Preliminary Exploration Of Species Identification Of Necrophagous Flies By HRM

[BACKGROUND]Accurate and rapid species identification of necrophagous insects is the key step to estimate the postmortem interval(PMI)by using the law of entomological succession.The species identification of necrophagous flies mainly depends on the methods of morphological and molecular identification.The former is limited by the developmental stage and sample integrity of flies,while the latter can compensate for these limitations of morphological identification,and sequencing is the mainstream detection method.However,sequencing is expensive and time-consuming,so it is not the best choice for detecting large quantities of samples in forensic investigations.High resolution melting curve(HRM)is a reliable,fast and cheap molecular technique for analyzing PCR products and sequence variations.Different DNA sequences can be distinguished from the shape of the melting curve and/or melting temperature(Tm)obtained by HRM.In this study,the common necrophagous Calliphoridae flies in Luoyang were taken as the research object,and we tried to establish a rapid,simple,accurate and reliable method to identify necrophagous flies based on HRM technology,in order to provide a new idea for the species identification of other necrophagous insects.[OBJECTIVES]1.To find potential polymorphic short fragments by analyzing the mitochondrial DNA cytochrome c oxidase subunit Ⅰ(CO Ⅰ)gene sequences of necrophagous Calliphoridae flies based on Dna SP 5.10 software.2.To establish the optimal reaction system for species identification of necrophagous flies based on PCR-HRM technique,and preliminarily explore validity of this technique.3.To establish a detection method for identification of the common necrophagous Calliphoridae flies by HRM based on polymorphic short fragments of mitochondrial COⅠgene.[METHODS]1.The Latin name of necrophagous Calliphoridae flies and COⅠwere used as key words,mitochondrial CO Ⅰ sequences worldwide were searched by NCBI database,and the length of the sequence was refined to more than 1300 bp.The obtained sequences were aligned and analyzed by MEGA7.0 and Dna SP 5.10 software.2.Based on COⅠgene,Chrysomya megacephala was taken as the research object,and the PCR-HRM reaction system was optimized under different primer concentration,different Mg2+ concentration and different Taq DNA polymerase.3.The common necrophagous Calliphoridae flies were detected by the built PCRHRM system.The obtained HRM profiles and melting temperature were analyzed,and compared with the results of morphological classification and DNA sequencing.[RESULTS]1.A total of 225 CO Ⅰ sequences of 18 species of Calliphoridae flies were obtained,411 variable sites were found in the final sequences,and 6 polymorphic regions were attained by sliding window analysis.2.Different primer and Mg2+ concentration had a great influence on HRM detection,but three kinds of Taq DNA polymerase had no significant effect on HRM detection.Considering the factor of PCR yields,the optimal primer concentration was0.4μM,Mg2+ concentration was 1.5m M and r-Taq DNA polymerase was 1U.3.Necrophagous Calliphoridae flies were classified into 8 species of 5 genera according to the shapes of melting curve and melting temperature in this study,which was consistent with morphology and DNA sequencing results.[CONCLUSIONS]In this study,6 polymorphic short fragments were obtained by analyzing the mitochondrial CO Ⅰ sequence,which could provide basic data for detection of degraded samples and application of short fragments detection techniques during identification of necrophagous flies.PCR-HRM technique based on polymorphic short fragments of COⅠgene was a convenient and effective detection method,which could be used for rapid species identification of necrophagous flies.