Dataset Information

Development of an Event-Specific Droplet Digital PCR Assay for Quantification and Evaluation of the Transgene DNAs in Trace Samples of GM PRNP-Knockout Goat.

ABSTRACT: The prion protein (PRNP) gene encoding prion protein is considered a prerequisite for the occurrence of scrapie disease, and knockout of the PRNP gene in transgenic goat is one effective approach to avoid scrapie. This study aims to establish an event-specific droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify the content of genetically modified (GM) PRNP-knockout goat event KoP1. The developed ddPCR assay presents high specificity, sensitivity, accuracy, precision and wide dynamic range. The limits of detection and quantification were as low as 1.44 and 7.2 haploid genome equivalent (HGE) per reaction, respectively. Furthermore, this assay was successfully applied in quantifying the goat KoP1 GM content in milk, feces and living environmental soil samples. We believe that the developed ddPCR assay has the potential to be used in the evaluation of horizontal gene transfer and the practical risk assessment of GM goat event KoP1 and its derivatives.

SUBMITTER: Xu W

PROVIDER: S-EPMC8953510 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Development of an Event-Specific Droplet Digital PCR Assay for Quantification and Evaluation of the Transgene DNAs in Trace Samples of GM PRNP-Knockout Goat.

Xu Wenting W Shen Ping P Li Rong R Liu Biao B Yang Litao L

Foods (Basel, Switzerland) 20220318 6

The prion protein (PRNP) gene encoding prion protein is considered a prerequisite for the occurrence of scrapie disease, and knockout of the PRNP gene in transgenic goat is one effective approach to avoid scrapie. This study aims to establish an event-specific droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify the content of genetically modified (GM) PRNP-knockout goat event KoP1. The developed ddPCR assay presents high specificity, sensitivity, a ...[more]

PMID: 35327291

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Project description:In goats, as in sheep, genotypes of the prion protein gene (PRNP) can influence animals' susceptibility to scrapie. Since the polymorphic codons in sheep are well known, a genetic selection plan has been implemented in Europe, in order to reduce the prevalence of susceptible genotypes to scrapie. In Italy, no breeding plan for scrapie resistance in goats has been adopted, yet. Likewise, according to the most recent modification of Regulation EU 999/2001 (Regulation EU 772/2020) of the European Commission (EU), based on all the available experimental and in field data, K222, D146 and S146 polymorphisms could be used as scrapie resistance alleles in genetic management both in scrapie outbreaks and in disease prevention. In order to collect data on the variability of PRNP, the present study aimed to analyze the sequence of the PRNP gene in eight Italian local goat populations/breeds reared in central and southern Italy (Bianca Monticellana, Capestrina, Facciuta della Valnerina, Fulva del Lazio, Garganica, Grigia Ciociara, Grigia Molisana, and Teramana), some of which were investigated for the first time; moreover, two cosmopolitan breeds (Alpine and Saanen) were included. Blood samples were collected from 219 goats. Genomic DNA was extracted from whole blood. DNA was used as template in PCR amplification of the entire PRNP open reading frame (ORF). Purified amplicons have been sequenced and aligned to Capra hircus PRNP. Particularly, the alleles carrying the resistance-related 222 K polymorphism occurred in all populations with a frequency between 2.5% and 12.5%. An additional resistance allele carrying the S146 variant was observed with a frequency of 3.7% only in the Alpine breed. For three of the estimated alleles, we could not establish if the found double polymorphisms in heterozygosis were in phase, due to technical limitations. In this context, in addition to selective culling in scrapie outbreaks according to the European regulation in force, in the future, selection plans could be adopted to deal with scrapie and to control its diffusion, meanwhile paying attention to preserve a high variability of PRNP.

Dataset Information

Development of an Event-Specific Droplet Digital PCR Assay for Quantification and Evaluation of the Transgene DNAs in Trace Samples of GM PRNP-Knockout Goat.

Publications

Development of an Event-Specific Droplet Digital PCR Assay for Quantification and Evaluation of the Transgene DNAs in Trace Samples of GM <i>PRNP</i>-Knockout Goat.

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