Methods for removing viral contaminants from pancreatic extracts
View Patent ↗Methods for screening pancrelipase for RNA virus contamination comprise removing free viral RNA from the pancrelipase, denaturing any viruses in the pancrelipase to release encapsidated RNA into the pancrelipase milieu, and detecting this released RNA. Removal of free viral RNA may comprise treating pancrelipase with RNase and DNase or precipitating the protein fraction of pancrelipase with a salt that precipitates the protein fraction while leaving nucleic acids such as RNA in solution. Pancrelipase substantially devoid of free nucleic acid is also provided.
1. A method for detecting Hepatitis E virus contamination in a sample comprising pancrelipase, the method comprising (a) treating the sample with a ribonuclease to catalyze degradation of substantially all free RNA in the sample comprising pancrelipase, (b) treating the sample with a ribonuclease inhibitor, (c) treating the sample with an agent to denature capsid of Hepatitis E virus, and (d) amplifying with PCR and detecting encapsidated Hepatitis E virus RNA, wherein the method reduces false positive results of Hepatitis E viral contamination in the sample.
2. The method of claim 1 , optionally comprising extracting the encapsidated Hepatitis E virus RNA and amplifying the extracted Hepatitis E virus RNA.
3. The method of claim 1 , further comprising treating the pancrelipase with a protease inhibitor.
4. The method of claim 1 , wherein the ribonuclease is ribonuclease A.
5. The method of claim 1 , wherein the sample of step a) further comprises free RNA and encapsidated Hepatitis E virus.
6. The method of claim 1 , wherein the ribonuclease inhibitor comprises 1,4-dithiothreitol (DTT).
7. The method of claim 1 , wherein b) further comprises heating pancrelipase and removing a cellular debris via centrifugation or molecular filters.