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- Detection of Hepatitis A Virus from Oyster by Nested PCR Using Efficient Extraction and Concentration Method
- Duwoon Kim , Seok-Ryel Kim , Ki-Sung Kwon , Ji-Won Lee , Myung-Joo Oh
- J. Microbiol. 2008;46(4):436-440. Published online August 31, 2008
- DOI: https://doi.org/10.1007/s12275-008-0131-1
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- 23 Citations
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Abstract
- The molecular methods using polymerase chain reaction have been proposed as useful tools for the identification of viral pathogens in food and water. However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification due to the low sensitivity of PCR in the presence of PCR inhibitors. We developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol in order to detect hepatitis A virus (HAV) inoculated in oyster digestive glands. The detection limit of HAV precipitated with zirconium hydroxide was 105 fold less sensitive in a nested PCR than that precipitated the HAV supernatant twice with PEG/NaCl (16% polyethylene glycol 6,000, 0.525 M NaCl) in a 1:2 (v/v) ratio, which provided an efficient detection of 0.0148 PFU/g from approximately 0.05 g of oyster homogenate. This method is efficient for potential use in the detection of HAV from shellfish and is more sensitive than most currently published tests.
- Removal and Inactivation of Hepatitis A Virus during Manufacture of a High Purity Antihemophilic Factor VIII Concentrate from Human Plasma
- In Seop Kim , Yong Woon Choi , Sung Rae Lee , Mahl Soon Lee , Ki Ho Huh , Soungmin Lee
- J. Microbiol. 2001;39(1):67-73.
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Abstract
- A validation study was conducted to evaluate the efficacy and mechanism of the cryo-precipitation, monoclonal anti-FVIIIc antibody (mAb) chromatography, Q-Sepharose chromatography, and lyophilization steps involved in the manufacture of high purity factor VIII (GreenMono) from human plasma, in the removal and/or inactivation of hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and subjected to scale-down processes mimicking the manufacture of the high purity factor VIII concentrate. Samples were collected at each step and immediately titrated using a 50% tissue culture infectious dose (TCID50) and then the virus reduction factors were evaluated. HAV was effectively partitioned from factor VIII during cryo-precipitation with the log reduction factor of 3.2. The mAb chromatography was the most effective step for removal of HAV with the log reduction factor of ³4.3. HAV infectivity was not detected in the fraction of factor VIII, while most of HAV infectivity was recovered in the fractions of flow through and wash during mAb chromatography. Q-Sepharose chromatography showed the lowest efficacy for partitioning HAV with the log reduction factor of 0.7. Lyophilization was an effective step in inactivating HAV with the log reduction factor of 2.3. The cumulative log reduction factor, ³10.5, achieved for the entire manufacturing process was several magnitudes greater than the potential HAV load of current plasma pools.
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