Rapid Microbiological Methods and the PAT Initiative - - BioPharm International

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Rapid Microbiological Methods and the PAT Initiative


BioPharm International


CELL-COMPONENT BASED

  • Biochemical Assays and Physiological Reactions Premise of Technology: Pure culture suspensions are tested with biochemical substrates or subjected to analysis to generate a spectrum. The results are compared to a database of expected results. Comparisons allow the user to identify the microorganism.
  • Endospore Detection Premise of Technology: A major component of the spore case is Ca (dpa). Dipicolinate anions (dpa2-) are only present in bacterial endospores. Ca (dpa) and (dpa2-), when dissolved, are not photoluminescent. It has been shown that Terbium (Tb3+) is able to complex with dpa2- forming a photoluminesecent complex.8
  • Enzyme Linked Immunosorbent Assay (ELISA) Premise of Technology: One can use an antigen-antibody reaction to detect unique microorganisms or cellular components.
  • Fatty Acid Profiles (Fatty Acid Methyl Esters [FAMEs]) Premise of Technology: Fatty acids are present in microorganisms. The fatty acid composition is typically homogeneous within different taxonomic groups. Isolates are grown on a standard media and selected for testing.The testing procedure includes saponification of fatty acids, methylation, and extraction, resulting in FAMEs. FAMEs are measured using gas chromatography. Measurements are then compared to a library of known organisms.
  • Fourier Transformed Infrared Spectroscopy (FTIR) Premise of Technology: An FTIR can generate an infrared spectrum of microorganisms. Patterns are generated and compared to a database of spectra of known microorganisms.
  • Gram Stains Premise of Technology: This technology uses a single solution without fixatives and washes. The method can be used with mixed cultures and results are obtained in a few minutes.
  • Immunological Methods Premise of Technology: Antigen-antibody reaction can be used to detect unique microorganisms or cellular components. These systems are useful for identification and pathogen detection. In some cases, the systems may not be able to distinguish whether the cells detected are viable.6
  • Limulus Amebocyte Lysate Endotoxin Testing (LAL) Premise of Technology: Amebocyte Lysate recovered from horseshoe crabs (Limulus) have similarities in blood coagulation to humans. This similarity has allowed the use of this reagent to detect the presence of bacterial endotoxins. This technology has been available for many years as a replacement for the rabbit pyrogen test. Many systems are available, which have widespread acceptance by regulators.
  • Mass Spectrometry (Matrix-Assisted Laser Desorption-Time Of Flight MALDI-TOF) Premise of Technology: When microbial isolates are heated in a vacuum, the gaseous breakdown products can be analyzed using mass spectrometry. A spectrum is generated. The spectrum is compared to a database of known organisms for identification. The size of the database is important in evaluating the effectiveness of system use. This technology has been used for microbial identifications.
  • RAMAN Spectroscopy Premise of Technology: A RAMAN Spectrophotometer can be used to generate a spectrum unique to the microorganism. Studies performed in clinical settings indicated that identifications could be made with about five hours incubation.

NUCLEIC ACID-BASED

  • Nucleic Acid Probes Premise of Technology: Data available from nucleic acid sequencing are used to select a desired nucleic acid. The desired nucleic acids are extracted, immobilized to a solid phase, and hybridized to a labeled probe.9
  • Polymerase Chain Reaction (PCR) Premise of Technology: PCR works like a copier machine, making "Xerox" copies of nucleic acid fragments. Nucleic acid fragments are amplified using polymerization techniques. This technology is widely used in other sciences such as anthropology and forensics.
  • Ribotyping/Molecular Typing Premise of Technology: This technology utilizes restriction fragment length polymorphisms (RFLPs) of nucleic acids from bacterial genomes. The size-separated RFLPs are hybridized to a ribosomal RNA probe. Digital information is captured, data are extracted and compared to a database of known patterns for identification. Molecular typing is considered the "gold standard" in identification of microorganisms.


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