Journal of Virological Methods 189 (2013) 311– 316
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Comparative analysis of rodent tissue preservation methods and nucleic acid extraction techniques for virus screening purposes
Ines N. Yamaa,*, Madougou Garbab,c, Janice Britton-Davidiand, Simon-Djamel Thibervillea, Gauthier Dobignyb,c, Ernest A. Goulda, Xavier de Lamballeriea,e, Remi N. Charrela,e
aAix-Marseille University, French Institute of Research for Development, EHESP French School of Public Health, UMR D 190 “Emergence des Pathologies Virales”, 13005 Marseille, France
b Institut de Recherche pour le Développement, Centre de Biologie pour la Gestion des Populations (UMR IRD-INRA-Cirad-SupAgro Montpellier), Campus International de Baillarguet, CS30016, 34988 Montferrier-sur-Lez, France
c Centre Régional Agrhymet, Rive droite, BP11011, Niamey, Niger
d UMR-ISEM-CNRS, Université de Montpellier II, Montpellier, France
e IHU Mediterranee Infection, APHM Public Hospitals of Marseille, 13005 Marseille, France
Abstract. The polymerase chain reaction (PCR) has become an essential method for the detection of viruses in tissue specimens. However, it is well known that the presence of PCR inhibitors in tissue samples may cause false-negative results. Hence the identification of PCR inhibitors and evaluation and optimization of nucleic acid extraction and preservation methods is of prime concern in virus discovery programs dealing with animal tissues. Accordingly, to monitor and remove inhibitors we have performed comparative analyses of two commonly used tissue storage methods and five RNA purification techniques using a variety of animal tissues, containing quantified levels of added MS2 bacteriophages as the indicator of inhibition. The results showed (i) no significant difference between the two methods of sample preservation, viz. direct storage at −80 ◦C or 4 ◦C in RNAlater, (ii) lung rodent tissues contained lower levels of inhibitor than liver, kidney and spleen, (iii) RNA extraction using the EZ1 + PK RNA kit was the most effective procedure for removal of RT-PCR inhibitors.
Keywords. Virus screening, PCR inhibitors, Organ storage, Proteinase K, Enterobacteria phage MS2
*Corresponding author at: UMR D 190, Faculté de Médecine, 28 boulevard Jean Moulin, 13005 Marseille, France. Tel.: +33 626719100.
E-mail address: rnabio@aol.fr (I.N. Yama).