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Research
(Published
online: 06-07-2014)
4. Comparison of three methods for
concentration of rotavirus from artificially spiked shellfish
samples - Vysakh Mohan, Shriya Rawat, K. M. Lokesh, H.
V. Mohan, D. Avinash Reddy, Ashok Kumar and K N. Bhilegaonkar
Veterinary World, 7(7): 463-466
doi:
10.14202/vetworld.2014.463-466
Vysakh
Mohan: Division of Veterinary Public Health, Indian Veterinary
Research Institute, Izatngar - 243122, Uttar Pradesh,
India; vysakhmohanvet@gmail.com
Shriya
Rawat: Division of Veterinary Public Health, College of
Veterinary and Animal Sciences, Sardar Vallabhbhai Patel
University of Agriculture and Technology, Modipuram, Meerut,
India; shriyarawat@gmail.com
K. M.
Lokesh: Division of Veterinary Public Health, Guru Angad Dev
Veterinary and Animal Sciences University, Ludhiana, Punjab,
India; lokeshvph@gmail.com
H. V.
Mohan:
Division of Veterinary Public Health, Veterinary college, Bidar,
Karnataka, India; mohanhv@gmail.com
D.
Avinash Reddy: Division of Veterinary Public Health, Indian
Veterinary Research Institute, Izatngar - 243122, Uttar Pradesh,
India; avi.incredible@gmail.com
Ashok
Kumar: Division of Veterinary Public Health, Indian Veterinary
Research Institute, Izatngar - 243122, Uttar Pradesh,
India; ashokakt@rediffmail.com
K N.
Bhilegaonkar: Division of Veterinary Public Health, Indian
Veterinary Research Institute, Izatngar - 243122, Uttar Pradesh,
India; kiranvph@rediffmail.com
Received: 01-03-2014, Revised: 03-06-2014, Accepted: 07-06-2014,
Published online: 06-07-2014
Corresponding author: Vysakh Mohan, email:
vysakhmohanvet@gmail.com
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.
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