Identification of forensically important blowflies in Thailand based on the second internal transcribed spacer region of ribosomal DNA and the NADH dehydrogenase subunit 5 gene.

Abstract

Problem/Background : Estimation of postmortem interval (PMI) is essential for supporting justice especially in legal cases. At early stage of death, the PMI can be evaluated using data from physical changes of the corpses. In case of the corpses that have died more than 48 hours, the physical change may be less accurate for prediction. The species identification of blowflies, early found on corpses, as well as the specific developmental stage are required for PMI estimation. However, it is not yet practical for morphological identification. To surpass these problems, DNAbased identification is preferentially applied.

Objective : To demonstrate the application of the second internal transcribed spacer (ITS2) region and the NADH dehydrogenase subunit 5 (ND5) gene for differentiation of forensically important blowflies in Thailand.

Design : Descriptive study

Setting : Department of Parasitology, Faculty of Medicine, Chulalongkorn University

Methods : Thirty-one samples of blowflies DNA from previous study were amplified for ITS2 and ND5 regions via PCR technique. Then, the PCR products were cloned using the pGEM ®-T vector and transformed into Escherichia coli strain DH5α competent cell. Three clones were sequenced from each sample. The DNA sequences were compared with GenBank database. For PCRRFLP analysis, the PCR products for ITS2 and ND5 were separately digested with restriction endonucleases DraI and VspI for ND5 gene, respectively. Also, phylogenetic tree was constructed by neighbor-joining method.

Results : Sequencing reactions revealed that PCR yielded the ITS2 product size of approximately 400 bp and the ND5 product size of 437 bp. Results from PCR-RFLP analysis could separate blowflies into 4 patterns. Additionally, sequence analysis showed no significant intraspecific divergence in the same species. Phylogenetic analysis by neighbor-joining (NJ) method indicated the close relationship among them.

Conclusions : These molecular methods serve as the promising tools for molecular identification of these common blowfly species especially in case of morphological difficulties.