title: An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products.
abstract: Real-time polymerase chain reaction (RT-PCR) enables effective and sensitive screening for infectious risk in the field of blood safety. However, when using RT-PCR to detect bacterial contamination, several intractable points must be considered, one of which is the lack of appropriate quality control. In this study, we developed a simplified RT-PCR assay in which the same primer set and two distinct probes were used to detect both, an internal reference control and the target in a reaction. The copy number of the internal reference control represents the positive detection limit of the assay; therefore, when the threshold-cycle value of the target is less than or equal to that of the internal reference control, the result obtained for the target can be considered to be a true positive. When human gDNA was spiked with Escherichia coli gDNA and the detection limit for the internal reference control was set to five copies, the measured detection limit for E. coli gDNA was two copies. The internal reference control duplex RT-PCR assay showed high efficiency (0.91-1.02), high linearity (R2 > 0.99), and good reproducibility in intra- and inter-assay comparisons. Lastly, when human platelet-rich plasma samples were spiked with E. coli or other bacterial species, all species were detected efficiently, and the results of a two-sample pooled t test showed that the limit of detection for E. coli was 1 cfu/mL. Here, we present a synthetic internal reference control molecule and a new statistical method for improving the reliability of RT-PCR assays when screening for bacterial contamination in blood products.
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Jin-Ju
authors.initials: JJ
authors.lastname: Zhang
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Tian
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Shuang-Shi
authors.initials: SS
authors.lastname: Wei
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Sheng-Bao
authors.initials: SB
authors.lastname: Duan
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Wang
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Ye-Zhou
authors.initials: YZ
authors.lastname: Chen
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Shao-Hua
authors.initials: SH
authors.lastname: Ding
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Qing-Lin
authors.initials: QL
authors.lastname: Meng
authors.affiliation: The Chinese Academy of Sciences Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Li
mesh_terms: D016470:Bacteremia / Q000382:microbiology*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D057230:Limit of Detection; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial