Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning.

OBJECTIVES: To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning. METHODS: Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated. RESULTS: The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05). CONCLUSIONS: MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Molecular characteristics of the HSP70 gene and its differential expression in female and male golden apple snails (Pomacea canaliculata) under temperature stimulation.

Heat-shock protein 70 (HSP70) is one of the most important heat-shock proteins that helps organisms to modulate stress response via over-expression. The HSP70 gene from Pomacea canaliculata was cloned using the RACE approach; the gene is 2,767 bp in length and contains an open reading frame of 1,932 bp, which is encoded by a polypeptide of 643 amino acids. BLAST analysis showed that the predicted amino acid sequence of the P. canaliculata HSP70 gene shared a relatively high similarity with that of other known eukaryotic species that display conserved HSP characteristics. The phylogeny demonstrated a separate clustering of the apple snail HSP70 with other constitutive members from other mollusk species. Quantitative real-time RT-PCR was used to detect the differential expression of HSP70 in both sexes of P. canaliculata at different temperature conditions. These results showed that HSP70 transcript levels decreased slightly under cold shock and increased significantly under heat-shock conditions in both sexes compared to normal temperatures (26 °C). Under cold-shock treatment, the sex effect was not significant. With heat treatment, HSP70 expression could be induced at 36 °C in both females and males, and it peaked at 42 and 39 °C in females and males, respectively. In addition, a clear time-dependent HSP70 expression pattern of the apple snail exposed to the same high temperature (36 °C) was observed at different time points. The maximal induction of HSP70 expression appeared at 12 and 48 h in males and females after heat shock, respectively. The maximal induction in females was significantly higher compared to males under heat stimulus. Taken together, these results strongly suggested that males were more susceptible to heat than females and provided useful molecular information for the ecological adaptability of P. canaliculata against extreme environmental stress.

Mitochondrial DNA as effective molecular markers for the genetic variation and phylogeny of the family Osteoglossidae.

The present study examined the genetic variation of the family Osteoglossidae from different geographical locations based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) and ATPase subunit 6 (ATPase6) genes; we then re-constructed the phylogenetic relationships using the two sequences in combination. The results showed that the partial sequences of mitochondrial ND2 and ATPase6 of the family Osteoglossidae were 813 bp and 669 bp, respectively. A total of 42 species-specific nucleotide positions of the family Osteoglossidae were found to be useful for molecular identification. The sequence variation showed greater differences (8.3%~28.1% for the combined sequences, 8.3%~26.7% for the ND2 gene, and 9.3%~28.7% for the ATPase6 gene) among the different species of Osteoglossidae, and there was a significant association between the genetic difference and geographical location. Phylogenetic analyses using neighbor-joining, Bayesian inference, and maximum parsimony (MP) methods based on the combined sequences of the two genes were able to distinguish the different species and were in agreement with the existing taxonomy based on morphological characters and in association with the geographical distribution among seven species of the family Osteoglossidae.