In the current research, brief contact with host cell monolayers, referred to as
"host cell capture", has been combined with quantitative sequence detection real-time
PCR (HCC-QSD) to develop a rapid (same day) method for the detection of potentially
infectious viruses. It differs from ICC-PCR in that replication of virus in cell culture is
not required. Attachment to host cells and intact nucleic acid are two requirements for
viral infection and form the basis of the HCC-QSD detection strategy. The HCC-QSD
method uses brief (2 hours or less) contact with cell culture monolayers to "capture" potentially infectious viruses, followed by washing to remove non-adsorbed viruses and
sample debris. RNA from "captured" viruses is then extracted using a commercial viral
RNA purification kit and quantified using QSD real-time PCR. These conditions,
combined with the specificity of molecular detection, are used to selectively recover,
detect, and quantify potentially infectious viruses.
HCC-QSD uses TaqMan probe chemistry which is commonly used for real-time
quantitative PCR. TaqMan quantitative PCR requires the use of primers similar to those
used in conventional PCR; however, unlike conventional PCR, TaqMan quantitative PCR
also requires an oligonucleotide probe labeled with 5' reporter and 3' quencher
fluorescent dyes and a thermal cycler equipped with a fluorometer. During each cycle of
the PCR, if the target of interest is present, the probe specifically anneals to the target
amplicon between the forward and reverse primer sites. Due to the 5' nuclease activity of
Taq DNA polymerase, the probe is cleaved during the polymerization step (PCR product
formation) of the PCR resulting in an increase in reporter fluorescence detected by the
instrument. Target signal increases in direct proportion to the concentration of the PCR
product being formed. The threshold cycle (CT) is the fractional PCR cycle number at
which a significant increase in target signal fluorescence above baseline is first detected
for a sample. By using amplification standards consisting of known quantities of target
nucleic acid or organisms to generate a standard curve, the starting copy number of
nucleic acid targets or target organisms for each sample can be estimated. Includes table, figure.
| Edition : | Vol. - No. |
| File Size : | 1
file
, 84 KB |
| Note : | This product is unavailable in Ukraine, Russia, Belarus |
| Number of Pages : | 4 |
| Published : | 11/01/2007 |
