An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products.
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 contro...
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2015-01-01
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doaj-b7bee82acd7d4091906f4ce486aa7ce72020-11-24T21:30:01ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01107e013474310.1371/journal.pone.0134743An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products.Jin-Ju ZhangJing-Jing TianShuang-Shi WeiSheng-Bao DuanHong-Mei WangYe-Zhou ChenShao-Hua DingChun ZhangQing-Lin MengYong LiReal-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.http://europepmc.org/articles/PMC4521875?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jin-Ju Zhang Jing-Jing Tian Shuang-Shi Wei Sheng-Bao Duan Hong-Mei Wang Ye-Zhou Chen Shao-Hua Ding Chun Zhang Qing-Lin Meng Yong Li |
spellingShingle |
Jin-Ju Zhang Jing-Jing Tian Shuang-Shi Wei Sheng-Bao Duan Hong-Mei Wang Ye-Zhou Chen Shao-Hua Ding Chun Zhang Qing-Lin Meng Yong Li An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products. PLoS ONE |
author_facet |
Jin-Ju Zhang Jing-Jing Tian Shuang-Shi Wei Sheng-Bao Duan Hong-Mei Wang Ye-Zhou Chen Shao-Hua Ding Chun Zhang Qing-Lin Meng Yong Li |
author_sort |
Jin-Ju Zhang |
title |
An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products. |
title_short |
An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products. |
title_full |
An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products. |
title_fullStr |
An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products. |
title_full_unstemmed |
An Internal Reference Control Duplex Real-Time Polymerase Chain Reaction Assay for Detecting Bacterial Contamination in Blood Products. |
title_sort |
internal reference control duplex real-time polymerase chain reaction assay for detecting bacterial contamination in blood products. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2015-01-01 |
description |
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. |
url |
http://europepmc.org/articles/PMC4521875?pdf=render |
work_keys_str_mv |
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