Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (22): 4352-4363.doi: 10.3864/j.issn.0578-1752.2018.22.013

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

Qualitative and Quantitative Detection Methods of Pork in Beef and Its Chinese Processing Products

ZHU Yang(),LIU YongFeng(),WEI YanChao,SHEN Qian,WANG YiFan   

  1. College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062
  • Received:2018-05-10 Accepted:2018-07-25 Online:2018-11-16 Published:2018-11-16

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Abstract:

【Objective】 The objective of this paper was to establish a qualitative and quantitative detection method for pork components in beef and Chinese processed products, so as to guarantee the purity of beef products. 【Method】 The pig genomic DNA from pork and different processed pork products were extracted, and then effects of processing methods on pig DNA quality, sensitivity and detection limit were analyzed through DNA quality testing, PCR amplification and sensitivity test. The raw beef was prepared, and the dried, steamed, boiled fried, stewed and roasted beef products were mixed with the binary mixed meat of different proportions (10%, 5%, 1%, 0.1%) of pork, and then the qualitative and quantitative detections of common PCR and real-time PCR were carried out. The application of DNA in adulteration identification. 【Result】 The DNA quality test results of different processing methods showed that different processing methods significantly affected the purity of DNA (P<0.05). The DNA purity (A260nm/A280nm) ranged from 1.893 to 1.977 in raw pork and seven kinds of pork products, which were higher than the theoretical value of 1.8. The DNA content ranged from 110 to 277 μg·g -1, and the DNA content of the processed pork products was significantly higher than that of the raw pork treatment group (P<0.05); Agarose electrophoresis showed that the DNA of raw pork and seven kinds of meat products was seriously degraded after 6 months of storage, but raw pork still obtained some unclear long-segment DNA, and the pig DNA of seven meat products all degraded into small fragments of DNA, indicating that long-term placement and heat treatment significantly affect the integrity of pig DNA; Although the degradation of DNA in pork products was serious, the mitochondrial genes were amplified by ordinary PCR, and the PCR products of all samples were presented as clear and single bands. It could be seen that the DNA extracted from the processed meat product could be tested for sensitivity and adulteration; The sensitivity test results showed that the common PCR was highly sensitive. The 10-fold gradient dilution showed that the minimum detection limit of pig DNA extracted from the eight test group samples was 0.005 ng. The standard curve formed by fluorescence quantitative PCR amplification of pig DNA was also formed, which had a good linear relationship. The slope of the standard curve was between -3.1 and -3.7, the coefficient of determination R 2 was greater than 0.99, the PCR amplification efficiency was between 89% and 100%, and the quantitative PCR could be detected 0.005 ng of pig DNA. Qualitative quantitative PCR test results of adulterated samples showed that the minimum detection limit of qualitative test for mixed raw meat and other six mixed meat products was 0.1% except for fried mixed meat (1%), indicating that ordinary PCR could detect trace amounts of pork composition. In the quantitative test of mixed meat, the coefficient of determination (R 2) of the standard curve of eight test groups established according to different adulteration ratios was more than 0.99, and the slope was -3.1--3.6. Each curve had a good linear relationship and could realize beef with quantitative detection of medium pork components. Comparing the quantitative results of raw meat and meat products, there was a difference of about 0.1 to 0.6 cycles between the intercepts of the standard curve of mixed raw meat and mixed meat products. 【Conclusion】 Different processing could significantly affect the content, purity and integrity of DNA in meat, but it did not affect the detection limit and sensitivity of DNA in meat products. Both ordinary PCR and quantitative PCR could detect the micro-content of adulterated meat. It could be seen that the detection method based on PCR technology had high sensitivity, high speed and high specificity, and the quantitative detection standard curve had high linear correlation and amplification efficiency, which could provide reliable quality control and inspection plan for meat industry and verification label declaration. The results could be applied to some commercial samples to ensure the purity of meat products.

Key words: beef, pork, processing technology, PCR, qualitative quantization, sensitivity

Table 1

Oligonucleotide primers"

引物名称
Primer name		 引物序列（5′–3′）
Primer sequence (5′-3′)		 片段大小
Fragment size (bp)		 资料来源
Source
Cytb F: ATGAAACATTGGAGTAGTCCTACTATTTACC
R: CTACGAGGTCTGTTCCGATATAAGG		 149 DOOLEY[19]
18S rRNA F: TCTGCCCTATCAACTTTCGATGG
R: TAATTTGCGCGCCTGCTG		 140 FAJARDO[20]

Table 2

DNA content and purity in pork and its meat products"

烹调方式
Cooking method		 浓度
Concentration (μg·g-1)		 A260nm/A280nm
生肉（对照组）
Raw meat (control group)		 110.6±18.8e 1.973±0.025a
干制 Dried 182.3±1.48d 1.893±0.289b
蒸制 Steaming 215.1±2.67c 1.873±0.006bc
煮制 Cooking 276.5±8.8b 1.810±0.010d
炖制 Stew 145.7±1.33a 1.853±0.006c
煎制 pan-Frying 222.3±6.04d 1.810±0.010d
炸制 Frying 275.9±7.71b 1.977±0.006a
烤制 Baked 153.1±19.7a 1.947±0.011b

Fig. 1

DNA agarose gel electrophoresis map of pork and its meat products Lane 1: Raw meat, 2: Dry, 3: Steamed, 4: Cooked, 5: Stewed, 6: Pan-fried, 7: Fried, 8: Baked; M: DL2000 Marker"

Fig. 2

Gel electrophoresis of PCR products of DNA in pork and its meat products Lane 1: Raw meat, 2: Dry, 3: Steamed, 4: Cooked, 5: Stewed, 6: Pan-fried, 7: Fried, 8: Baked; M: DL2000 Marker"

Fig. 3

Common PCR sensitivity test results for DNA in pork and its meat products Raw meat (A), Dried (B), Steamed (C), Cooked (D), Stewed (E), Pan-fried (F), Fried (G), Baked (H); M: DL2000 Marker, N: Negative control; Lanes 1-5: 50, 5, 0.5, 0.05, 0.005 ng"

Fig. 4

Standard curve corresponding to different concentration gradient DNA of pork and its meat products Raw meat (A), Dried (B), Steamed (C), Cooked (D), Stewed (E), Pan-fried (F), Fried (G), Baked (H)"

Fig. 5

Qualitative PCR for the determination of adulterated pork in beef Raw meat (A), Dried (B), Steamed (C), Cooked (D), Stewed (E), Pan-fried (F), Fried (G), Prepared baked (H); M : DL2000 Maker, N: Negative control; Lanes 1-5: 100%, 10%, 5%, 1%, 0.1%"

Fig. 6

Quantitative PCR to determine the standard curve of adulterated pork in beef Raw meat (A), Dried (B), Steamed (C), Cooked (D), Stewed (E), Pan-fried (F), Fried (G), Baked (H)"

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