Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR).
RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.
Jothikumar
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Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. jin2@cdc.gov
Sobsey
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Cromeans
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D002853:Chromatography, Liquid / Q000379:methods; D029822:Enterovirus B, Human / Q000302:isolation & purification; D005618:Fresh Water / Q000821:virology*; D030041:Hepatitis A virus / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D058428:Silica Gel; D014773:Virology / Q000379:methods*
D012367:RNA, Viral; D058428:Silica Gel