A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food.

Meat adulteration is a worldwide concern. In this paper, a new droplet digital PCR (ddPCR) method was developed for the quantitative determination of the presence of chicken in sheep and goat meat products. Meanwhile, a constant (multiplication factor) was introduced to transform the ratio of copy numbers to the proportion of meats. The presented ddPCR method was also proved to be more accurate (showing bias of less than 9% in the range from 5% to 80%) than real-time PCR, which has been widely used in this determination. The method exhibited good repeatability and stability in different thermal treatments and at ultra-high pressure. The relative standard deviation (RSD) values of 5% chicken content was less than 5.4% for ultra-high pressure or heat treatment. Moreover, we confirmed that different parts of meat had no effect on quantification accuracy of the ddPCR method. In contrast to real-time PCR, we examined the performance of ddPCR as a more precise, sensitive and stable analytical strategy to overcome potential problems of discrepancies in amplification efficiency discrepancy and to obtain the copy numbers directly without standard curves. The method and strategy developed in this study can be applied to quantify the presence and to confirm the absence of adulterants not only to sheep but also to other kinds of meat and meat products

Aptamer-based fluorometric determination of Salmonella Typhimurium using Fe3O4 magnetic separation and CdTe quantum dots.

Based on the high sensitivity and stable fluorescence of CdTe quantum dots (QDs) in conjunction with a specific DNA aptamer, the authors describe an aptamer-based fluorescence assay for the determination of Salmonella Typhimurium. The fluorescence detection and quantification of S. Typhimurium is based on a magnetic separation system, a combination of aptamer-coated Fe3O4 magnetic particles (Apt-MNPs) and QD-labeled ssDNA2 (complementary strand of the aptamer). Apt-MNPs are employed for the specific capture of S. Typhimurium. CdTe QD-labeled ssDNA2 was used as a signaling probe. Simply, the as-prepared CdTe QD-labeled ssDNA2 was first incubated with the Apt-MNPs to form the aptamer-ssDNA2 duplex. After the addition of S. Typhimurium, they could specifically bind the DNA aptamer, leading to cleavage of the aptamer-ssDNA2 duplex, accompanied by the release of CdTe QD-labeled DNA. Thus, an increased fluorescence signal can be achieved after magnetic removal of the Apt-MNPs. The fluorescence of CdTe QDs (λexc/em = 327/612 nm) increases linearly in the concentration range of 10 to 1010 cfu•mL-1, and the limit of detection is determined to be 1 cfu•mL-1. The detection process can be performed within 2 h and is successfully applied to the analysis of spiked food samples with good recoveries from 90% to 105%

Identification and quantification of chicken in commercial goat/sheep products.

<p>Identification and quantification of chicken in commercial goat/sheep products.</p

LOQ, repeatability and accuracy test.

<p>LOQ, repeatability and accuracy test.</p

Dynamic range of the ddPCR assay for quantification of chicken and sheep fractions.

<p>The vertical axis represents the measured fraction of chicken in sheep (w/w) by ddPCR and qPCR. The horizontal axis shows the actual fraction of chicken in sheep (w/w). Three replicates for each data point were analyzed. The points indicate the concentrations of 80%, 50%, 20%, 10%, 5% and 1% (w/w) of chicken in sheep. Linearity between the actual chicken fraction (w/w) and the measured chicken fraction (w/w) by ddPCR. The correlation coefficient (R²) for the weight of chicken was 0.9998 for ddPCR and 0.9878 for real-time PCR.</p

The repeatability results of 50% and 5% chicken in sheep processed under different temperature and pressure conditions for different durations.

<p>The target chicken fraction is indicated by a dotted line. The acceptance criterion for repeatability is ±25% of the target proportion, represented by the dashed lines. Error bars represent the standard deviation between the replicates for each treated condition. (<b>A</b>) The 50% chicken fraction processed at different temperatures for different durations and measured by ddPCR (three replicates in ten conditions). (<b>B</b>) The 5% chicken fraction processed at different temperatures for different durations and measured by ddPCR (three replicates in ten conditions). (<b>C</b>) The 50% chicken fraction processed at different pressure intensities and for different durations and measured by ddPCR (three replicates in ten conditions). (<b>D</b>) The 5% chicken fraction processed at different pressure intensities and for different durations and measured by ddPCR (three replicates in ten conditions).</p

The specificity results of primers and probes of chicken, sheep and goat.

<p>The horizontal axis indicates the event number of 13 kinds of meats. The vertical axis indicates the amplitude of samples. (A) Upper frame: droplet cluster positive for FAM (sheep, goat and other meats). Lower frame: droplet cluster negative for FAM (sheep, goat and any other meats) negative droplet cluster. Lanes: 1, sheep; 2, goat; 3, pork; 4, beef; 5, horse; 6, rabbit; 7, donkey; 8, dog; 9, chicken; 10, duck; 11, pigeon; 12, goose; 13, turkey. (B) Upper region: droplet cluster positive for FAM (chicken and any other meats). Lower region: droplet cluster negative for FAM (chicken and any other meats) negative droplet cluster. Lanes: 1, chicken; 2, sheep; 3, goat; 4, beef; 5, horse; 6, rabbit; 7, donkey; 8, dog; 9, chicken; 10, duck; 11, pigeon; 12, goose; 13, turkey.</p

Primer and probe sequences for quantitative PCR.

<p>Primer and probe sequences for quantitative PCR.</p

The ratios of copy numbers of unit mass with different proportions of chicken.

<p>The ratios of copy numbers of unit mass with different proportions of chicken.</p