Comparison of Extraction Methods for the Detection of Tick-Borne Encephalitis Virus RNA in Goat Raw Milk and Cream Cheese

Abstract

Infection with the tick-borne encephalitis virus (TBEV) can cause meningitis, meningoencephalitis and myelitis in humans. TBEV is an enveloped RNA virus of the family Flaviviridae, which is mostly transmitted via tick bites. However, transmission by consumption of virus-contaminated goat raw milk and goat raw milk products has also been described. Only a few methods have been reported for the detection of TBEV in food so far. Here, we compare different virus extraction methods for goat raw milk and goat raw milk cream cheese and subsequent detection of TBEV-RNA by RT-qPCR. Langat virus (LGTV), a naturally attenuated TBEV strain, was used for artificial contamination experiments. Mengovirus and the human coronavirus 229E were compared to assess their suitability to serve as internal process controls. Out of three tested extraction protocols for raw milk, sample centrifugation followed by direct RNA extraction from the aqueous interphase yielded the best results, with a recovery rate (RR) of 31.8 ± 4.9% for LGTV and a detection limit of 6.7 × 10³ LGTV genome copies/ml. Out of two methods for cream cheese, treatment of the samples with TRI Reagent® and chloroform prior to RNA extraction showed the best RR of 4.7 ± 1.6% for LGTV and a detection limit of 9.4 × 10⁴ LGTV genome copies/g. RRs of Mengovirus and LGTV were similar for both methods; therefore, Mengovirus is suggested as internal process control virus. The developed methods may be useful for screening or surveillance studies, as well as in outbreak investigations.

Keywords: Cheese; Detection method; Goat; Internal process control; Milk; Tick-borne encephalitis virus.

Fig. 1

Schematic illustration of the extraction methods tested for goat raw milk. A Method 1, based on direct RNA extraction from the sample, B method 2, based on PEG precipitation and chloroform/butanol cleanup prior to RNA extraction, C method 3, based on sample centrifugation prior to RNA extraction

Fig. 2

Schematic illustration of the extraction methods tested for goat raw milk cream cheese. A Method 4, based on sample centrifugation prior to RNA extraction, B method 5, based on extraction with TRI Reagent® and chloroform cleanup prior to RNA extraction

Fig. 3

Recovery rates for LGTV-RNA and process control virus-RNA resulting from different extraction methods. A Comparison of methods 1–3 applied to artificially contaminated goat raw milk samples. Statistically significant differences (p < 0.05) between the RRs of the distinct viruses comparing method 2 with method 1 and method 3 with method 1 (calculated by ANOVA with Bonferroni correction) are indicated with asterisks above the bars. B comparison of methods 4 and 5 applied to artificially contaminated goat raw milk cream cheese samples. Statistically significant differences (p < 0.05) between the RRs of the distinct viruses comparing both methods (calculated by unpaired Student’s t test) are indicated with asterisks above the bars. LGTV (Mengovirus): RR for LGTV-RNA from samples co-contaminated with LGTV and Mengovirus (black bars); Mengovirus: RR for Mengovirus-RNA from samples co-contaminated with LGTV and Mengovirus (dark gray); LGTV (HCoV-229E): RR for LGTV-RNA from samples co-contaminated with LGTV and HCoV-229E (white bars); HCoV-229E: RR for HCoV-229E-RNA from samples co-contaminated with LGTV and HCoV-229E (light gray bars). Mean values (bars) and standard deviations from three independent replicates per condition are shown