Analysis of 28S rRNA and COâ Gene Sequence of Nine Necrophagous Calliphorid Flies from Luoyang.

ABSTRACT: Objective To assess the feasibility of using 28S ribosomal RNA （28S rRNA） and mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） gene sequences of nine necrophagous Calliphorid flies for the identification of common necrophagous Calliphorid flies, and to provide technical support for postmortem interval （PMI） estimation. Methods Twenty-three Calliphorid flies were collected and identified morphologically, and DNA were extracted from legs. The gene fragments of 28S rRNA and COⅠ were amplified and sequenced, then the sequence alignment was performed with BLAST. The composition of obtained sequences was analyzed and evolutionary divergence rate between species and intraspecies were established. The phylogeny tree was constructed with neighbor-joining method. Results The 23 necrophagous Calliphorid flies were identified to 9 species of 5 genera. The 715 bp from 28S rRNA and 637 bp from COⅠ gene were obtained and the online BLAST result showed more than 99% of similarity. The phylogeny tree showed that the necrophagous flies could cluster well into 9 groups, which was consistent with morphological identification results. The intraspecific difference in 28S rRNA was 0 and the interspecific difference was 0.001-0.033. The intraspecific difference in COⅠ was 0-0.008 and the interspecific difference was 0.006-0.101. Conclusion Combined use of 28S rRNA and COⅠ gene sequence fragments can effectively identify the nine Calliphorid flies in this study. However, for closely related blowfly species, more genetic markers should be explored and used in combination in future.

Relationship between Electrical Conductivity and Chemical Content of Rat Skeletal Muscle Impregnating Solution and Postmortem Interval.

ABSTRACT: Objective To study the mechanism of change of the electrical conductivity （EC） of rat skeletal muscle impregnating solution that occurs with the change of postmortem interval （PMI）. Methods Healthy Sprague-Dawley rats were killed and kept at about 25 ℃. Skeletal muscles were extracted at different PMI--immediate （0 d）, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7 d, then mixed with deionized water to make impregnating solution with a mass concentration of 0.1 g/mL. The solution's EC and nine common chemicals in it, such as potassium ion, calcium ion, and chloride ion, were determined. Results EC increased gradually with the extending of PMI （P=0.024） during the 7 days after the rats' death. The content of uric acid （P=0.032）, urea nitrogen （P=0.013） and phosphorus （P=0.022） also increased during the extension. However, the content of magnesium ions decreased with extending of PMI （P=0.047）. The correlation between potassium ion, sodium ion, chlorine ion, calcium ion, creatinine and PMI were weak （P>0.05）. Conclusion The molecular basis of skeletal muscle EC change in rats after their death is the changes of uric acid, urea nitrogen, inorganic phosphorus and other chemical components. Furthermore, combine use of various indicators can improve the accuracy of the EC method to infer PMI.

[Molecular Identification of Sarcosaphagous Flies in Luoyang Based on 28S rRNA Gene Sequence].

OBJECTIVES: To detect 715 bp sequence of 28S rRNA in sarcosaphagous flies, and to identify their common species for solving the problem of morphological identification, as well as providing technical support for postmortem interval （PMI） estimation. METHODS: Twenty-nine common sarcosaphagous flies were collected in Luoyang and classified by morphological characteristics. The DNA was extracted from the fly's legs by Chelex-100 method and then the fragments of 28S rRNA were amplified and sequenced. The results were compared with twenty-eight corresponding fly species of GenBank and EMBL databases. All the sequences were analyzed by MEGA7.0 software, and sequence alignment was performed by the searching in BLAST. The nucleotide composition was analysed, and the intraspecific and interspecific genetic distance and phylogenetic tree were established. RESULTS: Twenty-nine sarcosaphagous flies were classified into 6 species of 5 genera, 3 families by morphological characteristics. In the obtained 715 bp sequence of 28S rRNA, the comparison result of online BLAST showed that the similarity was 100%. Five species were well clustered by a phylogenetic tree. Between different groups, the interspecific and intraspecific differences ranged from 0.007 to 0.045 and 0 to 0.001, respectively. CONCLUSIONS: The 28S rRNA target gene sequences shows a good identification capability, which can be a new genetic marker for the identification of sarcosaphagous flies.

Relationship between Early Postmortem Interval and Electrical Conductivity of Rat Liver, Spleen and Kidney after Death in Rats.

OBJECTIVES: To determine the electrical conductivity （EC） of the liver, spleen and kidney of rats at different postmortem intervals （PMIs） within 24 hours for investigating the relationship between EC of different organs and early PMI. METHODS: Totally 45 SD rats were sacrificed by cervical dislocation and kept at a constant temperature of 25 ℃. Tissues were taken from the liver, spleen, and kidney of rats at 0, 3, 6, 9, 12, 15, 18, 21 and 24 h. Impregnating solution with a mass concentration 0.1 g/mL was prepared using deionized water. The EC value of impregnating solution with different organs was separately determined. The regression equations of EC and PMI for different organs were established, respectively. The relationship between EC of different organs and early PMI was analysed in deceased rats. RESULTS: The relationship between PMI and EC of the liver and spleen was well fitted with the linear equation. The liver showed the best fitting degree followed by the spleen, while the EC of the kidney showed no significant changes within 24 h. There was a good linear relationship between early PMI and the EC of the liver and spleen. CONCLUSIONS: A good linear relationship between early PMI and the EC of the liver and spleen can be found in rats after death, which can be used for the early PMI estimation.

[Relationship between Electrical Conductivity and Decomposition Rate of Rat Postmortem Skeletal Muscle].

OBJECTIVES: To analyze the relationship among electrical conductivity （EC）, total volatile basic nitrogen （TVB-N）, which is an index of decomposition rate for meat production, and postmortem interval （PMI）. To explore the feasibility of EC as an index of cadaveric skeletal muscle decomposition rate and lay the foundation for PMI estimation. METHODS: Healthy Sprague-Dawley rats were sacrificed by cervical vertebrae dislocation and kept at 28 ℃. Muscle of rear limbs was removed at different PMI, homogenized in deionized water and then skeletal extraction liquid of mass concentration 0.1 g/mL was prepared. EC and TVB-N of extraction liquid were separately determined. The correlation between EC (x1) and TVB-N (x2) was analyzed, and their regression function was established. The relationship between PMI (y) and these two parameters were studied, and their regression functions were separately established. RESULTS: The change trends of EC and TVB-N of skeletal extraction liquid at different PMI were almost the same, and there was a linear positive correlation between them. The regression equation was x2=0.14x1-164.91(R²=0.982). EC and TVB-N of skeletal muscle changed significantly with PMI, and the regression functions were y=19.38x1³-370.68x1²+2 526.03 x1-717.06(R²=0.994), and y=2.56x2³-48.39x2²+330.60x2-255.04(R²=0.997), respectively. CONCLUSIONS: EC and TVB-N of rat postmortem skeletal muscle show similar change trends, which can be used as an index for decomposition rate of cadaveric skeletal muscle and provide a method for further study of late PMI estimation.