Guide to the Application of Genotyping to Tuberculosis Prevention and Control

CDC Tuberculosis Genotyping Laboratory Procedures

IS6110-based RFLP Analysis

TB programs can request the genotyping laboratories to perform IS6110-based RFLP analysis on isolates that match by both spoligotyping and MIRU analysis. See Chapter 5, Developing a Tuberculosis Genotyping Program, for a description of when to request RFLP analysis. The request for RFLP analysis must list the cluster number and the specific isolates in that cluster to be typed. If additional isolates in the cluster are identified at a later time, the program can request IS6110-based RFLP analysis of the additional isolates.

Only those isolates listed in the request will be typed by RFLP analysis. RFLP patterns will be analyzed for matches or non-matches among the requested isolates. The genotyping laboratory will not search the entire database for possible RFLP matches outside the requested cluster. Each isolate typed by RFLP analysis will be assigned an RFLP pattern designation number. If one of the requested isolates demonstrates an RFLP pattern that does not match the patterns of any of the other requested isolates, the isolate will be assigned a new RFLP pattern designation number, which will be the genotyping laboratory’s accession number for that isolate plus the band number for the RFLP pattern. For isolates with matching RFLP patterns, the lowest accession number in that cluster will be assigned to all isolates. Again, the RFLP pattern designation number applies only to isolates within a specific PCR cluster (i.e., the same spoligotype and MIRU type from the same TB program). Unfortunately, this may result in two isolates having the same RFLP pattern but different RFLP pattern designation numbers.