Objective: To establish a high coverage, sensitive and specific TaqMan probe real-time fluorescence quantitative PCR method for the detection of Hexon gene of fastidious human adenovirus (HAdV), and to determine the fastidious HAdV in human-derived cell substrates and raw materials. Methods: In this study, primers, probes, and plasmid controls were designed and synthesized based on the conserved sequences of Hexon genes. AAV-HAdV pseudovirus was used as a positive control, the plasmid containing the gene sequence of the F subgenus of human adenovirus (HAdV-F) was used as an amplification control. The linearity, sensitivity, specificity, robustness, and reproducibility of the detection method were fully verified to ensure that it could be applied to the safety evaluation of fastidious HAdV in biologics and their raw materials. Results: A high coverage rate for virus strains was achieved by this detection method, and 100% of HAdV-F strains (94 isolates) were covered. A good linear relationship was exhibited by the plasmid controls within the range of 20-2×108 copies · μL-¹, and the R² value could reach 1.000. The detection sensitivity was able to reach 4 copies · μL-¹. Good specificity was demonstrated by this method, as specific amplifications for human adenovirus type 5 and human adenovirus type 2 could not be performed. Good robustness was also possessed by this method. When two different models of instruments were used for detection, the R2 values of the standard curves were 0.999 and 1.000 respectively, the cycle threshold (Ct) values for sensitivity were 37.22±0.62 and 36.50±0.58 respectively, and the Ct values for the positive controls were 21.67±0.03 and 20.61±0.18 respectively. No significant differences were found in the results of various detections. When two reagents of different article numbers were used for testing, the R2 values of the standard curves were both 1.000. The Ct values for sensitivity were 37.99±1.19 and 38.31±0.97 respectively, and the Ct values for the positive controls were 19.69±0.09 and 20.58±0.05 respectively. No significant differences were observed in the results of various detections. The intermediate precision was verified under the conditions of different personnel or different instruments. The detection results of the HAdV-F plasmid amplification control in four experiments were 1.29×105 copies · μL-¹, 1.54×105 copies · μL-¹, 1.73×105 copies · μL-¹, and 1.37× 105 copies · μL-¹ respectively. The relative standard deviation was calculated to be 13.2%, indicating good intermediate precision. The AAV-HAdV pseudovirus was used as a positive control for the applicability verification in human mesenchymal stem cell samples. The detection result of the positive control was determined to be 137 copies · μL-¹, and the detection result of the applicability sample was found to be 133 copies · μL-¹. The spiked sample recovery rate was calculated as 97.1%. Conclusion: The assay has good linearity, sensitivity, specificity, robustness, and reproducibility, and has high coverage of the exacting HAdV strain, which can be used for the detection of exacting HAdV in cell matrix samples for production.
DONG Ying-ying
,
CHEN Xiao-fei
,
ZHANG Tong
,
LIU Ming-yue
,
ZHANG Rui-rui
,
CAO Yi-dan
,
LI Hui-ting
,
FU Xin-yue
,
WANG Yan-hui
,
WANG Xin-le
,
CUI Meng-shan
,
PANG Lin
,
RAO Chun-ming
. Establishment of a real-time fluorescence quantitative PCR assay for the detection of fastidious human adenovirus*[J]. Chinese Journal of Pharmaceutical Analysis, 2025
, 45(7)
: 1122
-1129
.
DOI: 10.16155/j.0254-1793.2024-1338
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