Advances in DNA sequencing technology have made it possible for scientists all over the world to sequence complete microbial genomes rapidly and efficiently. Access to the DNA sequences of entire microbial genomes offers new opportunities to analyze and understand microorganism at the molecular level. Scientists are able to detect pathogens in biological tissues and study variations in gene expression in response to the pathogenic invasion. These responses help in designing novel approaches for microbial pathogen detection and drug development. Identification of certain microbial pathogens as etiologic agents responsible for chronic diseases is leading to new treatments and prevention strategies for these diseases.
Pathogen Detection Mechanism
Each species of pathogens carries with it a unique DNA or RNA signature that differentiate it from other organisms. One of the challenges is to develop this DNA signature for each microorganism of interest for rapid and specific detection.
Pathogen Detection Applications
Pathogen detection has become an important part of research in many fields like:
# Animal health care
# Food safety
# Clinical research
# Drug discovery
For biodefense, accurate analytical techniques for discovering pathogenic agents are needed. Animal health care community uses pathogen detection to develop various diagnostic tests that are rapid, reliable and highly sensitive for effective control and treatment of diseases of animals. In diagnostics, the technique is employed to detect or identify infectious agents, toxins, parasites, metabolic disorders, and genetic susceptibility/resistance.
Challenges in Pathogen Detection
The predominant techniques currently used to identify microbial pathogens rely upon conventional clinical microbiology monitoring approaches that are well established suffer from a number of considerable drawbacks. Standard culture and susceptibility tests permit pathogen identification but is laborious, time-consuming, expensive and require labile natural products. More importantly, the tests that are routinely utilized for pathogen identification do not directly characterize virulence factors. Thus, these tests do not provide the needed information about the potential pathogenicity or virulence of the identified organisms. Conventional techniques also do not lend themselves well to managing large numbers of environmental or clinical samples. To quickly determine the presence of pathogen, researchers need reliable and accurate tools which can cater to the increasing need of finding faster, accurate analytical techniques for discovering agents.
Various Approaches towards Pathogen Detection
There are nearly as many approaches to rapidly detecting pathogens and diagnosing disease as there are companies and laboratories developing the technologies.
QPCR Based Pathogen Detection
Efforts to overcome problems like culturing of microorganisms, false positives and causative agent in pathogen detection have led to the development of DNA-based diagnostic methods such as polymerase chain reaction (PCR) amplification techniques. The use of DNA-based methods derives from the premise that each species of pathogen carries unique DNA or RNA signature that differentiates it from other organisms. Among the various PCR strategies available, those based on monitoring the amplification reaction in real time are probably the most promising and are increasingly used for rapid, sensitive, and specific detection of microbes.
The ideal solution for a real-time detector is a biological organism specific response that results in almost instantaneous, specific and repeatable identification. However, there are considerable technological and practical difficulties in the development of sensors that provide a real-time response for all three of these criteria.
Immuno-assay techniques also give a similar specific analysis. However, an additional drawback other than the long response time, is the requirement for special chemical consumables that add considerably to the logistic burden and costs. These can increase operational costs by hundreds of dollars per hour.
Optical technologies intrinsically result in real-time bio-detection and devices based on these technologies have been available to military and civil defense organizations for a number of years. However, the common drawback of this type of sensor is the lack of specificity. The sensors mostly offer a generic detection capability at best, since the optical similarity of the target particles with benign, naturally occurring backgrounds makes them difficult to distinguish. There are the some of the currently employed bio-agent detections strategies. Most represent a compromise between specificity, speed and cost.
Biotechnology offers the most specific detection approach. Quantitative Polymerase Chain Reaction (qPCR) technique is capable of amplification and detection of a DNA sample from a single bio-agent cell within 30 minutes. Knowing the pathogen nucleic acid sequence enables scientists to construct oligos to detect the pathogen. These oligos are at the basis of many highly specific analytical tests now on the market.
Microarray Based Pathogen Detection
The advantage of microarray-based detection is that it can combine powerful nucleic acid amplification strategies with the massive screening capability of microarray technology, resulting in a high level of sensitivity, specificity, and throughput. In addition to the previously mentioned caveats, the cost and organizational complexity of performing a large number of PCR reactions for downstream microarray applications render this option feasible but unattractive. This limitation has severely reduced the utility of this technique and impeded the continued development of downstream applications.
Researchers are often unsure of the validity of the microarray data and the often asked question is: Must you validate the data using an alternate technique? The answer is a resounding ‘Yes’. Simply because it will make the researcher feel more confident of the results, and more importantly the reviewers is sure to ask for it.
An excellent design software tool is crucial for success in assay development efforts. AlleleID , a new software tool, can play a vital role in easing the experimental burden and minimizing assay development as well as operational costs.
AlleleID is a pioneering software specially designed for meeting the challenges of pathogen detection, bacterial identification. species identification and taxa discrimination assay development. Its unique feature Minimal Set minimizes the number of probes required to identify a group of sequences. AlleleID starts by aligning sequences using the popular ClustalW algorithm, analyzes conserved and species specific regions and then designs primers and probes to amplify and detect only the species of interest from the mix.