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In the current research, brief contact with host cell monolayers, referred to as"host cell capture", has been combined with quantitative sequence detection real-timePCR (HCC-QSD) to develop a rapid (same day) method for the detection of potentiallyinfectious viruses. It differs from ICC-PCR in that replication of virus in cell culture isnot required. Attachment to host cells and intact nucleic acid are two requirements forviral infection and form the basis of the HCC-QSD detection strategy. The HCC-QSDmethod uses brief (2 hours or less) contact with cell culture monolayers to "capture" potentially infectious viruses, followed by washing to remove non-adsorbed viruses andsample debris. RNA from "captured" viruses is then extracted using a commercial viralRNA 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-timequantitative PCR. TaqMan quantitative PCR requires the use of primers similar to thoseused in conventional PCR; however, unlike conventional PCR, TaqMan quantitative PCRalso requires an oligonucleotide probe labeled with 5' reporter and 3' quencherfluorescent dyes and a thermal cycler equipped with a fluorometer. During each cycle ofthe PCR, if the target of interest is present, the probe specifically anneals to the targetamplicon between the forward and reverse primer sites. Due to the 5' nuclease activity ofTaq DNA polymerase, the probe is cleaved during the polymerization step (PCR productformation) of the PCR resulting in an increase in reporter fluorescence detected by theinstrument. Target signal increases in direct proportion to the concentration of the PCRproduct being formed. The threshold cycle (CT) is the fractional PCR cycle number atwhich a significant increase in target signal fluorescence above baseline is first detectedfor a sample. By using amplification standards consisting of known quantities of targetnucleic acid or organisms to generate a standard curve, the starting copy number ofnucleic acid targets or target organisms for each sample can be estimated. Includes table, figure.

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Edition: Vol. - No. Published: 11/01/2007 Number of Pages: 4File Size: 1 file , 84 KB