Comparison of two methods for the detection of hepatitis A virus in clam samples (Tapes spp.) by reverse transcription-nested PCR.
The detection of hepatitis A virus in shellfish by reverse transcription-nested polymerase chain reaction (RT-nested PCR) is hampered mainly by low levels of virus contamination and PCR inhibitors in shellfish. In this study, we focused on getting a rapid and sensitive processing procedure for the detection of HAV by RT-nested PCR in clam samples (Tapes spp.). Two previously developed processing methods for virus concentration in shellfish have been improved upon and compared. The first method involves acid adsorption, elution, polyethylene glycol (PEG) precipitation, chloroform extraction and PEG precipitation. The second method is based on elution with a glycine buffer at pH 10, chloroform extraction and concentration by ultracentrifugation. Final clam concentrates were processed by RNA extraction or immunomagnetic capture of viruses (IMC) before the RT-nested PCR reaction. Both methods of sample processing combined with the RNA extraction from the concentrates were very efficient when they were assayed in seeded and naturally contaminated samples. The results show that the first method was more effective in removal inhibitors and the second was simpler and faster. The IMC of HAV from clam concentrates processed by method 1 was revealed to be a very effective method of simultaneously removing residual PCR inhibitors and of concentrating the virus.
Pharmacy Faculty, University of the Basque Country, Vitoria-Gasteiz, Spain. oipsupae@vc.ehu.es
Ester
E
Suñén
Nerea
N
Casas
Belén
B
Moreno
Carmen
C
Zigorraga
D000818:Animals; D049872:Bivalvia / Q000821:virology*; D011232:Chemical Precipitation; D003257:Consumer Product Safety; D005516:Food Microbiology; D030041:Hepatitis A virus / Q000302:isolation & purification*; D018189:Immunomagnetic Separation / Q000379:methods; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*
D012367:RNA, Viral