Thursday, January 6, 2011

Detection of XMRV requires specific conditions

By Gerwyn:

For anyone wishing to get involved in this area, this extract from Danielson et al, 2010, will repay study
" We have found that detection of XMRV required rather specific conditions. For instance, at least 600 ng of prostate tissue DNA was necessary for reliable detection with our PCR assay.

XMRV was detected in 3.2% of the patients when we nitially used 100–140 ng of prostate tissue DNA, compared with 22.2% of the patients when we used 650 ng.

Additionally, we found that detection of XMRV from patient specimens, but not
from LNCaP cells infected in vitro, depended on the gene targeted in the PCR assay.

We were unable to detect XMRV in the patient tissue samples by nested PCR with primers specific for the gagand pol genes, regardless of whether 100 or 650 ng of DNA wasused as template. We found the gag primers to be at least 10-fold less sensitive than the env primers, and the pol primers tended to amplify a competing region from the human genome (data not shown).

It is unclear whether these deficiencies account for the inability to detect XMRV in patient samples or whether XMRV is mainly present as an incomplete provirus in the cells of these patients.

Nonetheless, the difficulty associated with detecting XMRV in patient samples may perhaps explain studies that do not detect the virus among large cohorts.

this is perhaps even more damning

To test for VP62 plasmid contamination in prostate specimens,a set of 4 primers were designed for nested PCR. The 2 forward primers (5 TCTGGCTAACTAGAGAACCCACTG-3 and 5 AATACGACTCACTATAGGGAGACC-3) were specific to the multiple cloning site of pCDA3.1(-) (Invitrogen). The 2 reverse primers
5 -GTTACGGTCTGTCCCATGATCTC-3) were specific to the 5 terminus of VP62 gag. The VP62 nested PCR assay was foundto be capable of detecting 10 plasmids diluted in 600 ng of uninfected LNCaP DNA in 3 of 3 samples and 1 plasmid in 600 ng of uninfected LNCaP DNA in 1 of 3 samples (data not shown).

In other words a PCR assay capable of detecting very low copy number of VP62 gag cannot detect XMRV in a known clinically positive sample."

BP Danielson, GE Ayala and JT Kimata; Detection of Xenotropic Murine Leukemia Virus-Related Virus in Normal and Tumor Tissue of Patients from the Southern United States with Prostate Cancer Is Dependent on Specific Polymerase Chain Reaction Conditions; J Infect Dis. (2010) 202 (10): 1470-1477. doi: 10.1086/656146

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