Molecular typing by DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE)
|Lab name||Nosocomial Infections Laboratory|
|Lab location||Kenepuru Science Centre, Porirua|
|Test name||Molecular typing by DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE)|
|Requested for surveillance||no|
|Routine turnaround time||15 working days for reference specimens|
|Culture requirements||Pure culture|
|Media requirements||A slope of any medium that supports the growth of the organism|
|Pre-transport requirements||Ambient temperature|
|Transport requirements||Ambient temperature|
|Unacceptable sample types||Clinical specimens will not be accepted. We will also not type single isolates that are unconnected to any situation.|
|How results reported||Report will state result as either distinct, indistinguishable or related to other isolates or a reference profile|
Macrorestriction analysis uses pulsed-field gel electrophoresis to separate large fragments of chromosomal DNA generated by restriction digestion with rare-cutting enzymes.
All isolates referred are retained in long-term storage.
Reasons for investigation
Typing of isolates is appropriate when the identification of similarities or differences between isolates may assist in the investigation of patterns of infection, common-source outbreaks, and routes of transmission of infection.
Restriction profiles are analysed using BioNumerics software to determine the percentage similarity between profiles and to produce a dendrogram.
In addition, restriction profiles, and the short-term epidemiologic relatedness of isolates, may be interpreted by determining the band differences between profiles as follows:
Restriction profiles have the same number of bands of the same size. Epidemiologically, indistinguishable isolates are considered to represent the same strain.
Closely related isolates
2 to 3 band difference between profiles (equates to the DNA of the isolates being different at one site).
Possibly related isolates
4 to 6 band difference between profiles (equates to the DNA of the isolates being different at two independent sites).
Profiles differ by 7 or more bands (equates to the DNA of the isolates being different at three or more independent sites). Epidemiologically, distinct isolates are considered unrelated.
System of DNA restriction pattern designation for outbreak strains
The designation includes the first letter (in upper case) of the genus name and the first letter (in lower case) of the species name, followed by an upper case letter which is assigned sequentially to the DNA restriction pattern of each new outbreak strain of the organism, eg Profiles SaA, SaB, SaC, etc for outbreak strains of Staphylococcus aureus and Profiles SmA, SmB, SmC, etc for outbreak strains of Serratia marcescens.