Development of a combined immunomagnetic separation and quantitative reverse transcription-PCR assay for sensitive detection of infectious rotavirus in water samples

• Published in: Journal of microbiological methods 2011-03, Vol.84 (3), p.447-453
• Main Authors: Yang, Wan, Gu, April Z., Zeng, Si-yu, Li, Dan, He, Miao, Shi, Han-chang
• Format: Article
• Language: English
• Subjects: antibodies; arginine; beef extracts; Biological and medical sciences; detection limit; Fundamental and applied biological sciences. Psychology; Group a rotavirus; Immunomagnetic separation; Immunomagnetic Separation - methods; Microbiology; phosphates; Quantitative polymerase chain reaction; rapid methods; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA; Rotavirus; Rotavirus - isolation & purification; Rotavirus A; Sensitivity and Specificity; Techniques used in virology; Time Factors; tissue culture; urea; Virology; Virology - methods; viruses; wastewater; Water Microbiology; Water quality monitoring
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2011.01.011

A quantitative and rapid detection method for rotavirus in water samples was developed using immunomagnetic separation combined with quantitative reverse transcription-polymerase chain reaction (IMS-RT-qPCR). Magnetic beads coated with antibodies against representative group A rotavirus were used to capture and purify intact rotavirus particles in both artificial and real environmental water sample matrix. Compared to extracting RNA using commercial kits and RT-qPCR assay, the developed IMS-RT-qPCR method increased the detection sensitivity by about one order of magnitude when applied in clean water, with a detection limit of 3.16 50% tissue culture infectious dose (TCID50)/mL within 5h. This method was compatible with various commonly used virus eluants, including beef extract (BE), beef extract with 0.05M glycine (BEG) and urea arginine phosphate buffer (UAPB). The recovery efficiencies from various eluants using IMS-RT-qPCR are higher than that using direct RT-qPCR method, demonstrating the effectiveness of the IMS step for eliminating inhibitors in the eluant matrix. This method was also successfully applied to purify and detect rotavirus particles seeded in 103-fold concentrated wastewater influent samples. It seemed to reduce the interference from complex sample background and increase the qPCR product reliability comparing to RT-qPCR method without the IMS step. The results indicated that IMS-RT-qPCR is a rapid, sensitive and reliable tool for detecting rotaviruses in complex water environments. ► A quantitative and rapid detection method for rotavirus in water samples was developed using immunomagnetic separation combined with quantitative RT-PCR (IMS-RT-qPCR). ► Compared to extracting RNA using commercial kits, the IMS increased the detection sensitivity by about one order of magnitude from clean water. ► The IMS-RT-qPCR was also successfully applied to purify and detect rotavirus seeded in 103-fold concentrated wastewater influents. ► The results of present study indicated that IMS-RT-qPCR is a rapid, sensitive and reliable tool for detecting rotaviruses in complex water environments.