HAV detection from milk-based products containing soft fruits: Comparison between four different extraction methods

Battistini, Roberta; Rossini, Irene; Listorti, Valeria; Ercolini, Carlo; Maurella, Cristiana; Serracca, Laura

doi:10.1016/j.ijfoodmicro.2020.108661

Cited by 11 publications

(15 citation statements)

References 28 publications

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“…In addition, the low level of viral contamination in food and the presence of PCR inhibitory substances in the food matrices affect the molecular detection of viral nucleic acid. Milk includes several components such as fats, casein, whey proteins, and lactose that affect the process of viral nucleic acid extraction and the recovery of enteric viruses [ 25 , 26 ]. The lack of standardized procedures for HEV detection in milk underestimates the risk of HEV infection in milk samples.…”

Section: Discussionmentioning

confidence: 99%

“…While method B did not involve the step of high-speed centrifugation. Battistini et al reported that high-speed centrifugation gave the best method of recovery HAV from the milk matrix [ 25 ]. Although the centrifuge speed and time needed could differ, the application of centrifugation in the extraction procedure could affect the recovery of enteric viruses from the milk matrix.…”

Section: Discussionmentioning

confidence: 99%

“…We used in this study whole milk, skim milk, and milk serum matrices. The skim milk and milk serum were prepared as described previously with a slight modification in the procedure [ 25 , 26 , 34 , 35 ]. The skim milk was prepared by centrifugation of the whole milk sample at 4000× g for 30 min at 4 °C to remove the cream layer that includes mainly the fat globules.…”

Section: Methodsmentioning

confidence: 99%

“…Milk is composed of different components such as fats, casein, whey proteins, and lactose that have different characteristics including adsorption, sedimentation, and separation. Several reports showed that the milk matrix and milk components affect the process of viral nucleic acid extraction and viral recovery yield [ 20 , 25 , 26 , 27 ]. Additionally, different processing and extraction procedures affect the detection and recovery of enteric viruses such as HAV and norovirus (NoV) from the milk matrix [ 20 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

“…Several reports showed that the milk matrix and milk components affect the process of viral nucleic acid extraction and viral recovery yield [ 20 , 25 , 26 , 27 ]. Additionally, different processing and extraction procedures affect the detection and recovery of enteric viruses such as HAV and norovirus (NoV) from the milk matrix [ 20 , 25 ]. It was reported that the milk matrix affected the sensitivity and performance of a closed automated system (The cobas ® 6800 system (Roche Diagnostics) that detects HEV RNA (gt1-4) in the human plasma with a LoD 95 % of 18.6 IU/mL.…”

Section: Introductionmentioning

confidence: 99%

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Enhancement of the Molecular and Serological Assessment of Hepatitis E Virus in Milk Samples

et al. 2020

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Hepatitis E virus (HEV) infection is endemic in developing and developed countries. HEV was reported to be excreted in the milk of ruminants, raising the possibility of transmission of HEV infection through the ingestion of contaminated milk. Therefore, the detection of HEV markers in milk samples becomes pivotal. However, milk includes inhibitory components that affect HEV detection assays. Previously it was reported that dilution of milk matrix improves the performance of HEV molecular assay, however, the dilution of milk samples is not the best strategy especially when the contaminated milk sample has a low HEV load. Therefore, the objective of this study is to compare the effect of extraction procedures on the efficiency of HEV RNA detection in undiluted milk samples. In addition, we assessed the effect of the removal of milk components such as fats and casein on the performance of the molecular and serological assays of HEV. Phosphate buffered saline (PBS) and different milk matrices (such as whole milk, skim milk, and milk serum) were inoculated with different HEV inoculums and subjected to two different extraction procedures. Method A includes manual extraction using spin column-based extraction, while method B includes silica-based automated extraction. Method A was more sensitive than method B in the whole milk and skim milk matrices with a LoD95% of 300 IU/mL, and virus recovery yield of 47%. While the sensitivity and performance of method B were significantly improved using the milk serum matrix, with LoD95% of 96 IU/mL. Interestingly, retesting HEV positive milk samples using the high sensitivity assay based on method B extraction and milk serum matrix increased the HEV RNA detection rate to 2-fold. Additionally, the performance of HEV serological assays such as anti-HEV IgG and HEV Ag in the milk samples was improved after the removal of the fat globules from the milk matrix. In conclusion, HEV RNA assay is affected by the components of milk and the extraction procedure. Removal of inhibitory substances, such as fat and casein from the milk sample increased the performance of HEV molecular and serological assays which will be suitable for the low load HEV milk with no further dilutions.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%