Emerging Approaches for Pathogen Detection
Year: 2024
Type: article
Abstract: The emergence of new and adaptive microbial strains has intensified the need for advanced pathogen detection methods. Over the past three decades, 33 novel pathogens, including severe acute respiratory syndrome (SARS) and HIV, have been identified. The delayed response to SARS in Canada, for instance, led to a 2.6-fold increase in the epidemic's scale and prolonged its duration by four weeks, highlighting the critical importance of timely pathogen detection and intervention. Recent advancements in DNA sequencing technology have revolutionized microbial genomics, enabling rapid and comprehensive sequencing of entire microbial genomes. This molecular-level insight facilitates precise identification of pathogens and opens avenues for developing innovative therapeutic and protective strategies against serious diseases. These emerging approaches are crucial for managing the growing challenge posed by virulent and antibiotic-resistant pathogens. Introduction Today there has been immense need for pathogen detection as newly strains have been appearing in the environment and moreover these newly strains are becoming adaptive in environment, virulent (dividing at a much greater rate) and resistant to most of the antibiotics. During the past three decades 33 new pathogens had been discovered which includes severe acute respiratory syndrome and HIV. This issue has been highlighted because it was from Canada that for acute respiratory syndrome, the control measures were not taken immediately but delayed for a week and as a result the epidemic increased to 2.6 fold and men epidemic extension of four weeks. [1] Fig 1: pathogen (HIV virus) Innovations in DNA sequencing technology have made it achievable for scientists all over the globe to sequence entire microbial genomes fast and effectively. At the molecular level access to DNA sequence of entire microbial genomes offers new possibilities to examine and recognize the entire microorganism. Detection of specific microbial pathogens as elements responsible for serious illnesses is leading to new remedies and protection techniques for these diseases. [3] Mechanism for Pathogen Detection Every species of pathogens bears with it a distinctive DNA or RNA signature that separates it from other microorganisms. One of the problems is to create this DNA signature for each organism of interest for quick and particular diagnosis. Representation of the rDna gene complex in bacteria and fungi denoting gene order and position of the internal transcribed spacer regions (ITS). Pathogen Detection Applications Pathogen detection has turn out to be an essential and significant part of research in many areas like: • Biodefense • Forensics • Animal health care • Pathology • Food safety • Diagnostics • Clinical research • Drug discovery For biodefense, precise systematic methods for finding pathogenic agents are required. Animal health care community uses pathogen detection to develop numerous diagnostic tests that are quick, reliable and extremely delicate for successful control and treatment of diseases of animals. In diagnostics, the method is used to diagnose or recognize infectious agents, parasites, toxins, metabolic problems, and genetic resistance. [3] Different Techniques towards Pathogen Diagnosis For the detection of pathogenic blood stream infections (BSI) many molecular diagnostic tools are been used for the detection of pathogens direct from blood culture in septic patients in time of less than 6 hours. The technique used is multiplex real-time PCR-based assay for the detection of 25 clinically based diseases. After 8 to 36h of sampling, the bacterial and fungal BSI becomes positive and then the therapy could be performed after the identification of bacteria after gram staining. The detection of pathogen with in the first 6 to 12 h is important for favorable out come with BSI. In molecular biology we use the following techniques a part from multiplex real-time PCR-based assay, for rapid detection of pathogenic strains in blood stream infections: Ø Nucleic acid based diagnostic systems. Ø Polymerase chain reaction (PCR). Ø Application of DNA and RNA probes. Ø Specific probe hybridization. Ø Sequencing of the genomic target. Ø DNA array technology Polymerase chain reaction (PCR) Over all the above techniques mentioned, theoretically PCR based techniques have immense importance due to specific detection of pathogen in very fewer times in blood cultures. The translation of blood culture to PCR based assay that cover nosocomical pathogens which uses universal primers for PCR amplification. They target that part of genome which is specific for certain species. It is performed by the following steps: Melting point analysis. Hybridization probes. Direct sequencing. Fig 2: Steps of PCR techniques Limitation and drawback in PCR: PCR based assay have certain limitations and some are mentioned as follows: v The preparation is done not by blood but from plasma, so some of the bacterial cells or DNA is engulfed by phagocytic cells such as macrophages and granulocytes. v Fungi species are not part of such pathogens. v There is no control to check the DNA preparation and PCR amplification in these assays. v Working conditions are less contaminant free in such assays. To, overcome all these limitations multiplex real-time PCR-based assay have been developed. This assay follows the following three steps: 1) Purification and mechanical lyses of DNA. 2) Target DNA is amplified in parallel reactions (fungi, gram-positive and gram negative) and PCR products are identified by specific hybridization probes. 3) Then the species are identified. So, from the study of multiplex real-time PCR-based assay following results could be derived by hit rate method: v 50% hit rate was obtained for E. aerogenes at a concentration of 3 CFU/ml. v 100% hit rate for S. marcescens, E. coli, P. mirabilis, P. aeruginosa,and, and A. fumigates at a concentration of 3 CFU/ml. v 75% hit rate was obtained by C. glabrata at a concentration of 30 CFU/ml. [2]. NANOBIOSENSORS Nanobiosensors basically detect very specific biological molecule interactions and in 21st century it is a highly emerging field. Advantages of Nanobiosensors: Nanobiosensors have many advantages over other methods of pathogen detection such as: Ø Few molecules could be detected by the use of Nanobiosensors even though the given volume of the solution is less. Ø They are highly sensitive to bulk measures for example in refractive index. Ø Nanobiosensors life time is so long that the entire platform of Nanobiosensors could be designed in a way that the entire volume of sample could be dipped in biosensor. Ø There are many different types of Nanobiosensors which could be designed according to our need and depending upon the conditions, such as in highly damp conditions 1D nanostructure electrical detection could be designed and used. It is to increase the sensitivity. Ø Label free and multiplexed biosensing could also be developed for example in Surface Plasmon resonance imaging. Ø Chip based devices could also be developed in Nanobiosensors and the benefit obtained from it is that they could be operated in a single plane. Thus the major advantage gained by single plane operation of Nanobiosensors is that the electrical measurements could be taken easily and their sensitivity is un-doubted. Types of Nanobiosensors There are many types of Nanobiosensors and some are as follows: 1. Nanowire biosensors: Fig 3: Nanowire biosensor Nanowire could be designed with almost any prospective chemical substance or biological molecular identification unit, making the cables component impartial. The smaller size and ability of these semiconductor nanowires for delicate, label free, real time detection of a broad range of chemical and biological species could be used in variety of based testing and in vivo diagnostics. [9] 2. Homogeneous phase biosensors: This type of Nanobiosensors is used in in-vivo detection of pathogenic organisms and their feedback mechanism is much easier as compared to other Nanobiosensors. The efficiency of target binding is increased by concentrating the sensing units in a defined volume. It is because the nanoparticles which are functional in solution perform dual function: 1) They act as binding platform. 2) They act as detecting platform. 3. Nanoscale mechanical biosensors: These are those types of biosensors which make use of mechanical effect. The mode of action of some of their types is as the mass is adsorbed on the surface and these changes are induced in piezoelectric crystals which are detected by changes in resonant frequency produced by piezoelectric crystal. 4. Optofluidic biosensors: This is an optical biosensing technique, which is usually label free. It have many types and each type uses different mechanism for pathogen detection. Such as refractive index have localizes changes and in di-electric field structure the bi-molecular binding is induced. 5. Surface Plasmon resonance biosensing Surface Plasmon are basically the electromagnetic waves which are propagate along the metal/dielectric interfaces. The conditions that are used for the excitation of these optical modes are extremely sensitive to the dielectric environment very near this interface. As a result SPR is one of the most commonly exploited label-free optical biosensing technique in use today.
Author: Hussain, Zahid
Institution: Government College University, Lahore
Language: English
Subfield: Clinical Biochemistry
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6968324310
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