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 

 

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 basically detect very specific biological molecule interactions and in 21^st 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.

 

 

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.

 

 

 

Cystic fibrosis is a genetic disorder at chromosome no 7, it is due to the occurrence of mutation in CFTR (Cystic Fibrosis Tran membrane Conductance Regulator). Selective media was used for the isolation or detection of bacteria, simple hybridization probe methods, using 16S RFLP (restriction fragment length polymorphism) were used but these techniques failed to isolate and identify unknown bacterial strains. So, new isolation and identification methods had to be used.

 

By using the following method, many strains of pathogenic bacteria were isolated and identified:

Ø  Sample were collected from sputum and then stored at -20 degree Celsius.

Ø  Then all the sputa were inoculated on different agar plates, all of which contained different medium. Then incubated at 37degree Celsius for 48 h.

Ø  Identification of colonies was done by doing Gram-staining, optochin susceptibility test, and catalase test and oxydase activity. Antibiotic resistivity test was also done.

Ø  Amplification and sequencing of 16S rRna was done if our expected identification test was not obtained in accordance to antibiotic resistivity test.

Ø  Then DNA was extracted, leading with gene amplification, then cloning, insertion of amplification and then finally sequencing was done. [5]

 

Genetic sequence features are used for the identification of uncharacterized microorganisms:

Ø   The genetic sequence should be conserved in large number of known organisms.

Ø  Secondly the rate of change should be constant in diverse organisms over the long periods and they should be allowed to interfere the evolutionary distance among wide range of the life forms.

Ø   Thirdly the sequence shouldn’t have been shared by horizontal transmission among the different organisms.

Ø  Fourthly, sequence should be amenable for broad range of amplification or either detection.

 

These criteria are met by the sequence of the small subunit ribosomal RNA or DNA (ssu rDna). The priming sites are provided by the ssu rDna and ssu rRna regions which are highly conserved sites. These priming sites are used for broad-range polymerase chain reaction (PCR) (or RT-PCR). So the previously uncharacterized bacterium, now could be identified, for example, in bacterium16S rDna from an infected site or tissue by the broad range bacterial amplification, leading to sequencing, and the finally to phylogenetic analysis. Broad-range PCR is as a method used for the “pathogen discovery” .It is not limited to only ssu rDna as the target or only to cellular life.

The gene sequence of any phylogenetically reliable family of orthologous among the coherent group of the microorganisms could be targeted. But the point to be kept in mind is that the priming sites should be remained conserved. For example, hantavirus which was newly discovered virus was identified as the cause for the acute pulmonary disease by using the broad-range primers which were directed at a conserved region of a coat protein-encoding genomic segment.

 

 There are also two other independent sequence-based methods which are available for the pathogen discovery.

 It relies upon subtractive hybridization and it is used to isolate the fragments of nucleic acid which are unique to one member. These molecules are amplified selectively by using the linker sequences which had been ligated to all of the fragments which were derived from the infected specimen. Multiple rounds of the amplification and subtraction were required to find the rare fragments. This method of analysis is very cumbersome. RDA was first identified in herpes virus and as a causative agent of Kaposi sarcoma. Any class of microorganisms could be identified by this method but mostly used for viruses.

The other sequence-based method is the one which takes advantage of the host immunologic recognition for an exogenous microbial agent. Immune sera are used for screening any infected specimen. Although this method is time-consuming but it had helped in the identification of an hepatitis C virus in humans. The advantage of Sequence-based methods is that the speed and sensitivity as well as specificity in genotypic characterization of newly approaching molecular biological methods is very high

The additional advantage gained by Consensus PCR is that those sequences are selected which are reliable in the evolutionary relationships.

Limitations for Sequence Based Methods.

·         Clinical specimens cannot be processed.

·         Many microorganisms gained resistance to digestion.

·         Sometimes the sample is heterogenic.

·         PCR inhibitors may be present which cause problem in PCR methods.

Where as many virulence-associated genes and their products could be recognized by their sequence.

These all problems could be over-come by making our procedures more sophisticated. It could be done by detecting many different molecular markers such as specific mRNAs, rRna/

RDna ratio, resistance-encoding loci.

 

Table 1.

 

Newer diagnostic technologies:

1.High-density DNA microarrays

• broad-based pathogen detection and characterization: bacteria, eukaryote, viruses

• virulence-associated gene families

• Comprehensive host gene expression profiles.

2. Improved nucleic acid subtractive methods.

3.  Novel bioassays for toxin activity.

• neurons or myocytes on a chip

 

Table 2

 

Pathogens that may be difficult to detect or identify.

1. Pathogens that establish intimate relationships With the host.

• endosymbionts and intracellular organisms

2. Chimeras: natural versus man-made?

3. “Non pathogens” that acquire virulence-associated Genes.

 

 [6]

This system, the autonomous pathogen detection system (APDS), acts as a biological “smoke alarm” and is targeted for domestic applications in which the public is at high risk of exposure to covert releases of bioagent (such as mass transit, office complexes, and convention centers), and as part of a monitoring network for urban areas and major gatherings. The APDS is completely automated, of­fering aerosol sampling, in-line sample preparation fluidics, multiplex flow cytometer detection and identification assays, and orthogonal, flow-through PCR (nucleic acid) amplifica­tion and detection. For the flow-cytometer subsystem, small “capture” beads 5 μm in diameter are coated with antibodies specific to the target pathogens. The beads are color-coded according to which antibodies they hold. Once the pathogens attach to their respective antibodies, more antibodies (labeled with a fluorescent dye), are added to the mixture. A labeled antibody will stick to its respective pathogen, creating a sort of bead sandwich—antibody, pathogen, and labeled antibody. The beads flow one by one through a flow cytometer, which illuminates each bead in turn with a laser beam. Any bead with labeled antibodies will fluoresce. The system can then identify which agents are present, depending on the color of the capture bead, and have several key advantages over competing technologies:

 

(i) The ability to measure up to 100 different agents and controls in a single sample

(ii) The flex­ibility and ease with which new bead-based assays can be developed and integrated into the system

(iii) Low false-posi­tive and false-negative detection due to the presence of two orthogonal detection methods

(iv)The ability to use the same basic system components for multiple deployment architec­tures

(v) The relatively low cost per assay and minimal consumables.

 

 

Miniaturization of biodetectors into a single, integrated, “lab-on-a-chip” system offers great potential for environ­mental monitoring, which includes improved accuracy, lower power and sample consumption, disposability, and automation. This technology adapts micro fabrication techniques used in semiconductor manufacture to convert experimental and analytical protocols into chip architectures. Already chips are being fabricated with picoliter-size wells and 10-μL-size chambers for sample preparation and detection. The integration of micro fluidic transport, total automation, and materials handling contributes to a major reduction in sys­tem retention and material transfer losses, which increases accuracy and reduces sample size requirements. However, one of the remaining barriers to achieving true miniaturized total analysis systems is the integration of sample pretreat­ment for micro fluidic devices. The challenge is complicated by the complexity and variation in prospective samples and analytes. There is an issue of integration and interfacing the pretreatment operation to the analysis device with which it is coupled and codependent in terms of sample volume, time, and reagent and power consumption. The majority of published work has concentrated on using electro kinetically driven separation schemes to separate and detect analytes of interest. The electro kinetic phenomenon occurs due to the interaction of induced dipole in the bioparticles with electric fields and is used for movement of fluids and other materials through a network of fluid channels. In this case, external pumps or valves are not needed. Precise control of fluid motion and reaction timing is achieved by changing parameters such as the current or voltage. The chip-based capillary electrophoresis system has the capacity to perform the following functions: reagent dispensing, mixing, incuba­tion, reaction, and sample partition and analytes detection. Evidence in the experimental data produced by different organizations has shown that microchip electrophoresis is an effective process for analyzing biological agents at very high speeds and low concentrations. Chip-based capillary electrophoresis technology has many benefits if compared to conventional methods of analysis. For example, the chip can analyze a mixture in seconds where it would take capillary electrophoresis at least 20 min and gel electrophoresis 1 h to do the same analysis.

 

 

The microchip can detect a sample con­centration in the range of 100 pM, which is at least two orders of magnitude greater than conventional capillary electropho­retic analysis. PCR amplification of single DNA template molecules, followed by capillary electrophoretic analysis of the products, has been demonstrated in an integrated micro ­fluidic device. The micro device consists of submicroliter PCR chambers, etched into a glass substrate, which are di­rectly connected to a micro fabricated capillary electrophore­sis system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers, low volume reactor, and low thermal mass. The use of thin-film heaters permits cycle times as fast as 30 s. In operation, an amplified product, labeled with an intercalating fluorescent dye, is directly injected into a gel-filled capillary channel for electrophoretic analysis. This microchip electrophoresis has proved to be quicker, more sensitive, and cheaper than con­ventional techniques. [7]

An exceptional style and design software tool is vital for achievements in analysis progress attempts. AlleleID®, a new application tool, can perform an essential role in reducing the experimental problem as well as pressure and reducing analysis progress along with working expenses.

[8]

AlleleID® is a revolutionary application exclusively developed for interacting with the difficulties of pathogen diagnosis, microbe recognition, and species recognition and taxa discrimination analysis development. AlleleID® begins by straightening sequences applying the use of common ClustalW algorithm, evaluates conserved and species particular regions and then patterns primers and probes to improve and identify only the variety of species of interest from the mixture. [9]

 

 

 

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