Detection of Rotavirus from stool samples using a standardized immuno-PCR ("Imperacer") method with end-point and real-time detection.
Immuno-PCR (IPCR) has been studied to increase the detection sensitivity of current enzyme-linked immuno-sorbent assays (ELISA) as a novel approach for the early detection of Rotavirus infection, a major source for serious diarrhoea for susceptible risk groups. IPCR utilizes specific antibody-DNA conjugates with subsequent amplification of the marker-DNA. An antibody-DNA conjugate specific for Rotavirus antigen VP6 was synthesized and used in combination with a commercially available Rotavirus-ELISA kit. IPCR was carried out using reagents and protocols of the standardized Imperacer system. Real-time PCR monitoring of the marker-DNA amplification was compared to endpoint quantification of amplified haptene-labeled PCR products, using a microtiterplate-based PCR-ELISA. In spiked calibration samples, as few as 100 virus particles/ml could be clearly detected using the IPCR method and either real-time or end-point quantification compared to about 100,000 virus particles/ml in ELISA. Rotavirus positive and negative stool samples were correctly identified by IPCR with a clear separation even of a 10,000-fold dilution of the positive stool samples from the negative control.
Adler
Michael
M
Chimera Biotec GmbH, Emil-Figge-Str. 76 A, D-44227 Dortmund, Germany. adler@chimera-biotec.com
Schulz
Sven
S
Fischer
Renate
R
Niemeyer
Christof M
CM
D003199:Computer Systems; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005243:Feces / Q000821:virology*; D006801:Humans; D008403:Mass Screening / Q000379:methods; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics* / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis* / Q000821:virology*; D012680:Sensitivity and Specificity
D011933:Reagent Kits, Diagnostic