Analysis of Salivary Bacterial Community by Direct PCR and High Resolution Melting Curve and Its Forensic Applications

doi:10.12116/j.issn.1004-5619.2021.511006

Abstract

Abstract:

Objective To develop a rapid test for salivary bacterial community based on direct PCR （dPCR） and high resolution melting （HRM） curve analysis, to evaluate its application value in forensic medicine. Methods The salivary bacteria were collected by centrifugation and then resuspended in Tris-EDTA （TE） buffer, and directly used as the template for amplification and HRM curve analysis （dPCR-HRM） of the 16S rDNA V4 region. The genotype confidence percentage （GCP） of the HRM profiles compared with the reference profile was calculated. The template DNA was extracted by traditional kit and then PCR-HRM （namely kPCR-HRM） was used as reference to validate the feasibility of dPCR-HRM. The gradient dilution templates, population samples and simulated salivary stains were analyzed by dPCR-HRM to evaluate its sensitivity, typing ability and adaptability. Results Using dPCR-HRM method, the HRM profiles of salivary bacterial community were obtained within 90 minutes. The GCP between dPCR-HRM and kPCR-HRM was greater than 95.85%. For general individuals, the HRM type of bacterial community could be determined with 0.29 nL saliva by dPCR-HRM. The 61 saliva samples could be divided into 10 types. The typing of salivary stains deposited within 8 h was the same as those of fresh saliva （GCP>90.83%）. Conclusion dPCR-HRM technology can be used for rapid typing of salivary bacterial community, and has the advantage of low cost and simple operation.

Key words: forensic genetics, high resolution melting, polymerase chain reaction （PCR）, saliva, bacterial community

CLC Number:

DF795.2

Rui YANG, Jiong CHEN, Gui-sen ZHAO. Analysis of Salivary Bacterial Community by Direct PCR and High Resolution Melting Curve and Its Forensic Applications[J]. Journal of Forensic Medicine, 2022, 38(6): 709-718.

Figures/Tables 8

Fig. 1 PCR-HRM profiles of 16S rDNA V4 region of Pasteurella multocida

Fig. 2 kPCR-HRM profiles of salivary bacterial community

Fig. 3 dPCR-HRM and kPCR-HRM profiles of the same sample

Fig. 4 dPCR-HRM profiles of the mixed sample with gradient dilution

Fig. 5 dPCR-HRM profiles of salivary bacterial community of different individuals

Fig. 6 dPCR-HRM profiles of salivary stains with different deposition times

Fig. 7 Normalized HRM profiles of 10 bacterial community types in saliva

Fig. 8 Melting curves of 10 bacterial community types in saliva

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