Fa yi xue za zhi · 2025
Establishment and Application of TaqMan qPCR Detection Method for Human DNA Contamination in DNA Laboratory.
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Shen Gao-Fang, Zhou Yong-Song, Zhang Jian-Qiu, Ji Shi-You, Wu Ying-Feng, Shang Hao, Zhu Bo-Feng
Аннотация
OBJECTIVES: 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 ExpressTM 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 the Minimum Information for Publication of Quantitative Real-time PCR Experiments (MIQE) guidelines, the specificity, sensitivity, repeatability 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%.
CONCLUSIONS: The established human DNA detection method based on qPCR technique has good specificity, high sensitivity, and robust stability. It can be used for rapid detection of DNA contamination and daily monitoring of the accumulated human DNA in the laboratory environment.
METHODS: Primers and probes were designed with Primer ExpressTM 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 the Minimum Information for Publication of Quantitative Real-time PCR Experiments (MIQE) guidelines, the specificity, sensitivity, repeatability 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%.
CONCLUSIONS: The established human DNA detection method based on qPCR technique has good specificity, high sensitivity, and robust stability. It can be used for rapid detection of DNA contamination and daily monitoring of the accumulated human DNA in the laboratory environment.