Fluorescence-based PCR Detection of Goat Milk Powder Mixed with Cow Milk

Yunxia WANG, Lijuan JING, Zhijun LI, Cuizhi LI, Zhiyong LU, Lijun LIU*

1. Quality Management Department, Inner Mongolia Yili Industrial Group Co., Ltd., Hohhot 010110, China; 2. Quality Management Department of Yogurt Division, Inner Mongolia Yili Industrial Group Co., Ltd., Hohhot 010110, China

Abstract [Objectives] To amplify and identify the specific gene fragments of cattle and sheep by real-time fluorescence-based PCR, and quickly and accurately identify the adulteration problem of goat milk powder according to the differences of mitochondrial genes among different species. [Methods] The specificity, limit of detection and repeatability test were carried out by isolating somatic cells, extracting template DNA and determining the reaction conditions of real-time fluorescence-based PCR. [Results] The real-time fluorescence-based PCR method could be used to identify the bovine and ovine-derived materials of milk powder with high specificity, high sensitivity and good repeatability, and the limit of detection of milk components in goat milk powder could reach 0.01%. [Conclusions] The real-time fluorescence-based PCR method can be used to quickly and accurately detect the milk components in goat milk powder, and provide some technical support for the adulteration detection of dairy industry.

Key words Goat milk powder, Cow milk, Real-time fluorescence-based PCR, Animal origin

1 Introduction

With the rapid development of China’s dairy industry and the improvement of people’s health awareness, dairy products have become an important source of nutrition in healthy meals in China. goat milk and its milk powder are sought after by more and more consumers for their advantages of rich nutrition and easy absorption. Although the main components of goat milk and cow milk are similar, the fat granule volume of goat milk is smaller than that of cow milk, which is beneficial to human absorption and closer to human milk. At the same time, goat milk also contains some special nutrients that cow milk does not have, such as epidermal growth factor, and immunoglobulin, which can significantly improve human immunity. In addition, goat milk does not contain proteins that can cause allergies in cow milk, and sensitive people avoid the risk of allergies, so it is ideal nutrient for people.

However, the lactation period of milk goat is affected by the season, and the milk yield is also low, so that the supply of goat milk is limited and the price is much higher than that of cow milk. Therefore, some illegal traders in the market add cow milk to goat milk to reduce costs and reap exorbitant profits, which seriously affects the quality and safety of products, damages the rights and interests of consumers and disrupts market order. This shows the necessity of detection of adulteration of goat milk and its products.

Recently, with the rapid development of molecular biology technology, real-time fluorescence-based PCR assay built upon nucleic acid has the advantages of high sensitivity and specificity, so it can be used for rapid, batch, automatic and real-time detection, and is widely used in adulteration detection of all kinds of food. According to the specific gene sequences of mitochondrial genes among different animal species, the animal-derived materials of goat milk and cow milk were detected and identified by PCR. This method can be used to quickly detect the adulteration behavior in goat milk products and ensure the quality and safety of the products.

2 Materials and methods

2.1 Materials and reagents

Formula goat milk powder for infant and full-fat cow milk powder samples were purchased from the supermarket. Self-made adulterated samples (add 50%, 10%, 1%, 0.1%, 0.01%, 0.001% cow milk powder to formula goat milk powder for infant, respectively).

The broad-spectrum genomic DNA extraction kit (MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0), bovine genomic DNA detection kit and ovine genomic DNA detection kit were all purchased from Takara Bio; p-Octyl polyethylene glycol phenyl ether (Triton-X100) was purchased from Beijing Solarbio Technology Co., Ltd.; anhydrous ethanol and sodium chloride were analytically pure and purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; phosphate buffer (PBS) was analytically pure and purchased from Beijing Luqiao Technology Co., Ltd.

Emulsifying buffer: 430 mL of sodium chloride solution with a concentration of 0.9 g/L was taken and mixed with 10 mL of p-Octyl polyethylene glycol phenyl ether (Triton-X100) and 60 mL of absolute ethanol for later use.

2.2 Instruments and equipment

The analytical balance was purchased from Mettler Toledo in Switzerland; the real-time fluorescence-based PCR instrument was purchased from Bio-Rad Company in the United States; the micro nucleic acid and protein analyzer was purchased from Bio-Drop Company in the United States; high-speed freezing centrifuge and mini desktop centrifuge were purchased from Thermo-Fisher Company in the United States; the micro pipette was purchased from Eppendorf Company in Germany.

2.3 Methods

2.3.1

Isolation of somatic cells. The formula goat milk powder for infant, whole cow milk powder and self-made adulterated samples were prepared into restored milk at 1∶8, and 40-50 mL of restored milk was pipetted and placed in a 4 ℃ freezing centrifuge. The supernatant was discarded after centrifugation with 3 000 r/min for 15 min. Precipitate at the bottom was suspended with 1 mL of PBS buffer and transferred to the 2 mL centrifuge tube, centrifuged with 3 000 r/min for 10 min, and the supernatant was discarded. 1 mL of PBS buffer was added to the centrifuge tube to suspend and precipitate, and 1 000 r/min was used to centrifuge for 10 min, and the supernatant was discarded after 1 000 r/min centrifugation for 10 min. After 150 μL of emulsified buffer and 1 mL of PBS buffer were added to suspend precipitate, it was heated 10 min in a thermostatic water bath at 40 ℃, and centrifuged at 3 000 r/min for 10 min, and the supernatant was discarded. The somatic cells were obtained by adding 1 mL of PBS buffer to wash the precipitate and centrifuging at 12 000 r/min for 10 min.

2.3.2

Template DNA extraction. The above somatic cells were placed in a 2 mL centrifuge tube, and the broad-spectrum genomic DNA extraction kit was used to extract and purify the template DNA. It could be used for fluorescence-based PCR detection when its mass and concentration were determined by micro nucleic acid protein analyzer (

OD

/

OD

value is between 1.7 and 2.0).

2.3.3

Fluorescence-based PCR detection. Using bovine genomic DNA detection kit, a 25 μL reaction system was prepared. 2×Premix for Bovine: 12.5 μL; Primer Mix for Bovine: 1 μL; Probe Mix for Bovine: 1 μL; template DNA: 1 μL; ddHO: 9.5 μL.

Using goat-derived genomic DNA detection kit, a 25 μL reaction system was prepared. 2×Premix for Ovine: 12.5 μL; Primer Mix for Ovine: 1 μL; Probe Mix for Ovine: 1 μL; template DNA: 1 μL; ddHO: 9.5 μL.

The fluorescence-based PCR reaction conditions for the above two reaction systems were as follows: 95 ℃ 10 s, 95 ℃ 5 s, 60 ℃ 30 s, 40 cycles; FAM fluorophore was selected, and positive control, negative control and blank control were set at the same time in each experiment.

2.3.4

Result determination. After the end of the fluorescence-based PCR reaction, the results were determined according to the

C

value and the amplification curve. Under the condition that the positive control, negative control and blank control were all normal, the animal-derived component was determined to be detected if FAM fluorescence was detected and

C

≤35. If no FAM fluorescence was detected or

C

>35, it was determined that the animal-derived component was not detected.

3 Results and analysis

3.1 Extraction result

The genomic template DNA of formula goat milk powder for infant, full-fat cow milk powder and self-made adulterated samples was extracted. The

OD

/

OD

was 1.84, 1.79 and 1.82, respectively. The test of specificity, limit of detection and reproducibility was carried out.

3.2 Specificity test

The template DNA of formula goat milk powder for infant and full-fat cow milk powder was used for the specificity test of real-time fluorescence-based PCR. At the same time, sterile water was used as blank control, DNA of chicken samples as negative control, control DNA for Ovine and control DNA for Bovine in the kit as positive control. The results are shown in Fig.1-2.

Note: 1-2: ovine-derived positive control; 3-4: sample of formula goat milk power for infant; 5-6: bovine-derived positive control; 7-8: full-fat milk powder sample; 9-10: blank control; 11-12: negative control.

Note: 1-2: bovine-derived positive control; 3-4: full-fat milk powder sample; 5-6: ovine-derived positive control; 7-8: sample of formula goat milk power for infant; 9-10: blank control; 11-12: negative control.

From Fig.1 to 2, we can see that the test results of blank control, negative control and positive control were all normal, ovine-derived materials were detected in the sample of formula goat milk power for infant, but no bovine-derived materials were detected. Bovine-derived materials were detected in full-fat cow milk powder sample, but no ovine-derived materials were detected, indicating that this method had good specificity for the detection of bovine-derived and ovine-derived materials.

3.3 Limit of detection of bovine-derived materials

The template DNA of bovine-derived positive quality control and self-made adulterated sample was taken for test of limit of detection, with sterile water as blank control, chicken sample DNA as negative control, and control DNA for Bovine in the kit as positive control. The results are shown in Fig.3.

It can be seen from Fig.3 that the test results of blank control, negative control and positive control were all normal, and the limit of detection of bovine-derived materials in self-made adulterated sample could reach 0.01%. It was determined that this method had high sensitivity for detecting goat milk powder mixed with bovine-derived materials.

3.4 Repetition detection

The template DNA of formula goat milk powder for infant and full-fat cow milk powder was taken for replication experiment, respectively, and each sample was tested 6 times. At the same time, sterile water was used as blank control, chicken sample DNA as negative control, control DNA for Bovine and control DNA for Ovine in the kit as positive control. The statistical results are shown in Table 1.

Note: 1-2: bovine-derived positive quality control; 3-4: 50% cow milk powder sample; 5-6: 10% cow milk powder sample; 7-8: 1% cow milk powder sample; 9-10: 0.1% cow milk powder sample; 11-12: 0.01% milk powder sample; 13-14: 0.001% milk powder sample; 15-16: blank control; 17-18: negative control.

Table 1 Repetition detection of bovine-derived and ovine-derived materials by fluorescence-based PCR

It can be seen from Table 1 that the test results of blank control, negative control and positive control were all normal, and each sample was tested for 6 times, and the results were accurate and stable, indicating that the stability and repeatability of this method were good.

3.5 Actual testing of products sold in the market

5 batches of products sold in the market were purchased (No.1: formula goat milk powder for infant, No.2: full-fat nutritious cow milk powder, No.3: skimmed cow milk powder, No.4: pure cow milk, No.5: pure goat milk), and the above real-time fluorescence-based PCR detection method was used to verify the actual detection effect. The results are shown in Table 2.

Table 2 Testing of products sold in the market

It can be seen from Table 2 that the test results of blank control, negative control and positive control were all normal, and the test results of 5 batches of products sold in the market were consistent with the ID of samples and the actual animal-derived materials, indicating that the method was accurate, sensitive, stable and applicable.

4 Conclusions

In this paper, bovine-derived and ovine-derived materials of milk powder were identified by real-time fluorescence-based PCR. The above verification experiments show that this method is fast, accurate, specific, repeatable and sensitive. The tested market samples are completely consistent with the actual animal-derived materials contained in each sample. This proves that the method has good applicability and is suitable for raw materials of fresh goat milk, sterilized milk after processing and milk powder products. This provides an effective technical means for dairy enterprises to monitor their products and raw materials, and effectively ensure the quality and safety of food.