PCR-RFLP identification of Diptera (Calliphoridae, Muscidae and Sarcophagidae) - A generally applicable method

A simple, rapid method using restriction fragment length polymorphisms (RFLPs) within the internal transcribed spacer (ITS) regions of the ribosomal DNA gene repeat allows identification of insects and other organisms. We used the method to identify the morphologically similar Diptera larvae that are important in forensic entomology for estimating the time and location of death. Polymerase chain reaction (PCR) was used to amplify a region from the 18S to the 28S rRNA genes. The ITS1 and ITS2 regions provided variation between species and homogeneity within species, with the exception of Cochliomya macellaria. Combinations of the restriction enzymes DdeI, HinfI and Sau3AI provided diagnostic bands for identification of the ten species from three families of Diptera (Calliphoridae, Muscidae and Sarcophagidae).