Molecular biology tools used for epidemiological investigations
B.V. Sunil Kumar
ICAR-National Institute on Foot and Mouth Disease, Bhubaneswar-752050
Corresponding author: drbvskumar@gmail.com
Abstract
Epidemiology aims to review the distribution, determinants, and causes of health related problems to stop and control them effectively. Despite the beneficial effects of traditional epidemiological studies on the prevention and control of the many diseases, there remain important questions on the biological mechanisms underlying either infectious or chronic diseases. Molecular epidemiological studies have an excellent potential to deal with these issues. Molecular epidemiology may be a branch of epidemiology developed by merging molecular biology into epidemiological studies. It involves high throughput laboratory techniques, applicable to large sample sizes that enabled epidemiologists to conduct large scale epidemiological studies at molecular levels.
Key words: Epidemiology, Molecular biology, Laboratory techniques, Investigation
Introduction
Over the previous couple of decades, epidemiology has evolved into a dynamic science involving the biological sciences, mathematics, and computing to review the diseases or health related events within the population along side the factors that determine their occurrence. Within the event of a disease outbreak, epidemiologists/specialists of infectious diseases working within the veterinary field sometimes find themselves lacking the accurate diagnosis procedure and control the spread of the pathogen. Surveillance and diagnosis with molecular and immunological tools like Immunoassays, PCR, Real-time PCR, flow cytometry, microscopy, recombinant deoxyribonucleic acid technology, gene cloning, and expression, western blot analysis, Restriction analysis, and AFLP, etc
Methods used to study biological variations in molecular epidemiology
All laboratory-based methods used to type microorganisms (typing systems) fall into phenotypic and genotypic methods. Most of the “conventional” laboratory typing methods fall into phenotypic methods, which are based on the detection of phenotypes or characteristics expressed by an organism. Strain typing methods based on genotypes rely on the analysis of nucleic acid contents and gene sequence polymorphisms (chromosomal DNA, extrachromosomal DNA, and RNA).
Molecular techniques in epidemiologic studies:
Phenotyping Methods
The first step in data stratification as applied to bacterial infectious disease agents is differentiating pathogens from normal commensal or saprophytic bacterial organisms. Commensal organisms reside, metabolize, and replicate in a healthy, immunocompetent host without causing disease. They colonize the skin, mouth, nasopharynx, gastrointestinal tract, vaginal mucosa, and others. This initial step separates organisms into species. Microorganism characteristics used to define species include growth, morphologic, biochemical, serologic, functional, and physiologic properties
Differentiation by Growth and Morphologic Characteristics
Differences in the nutritional requirements of bacterial organisms determine their growth characteristics. They can be classified according to whether they grow at all or the way they grow on solid agar culture plates or in liquid media. Morphologically, they may be differentiated by their colony shape, color, texture, smell, or whether they swarm on the plate. They are also differentiated by the way they stain (e.g., Gram stain, acid-fast stain) as observed under a light microscope, or their shape and size as observed under an electron microscope.
Typing Based on Biochemical Characteristics
Biochemical tests wont to differentiate bacteria are supported the organism’s metabolic activities. Strains are differentiated by a panel of biochemical tests group into biotypes, and therefore the tests are thus mentioned as biotyping tests. The tests depend upon an organism’s metabolic activities, which are sometimes subject to minor changes in growth condition (e.g., nutrient concentration, pH, and temperature) used, and hence the knowledge that’s generated might not always be reliable for epidemiologic studies.
Typing by Serologic Characteristics
The frequently used phenotyping method is predicated on a characteristic antigen specific serologic (antibody) response induced by an invading organism during a mammalian host. Bacterial antigens that induce antibody response used for serologic classification include proteins and polysaccharides. the precise sero grouping or serotyping assays include bacterial blood test with a panel of antigen specific antisera, enzyme linked immune sorbent assay (ELISA), latex particle agglutination, Western blot (or immune blot) analysis, or a modification of those tests. One limitation of serotyping is that different strains from an equivalent species or maybe strains from different species could carry cross reacting antigens, which may end in false positive tests.
Typing by Functional or Physiologic Characteristics
Other ways to differentiate bacterial isolates involve examination of their response to specific manipulations. They include identifying differences in susceptibility to antimicrobial agents (antibiograms), lysis by or susceptibility to bacteriophages (phage typing), patterns of association with tissue culture cells (cell culture assays), toxigenicity, survival under in vitro or in vivo stress, and metabolic enzyme expressions (multilocus enzyme electrophoresis, or MLEE).
Genotyping Methods
Genotyping or macromolecule based typing methods are ultimately supported the macromolecule sequence differences within the chromosomal or extra chromosomal nucleic acid contents. Three basic analytical procedures (a) hybridization, (b) gel electrophoresis, and (c) macromolecule sequencing. This enables for the utilization of common equipment and standard reagents to research many various sorts of infectious agents. Furthermore, genotypic characterizations of pathogens facilitate standard information storage and data analyses, interpretation, and communication, which are all amenable to computer assisted manipulations.
In bacteria, genotyping analyses are supported the comparison of two sorts of DNA: the genome and therefore the extra chromosomal DNA. Typing systems supported the analysis of the genome fall under those designed to match differences supported nucleotide changes that occur in parts of the genome (micro diversity) or the whole genome (macro diversity). These typing methods, had to be developed in situ of direct whole genome DNA sequence analysis, which, despite recent advances within the sequencing technology, remains cumbersome and expensive for bacterial organisms (although not the case with viruses).
Extrachromosomal DNA Genotyping Methods
Plasmids are covalently closed circular DNA that carries genes which will encode characteristics like antibiotic resistance, virulence factors, or other functions that provide a selective advantage to the organism but aren’t essential for its growth or survival. In fungi or protozoa, extra chromosomal elements used for typing may include mitochondria and kinetoplasts.
Plasmid based typing is an example of an electrophoresis based strain typing procedure. It compares differences in relative molecular mass and the amount of various plasmids a bacterium may carry. Nucleic acids and proteins are charged. Plasmids, thus, are often resolved electrophoretically in an agarose gel matrix consistent with relative molecular mass. That is, the separation of those molecules depends on their drag through the gel matrix as they migrate toward the charge pole. Large relative molecular mass plasmids migrate more slowly within the gel matrix than low relative molecular mass plasmids. Thus, counting on the amount of various relative molecular mass plasmids, each strain represented by one electrophoretic lane will generate a banding pattern, a “fingerprint”, or a profile, which will then be compared across multiple lanes.
Genome-Based Typing Methods
In genome based typing methods, the whole genomic content of a microorganism is compared among different strains (macro diversity analysis). There are three basic genome based typing methods utilized in epidemiologic investigations, which are supported (1) allelic differences that occur within restriction nuclease recognition sites during a genome, (2) comparison of multiple nucleotide sequences of whole genomes (called comparative genomics), and (3) whole genome hybridization patterns, obtained from arrays of thousands of distinct short DNA fragments representing the entire genome fixed onto a glass slide or nitrocellulose filter (“gene chip” or microarray).
Restriction Endonuclease Analysis (REA)
Bacterial organisms express enzymes called restriction endonucleases that recognize unique sequences of several nucleotides long and cut the DNA molecule at a selected position. They’re called restriction enzymes because bacteria use these enzymes to eliminate (restrict) foreign DNA accidentally introduced into them. Thus one enzyme may generate distinct sets of DNA fragments from two different strains if the strains have a sequence difference at the corresponding restriction sites. Comparison of electrophoretic patterns supported the amount and differences in relative molecular mass cut DNA fragments is named restriction nuclease analysis of fragment length polymorphism (RFLP) analysis.
Southern Blot Hybridization
Southern blot hybridization analysis, facilitates detection of a specific DNA fragment of interest among many other DNA fragments generated by REA. DNA fragments separated in an agarose gel are first transferred onto a bit of nitrocellulose or nylon membrane. The membrane is then exposed to a bit of single stranded DNA (probe) labeled with a molecule that facilitates visual detection of a specific target DNA fragment. The probe will specifically bind (hybridize) to its complementary DNA sequence embedded within the membrane.
Pulsed Field Gel Electrophoresis (PFGE)
PFGE was developed during which the orientation of the electrical field across the gel is modified (pulsed) periodically. This enables the DNA fragments to maneuver in several directions, releasing them from the stuck position. The DNA fragment will thus take a path of least effort alongside its gel migration toward the charge pole. The main advantage of PFGE is that virtually all genome containing organisms are typeable by this method. One major limitation of PFGE is that it’s technically demanding, requiring an upscale electrophoresis apparatus. Thus, this typing system is currently used only by reference or research laboratories.
Whole-Genome Sequence Comparison
The reduced cost and skill to rapidly sequence large pieces of DNA engendered a replacement discipline called comparative genomics. With viruses, whose genomes are considerably smaller than those of bacteria, comparative genomics is already applied to directly genotype clinical isolates. That is, many genomes of viral strains belonging to at least one species can now be compared rapidly to every other. For instance, it had been the comparative genomics analyses that enabled investigators to conclude that the agent of the severe acute respiratory syndrome (SARS) belonged to a gaggle of coronaviruses. With bacteria, due to the massive genome, such comparisons are still impossible for an outsized number of strains. That is, many genomes of one bacterial species can’t be compared because they need not been sequenced.
PCR-Based Methods
Improper storage of bacterial isolates can also cause them to die, during which case genotyping by any of the above methods won’t be possible. Polymerase chain reaction (PCR) based tests can overcome these limitations. The PCR technology may be a powerful and straightforward biology tool, which is now applied during a sort of ways to genotype microorganisms in taxonomic also as epidemiologic studies. PCR is merely one among several methods called macromolecule amplification tests (NAAT) wont to make multiple copies of a target DNA or RNA of an organism or cells in vitro without culturing the organism or cells. PCR is probably the foremost frequently used NAAT and therefore the commonest method applied to subtype microorganisms.
PCR based genotyping methods that compare electrophoretic bands are classified into two general categories: (1) those supported relative molecular mass polymorphism of one amplified product and (2) people who display band patterns (fingerprints) from multiple amplified products. The main advantage is that this PCR based method is considerably simpler than the traditional methods.
PCR Restriction Enzyme Analysis
The same principles behind REA, RFLP, and PFGE analyses of genomes of pathogens are often applied to PCR products. A PCR product, if it’s sufficiently large, 1000–2000bp, are often digested with an endonuclease, and therefore the generated DNA fragments are often resolved by gel electrophoresis and visualized. If such amplified products contain nucleotide changes at the restriction sites, varied electrophoretic band patterns will result from the digested PCR products. One drawback of this PCR-REA (or PCR- RFLP analysis) method is that a part of the target sequence to be amplified has got to be known before time.
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