Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (1): 156-164.doi: 10.3864/j.issn.0578-1752.2023.01.012

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

Quantitative Detection Technology of Porcine-Derived Materials in Meat by Real-time PCR

ZHAI XiaoHu1(),LI LingXu2(),CHEN XiaoZhu2,JIANG HuaiDe2,HE WeiHua1,YAO DaWei2()   

  1. 1. Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, Jiangsu
    2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095
  • Received:2022-03-26 Accepted:2022-11-01 Online:2023-01-01 Published:2023-01-17
  • Contact: DaWei YAO;;


【Objective】The aim of this study was to develop a rapid and accurate quantitative method for identifying porcine-derived materials. 【Method】Porcine microsatellites DNA were selected from GenBank nucleotide database. Primers specific for porcine were designed based on the sequences of microsatellite DNA. Genomic DNA from 10 kinds of common animals was amplified by PCR method. The specificity of selected microsatellite DNA to porcine-derived materials was judged by the amplification products. According to the microsatellite sequence, the specific primers and probes were designed to establish a Real-time PCR method for identifying porcine-derived materials. The double standard curve was used to quantify the porcine-derived materials and total animal-derived materials, respectively, and the percentage content of porcine-derived materials was calculated. 【Result】Porcine specific microsatellite DNA with the accession number EF172428 was selected. Only porcine DNA gave a fragment through PCR assay, while there was no amplification for other non-target animal species DNA. The limit of detection was 0.02 ng in a 25 μL reactive system using the Real-time PCR method. This method could accurately detect porcine-derived components in mixed DNA samples and mixed meat samples with 1.32% percent error and 1.06%-7.12% percent error, respectively. 【Conclusion】The quantitative detection method of porcine-derived materials by Real-time PCR in this research could be used to detect the percentage content of porcine-derived materials in mixed samples.

Key words: meat, swine, animal-derived materials, Real-time PCR, quantitation

Table 1

Information of primers and Taqman probes"

引物名称 Primers name 序列 Sequence (5′-3′) 片段大小 Sizes (bp) 基因编号Accession
Common-Taqman-probe Texas red-TAACCTCTTGTCTCTTCGGCTGATG-BHQ2 AB584373

Fig. 1

Specificity of porcine primers SEQ-sus1-F and SEQ- sus1-R 1: DNA Marker; 2: Pork; 3: Beef; 4: Goat; 5: Chicken; 6: Duck; 7: Goose; 8: Rabbit; 9: Donkey; 10: Horse; 11: Deer; 12: Blank control. The same as below"

Fig. 2

Specificity of porcine primers SEQ-sus2-F and SEQ- sus2-R"

Fig. 3

Specific detection of porcine primer and Taqman Dotted arrow: DNA form swine; Hollow arrow: DNA form other animals; Solid arrow: NTC"

Table 2

Annealing temperature optimization of porcine primer"

Annealing temperature (℃)
Efficiency (%)
Coefficient of correlation (R2)
Non-specific amplification Ct
Blank control Ct
57.0 95.51±3.51 0.9987±0.0015a 21.75±0.73c 33.65±1.07c 未检测到Undetected
58.5 94.96±5.39 0.9990±0.0007a 22.40±0.31b 35.70±1.90b 未检测到Undetected
60.0 97.73±1.10 0.9979±0.0015a 22.71±0.37b 35.52±0.71b 未检测到Undetected
61.5 97.76±1.09 0.9909±0.0017b 24.47±0.80a 39.17±0.15a 未检测到Undetected

Table 3

Optimization of porcine primer and Taqman concentration"

Probe and primer content (nmol·L-1)
Coefficient of correlation (R2)
Non-specific amplification Ct
Blank control Ct
150-150 91.18±5.55ab 0.9973±0.0018 23.55±0.50a 35.89±0.64bc 未检测到Undetected
150-250 95.37±0.63ab 0.9998±0.0001 22.56±0.31bc 34.77±0.10c 未检测到Undetected
150-300 96.08±3.81ab 0.9996±0.0001 22.52±0.28bc 35.28±0.13bc 未检测到Undetected
200-150 92.92±5.07ab 0.9970±0.0013 23.54±0.42a 35.03±0.82bc 未检测到Undetected
200-250 95.04±1.46ab 0.9989±0.0002 22.19±0.07c 37.85±0.35a 未检测到Undetected
200-350 97.31±1.80a 0.9993±0.0006 22.33±0.14c 34.57±0.66c 未检测到Undetected
250-150 97.71±0.86a 0.9943±0.0052 23.32±0.31a 36.09±0.35b 未检测到Undetected
250-250 95.58±2.37a 0.9985±0.0004 22.45±0.12bc 34.62±0.70c 未检测到Undetected
250-350 88.20±7.57b 0.9928±0.0080 22.83±0.68b 35.20±0.63bc 未检测到Undetected

Fig. 4

Real-time PCR amplification of animal-derived materials"

Table 4

Annealing temperature optimization of common primer"

Annealing temperature (℃)
Efficiency (%)
Coefficient of correlation (R2)
Blank control Ct
55.5 91.40±1.93ab 0.9963±0.0012 22.55±0.64d 未检测到Undetected
57.0 93.66±1.37ab 0.9969±0.0001 22.59±0.62d 未检测到Undetected
58.5 95.92±3.06a 0.9961±0.0019 22.849±0.65c 未检测到Undetected
60.0 96.77±1.86a 0.9951±0.0009 23.68±0.59b 未检测到Undetected
61.5 95.45±2.78a 0.9691±0.0296 26.70±0.96a 未检测到Undetected

Table 5

Optimization of common primer and TaqMan concentration"

Probe and Primer (nmol·L-1)
Coefficient of correlation (R2)
Non-specific amplification Ct
Blank control Ct
150-150 95.8183±3.4270 0.9990±0.0001 22.07±0.20a 36.537950 未检测到Undetected
150-250 94.6394±0.2007 0.9983±0.0001 21.64±0.07b 36.072550 未检测到Undetected
150-300 96.1199±4.7783 0.9980±0.0017 21.60±0.11b 36.309250 未检测到Undetected
200-150 97.3010±0.2045 0.9979±0.0004 22.12±0.30a 37.725000 未检测到Undetected
200-250 97.0340±1.1993 0.9983±0.0007 21.70±0.13b 35.923600 未检测到Undetected
200-350 96.7049±3.9347 0.9967±0.0020 21.63±0.15b 36.617600 未检测到Undetected
250-150 97.1256±2.4211 0.9976±0.0011 21.97±0.42a 36.158100 未检测到Undetected
250-250 93.8683±6.0015 0.9952±0.0054 21.65±0.13b 36.344000 未检测到Undetected
250-350 94.6619±3.6286 0.9956±0.0059 21.56±0.15b 36.441800 未检测到Undetected

Fig. 5

Standard curve of porcine-derived genomic DNA"

Fig. 6

Standard curve of animal-derived genomic DNA"

Table 6

Results of porcine-derived composition detection in mixed meat samples"

Actual value
Measured value
Percentage error (%)
样品1 Sample 1 40.00 42.20±1.66 5.50
样品2 Sample 2 70.05 70.79±11.06 1.06
样品3 Sample 3 63.96 60.10±10.60 1.34
样品4 Sample 4 60.34 64.64±15.36 7.12
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