Use of Molecular Methods to Authenticate Animal Species and Tissue in Bovine Liver Dietary Supplements.

Dietary supplements containing bovine (subfamily Bovinae) liver are susceptible to fraud due to their high value and the lack of modern detection methods available for processed animal tissues. The objective of this research was to use molecular methods to authenticate dietary supplements claiming to contain bovine liver or beef liver through the verification of animal species and tissue type. A total of 53 bovine/beef liver dietary supplements were purchased from online sources. The presence of liver was verified with reverse transcription and real-time PCR testing for microRNA-122 (miR-122), which is highly expressed in liver tissue. Multiplex real-time PCR targeting domestic cattle (Bos taurus), horse (Equus caballus), sheep (Ovis aries), and pork (Sus scrofa) was used to verify species. Samples that failed species identification with multiplex real-time PCR underwent DNA mini-barcoding. Overall, bovine species were detected in 48/53 liver supplements: 35 samples were confirmed as domestic cattle with multiplex real-time PCR and an additional 13 samples were confirmed as domestic cattle or Bos spp. with DNA mini-barcoding. One of these samples was also positive for sheep/lamb, which was declared on the label. One product contained undeclared pork in addition to beef. MiR-122 was detected in 51 out of 53 supplements, suggesting the presence of liver. While this study demonstrates the potential use of tissue-specific microRNAs in verifying tissues in dietary supplements, more research is needed to evaluate the specificity of these markers.

Application of whole-genome sequencing for norovirus outbreak tracking and surveillance efforts in Orange County, CA.

Noroviruses are the leading cause of acute gastroenteritis and foodborne illness in the United States. Traditional Sanger sequencing of short genomic regions (~300-600 bp) is the primary method for differentiation of this pathogen; however, whole-genome sequencing (WGS) offers a valuable approach to further characterize strains of this virus. The objective of this study was to investigate the ability of WGS compared to Sanger sequencing to differentiate norovirus strains and enhance outbreak investigation and surveillance efforts. WGS results for 41 norovirus-positive stool samples from 15 different outbreaks occurring from 2012 to 2019 in Orange County, CA, were analyzed for this study. All samples were genotyped with both WGS and Sanger sequencing based on the B-C region. WGS generated nearly full-length viral genome sequences (7029-7768 bp) with 4x to 35,378x coverage. Phylogenetic analysis of WGS data enabled differentiation of genotypically similar strains from separate outbreaks. Single nucleotide variation (SNV) analysis on a subset of strains revealed nucleotide variations (15-79 nt) among isolates from multiple outbreaks of GII.4 Sydney_2015[P31] and GII.17[P17]. Overall, the results demonstrated that coupling norovirus genotype identification with WGS enables enhanced genetic differentiation of strains and provides valuable information for outbreak investigation and surveillance efforts.

DNA-based techniques for seafood species authentication.

Global trade of seafood has increased in the last decade, leading to significant concerns associated with seafood fraud. Seafood fraud involves the intentional misrepresentation of fish or shellfish for the purpose of economic gain and includes acts such as species substitution, illegal transshipment, overtreatment/short weighting, and mislabeling country of origin or production method. These fraudulent acts have had economic, environmental, and public health consequences on a global level. DNA-based techniques for seafood authentication are utilized by regulatory agencies and can be employed as part of a food fraud risk mitigation plan. This chapter will focus specifically on the use of DNA-based methods for the detection of seafood species substitution. Various methods have been developed for DNA-based species identification of seafood, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), species-specific PCR, real-time PCR, Sanger sequencing, microarrays, and high-resolution melting (HRM). Emerging techniques for seafood authentication include droplet digital PCR, isothermal amplification, PCR-enzyme-linked immunosorbent assay (ELISA), and high-throughput or next-generation sequencing. Some of these DNA-based methods target specific species, such as real-time PCR and droplet digital PCR, while other methods allow for simultaneous differentiation of a wide range of fish species, including Sanger sequencing and high-throughput sequencing. This chapter will begin with an introduction on seafood fraud and species substitution, followed by an analysis of the main DNA-based authentication methods and emerging techniques for species identification.