[Identification of sarcosaphagous Calliphorid flies by analyzing the sequence of 16S rDNA].

OBJECTIVE: To explore the application of a 289bp fragment of the 16S rDNA gene to identify various species of sarcosaphagous Calliphorid flies. METHODS: Twenty-six Calliphorid flies were collected from 14 Chinese provinces. All specimens were properly assigned into three genera and six species. The DNA of the pectoralis was extracted using CTAB method. Then PCR amplification was done for the 289 bp fragment of the 16S rDNA gene. The PCR products were then purified and sequenced, and the obtained sequences were uploaded to GenBank. The phylogenetic tree was built by the neighbor-joining method and intraspecific and interspecific divergences were calculated by sequence analysis. RESULTS: The above 26 sarcosaphagous flies could be well clustered according to different genera and species. The evolutional intraspecific values were all zero, the evolutional interspecific variations varied from 0.3% to 6.5%. CONCLUSION: The 289 bp fragment of the 16S rDNA of sarcosaphagous flies can be effectively used to identify most of the flies at species level. This method appears to be fast and low dissipative, which might be used to estimate postmortem interval by sarcosaphagous flies.

[Recombinant plasmid ZLW/pEGFP-C2 transfection into schistosomula of Schistosoma japonicum].

OBJECTIVE: To study the efficiency of ZLW/pEGFP-C2 plasmid transfection into Schistosoma japonicum schistosomula and observe its in vitro effect of anti-schistosomula. METHODS: Recombinant plasmid ZLW/pEGFP-C2 was transfected into mechanically transformed schistosomula by immersion in 0.75% DMSO and high concentration of plasmid. Enhanced green fluorescent protein (EGFP) transfected cells were observed under inverted fluorescence microscope. At 48 hours after culture, total RNA and proteins from transfected schistosomula were extracted, and the presence of the transgenes (ZLW and EGFP) in schistosomula were confirmed by RT-PCR and Western blotting. At 24, 48, 72, and 96 hours after transfection, the schistosomula were counted by light microscope with methylene blue staining. pEGFP-C2 empty plasmid group and TBS group served as controls. RESULTS: The transfection rate was about 10%. The fluorescence of ZLW/EGFP protein was mainly localized in the tegument of the worms, especially abundant around oral sucker and ventral sucker. The expected size of 259 bp fragment was successfully amplified by RT-PCR and confirmed by DNA sequencing. Western blotting analysis showed that ZLW/EGFP was expressed in schistosomula. No statistically significant difference was established for schistosomula mortality among ZLW/pEGFP-C2 group (14.0%, 48.8%), pEGFP-C2 group (15.9%, 45.7%) and TBS group (16.9%, 50.3%) at 24 and 48 hours after transfection (P > 0.05). At 72 hours after transfection the mortality rate of ZLW/pEGFP-C2 group (92.7%) was significantly higher than that of pEGFP-C2 group (73.2%) (P < 0.01), and after 96 h the mortality in ZLW/pEGFP-C2 group increased to 100%. CONCLUSION: ZLW/pEGFP-C2 plasmid has been introduced into juvenile S. japonicum by immersion in 0.75% DMSO and high concentration of plasmid, and was expressed in the parasite.

[Succession of sarcosaphagous insects at summer and autumn in Shijiazhuang area].

OBJECTIVE: To study the succession of sarcosaphagous insects and their regular activity on carcass in Shijiazhuang area. METHODS: Nine rabbits were sacrificed and placed at the same site during June to September in 2007-2009. The common species of sarcosaphagous insects were observed. RESULTS: Nine main species could be identified belonging to 3 families and 4 genera from Diptera, including Musca domestica (Linnaeus), Muscina stabulans (Fall én), Hydrotaea (Ophyra) capensis (Wiedemann), Hydrotaea (Ophyra) spinigera (Stein), Lucilia sericata (Meigen), Chrysomya megacephala (Fabricius), Boerttcherisca Peregrina (Robineau-Desvoidy), Parasarcophaga crassipalpi (Macquart) and Helicophagella melanura (Meigen). Eleven main species belonging to 4 families from Coleoptera include Nicrophorus concolor (Kraatz), Silpha carinata(Herbst), Nicrophorus fossor (Eneshas), Ptomascopus morio (Kraatz), Eusilpha bicolor (Fairmaire), Scarabaeus rugosus (Hausmann), Harpalus rufipes (DeGeer), Dolichus halensis (Schaller), Goncephalum pusillum (Fabricius), Cafius seminitens (Horn) and Aleochara pacifica (Casey). Two main species from 2 families were Tetramorium caespitum (Linnaeus) and Vespa velutina(Lepeletier). CONCLUSION: It is evident that the succession of sarcosaphagous flies in Shijiazhuang with its unique geographical features. It may be used for estimating postmortem interval in Shijiazhuang area.

[Screening and characterization of peptides specifically binding to the schistosomulum tegument of Schistosoma japonicum].

OBJECTIVE: To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum tegument of Schistosoma japonicum. METHODS: A 12 phage-display peptide library was screened with the S. japonicum schistosomula as the target cells for biopanning by degrees, positive clones picked randomly were deduced by DNA sequencing. According the sequence seeing result, immunohistochemical staining was performed to determine the specificity of the phages to the tegument. To test their targeting efficacy, the interested phage clones were infused back to the mice infected with S. japonicum, mice were sacrificed 2.5 hours later, and the phage distribution in the liver and the tegument of schistosomula was appraised, respectively. RESULTS: After 3 rounds of biopanning, the phage recovery rate increased from 0.77 x 10(-8) to 0.75 x 10(-5), indicating that the phage library was successfully enriched in the tegument of schistosomula. Seventy-five percent (15/20) of the analyzed sequences were identical with a sequence of QHPRIRKOOOOO. The immunohistochemical stainings showed this sequence specifically binding to the tegument. In vivo titering displayed that this sequence selectively targeted the tegument. CONCLUSION: The peptide of QHPRIRKOOOOO specifically binds to the schistosomulum tegument.

[Comparison of mtDNA extraction from different parts of sarcosaphagous insects].

OBJECTIVE: To explore mitochondrial DNA (mtDNA) extraction effects of different parts from sarcosaphagous insects using improved cetyltriethylammnonium bromide (CTAB) method. METHODS: Thirteen Lucilia sericata (Meigen) and 13 Nicrophorus fossor (Erichson) were collected from the corpses of rabbits placed on the outdoor lawn in Huhehot district. Four parts (head, chest muscle, legs and wings) of insect were collected, and the mtDNA of all samples were extracted using CTAB method. The purity and concentration were tested using protein and nucleic acid spectrophotometry. The integrity of the extracted mtDNA and PCR products were checked by agarose gel electrophoresis. The PCR products were sequenced and the obtained sequences were imputed into GenBank for comparison. RESULTS: mtDNA were successfully extracted from 10 head samples, 6 legs samples, 4 wing samples and 13 chest muscle samples of the Lucilia sericata (Meigen). Also, mtDNA were successfully extracted from 5 head samples, 8 legs samples, 3 wing samples and 13 chest muscle samples of the Nicrophorus fossor (Erichson). CONCLUSION: mtDNA can be obtained from chest muscle and other parts of sarcosaphagous insects using the improved CTAB method.

[Extract human DNA from maggot crop contents by phenol-chloroform method coupled with paramagnetic particle method].

OBJECTIVE: To establish an effective phenol-chloroform method coupled with paramagnetic particle method for human DNA extraction from maggot crop contents in STR genotyping. METHODS: Human DNA was extracted from the maggot crop contents using phenol-chloroform method and purified by paramagnetic particle method. DNA was quantified by PCR with Quantifiler Human DNA Quantification Kit using 7500 real-time fluorescence quantitative PCR instrument. PCR products were genotyped by AmpFlSTR Identifiler PCR Amplification Kit using 3130XL-Avant genetic analyzer. RESULTS: The template DNA yield by the method described were increased at least 2 times than the phenol-chloroform extraction method alone. All of the full 16 STR profiles could be obtained with the samples extracted by this method when the DNA yield reached (0.218 +/- 0.041) ng/microL. CONCLUSION: Phenol-chloroform method coupled with paramagnetic particle method can effectively increase the sensitivity of STR analysis of human DNA recovered from maggot crop contents and is a valuable tool for forensic entomology.

[The development and application of lie detection in forensic science].

Lie detection technology has been applied increasingly to investigate and solve criminal cases. This article explores the evolvement of lie detection technology in the ancient times and the application of the psychological and physiological parameters which have become more accurate with the introduction of modern polygraph. The cognitive exploration and the application of Event Related Potentials (ERPs), functional Magnetic Resonance Imaging (fMRI), and Event-Related functional Magnetic Resonance Imaging (E-R fMRI) have made detection technology focus on the brain activities, which produce more objective results by tracing the original state of lying. In summary, this article describes different types of lie detections, simple and complex, their working principles, the latest development, and the prospect of their application in forensic science.

[Relationship between CO II gene of mtDNA of Lucilia sericata and latitude interval].

OBJECTIVE: To deduce the region that the geographical species of Lucilia sericata come from and determine the scene of crime (SOC) based on the gene analysis of mtDNA CO II. METHODS: A 635 bp region for CO II of 4 Lucilia sericata (belong to 2 geographical species) were collected and sequenced, compared with the data of GenBank. A neighbour-joining tree with the Tamura and Nei model was constructed by MEGA2.1 package. The number of inherit intervals of inner-species were analyzes by Kimura's two-parameter model and used for construction the relationships between hereditary and latitude interval by SPSS10.5 soft. RESULTS: It showed that they had the relationships between inherit and latitude interval for the 8 geographical species of Lucilia sericata for CO II. CONCLUSION: This method can be the evidence deducing the region that the geographical species of Lucilia sericata come from and further to determine the scene of crime (SOC).

[Advances of forensic entomology in China].

Forensic entomology is a branch of forensic medicine, which applies studies of insects and arthropods to getting evidence for court and has an analogous advantage in the estimation of the postmortem interval (PMI) and other questions of forensic relevance. The paper expounds its definition and contents and reviews some progress of the studies in some aspects in China such as the constitution and succession of insect community on the different cadavers, the applications of morphological features of insects and the technology of analysis of deoxyribonucleic acid (DNA) in forensic entomology, and forensic entomological toxicology etc.