核桃乳DNA提取方法优化与DNA条形码鉴定

doi:10.3969/j.issn.1004-1524.2022.08.19

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (8): 1752-1761.DOI: 10.3969/j.issn.1004-1524.2022.08.19

核桃乳DNA提取方法优化与DNA条形码鉴定

黄灵芝¹(), 姜广泽², 丁艳菲¹, 朱诚¹^,^*()

1.中国计量大学生命科学学院,浙江省特色农产品品质及危害物控制技术重点实验室,浙江杭州 310018
2.杭州迪安医学检验中心有限公司,浙江杭州 310030

收稿日期:2021-11-03 出版日期:2022-08-25 发布日期:2022-08-26
通讯作者: 朱诚
作者简介:*朱诚,E-mail: pzhch@cjlu.edu.cn
黄灵芝(1998—),女,浙江金华人,硕士研究生,主要从事食品安全控制技术研究。E-mail: Lindseyhlz98@163.com
基金资助:
浙江省高校基本科研业务费(2022YW79);台州市“500精英计划”人才项目

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

摘要/Abstract

摘要：

随着乳饮料行业快速发展,掺杂使假现象层出不穷,对核桃乳真实成分的准确鉴定变的尤为重要。核桃乳属深加工食品,DNA破坏严重,DNA提取是开展核桃乳DNA条形码鉴定的首要环节。为优化核桃乳DNA提取方法,并基于psbA-trnH基因DNA条形码建立核桃乳的掺假造假鉴定方法。以10种不同品牌的核桃乳为样品,采用3种方法(静置抽提、异丙醇沉淀、抽真空冻干)进行预处理,再用2种CTAB裂解沉淀方法和3种试剂盒方法(康为新型植物基因组DNA提取试剂盒、爱思进植物基因组DNA提取试剂盒和天根深加工食品DNA提取试剂盒)提取核桃乳DNA,并在此基础上创新尝试了CTAB与爱思进试剂盒结合方法。以提取的市售核桃乳DNA为模板,利用自行设计的特异性基因psbA-trnH-wal鉴定样品中是否含有核桃成分,确定造假情况;选择常见植物候选基因psbA-trnH鉴定样品中是否含有其他成分,确定掺假情况。结果表明,抽真空冻干预处理方法优于其他2种预处理方式。CTAB与爱思进试剂盒结合方法能提取到纯度好、得率高、扩增能力强的核桃乳DNA,是最佳的核桃乳DNA提取方法。扩增及比对结果显示,1款核桃乳样品中含有花生,属于掺假产品。抽真空冻干预处理、CTAB与爱思进试剂盒结合为优化后的核桃乳DNA提取方法,psbA-trnH-wal基因和psbA-trnH基因结合能够对核桃乳及其掺假造假品进行快速准确的鉴定。

关键词: 核桃乳, DNA提取, DNA条形码, psbA-trnH, 标签验证

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

中图分类号:

TS207.7

黄灵芝, 姜广泽, 丁艳菲, 朱诚. 核桃乳DNA提取方法优化与DNA条形码鉴定[J]. 浙江农业学报, 2022, 34(8): 1752-1761.

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.

图/表 9

参考文献 34

[1]	魏晓璐, 黄鑫, 冯悦, 等. 核桃乳(露)饮品中花生、大豆成分的PCR检测方法[J]. 食品工业科技, 2014, 35(13): 288-291.
	WEI X L, HUANG X, FENG Y, et al. Detection of peanut and soybean in walnut milk using PCR[J]. Science and Technology of Food Industry, 2014, 35(13): 288-291. (in Chinese with English abstract)
[2]	BHARDWAJ R, DOD H, SANDHU M S, et al. Acute effects of diets rich in almonds and walnuts on endothelial function[J]. Indian Heart Journal, 2018, 70(4): 497-501. DOI URL
[3]	HAIDER S, BATOOL Z, TABASSUM S, et al. Effects of walnuts (Juglans regia) on learning and memory functions[J]. Plant Foods for Human Nutrition (Dordrecht, Netherlands), 2011, 66(4): 335-340. DOI URL
[4]	SUH K I, HWANG J M, BAE Y J, et al. Comprehensive DNA barcodes for species identification and discovery of cryptic diversity in mayfly larvae from South Korea: implications for freshwater ecosystem biomonitoring[J]. Entomological Research, 2019, 49(1): 46-54. DOI URL
[5]	HEBERT P D N, CYWINSKA A, BALL S L, et al. Biological identifications through DNA barcodes[J]. Proceedings Biological Sciences, 2003, 270(1512): 313-321. DOI URL
[6]	MARALIT B A, AGUILA R D, VENTOLERO M F H, et al. Detection of mislabeled commercial fishery by-products in the Philippines using DNA barcodes and its implications to food traceability and safety[J]. Food Control, 2013, 33(1): 119-125. DOI URL
[7]	SMITH P J, MCVEAGH S M, STEINKE D. DNA barcoding for the identification of smoked fish products[J]. Journal of Fish Biology, 2008, 72(2): 464-471. DOI URL
[8]	STOECKLE M Y, GAMBLE C C, KIRPEKAR R, et al. Commercial teas highlight plant DNA barcode identification successes and obstacles[J]. Scientific Reports, 2011, 1: 42. DOI URL
[9]	RASMUSSEN R S, MORRISSEY M T, HEBERT P D N. DNA barcoding of commercially important salmon and trout species (Oncorhynchus and Salmo) from North America[J]. Journal of Agricultural and Food Chemistry, 2009, 57(18): 8379-8385. DOI URL
[10]	KUNDU S, SHARMA G, BALAKRISHNAN S, et al. DNA barcoding identified two endangered dolphins: threats on living aquatic mammals in India[J]. Mitochondrial DNA Part B, 2019, 4(1): 77-80. DOI URL
[11]	李梅阁. 基于DNA条形码的浆果及其制品真伪鉴别技术研究[D]. 南京: 南京农业大学, 2016: 29-40.
	LI M G. Authentic identification technique of berry or berry products on DNA barcoding[D]. Nanjing: Nanjing Agricultural University, 2016: 29-40. (in Chinese with English abstract)
[12]	ABDALLAH I B, TLILI N, MARTINEZ-FORCE E, et al. Content of carotenoids, tocopherols, sterols, triterpenic and aliphatic alcohols, and volatile compounds in six walnuts (Juglans regia L.) varieties[J]. Food Chemistry, 2015, 173: 972-978. DOI URL
[13]	PANTH N, PAUDEL K R, KARKI R. Phytochemical profile and biological activity of Juglans regia[J]. Journal of Integrative Medicine, 2016, 14(5): 359-373. DOI URL
[14]	PSIFIDI A, DOVAS C I, BANOS G. A comparison of six methods for genomic DNA extraction suitable for PCR-based genotyping applications using ovine milk samples[J]. Molecular and Cellular Probes, 2010, 24(2): 93-98. DOI URL
[15]	D'ANGELO F, SANTILLO A, SEVI A, et al. Technical note: a simple salting-out method for DNA extraction from milk somatic cells: investigation into the goat CSN1S1 gene[J]. Journal of Dairy Science, 2007, 90(7): 3550-3552. DOI URL
[16]	CAPUANO F, PROROGA Y, MANCUSI A, et al. Evaluation of DNA preparation methods combined with different PCR-based assays for Coxiella burnetii detection in milk[J]. Large Animal Review, 2016, 22(2): 59-62.
[17]	USMAN T, YU Y, LIU C, et al. Comparison of methods for high quantity and quality genomic DNA extraction from raw cow milk[J]. Genetics and Molecular Research: GMR, 2014, 13(2): 3319-3328. DOI URL
[18]	郭梁, 刘国强, 罗建兴, 等. 乳及乳制品中DNA提取方法的比较研究[J]. 农业与技术, 2021, 41(20): 46-50.
	GUO L, LIU G Q, LUO J X, et al. Comparative study on DNA extraction methods from milk and dairy products[J]. Agriculture and Technology, 2021, 41(20): 46-50. (in Chinese)
[19]	任君安, 王国兰, 程曦, 等. 果蔬汁饮料DNA提取方法的比较研究[J]. 食品科技, 2013, 38(2): 42-45.
	REN J N, WANG G L, CHENG X, et al. Comparison of DNA extraction method for fruit and vegetable juices[J]. Food Science and Technology, 2013, 38(2): 42-45. (in Chinese with English abstract)
[20]	韩晴, 王赞, 章晶晶, 等. 基于植物DNA条形码技术对杏仁露中花生源性成分的鉴别研究[J]. 现代食品科技, 2018, 34(2): 232-240.
	HAN Q, WANG Z, ZHANG J J, et al. Identification of peanuts in almond juice based on plant DNA barcode technology[J]. Modern Food Science and Technology, 2018, 34(2): 232-240. (in Chinese with English abstract)
[21]	杨硕, 江丰, 刘艳, 等. 多重微滴式数字PCR定量检测市售核桃乳中核桃、大豆源性成分[J]. 食品科学, 2017, 38(16): 280-286.
	YANG S, JIANG F, LIU Y, et al. Duplex digital droplet PCR for the determination of walnut-derived and soybean-derived ingredients in walnut protein drink[J]. Food Science, 2017, 38(16): 280-286. (in Chinese with English abstract)
[22]	YANO T, SAKAI Y, UCHIDA K, et al. Detection of walnut residues in processed foods by polymerase chain reaction[J]. Bioscience, Biotechnology, and Biochemistry, 2007, 71(7): 1793-1796.
[23]	KURZ C, LEITENBERGER M, CARLE R, et al. Evaluation of fruit authenticity and determination of the fruit content of fruit products using FT-NIR spectroscopy of cell wall components[J]. Food Chemistry, 2010, 119(2): 806-812. DOI URL
[24]	FÜGEL R, CARLE R, SCHIEBER A. Quality and authenticity control of fruit purées, fruit preparations and jams: a review[J]. Trends in Food Science & Technology, 2005, 16(10): 433-441.
[25]	POLLACK S J, KAWALEK M D, WILLIAMS-HILL D M, et al. Evaluation of DNA barcoding methodologies for the identification of fish species in cooked products[J]. Food Control, 2018, 84: 297-304. DOI URL
[26]	CALDWELL J M. Food analysis using organelle DNA and the effects of processing on assays[J]. Annual Review of Food Science and Technology, 2017, 8: 57-74. DOI URL
[27]	邢冉冉, 吴亚君, 陈颖. 宏条形码技术在食品物种鉴定中的应用及展望[J]. 食品科学, 2018, 39(13): 280-288.
	XING R R, WU Y J, CHEN Y. DNA metabarcoding in food species identification: current applications and future prospects[J]. Food Science, 2018, 39(13): 280-288. (in Chinese with English abstract)
[28]	PARDO M Á, JIMÉNEZ E, VIë ARSSON J R, et al. DNA barcoding revealing mislabeling of seafood in European mass caterings[J]. Food Control, 2018, 92: 7-16. DOI URL
[29]	LEE S C, WANG C H, YEN C E, et al. DNA barcode and identification of the varieties and provenances of Taiwan's domestic and imported made teas using ribosomal internal transcribed spacer 2 sequences[J]. Journal of Food and Drug Analysis, 2017, 25(2): 260-274. DOI URL
[30]	UNCU A T, UNCU A O, FRARY A, et al. Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil[J]. Food Chemistry, 2017, 221: 1026-1033. DOI URL
[31]	夏义苗, 陈复生, 郝莉花, 等. 大豆油DNA提取及检测研究进展[J]. 中国食品学报, 2019, 19(10): 351-357.
	XIA Y M, CHEN F S, HAO L H, et al. Research progress of DNA extraction and detection for soybean oil[J]. Journal of Chinese Institute of Food Science and Technology, 2019, 19(10): 351-357. (in Chinese with English abstract)
[32]	董蕾. 不同食品加工工艺对DNA提取和品质的影响[J]. 现代农业科技, 2019(5): 212-215.
	DONG L. Effect of food processing on DNA extraction and quality[J]. Modern Agricultural Science and Technology, 2019(5): 212-215. (in Chinese with English abstract)
[33]	IKEDA S, TSURUMARU H, WAKAI S, et al. Evaluation of the effects of different additives in improving the DNA extraction yield and quality from andosol[J]. Microbes and Environments, 2008, 23(2): 159-166. DOI URL
[34]	SUN Z Y, GAO T, YAO H, et al. Identification of Lonicera japonica and its related species using the DNA barcoding method[J]. Planta Medica, 2011, 77(3): 301-306. DOI URL

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

核桃乳DNA提取方法优化与DNA条形码鉴定

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 34

相关文章 8

编辑推荐

Metrics

本文评价

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

[1]	陈文强, 汪小福, 陈笑芸, 彭城, 徐俊锋, 蔡健. 基于ITS2和SNP技术鉴定浙江铁皮石斛的初步研究[J]. 浙江农业学报, 2021, 33(1): 69-76.
[2]	纪艺, 姜媛媛, 汪小福, 徐晓丽, 徐俊锋, 李玥莹, 陈笑芸. 几种动物生鲜肌肉组织样品DNA提取方法的比较研究[J]. 浙江农业学报, 2019, 31(9): 1471-1477.
[3]	巫伟峰, 陈孝丑, 陈发兴, 陈春, 张毅智. 基于ITS2序列探讨兰属的DNA条形码鉴定和系统发育关系[J]. 浙江农业学报, 2019, 31(8): 1295-1304.
[4]	赵孟良;韩睿;李莉*. 菊芋不同部位DNA提取的比较研究[J]. , 2013, 25(5): 0-996.
[5]	张巧艳;;陈亭亭;陈笑芸;杨胜利;;缪青梅. 基于SYBR Green I荧光定量PCR建立生乳及乳制品沙门氏菌快速检测技术[J]. , 2012, 24(5): 0-921.
[6]	汤江武;王新;姚晓红;吴逸飞;许尧兴;柳永. 棉粕发酵样品总DNA的提取方法[J]. , 2010, 22(4): 0-413.
[7]	魏战勇;梁静怡;李厚伟;王东方;陈红英;崔保安. 精液中猪细小病毒DNA三种提取方法效果比较[J]. , 2009, 21(05): 0-458.
[8]	孙志栋;王学德;倪西源;黄坚 . 棉花DNA提取方法的探讨[J]. , 2004, 16(4): 0-181.