A real-time qPCR method to identify diatom UPA gene for the drowning diagnosis
10.13618/j.issn.1001-5728.2018.02.003
- VernacularTitle:实时荧光定量PCR检测硅藻UPA基因在溺死诊断中的研究
- Author:
Xiangdong LIU
1
;
Chao LIU
;
Quyi XU
;
Fan PENG
;
Sunlin HU
;
Baishen MAI
;
Hong LIU
;
Yue LI
;
Huiying HU
;
Jichao XU
;
Shurui ZHANG
;
Yali HAN
;
Zhujun TAN
Author Information
1. 广东工业大学轻工化工学院
- Keywords:
forensic pathology;
drowning;
diatom test;
real-time quantitative PCR;
UPA
- From:
Chinese Journal of Forensic Medicine
2018;33(2):124-129
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a Real-time quantitative PCR method (qPCR) for the detection of diatom UPA barcoding genes and evaluate its application in the drowning diagnosis. Methods The homologous sequences of diatoms UPA gene was obtained by Blast from GeneBank, based on which the universal primers for diatoms were designed. DNA were extracted from 2 common human symbiotic bacteria (Escherichia coli, Bifidobacterium longum), 3 species of planktonic bacteria, 15 species of planktonic algae, tissue samples (lung, liver and kidney) from human cadavers (28 drowning victims, 1 victims by non-drowning in the water, 3 victims deaths on land) in 32 cases. The specificity, sensitivity and repeatability of the designed primers were tested. The positive rates of diatoms detection in the drowning cases were calculated. The results of the real-time quantitative method were evaluated comparatively by Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) and PCR-Capillary Electrophoresis (PCR-CE). Results The results showed that the primers UPA99 had strong specificity for the diatomaceae (Synedra radians, Navicula sp., Melosira varians, Cyclotella sp. and Nitzschia sp.) DNA. The melting curve of the amplified product was smooth; the peak was narrow; the melting temperature was (87±1)℃. The sensitivity of qPCR method was 1.56×10-5ng/μL with the detection range of 1.56×102ng/mL~1.56×10-5ng/μL, in contrast with the PCR-CE method (1.56×10-3ng/μL). This real-time PCR method showed high repeatability and stability with the coefficient of variation less than 2%. The detection rate of lung, liver and kidney was 89.3%, 71.4% and 64.3% respectively. Conclusion The established qPCR method, based on the universal primers designed for diatom UPA gene, has high specificity, high sensitivity and good repeatability. With a promising prospect for application, qPCR is suitable for drowning diagnosis.
