• Journal of Microbiology and Biotechnology
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• v.20 no.10
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• pp.1463-1470
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• 2010

E. coli has long been widely used as a host system for the manufacture of recombinant proteins intended for human therapeutic use. When considering the impurities to be eliminated during the downstream process, residual host cell DNA is a major safety concern. The presence of residual E. coli host cell DNA in the final products is typically determined using a conventional slot blot hybridization assay or total DNA Threshold assay. However, both the former and latter methods are time consuming, expensive, and relatively insensitive. This study thus attempted to develop a more sensitive real-time PCR assay for the specific detection of residual E. coli DNA. This novel method was then compared with the slot blot hybridization assay and total DNA Threshold assay in order to determine its effectiveness and overall capabilities. The novel approach involved the selection of a specific primer pair for amplification of the E. coli 16S rRNA gene in an effort to improve sensitivity, whereas the E. coli host cell DNA quantification took place through the use of SYBR Green I. The detection limit of the real-time PCR assay, under these optimized conditions, was calculated to be 0.042 pg genomic DNA, which was much higher than those of both the slot blot hybridization assay and total DNA Threshold assay, where the detection limits were 2.42 and 3.73 pg genomic DNA, respectively. Hence, the real-time PCR assay can be said to be more reproducible, more accurate, and more precise than either the slot blot hybridization assay or total DNA Threshold assay. The real-time PCR assay may thus be a promising new tool for the quantitative detection and clearance validation of residual E. coli host cell DNA during the manufacturingprocess for recombinant therapeutics.

• Journal of Microbiology and Biotechnology
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• v.22 no.9
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• pp.1301-1306
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• 2012

We combined real-time RT-PCR and real-time PCR (R/P) assays using a hydrolysis probe to detect Mycobacterium tuberculosis complex (MTBC)-specific 16S rRNA and its rRNA gene (rDNA). The assay was applied to 28 non-respiratory and 207 respiratory specimens from 218 patients. Total nucleic acids (including RNA and DNA) were extracted from samples, and results were considered positive if the repeat RT-PCR threshold cycle was ${\leq}35$ and the ratio of real-time RT-PCR and real-time PCR load was ${\geq}1.51$. The results were compared with those from existing methods, including smear, culture, and real-time PCR. Following resolution of the discrepant results between R/P assay and culture, the overall sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) of all samples (including non-respiratory and respiratory specimens) were 98.2%, 97.2%, 91.7%, and 99.4%, respectively, for R/P assay, and 83.9%, 89.9%, 72.3%, and 94.7%, respectively, for real-time PCR. Furthermore, the R/P assay of four patient samples showed a higher ratio before treatment than after several days of treatment. We conclude that the R/P assay is a rapid and accurate method for direct detection of MTBC, which can distinguish viable and nonviable MTBC, and thus may guide patient therapy and public health decisions.

• Food Science of Animal Resources
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• v.31 no.6
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• pp.827-833
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• 2011

The objective of this study was to develop a fluorogenic real-time PCR-based assay for detecting and quantifying amounts of cow milk in cow/goat milk mixtures or goat milk products. In order to quantify the exact amount of cow milk in cow/goat raw milk mixtures and commercial goat milk products, it was necessary to achieve quantitative extraction of total genomic DNA from the raw milk matrix. Both mammalian-specific PCR and cow-specific PCR were performed. A cow-specific 252 bp band obtained from the raw cow milk and raw goat milk mixtures, commercial goat milk, and two goat milk powders was identified, along with the relationship between the cow milk amount and band intensity of the electrophoresis image. The detection threshold was found to be 0.1%. The expression of cow's 12S rRNA in the cow/goat milk mixtures, commercial goat milk, and two goat milk powders was identified. The expression quantity of the milk 12S rRNA increased with increasing ratios of the cow/goat milk mixtures. Using these calibrated relative expression levels as a standard curve in the cow/goat raw milk mixtures, the contents of cow milk were 1.8% in the commercial goat milk, 9.6% in goat milk powder A, and 11.6% in goat milk powder C. However, cow milk was not detected in goat milk powder B.