult stage and may be damaged, which seriously handicaps correct identification. Molecular tools now enable precise and rapid identification, irrespective of the developmental stage and condition of the samples. Furthermore, molecular tools also enable genetic characterisation of specific attributes of an organism and, if combined with a high-throughput technology such as microarrays, they are ideally suited for molecular genetic screening. In the future molecular diagnostics will therefore play a vital role in addressing the growing problem of pesticide resistance, compounded by the extent and ease of international transport (an ideal dispersal mechanism). Molecular diagnostics will also facilitate the acquisition of epidemiological information, such as that relating to pesticide resistance genes, and thus become an important component of sustainable pest management strategies. Here we present a short overview on the problems related to molecular species identification and on the potential of molecular screening for crop protection. We describe the most common molecular techniques currently in use and offer a practical example for the application of microarray technology to species identification of fruit flies.

Applications to Biomedical and Clinical Sciences

Chapter 7
Biosensor Applications of Peptide Nucleic Acid for Direct Detection of PCR Amplified DNA of Pathogens E. coli 0157:H7 and MRSA
Tomohiko Nishino, Mifumi Shimomura-Shimizu and Isao Karube

Peptide nucleic acid (PNA) probes are useful for detecting target DNA because they have high thermal stability, strong binding capacity, and high binding specificity. PNA probes would thus be powerful tools for detection of PCR amplified pathogenic DNA from clinical samples or direct in situ detection using microscopy. In this chapter, we have used E. coli 0157:H7 or MRSA as specific examples to demonstrate the versatility of PNA probes in detection of the PCR products using SPR systems. PNA is advantageous because of its compatibility with most molecular techniques, and can be applied for the detection of pathogenic bacterium of concern to human health and food safety.

Chapter 8
Molecular Diagnostics of Medically Important Bacterial Infections
Beverley Cherie Millar, Jiru Xu and John Edmund Moore

Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world's biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood. In developed countries, the emergence of new, rare or already-forgotten infectious diseases, such as HIV/AIDS, Lyme disease and tuberculosis, has stimulated public interest and inspired commitments to surveillance and control. Recently, it is reported that infectious diseases are responsible for more than 17 million deaths worldwide each year, most of which are associated with bacterial infections. Hence, the control of infectious diseases control is still an important task in the world. The ability to control such bacterial infections is largely dependent on the ability to detect these aetiological agents in the clinical microbiology laboratory.

Chapter 9
Molecular Diagnostic Tools for the Detection of Bacteria in Dental Plaque
Wilson A. Coulter, Charles E. Shelburne and Denis E. Lopatin

The accumulation of bacteria on the hard surfaces of the mouth is the cause of both caries and periodontal diseases. The species that makeup this biofilm have been the subjects of intense research for the last 30 years, as has speculation about their relationship to the disease process. Recently, there has been increasing interest in the detection and characterization of bacterial phylotypes by exclusively molecular techniques. This has been prompted by several studies of both medically important and environmentally interesting samples, which have yielded evidence that current cultivable techniques may substantially underestimate the number and types of bacteria in such samples. While several non-culture based methods have emerged in the last 15 years the most promising seems to be real-time Polymerase Chain Reaction (PCR). The ability of this technique to use the great sensitivity of the PCR and simultaneously quantitate bacteria at the species level and below makes real-time PCR the current methodology of choice. In addition to real-time PCR several other techniques that can evaluate bacterial expression analysis of virulence factors and antimicrobial resistance promise to extend the value of nucleic acid based technologies into both the clinical evaluation and scientific investigation of oral bacterial communities.

Chapter 10
Molecular Investigations of Haematological Malignancies
H. Denis Alexander and Mark A. Catherwood

An overview of the application of molecular studies in haematological malignancies is presented. This includes investigations to differentiate between reactive and malignant proliferations, identification of acquired chromosomal aberrations defining specific disease sub-types, study of altered gene expression influencing the biology of the disease and detection of minimal residual disease following treatment. The contribution of molecular studies to enhanced understanding of alterations in the cellular and molecular characteristics of malignant cells, compared to their normal counterparts, is discussed. The contribution this is making to the design of new drugs, with activity against the tumour cells but minimal toxicity to normal tissues, is addressed. Summaries of methodological approaches to these molecular investigations are given and future developments anticipated.

Chapter 11
Molecular Diagnosis of Medical Viruses
Rodney M. Ratcliff, Grace Chang, Tuck Weng Kok and Theo P. Sloots

The diagnosis of human virological disease has been revolutionised by the development of molecular techniques. Foremost have been the applications of the polymerase chain reaction (PCR), the consequence of the achievable increase in sensitivity and specificity, and the ease with which the method can be adapted to detect any known genetic sequence. Real-time PCR assays are now making a major contribution, because of the additional increase in sensitivity over conventional PCR, and the ability to confirm the amplification product and quantitate the target concentration afforded by the fluorescent probe detection system. Moreover, sequencing of the amplification product has facilitated the epidemiological analysis of infectious outbreaks and the assessment of treatment outcomes for infections with highly mutable viruses. However, the frequency with which tests are performed in a diagnostic laboratory creates challenges to ensure 'result quality'. This chapter discusses the importance of these quality assurance issues, and then presents specific examples of nested RT-PCR, qualitative and quantitative real-time PCR, multiplex PCR, and two examples of the use of sequence analysis of the amplified product.

Chapter 12
Molecular Diagnosis of Parasites of Medical Importance
Lihua Xiao

Recently, molecular tools have been developed to detect and differentiate parasites at the species, genotype, and subtype levels. These tools now make it possible to detect parasites with increased sensitivity, determine the identity of parasites that infect humans, track the source of contamination in outbreaks and environment and infections in humans, compare the pathogenicity, infection patterns, and disease spectrum among species/genotypes, assess the human infection potential of animal parasites, help to characterize the transmission dynamics of infection in endemic areas, and monitor the development of drug resistance. Some of these tasks are too expensive or time-consuming, or simply impossible to achieve with traditional diagnostic tools based on microscopy or biological characterizations. Thus, molecular diagnosis has become an important element in public health research and practice of parasitic and tropical diseases.

Applications to Biothreats, Drugs and Forensics

Chapter 13
Applications of Biosensor Technology for Molecular Recognition and Detection
James P. Chambers

Biosensors are devices that transduce a recognition event via a recognition element, e.g., an antibodys binding to a specific target, i.e., antigen, the binding of which is converted into a measurable signal, i.e., chemical, electronic or opto-electronic. The marriage of biology and micro- and nanofabrication has revolutionized biosensing and led to integration of biological recognition elements into devices that will with development significantly impact commercially available detection/diagnostic (static 'snapshots' and continuous real time) sensing at the genome, proteome, and metabalome levels. The development of biosensors was until recently motivated mainly by medical applications. However, 9/11 and the subsequent anthrax letter events have thrust the applications of biosensors for detection of chemical and biological agents of mass destruction. In both medical and national security applications, one must be able to detect biological agents together with pre-clinical, clinical, and agricultural surveillance and diagnosis. Biosensors involve three different empirical activities, i.e., development of 1) molecular recognition elements, 2) techniques and tools for biosensor construction, and 3) sensor devices. Because biology contributes the materials that offer unique and important structural recognition and catalytic functions distinguishing the 'bio'-sensor, the focus of this chapter is that of selected recognition elements currently shaping the biosensing arena.

Chapter 14
Molecular Diagnosis and Bioterrorism: An Update
James Dooley and Colm Lowery

Molecular diagnostic technology offers a rapid, specific and adaptable approach to detecting pathogens that could be used as bioweapons. At present one of the major challenges is to decide upon how this technology can be best applied and what agents should be targeted for surveillance. Human, animal or even plant pathogens could be -useful to a terrorist seeking to cause major disruption within a target population. It has even been speculated that genetically altered pathogens could be produced and deployed. Microarray systems have the ability to screen for multiple agents at one time and can be regarded as front line technology for the investigation of an incident. Prompted by recent advances in MALDI-TOF spectrometry, an alternative strategy is the detection of microbial signature sequences which may allow rapid environmental analysis. The integration of diagnostic systems with databases of genomic, clinical and biological information will be necessary to ensure that the full range of possible threats can be foreseen and efficiently monitored.

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