The PCR-HRM Fingerprint Of Human Bacterial Community And Its Forensic Use

[BACKGROUND]Human health and disease are both linked to the human bacterial community.Due to gender,age,living environment,eating habits,health status and other differences,the composition of bacterial community in different individuals and different parts of the same individual is different.Human bacterial community can be easily transferred to objects on the spot,or left on the spot with the secretion and excrement,even more than the number of human cells,and can maintain the original characteristics of flora in a certain period of time.After death,the flora in all parts of the cadaver changes regularly with the progress of corruption.The flora characteristics of scene samples or corpses can provide related information such as the type of stain,the source of individual and the postmortem interval(PMI).Flora analysis has attracted more and more attention as a new approach to solve forensic problems.The flora can be analyzed by morphological,biochemical and genetic methods.However,due to the limitation of sample conditions,forensic flora analysis is mostly DNA analysis of flora.However,traditional DNA analysis of flora requires cultivation,extraction and electrophoresis,which is time-consuming and laborious.In addition,its resolution and sensitivity is lower,the resulting information is less,and the information is more easily distorted.Therefore,it is difficult to meet the requirements of forensic application.Metagenomics of the flora high throughput sequencing can provide a large amount of information,but is expensive,equipment and technical requirements are high,making it difficult to spread or directly use it for sample screening.The metagenomic high-throughput sequencing of flora can provide a large amount of information,but it is difficult to popularize or be directly used for sample screening because of its high price and high requirements of equipment.High resolution melting curves are simple and rapid,with high sensitivity and information content,and have been applied to variant or mutation screening,rapid pathogen detection,forensic DNA typing,and so on.Its application in the analysis of flora,has been preliminarily explored so far.Direct PCR is a mature forensic DNA typing technology,and it is also used in colony screening and pathogen diagnosis.It can simplify the operation and shorten the time,and it is another potential way for rapid metagenomic analysis.[OBJECTIVE]To develop a direct PCR(dPCR)and high resolution melting(HRM)based,rapid test for saliva bacterial community,and evaluate its forensic use.[METHODS]Human saliva were used as exemplary samples.After centrifugation,the bacteria in the saliva were collected,resuspended in TE buffer,and directly used as template for fluorescence PCR and HRM analysis(dPCR-HRM).For comparison,the bacterial DNA extracted by kit was also tested(k PCR-HRM)as above.Samples from different individuals,diluted samples and stain samples were investigated.The HRM profiles obtained were compared.The genotype confidence percentage(GCP)between profiles was used as the objective index to evaluate the feasibility of dPCR-HRM.[RESULTS]1.By using the DNA fragment of the 16 SrDNAV4 region of a single bacterial strain Pasteurella multocida as a template,and continuously optimizing the volume parameters,a PCR-HRM method for detecting bacterial flora was initially established.2.By dPCR-HRM,a sample specific profile could be obtained within 90 min,which were similar to that of k PCR-HRM(GCP > 95.85%).When the same saliva sample was diluted(35-3500times)or detected at different times,the HRM profiles obtained were basically consistent(GCP > 92.11%%).3.Seting GCP≥80.00% as the threshold,61 saliva samples can be divided into10 basic types.Saliva stains with different deposition times were also investigated,and the HRM profiles were relatively stable at least within 8hs(GCP >90.83%).[CONCLUSIONS]1.dPCR-HRM can obtain the HRM map of saliva in 90 min.2.dPCR-HRM can be used for economic and rapid typing of saliva bacterial community,with relatively high sensitivity,sample adaptability.