Activity NO.2 ( Molecular Basis OF Detection) PDF

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Navas, Kimberly S. MODX 311 LEC BSMLS-3B-4 MS. SANDRA LUTOVA, RMTACTIVITY NO (MOLECULAR BASIS OFDETECTION)1. What are the traditional basis of detection and identification of pathogenic microorganisms. Explain each.Microbial cultivation, immunological (ex. antibody-based) assays, and nucleic acid de...

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Navas, Kimberly S. BSMLS-3B-4

MODX 311 LEC MS. SANDRA LUTOVA, RMT

ACTIVITY NO.2 (MOLECULAR BASIS OF DETECTION)

1. What are the traditional basis of detection and identification of pathogenic microorganisms. Explain each. Microbial cultivation, immunological (ex. antibody-based) assays, and nucleic acid detection schemes—particularly amplification methods like PCR—are all traditional methodologies for microbial detection and identification. Microbial cultivation, immunological (e.g., antibody-based) assays, and nucleic acid detection schemes— particularly amplification methods like PCR—are all traditional methodologies for microbial detection and identification. Cultivation is the most extensively utilized method in laboratories, clinics, and healthcare institutions around the world, particularly in developing countries, and is thus the most widely used microbial detection platform for international surveillance. Despite being sluggish, restricted in sensitivity for some therapeutically relevant microorganisms, and technologically primitive, cultivation allows for the most rapid examination of complex microbial phenotypes (behaviors) such as drug resistance. Solid-phase immunological tests, such as dipsticks and optical immunoassays, have found a role in the clinical context, although their efficacy has only been proved in a few infectious disease conditions. PCR is the most widely used method for microbial nucleic acid detection; other signal and target amplification techniques for nucleic acid detection, such as ligase chain reaction, have generated more limited commercial markets. Polymerase chain reaction assays have been routinely used for rapid detection, identification and differentiation of foodborne pathogens. They have been used in areas such as DNA cloning, diagnosis of hereditary and infectious diseases, identification of genetic fingerprints, and detection and diagnosis of infectious diseases. Polymerase chain reaction technique plays an important role in the identification of typical bacterial strains that exist in viable but nonculturable coccoid forms

For Bacteria, Laboratory detection and identification of these organisms by nonmolecular methods often lack sensitivity and/or are time-consuming. Primer and probe ASR for B. pertussis and Bordetella parapertussis detection by qPCR targeting IS 481 and IS 1001, respectively, have been available. 18 PCR tests for Legionella have targeted the macrophage infectivity potentiator (mip) gene and 16S and 5S rRNA genes. For Urogenital Tract Pathogens, nucleic acid amplification assays have the advantages of being rapid, and testing can be batched and automated, resulting in further savings for the laboratory, a nonamplification-based nucleic acid hybridization method that detected the rRNA with an acridinium-labeled single-stranded DNA probe. For Viruses, The classical method for detection and identification of viruses in body fluids is tissue or cell culture. Monolayers of host cells are grown in vitro, the patient’ s specimen is inoculated onto the cells, and changes in the cells due to viral infection, called cytopathic effect, are observed microscopically by the technologist. For Fungi, Traditional methods of identification by phenotype have become more difficult with the expanding diversity of organisms. Fungal infections are most often diagnosed by direct staining methods and isolation of the causative agent in culture. For Parasites, nucleic acid template preparation from oocysts and spores of protozoan parasites is complicated by the nature of the organism and its resistance to disruption and lysis. Parasites found in complex matrices such as stool samples present a further difficulty due to inhibition of PCR and other enzymatic assay. PCR assays have been developed for the detection of Trypanosomes, Plasmodia, Toxoplasma, Entamoebae, and Cryptosporidium in hosts and water sources. Multiplex qPCR methods can also measure infection rates and organism loads in hosts and nonhuman vectors (carriers of infection). In-house-developed real-time PCR methods are used in clinical laboratories for identification of Babesia, Trichomonas microti in blood and from ticks ; Encephalitozoon species microsporidia, and Trichomonas vaginalis. multiplex real-time PCR assays for detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum simultaneously or E. histolytica, Giardia intestinalis, and Cryptosporidium spp. simultaneously in stool samples. Plasmid analysis involves isolation and restriction mapping of bacterial plasmids. The same bacterial strain can have different plasmids carrying different phenotypes or resistance patterns. Plasmids are distinguished by gel electrophoresis patterns of fragments generated when cut with the appropriate enzymes.  Pulsed-Field Gel Electrophoresis Most molecular epidemiological tests are performed using PFGE, which can identify organisms with larger genomes or multiple chromosomes. For PFGE analysis, the DNA is digested with restriction enzymes that cut infrequently within the genomic sequences. The resulting large fragments (hundreds of thousands of base pairs) are resolved by PFGE.  Restriction Fragment Length Polymorphism Analysis RFLP analysis by Southern blot is the same technique first used to identify and investigate human genes.

involves cutting DNA with restriction enzymes, resolving the resulting fragments by gel electrophoresis, and then transferring the separated fragments to a membrane for probing with a specific probe.  Arbitrarily primed PCR, or random amplified polymorphic DNA (RAPD) assay, is a modified PCR using short (e.g., 10-base-long) oligonucleotides of random 2. What are the molecular basis of detection and identification of pathogenic microorganisms. Explain each. Target detection is accomplished by a variety of methods, including agarose gel electrophoresis, amplification methods (PCR, TMA, loop-mediated isothermal amplification [LAMP]), sequencing, immunoassays, western blots, and mass spectrometry. Real-time PCR, or quantitative PCR (qPCR), is used frequently for the detection of infectious agents because it provides a sensitive, safe closed-tube assay with quantitative information not available from conventional PCR or other Phenotypic Methods of Identification They are called the 5 ‘I’s: inoculation, incubation, isolation, inspection and identification.  Inoculation: to culture microorganisms a tiny sample (inoculum) is introduced into medium (inoculation).  Isolation involves the separating one species from another.  Incubation: once the media is inoculated it is incubated which means putting the culture in a controlled environment (incubation) to allow for multiplication.  After incubation the organisms are inspected and identified phenotypically, immunologically or genetically.  Microscopic Morphology include a combination of cell shape, size, Gram stain, acid fast reaction, special structures e.g. endospores, granule and capsule can be used to give an initial putative identification

Biochemical Tests The microbe is cultured in a media with a special substrate and tested for an end product. Prominent biochemical tests include carbohydrate fermentation, acid or gas production and the hydrolysis of gelatin or starch. Many of these test in rapid miniaturized system that can detect for 23 characteristics in small cups called Rapid test.

The info from the rapid test are input into a computer to help in identification of the organisms.

Rapid Tests a biochemical system for the identification of Enterobacteriaceae and other Gram negative bacteria.  It consist of plastic strips with 20 μl of dehydrated biochemical substrates used to detect biochemical characteristics.  The biochemical substrates are inoculated with pure cultures and suspended in physiological saline.  After 5 hrs-overnight the 20 tests are converted to 7-9 digital profile. Flow cytometry (FCM) allows single- or multiple- microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometry parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way Genotypic methods of microbe identification include the use of: Currently many PCR tests employ real time PCR. This involves the use of fluorescent primers. The PCR machine monitors the incorporation of the primers and display an amplification plot which can be viewed continuously thru the PCR cycle. Real time PCR yields immediate results. Another application of PCR is RT-PCR (reverse trancriptase PCR). During RT-PCR an RNA template is used to generate cDNA and from this dsDNA is generated. The enzyme used is reverse transciptase. RT-PCR is used to detect for HIV and to monitor the progress of the disease. RAPD-PCR Random amplified polymorphic DNA PCR uses a random primer (10-mer) to generate a DNA profile. Computer and Bacteria Identification Computers improve the efficiency of the lab operations and increase the speed and clarity with which results can be reported. Computers are also important for the result entry, analysis and preparation

3. Compare the traditional and molecular basis of detection and identification of pathogenic microorganisms.

Traditional detection and identification of pathogenic microorganisms

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Growth of fastidious pathogens – Delay in cultivation and takes more time Non-culturability of certain organisms Maintenance of viability Hazardous to propagate in laboratory Technical Expertise & Cost

Molecular basis of detection and identification of pathogenic microorganisms  The biological technique in which macromolecules like nucleic acid and proteins are used – known as Molecular technique.  Faster and simpler  Increased sensitivity and specificity.  Better adapted to instrumentation-decrease need for manpower.  Identification of epidemiologically important strain....