Comparison of three methods for concentration of rotavirus from artificially spiked shellfish samples

Abstract

Background: Shellfish are a nutritious food source whose consumption and commercial value have risen dramatically worldwide. Shellfish being filter feeders concentrate particulate matters including microorganisms such as pathogenic bacteria and viruses and thus constitute a major public health concern. Effective preliminary sample treatment steps such as concentration of virus from shellfish are essential before RNA/DNA isolation for final PCR accuracy and reproducibility due to presence of PCR inhibitors in shellfish. Aim: The current study was done to compare three methods for concentration of rotavirus from shellfish samples. Materials and Methods: Shellfish samples artificially spiked with tenfold serial dilutions of known concentration of rotavirus were subjected to three different concentration methods namely; proteinase K treatment, precipitation with polyethylene glycol 8000 and use of lysis buffer. RNA was isolated from the concentrated samples using phenol chloroform method. Rota viral RNA was detected using RT-PCR. Results: Concentration of virus using proteinase K and lysis buffer yielded better result than concentration by PEG 8000 in samples with lowest concentration of virus. Among these two methods proteinase K treatment was superior as it showed better amplification of the highest dilution (107) used. Conclusion: Treatment with proteinase K was better than other two methods as it could detect the viral RNA in all three tenfold serial dilutions.

Keywords

• concentration
• lysis buffer
• polyethylene glycol 8000
• proteinase K
• RT-PCR
• shellfish