[Rapid detection of rotavirus in water samples using immunomagnetic separation combined with real time PCR].
A quantitative and rapid detection method for rotavirus in water samples was developed, by using immunomagnetic separation combined with reverse transcription and real time polymerase chain reaction (IMS-RT-real time PCR). Magnetic beads coated with antibodies directed against group A rotavirus were used to capture and purify the virus in water samples. The experimental results showed that IMS was optimized when 1 mL samples were supplemented with 10 microL of immunomagnetic beads, 2.5 microL of Tween 20 and incubated for 2 h. The IMS method was employed in the detection of rotavirus in seeded virus eluant such as 3% beef extract successfully and thus manifested its compatibility with established virus concentration methods. The IMS-RT-real time PCR method could yield quantitative results within about 5 h with a detection limit at 1 x 10(4) copies/mL (equivalent to 3-4 PFU/mL). The method exhibited a high level correlation (R2 = 0.9816) with cell culture assay, indicating that it could perform as well as cell culture assay does in infection tests. And the method functioned satisfactorily in seeded concentrate of secondary waste water treatment plant effluent, reclaimed water, surface water and tap water.
Yang
Wan
W
State Key Joint Laboratory of Environment Simulation and Pollution Control, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China. wanyang08@gmail.com
He
Miao
M
Li
Dan
D
Shi
Han-Chang
HC
Liu
Li
L
D000956:Antigens, Viral / Q000276:immunology; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D000956:Antigens, Viral