Info

outbreak strain

Different

>3

>6

Test isolate unrelated to the outbreak

*Compared with the outbreak strain

*Compared with the outbreak strain performing PFGE to type strains is the time involved to perform the assay. It can take 2-3 days to complete one analysis.91

Restriction Fragment Length Polymorphism Analysis

Restriction fragment length polymorphism (RFLP) analysis by Southern blot is the same technique first used to identify and investigate human genes (see Chapter 6). This method is performed by 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. Gene-specific probes are used to identify or subtype microorganisms such as P. aeruginosa in cystic fibrosis patients and nosocomial L. pneumophila infections.96,97

Insertion elements are segments of DNA that can move independently throughout the genome and insert themselves in multiple locations. Strains can be typed based on how many insertions are present and where they are located. Strains that are the same will have the same number and location of elements. For strain typing of M. tuberculosis by this method, the probe is complementary to IS6110 and will bind to restriction fragments on the membrane that contain the insertion sequence, resulting in a series of bands that can be easily analyzed and com-pared.98 This is the preferred method for typing M. tuberculosis isolates using the IS6110 insertion element.99

The gene targets selected for this procedure depend on the organism under investigation and which genes will be most informative. Ribosomal RNA genes are highly informative over a range of microorganisms, which is the basis for a modification of the RFLP procedure called ribotyping. For this method, probes target the 16S and 23S rRNA genes. RFLP and ribotyping have been applied in industrial as well as clinical microbiology.100,101

RFLP can be investigated more rapidly using PCR amplification with gene-specific primers (locus-specific RFLP, or PCR-RFLP). This method requires amplification of specific regions by PCR (see Chapters 7 and 9 for more details on this PCR and PCR-RFLP). The ampli-cons are then cut with restriction enzymes, yielding bands of informative size. The advantage of this procedure, in addition to its speed, is the simple band patterns, which are much easier to interpret. Although the method is limited by the sequences that can be amplified and differentiated through restriction enzyme digestion, proper gene selection provides a highly reproducible and discriminatory test.102 In one study, an 820-bp amplified fragment of the ureC gene from Helicobacter pylori digested with Sau3A and Hhal yielded 14 different Sau3A patterns and 15 different Hhal patterns. These patterns were informative as to antibiotic sensitivity of the various types to clar-ithromycin or clarithromycin-omeprazole dual therapy.103

Arbitrarily Primed PCR

Arbitrarily primed PCR, or random amplified polymorphic DNA (RAPD) assay, is a modified PCR using 10-base-long oligonucleotides of random sequences to prime DNA amplification all over the genome.104,105 The gel pattern of amplicons produced is characteristic of a given organism. If two organisms have the same pattern, they are considered the same type. If the patterns differ, they are different types. The RAPD assay is relatively rapid and inexpensive; however, producing consistent results may be technically demanding. Accurate interpretation of RAPD raw data requires that the procedure con-

Detection and Identification of Microorganisms Chapter 12 287 (A)

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