Improvement of Virus Extraction from Soft Fruit by Implementing a PCR Inhibitor Removal Kit

Introduction: Soft fruit has been associated with food-borne outbreaks. A horizontal method for the detection of norovirus (NoV) GI and GII and hepatitis A virus (HAV) in food using one-step RT real-time PCR has been published as ISO15216. Inhibitory substances from fruit often require the analyses of both undiluted and 10-1 diluted RNA.

Purpose: To examine whether the OneStep PCR Inhibitor Removal Kit (Zymo Research) can reduce RT-PCR inhibition levels in soft fruit samples.

Methods: Samples were analyzed according to ISO15216-2 with or without application of the kit just prior to RT real-time PCR. Inhibition was defined as amplification of 2x10² DNA equivalents of in vitro transcribed HAV ssRNA external amplification control (EAC) with more than 2 Ct in undiluted sample RNA than in water.

Results: Selected RNAs derived from frozen soft fruit, previously demonstrated to contain inhibitory substances, were pooled per fruit type, resulting in 15 samples. Half of each pooled RNA sample was left untreated, the other half was cleaned-up. All treated samples had acceptable inhibition levels (< 2 Ct), whereas too much inhibition (>2 Ct) was shown for 11/15 (73%) of the untreated samples. Using artificially contaminated fresh soft fruit samples it was shown that the kit had no (substantial) negative effect on the detectability of NoV GI, NoV GII and HAV. Therefore, the kit was implemented in routine analyses. Without clean-up, 43% of the 145 samples collected in 2014 showed too much inhibition (>2 Ct) (17/47 strawberries; 4/15 blue berries, 14/34 blackberries and 27/49 raspberries). After implementation, all of the 115 soft fruit samples collected in 2015 (35 strawberries, 28 blue berries, 24 blackberries and 28 raspberries) had acceptable inhibition levels (<2 Ct).

Significance: With the clean-up method for RNA, all soft fruit samples had acceptable inhibition levels, which is an improvement of the method.