Dataset Information

Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products.

ABSTRACT: Poultry meat is consumed worldwide and is prone to food fraud because of large price differences among meat from different poultry species. Precise and sensitive analytical methods are necessary to control poultry meat products. We chose species-specific sequences of the cytochrome b gene to develop two multiplex real-time polymerase chain reaction (real-time PCR) systems: one for chicken (Gallus gallus), guinea fowl (Numida meleagris), and pheasant (Phasianus colchicus), and one for quail (Coturnix japonica) and turkey (Meleagris gallopavo). For each species, added meat could be detected down to 0.5 % w/w. No cross reactions were seen. For these two real-time PCR systems, we applied three different quantification methods: (A) with relative standard curves, (B) with matrix-specific multiplication factors, and (C) with an internal DNA reference sequence to normalize and to control inhibition. All three quantification methods had reasonable recovery rates from 43% to 173%. Method B had more accepted recovery rates, i.e., in the range 70-130%, namely 83% compared to 75% for method A or C.

SUBMITTER: Dolch K

PROVIDER: S-EPMC7466254 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products.

Dolch Kerstin K Andrée Sabine S Schwägele Fredi F

Foods (Basel, Switzerland) 20200803 8

Poultry meat is consumed worldwide and is prone to food fraud because of large price differences among meat from different poultry species. Precise and sensitive analytical methods are necessary to control poultry meat products. We chose species-specific sequences of the cytochrome b gene to develop two multiplex real-time polymerase chain reaction (real-time PCR) systems: one for chicken (Gallus gallus), guinea fowl (Numida meleagris), and pheasant (Phasianus colchicus</ ...[more]

PMID: 32756511

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Project description:Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N?=?34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2?=?0.97, msRNA: R2?=?0.92) and patient-derived samples (usRNA: R2?=?0.51, msRNA: R2?=?0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p?=?0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias?=?0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use.

Dataset Information

Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products.

Publications

Comparison of Real-Time PCR Quantification Methods in the Identification of Poultry Species in Meat Products.

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