Establishment and Application of TaqMan qPCR Detection Method for Human DNA Contamination in DNA Laboratory

doi:10.12116/j.issn.1004-5619.2023.531004

Abstract

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 contamination sources in DNA laboratories. Methods Primers and probes were designed with Primer Express^® software using the reference sequence of human 18S rRNA gene as a template, and the optimal prime-probe combination was screened by matrix method. The PCR products of the target sequence 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 MIQE guidelines, the specificity, sensitivity, repeatability, and application effect of the qPCR system were evaluated. Results The sensitivity of 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 sensitivity, and good stability. It can be used for rapid detection of DNA contamination and daily monitoring of the accumulated human DNA in the laboratory environment.

Key words: forensic genetics, DNA contamination, laboratory, real time quantitative PCR （qPCR）, TaqMan probe, forensic DNA analysis

CLC Number:

Gao-fang SHEN, Yong-song ZHOU, Jian-qiu ZHANG, Shi-you JI, Ying-feng WU, Hao SHANG, Bo-feng ZHU. Establishment and Application of TaqMan qPCR Detection Method for Human DNA Contamination in DNA Laboratory[J]. Journal of Forensic Medicine, DOI: 10.12116/j.issn.1004-5619.2023.531004.

Figures/Tables 6

Fig. 1 Specificity test results of the qPCR system

Tab. 1 Sensitivity test results of the qPCR system

质粒质量浓度/（ng·μL^-1）	平均Ct值	检出率/%
5.3×10¹	13.59	100
5.3×10⁰	16.93	100
5.3×10^-1	20.24	100
5.3×10^-2	23.67	100
5.3×10^-3	26.91	100
5.3×10^-4	30.20	100
5.3×10^-5	33.51	100
5.3×10^-6	34.21	88
5.3×10^-7	35.40	50
5.3×10^-8	35.87	31

Fig. 2 Amplification plot of the plasmid samples（5.3×10-5 ng/μL， 16 replicates）

Tab. 2 Repeatability test results of the qPCR system

质粒质量浓度（ng·μL^-1）	批次内		批次间
质粒质量浓度（ng·μL^-1）	$x ¯$ ±SD	CV/%	$x ¯$ ±SD	CV/%
5.3×10¹	13.07±0.05	0.85	12.96±0.22	1.07
5.3×10^-2	22.76±0.14	0.74	23.05±0.17	1.13
5.3×10^-5	34.31±0.12	0.69	33.92±0.24	1.56

Tab. 2 Repeatability test results of the qPCR system

质粒质量浓度（ng·μL^-1）	批次内		批次间
质粒质量浓度（ng·μL^-1）	$x ¯$ ±SD	CV/%	$x ¯$ ±SD	CV/%
5.3×10¹	13.07±0.05	0.85	12.96±0.22	1.07
5.3×10^-2	22.76±0.14	0.74	23.05±0.17	1.13
5.3×10^-5	34.31±0.12	0.69	33.92±0.24	1.56

Fig. 3 Amplification efficiency of the qPCR system

Tab. 3 qPCR detection results of the collected real samples

实验室区域	样本数/份	检出率/%	Ct值
案件受理室	7	100.00	36.90~38.63
物证预处理室	4	100.00	36.92~40.62
物证临时保管室	12	66.67	0.00~41.05
人员样本采样室	4	50.00	0.00~37.32
常规物证DNA提取室	16	43.75	0.00~39.28
微量物证DNA提取室	5	20.00	0.00~38.30
PCR体系配制室	7	28.57	0.00~41.06
PCR扩增室	12	41.67	0.00~40.81
PCR产物检测室	12	83.33	0.00~40.85
技术大楼顶层天台（对照区域）	3	0	0

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