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36 Review of Microbiology and Immunology Section 1 • More sensitive: Double round of amplification yields high quantity of DNA. • More specific: Use of two primers targeting same organisms makes more specific. • Disadvantage: There is more chance of contamination of the PCR tubes, which may lead to false positive results. 4. Multiplex PCR: It uses more than one primer which can detect many DNA sequences of several organisms in one reaction. • Syndromic approach: To diagnose infectious syndrome caused by more than one organism. • Contamination chances of reaction tubes with environmental DNA. Real-time PCR (rt-PCR) Real time PCR, though expensive, but has many advantages over a conventional PCR: • Quantitative, hence can be used for monitoring treatment response, e.g. in HIV or HBV. • Takes less time: As the amplification can be visualized even when the amplification cycle is going on. • Contamination rate is extremely less. • Sensitivity and specificity of rt-PCR assays are extremely higher. Detection of amplification products of real time PCR • Nonspecific methods use SYBR green dye that stains any nucleic acid nonspecifically. • Specific methods use fluorescent labelled DNA probe such as (i) TaqMan or hydrolysis probe, (ii) Molecular beacon and (iii) FRET (Fluorescence Resonance Energy Transfer) probe. MICROBIAL TYPING Microbial typing refers to characterization of an organism beyond its species level. It is used to determine the relatedness between different microbial strains of the same species and thereby helps to (i) investigate outbreaks, (ii) determine the source and routes of infections. Genotypic methods are more reliable and have better reproducibility and discriminative power than phenotypic methods, however they are expensive. Table 1.3.3: Typing methods Phenotypic methods Genotyping methods Bacteriophage typing: Based on their susceptibility to bacteriophages. It is done for • Staphylococcus aureus and SalmonellaTyphi • Vibrio cholerae, Brucella and Corynebacterium diphtheriae Non Amplification based methods 1. Plasmid profile analysis 2. Chromosomal DNA analysis 3. RFLP (Restricted fragment length polymorphism) 4. Ribotyping (RFLP analysis of ribosomal DNA) 5. Pulse field gel electrophoresis (PFGE)-Gold standard method Amplification based methods 1. PCR-RFLP 2. Amplified Fragment Length Polymorphism (AFLP) 3. Sequencing-based methods 4. Microarrays Bacteriocin typing: Based on the ability of a strain to produce particular bacteriocin which inhibits the growth of a set of selected indicator strains. It is done for: • Shigella sonnei (colicin typing) • Klebsiella (klebocin typing), E.coli (colicin typing) • Proteus (proticin typing),Pseudomonas (pyocin typing) Biotyping: Based on different biochemical properties of the organism. It is done for • C.diphtheriae (gravis, intermedius and mitis • Vibrio choleraeO1(classical and El Tor) and Yersinia pestis Antibiogram typing: Based on their resistance pattern to different antimicrobials. It is the most commonly used typing method. Auxotyping: Based on nutritional requirement of the organism (e.g. Gonococcus) Morphotyping: Based on different type of colonies in culture (e.g. Pseudomonas) Serotyping: Based on the antigenic property of an organism. • Streptococcus (Lancefield grouping) • Based on capsular antigen, e.g. pneumococcus, meningococcus and H. influenzae • Based on somatic antigen- E. coli, Shigella, Salmonella and Vibrio cholerae. Bacteriophage typing is done for: • Staphylococcus aureus • SalmonellaTyphi • Vibrio cholerae • Brucella • Corynebacterium diphtheriae Bacteriocin typing is done for: • Shigella sonnei (colicin typing) • Klebsiella (klebocin typing), • E.coli (colicin typing) • Proteus (proticin typing), • Pseudomonas (pyocin typing) Biotyping is done for: • C. diphtheriae • Vibrio cholerae mebooksfree.com