Optimization of DNA extraction method and DNA barcoding identification of walnut milk

doi:10.3969/j.issn.1004-1524.2022.08.19

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (8): 1752-1761.DOI: 10.3969/j.issn.1004-1524.2022.08.19

• Food Science • Previous Articles Next Articles

Optimization of DNA extraction method and DNA barcoding identification of walnut milk

HUANG Lingzhi¹(), JIANG Guangze², DING Yanfei¹, ZHU Cheng¹^,^*()

1. Key Laboratory of Specialty Agri-Product Quality and Hazard Controlling Technology of Zhejiang Province, College of Life Sciences, China Jiliang University, Hangzhou 310018, China
2. Hangzhou Dean Medical Testing Center Co., Ltd., Hangzhou 310030, China

Received:2021-11-03 Online:2022-08-25 Published:2022-08-26
Contact: ZHU Cheng

Abstract

Abstract:

With the rapid development of milk beverage industry, adulteration of milk beverage was common, thus it is particularly important to accurately identify the true ingredients of walnut milk. Walnut milk is a deep-processed food with severe DNA damage. DNA extraction is the first step in DNA barcode identification of walnut milk. The purpose of this experiment was to optimize the DNA extraction method of walnut milk, and to establish an adulteration and fraud identification method based on psbA-trnH gene DNA barcoding. Taking 10 different brands of walnut milk as samples, three methods (static extraction, isopropanol precipitation and vacuum lyophilization) were used for pretreatment. Walnut milk DNA was extracted using two CTAB lysate precipitation methods and three kit methods (Kangwei Nuclean Plant Genomic DNA Kit, AxYGen AxyPrep Multisource Genomic DNA Miniprep Kit, and TianGen Deep Processing Food DNA Extraction Kit). Furthermore, CTAB and AxYGen kit was combined for DNA extraction. Using the extracted commercial walnut milk DNA as a template, the self-designed specific gene psbA-trnH-wal was used to identify whether the sample contained walnut components, and the fraud situation was determined. The common plant candidate gene psbA-trnH was selected to identify whether the sample contained other components, to determine adulteration. Vacuum freezing method was superior to the other two pretreatment methods. Walnut milk extracted DNA using CTAB and AxYGen kit combination method was in good purity, high yield and strong amplification ability, thus it was the best method for walnut milk DNA extraction. Amplification and comparison results showed that one walnut milk sample contained peanuts, which belonged to adulterated products. The vacuum freezing method and the combination of CTAB and AxYGen kit combination method were the optimized methods for DNA extraction from walnut milk. The combination of psbA-trnH-wal gene and psbA-trnH gene could quickly and accurately identify walnut milk and its adulterated fakes.

Key words: walnut milk, DNA extraction, DNA barcode, psbA-trnH, label verification

CLC Number:

TS207.7

HUANG Lingzhi, JIANG Guangze, DING Yanfei, ZHU Cheng. Optimization of DNA extraction method and DNA barcoding identification of walnut milk[J]. Acta Agriculturae Zhejiangensis, 2022, 34(8): 1752-1761.

Figures/Tables 9

References 34

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候选DNA条形码 Candidate DNA barcodes	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')	退火温度 Annealing temperature/℃	扩增大小 Amplification size/bp
CP-03 ^[22]	CGGACGAGAATAAAGATAGAGT	TTTTGGGGATAGAGGGACTTGA	55	123
psbA-trnH-wal	ATCAAGAGGATTGGTATTGCTCC	TCCGCCCCTATACTCCATGT	64	129
psbA-trnH ^[11]	TGTAAAACGGCCAGTGTTATGCATGAACGTAATGCTC	CAGGAAACAGCTATGACCGCGCATGG- TGGATTCACAATCC	64	350

候选DNA条形码 Candidate DNA barcodes	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')	退火温度 Annealing temperature/℃	扩增大小 Amplification size/bp
CP-03 ^[22]	CGGACGAGAATAAAGATAGAGT	TTTTGGGGATAGAGGGACTTGA	55	123
psbA-trnH-wal	ATCAAGAGGATTGGTATTGCTCC	TCCGCCCCTATACTCCATGT	64	129
psbA-trnH ^[11]	TGTAAAACGGCCAGTGTTATGCATGAACGTAATGCTC	CAGGAAACAGCTATGACCGCGCATGG- TGGATTCACAATCC	64	350

样品编号 Sample serial number	方法Method 1		方法Method 2		方法Method 3
样品编号 Sample serial number	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀
1-3	90.85±15.05	2.27±0.03	83.42±10.00	2.36±0.10	154.26±35.66	1.70±0.04
4-6	13.95±12.80	1.79±0.07	77.31±6.26	2.04±0.01	81.56±3.93	1.53±0.06
7-9	204.51±25.86	2.05±0.03	190.12±21.57	2.12±0.04	173.21±28.87	1.78±0.01
10-12	184.10±64.73	2.07±0.06	128.25±35.41	2.16±0.05	164.27±18.64	1.83±0.04
13-15	142.73±49.20	2.10±0.04	122.97±38.71	2.69±0.46	242.48±37.59	1.71±0.06
16-18	105.72±1.53	1.95±0.09	66.73±5.25	2.32±0.12	113.45±22.57	1.43±0.05
19-21	180.61±23.60	2.17±0.03	96.42±10.30	2.39±0.02	160.94±21.39	1.90±0.01
22-24	81.10±12.95	2.51±0.12	65.09±1.80	2.66±0.03	40.31±5.88	1.88±0.05
25-27	107.79±12.40	2.30±0.05	101.78±14.96	2.34±0.05	137.01±24.89	1.50±0.13
28-30	165.78±18.69	2.06±0.02	115.58±3.38	2.26±0.03	172.21±30.21	1.62±0.01

样品编号 Sample serial number	方法Method 1		方法Method 2		方法Method 3
样品编号 Sample serial number	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀
1-3	90.85±15.05	2.27±0.03	83.42±10.00	2.36±0.10	154.26±35.66	1.70±0.04
4-6	13.95±12.80	1.79±0.07	77.31±6.26	2.04±0.01	81.56±3.93	1.53±0.06
7-9	204.51±25.86	2.05±0.03	190.12±21.57	2.12±0.04	173.21±28.87	1.78±0.01
10-12	184.10±64.73	2.07±0.06	128.25±35.41	2.16±0.05	164.27±18.64	1.83±0.04
13-15	142.73±49.20	2.10±0.04	122.97±38.71	2.69±0.46	242.48±37.59	1.71±0.06
16-18	105.72±1.53	1.95±0.09	66.73±5.25	2.32±0.12	113.45±22.57	1.43±0.05
19-21	180.61±23.60	2.17±0.03	96.42±10.30	2.39±0.02	160.94±21.39	1.90±0.01
22-24	81.10±12.95	2.51±0.12	65.09±1.80	2.66±0.03	40.31±5.88	1.88±0.05
25-27	107.79±12.40	2.30±0.05	101.78±14.96	2.34±0.05	137.01±24.89	1.50±0.13
28-30	165.78±18.69	2.06±0.02	115.58±3.38	2.26±0.03	172.21±30.21	1.62±0.01

样品编号 Sample serial number	CTAB方法1 CTAB method 1		CTAB方法2 CTAB method 2		康为试剂盒方法 Kangwei kit
样品编号 Sample serial number	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀
1-3	121.17±71.21	2.51±0.14	54.26±35.66	1.70±0.04	57.64±2.55	1.51±0.07
4-6	500.00±55.96	1.12±0.05	181.56±3.93	1.81±0.06	15.96±5.87	1.66±0.24
7-9	318.33±31.75	1.31±0.08	173.21±28.87	1.78±0.01	9.66±0.06	2.20±0.01
10-12	774.85±287.93	2.64±0.13	164.27±18.64	1.83±0.04	27.78±0.36	1.53±0.07
13-15	848.45±37.32	0.95±0.01	242.48±37.59	1.43±0.05	30.35±2.23	1.80±0.02
16-18	309.10±53.07	1.35±0.05	113.45±22.57	1.71±0.06	10.59±0.77	1.87±0.05
19-21	1 234.48±222.30	2.24±0.10	160.94±21.39	1.90±0.01	8.12±0.29	1.99±0.05
22-24	495.23±162.69	2.84±0.06	40.31±5.88	1.50±0.13	21.75±1.56	1.79±0.05
25-27	649.18±32.82	2.80±0.18	137.01±24.89	1.88±0.05	14.75±1.49	1.81±0.06
28-30	542.05±216.55	2.81±0.03	172.21±30.21	1.62±0.01	14.40±0.90	1.96±0.02
样品编号 Sample serial number	爱思进试剂盒方法 AxYGen kit		天根试剂盒方法 TianGen kit		CTAB与爱思进试剂盒结合方法Combination method of CTAB and AxYGen
样品编号 Sample serial number	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	浓度Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀
1-3	20.38±2.26	1.73±0.02	37.48±4.856	2.05±0.034	107.72±10.263	1.74±0.042
4-6	10.46±1.25	1.70±0.01	37.86±5.121	1.39±0.097	79.89±2.107	1.9±0.06
7-9	16.45±3.49	1.84±0.06	36.24±12.417	1.63±0.135	88.22±2.296	1.80±0.057
10-12	12.84±2.11	1.90±0.09	39.01±7.57	1.70±0.02	83.56±8.10	1.80±0.03
13-15	32.21±4.59	1.88±0.08	72.11±7.22	1.96±0.15	128.35±10.96	1.78±0.10
16-18	13.77±2.44	1.83±0.01	40.21±4.02	1.71±0.10	64.99±4.27	1.74±0.02
19-21	38.48±10.08	1.88±0.06	51.99±8.40	1.99±0.01	95.82±1.89	1.83±0.03
22-24	13.18±0.72	1.75±0.03	91.33±0.47	1.80±0.03	76.81±10.01	1.80±0.02
25-27	14.06±1.23	1.75±0.04	14.38±1.80	1.45±0.05	134.88±4.10	1.84±0.06
28-30	23.83±2.68	1.81±0.06	61.21±10.18	1.87±0.01	118.20±7.02	1.75±0.03

Optimization of DNA extraction method and DNA barcoding identification of walnut milk

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样品编号 Sample serial number	配料表源性成分 Ingredient table source ingredients	psbA-trnH比对结果 psbA-trnH alignment results	相似度 Similarity/%	NCBI相似物种号 NCBI similar species number	拉丁名 Latin name	标签鉴定 Label identification
1-3	优质核桃仁 High-quality walnut kernels	核桃 Walnut	100	MF167464.1	Juglans regia	一致 Consistence
4-6	优质核桃仁 High-quality walnut kernels	核桃 Walnut	100	MF167464.1	Juglans regia	一致 Consistence
7-9	优质核桃仁 High-quality walnut kernels	核桃 Walnut	100	MF167464.1	Juglans regia	一致 Consistence
10-12	优质核桃仁 High-quality walnut kernels	核桃 Walnut	100	MF167464.1	Juglans regia	一致 Consistence
13-15	优质核桃仁 High-quality walnut kernels	核桃 Walnut	100	MF167464.1	Juglans regia	一致 Consistence
16-18	优质核桃仁 High-quality walnut kernels	核桃 Walnut	100	MF167464.1	Juglans regia	一致 Consistence
19-21	优质核桃仁 High-quality walnut kernels	核桃 Walnut	100	MF167464.1	Juglans regia	一致 Consistence
22-24	优质核桃仁 High-quality walnut kernels	花生 Peanut	100	CP030998.1	Arachis hypogaea	不一致 Non-consistence
25-27	优质核桃仁 High-quality walnut kernels	核桃 Walnut	100	MF167464.1	Juglans regia	一致 Consistence
28-30	优质核桃仁 High-quality walnut kernels	核桃 Walnut	99	MF167464.1	Juglans regia	一致 Consistence

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