Microbiology

The terminal restriction fragment length polymorphism (T-RFLP) method to identify pathogenic bacteria is described in the following citation:

Nilsson, W. B. and M. S. Strom. 2002. Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16s rRNA genes. Diseases of Aquatic Organisms 48:175-185.

To download the T-RFLP Excel Spreadsheet Library that is described in the publication, use one of the following links:

Zipped format (Windows) | Binhexed format (Macintosh)

The RFLP Library contains a tabulation of expected terminal RFLP fragment sizes predicted from GenBank entries for the 16S rRNA gene of a number of common bacterial pathogens of fish (see the publication listed above). Columns Q-AB contain the terminal fragment sizes expected from endonuclease digestions based upon the GenBank Accession submission specified in column P. The user simply enters observed fragment lengths (one blue fragment and one yellow fragment for each digestion) in cells B4:M4. If an observed fragment length from a given digest matches a library entry prediction for a submission in columns Q-AB (�10 bases), the routine will enter a "1" in the appropriate row of columns B:M. Otherwise, "0" is entered. Column N is a tabulation of the "Score", i.e., the sum of sum of the number of observed fragment lengths that match the predicted lengths of a given GenBank submission. A score of 12 indicates that all entries match (�10 bases) those predicted by the submission given in column P and provides a presumptive identification of organism. In some cases, more than one closely related species may give a "perfect" match and further testing (e.g., PCR using species specific primers) will be required. All cells but B4:M4 are protected and cannot be changed.

This routine is available free of charge to the public. Send questions to william.nilsson@noaa.gov. To unprotect the spreadsheet routines, the password is "sophie". This library will be updated periodically. Suggestions concerning the content or organization of this library are appreciated. This library is based upon a design by Jeff Engebretson.