[Species Identification of Puparium Samples of Common Sarcosaphagous Flies Based on Molecular Marker Analysis].

OBJECTIVE: To investigate the application value of empty puparia in species identification of common sarcosaphagous flies. METHODS: Fifty-five samples of adult flies and their empty puparia were collected. All the samples were identified as 2 families, 6 genera and 8 species by morphological characteristics. The samples were divided into 3 groups according to their time period between eclosion and our analyses: less than 2 years (n = 23), 2-5 years (n = 20), and more than 5 years (n = 12). The mtDNA of each sample was extracted by CTAB method. The purity and concentration of DNA were tested. PCR products were amplified using two sets of primers. Two sequences of CO I gene (sequence I: 498 bp, sequence II : 841 bp) from each sample were compared to the sequences in GenBank using BLAST for species identification. RESULTS: The mtDNA was extracted successfully from all the samples. DNA concentration of adult chest muscle preserved less than or equal to 5 years and empty puparia preserved less than 2 years ranged from 1.0 to 3.0 µg/µl, and the value of A260/A280 ranged from 1.6 to 1.8. The purity and concentration was lower than 1.6 and 1.0 µg/µl, when the adult chest muscle and empty puparia preserved more than 5 years and 2 years, respectively. DNA concentration of the samples significantly decreased with the prolonged preservation time (P < 0.01). Two sequences of CO I gene was amplified in adult chest muscle and empty puparia which preserved less than 2 years. The success rates of amplification decreased with the prolonged preservation time, especially for the sequence II (P < 0.01). The morphological identification of 8 species did not match exactly with the results based on the COI gene, correct species identification occurred in 6 and 7 species out of 8 based on the two sequences, respectively, and their Max ident value exceeded 97% CONCLUSION: Empty puparium samples can be used to extract mtDNA and identify species.

Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3.

Forensic DNA analysis of sexual assault evidence requires unambiguous differentiation of DNA profiles in mixed samples. To investigate the feasibility of magnetic bead-based separation of sperm from cell mixtures using a monoclonal antibody against MOSPD3 (motile sperm domain-containing protein 3), 30 cell samples were prepared by mixing 10(4) female buccal epithelial cells with sperm cells of varying densities (10(3), 10(4), or 10(5) cells/mL). Western blot and immunofluorescence assays showed that MOSPD3 was detectable on the membrane of sperm cells, but not in buccal epithelial cells. After biotinylated MOSPD3 antibody was incubated successively with the prepared cell mixtures and avidin-coated magnetic beads, microscopic observation revealed that each sperm cell was bound by two or more magnetic beads, in the head, neck, mid-piece, or flagellum. A full single-source short tandem repeat profile could be obtained in 80% of mixed samples containing 10(3) sperm cells/mL and in all samples containing ≥10(4) sperm cells/mL. For dried vaginal swab specimens, the rate of successful detection was 100% in both flocked and cotton swabs preserved for 1 day, 87.5% in flocked swabs and 40% in cotton swabs preserved for 3 days, and 40% in flocked swabs and 16.67% in cotton swabs preserved for 10 days. Our findings suggest that immunomagnetic bead-based separation is potentially a promising alternative to conventional methods for isolating sperm cells from mixed forensic samples.