Establishment and Application of TaqMan qPCR Detection Method for Human DNA Contamination in DNA Laboratory
10.12116/j.issn.1004-5619.2023.531004
- VernacularTitle:DNA实验室人源性DNA污染TaqMan qPCR检测方法的建立及应用
- Author:
Gao-Fang SHEN
1
;
Yong-Song ZHOU
;
Jian-Qiu ZHANG
;
Shi-You JI
;
Ying-Feng WU
;
Hao SHANG
;
Bo-Feng ZHU
Author Information
1. 扬州市公安局,江苏 扬州 225100
- Keywords:
forensic genetics;
DNA contamination;
laboratory;
real time quantitative PCR(qPCR);
TaqMan probe;
forensic DNA analysis
- From:
Journal of Forensic Medicine
2025;41(1):66-73
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a highly sensitive and specific method for detecting human DNA based on real time quantitative PCR(qPCR)technique for the rapid detection of potential DNA con-tamination sources in DNA laboratories.Methods Primers and probes were designed with Primer Ex-pressTM software using the reference sequence of human 18S rRNA gene as a template,and the opti-mal prime-probe combination was screened by matrix method.The PCR products of the target se-quence of human 18S rRNA gene were used to construct the plasmid,and a plasmid standard was used to draw the standard curve of the qPCR system.According to the Minimum Information for Pub-lication of Quantitative Real-time PCR Experiments(MIQE)guidelines,the specificity,sensitivity,re-peatability and application effect of the qPCR system were evaluated.Results The sensitivity of the qPCR system established in this study was 5.3×10-5 ng/μL,which showed good specificity for human DNA samples.The correlation coefficient of the qPCR system was-0.999,and amplification efficiency was 100％.Both the intra-batch and inter-batch variation coefficients were less than 2％.Conclusion The established human DNA detection method based on qPCR technique has good specificity,high sen-sitivity,and robust stability.It can be used for rapid detection of DNA contamination and daily moni-toring of the accumulated human DNA in the laboratory environment.
