食品香菇源性成分荧光PCR检测方法研究

doi:10.19586/j.2095-2341.2025.0075

生物技术进展 ›› 2026, Vol. 16 ›› Issue (1): 149-156.DOI: 10.19586/j.2095-2341.2025.0075

食品香菇源性成分荧光PCR检测方法研究

苏丽娜¹(), 李凤新², 吴杰³, 曾宪东¹(), 郑剑¹()

^1.武汉海关技术中心，武汉 430050
^2.玉林海关综合技术服务中心，广西玉林 537000
^3.中国检验认证集团湖北有限公司，武汉 430000

收稿日期:2025-07-03 接受日期:2025-09-05 出版日期:2026-01-25 发布日期:2026-02-12
通讯作者: 曾宪东,郑剑
作者简介:苏丽娜 E-mail: sulinalove2008@163.com
基金资助:
武汉海关科研项目(2023WK002)

Fluorescence PCR Detection Method for Shiitake-derived Components in Food

Lina SU¹(), Fengxin LI², Jie WU³, Xiandong ZENG¹(), Jian ZHENG¹()

^1.Technical Center of Wuhan Customs，Wuhan 430050，China
^2.Comprehensive Technical Service Center of Yulin Customs，Guangxi Yulin 537000，China
^3.China Certification & Inspection Group Hubei Co. ，Ltd. ，Wuhan 430000，China

Received:2025-07-03 Accepted:2025-09-05 Online:2026-01-25 Published:2026-02-12
Contact: Xiandong ZENG,Jian ZHENG

摘要/Abstract

摘要：

香菇具有重要的食药用价值，市场上掺假的情况屡有发生，传统的形态学鉴定方法难以对香菇深加工产品进行鉴定，建立香菇源性成分分子生物学检测方法具有重要意义。根据香菇线粒体烟酰胺腺嘌呤二核苷酸脱氢酶亚单位1(nicotinamide adenine dinucleotide dehydrogenase subunit 1, ND1)基因序列设计特异性引物和探针，建立食品中香菇源性成分实时荧光PCR检测方法。结果表明，方法仅对花菇、厚菇、薄菇、野生香菇有扩增，对其他常见的28种食用菌均无交叉反应，方法的检出限分别为100 copies·μL^-1和0.05%(质量分数)，且具有很好的重复性，实际应用检测准确。该方法特异性好、灵敏度高，稳定且实用，可以满足食品中香菇源性成分的检测需求。

关键词: 香菇, 烟酰胺腺嘌呤二核苷酸脱氢酶亚单位1, 实时荧光PCR, 源性成分检测

Abstract:

Lentinula edodes （Shiitake mushrooms） possess significant edible and medicinal value. However， adulteration frequently occurs in the marketplace， and traditional morphological identification methods struggle to authenticate L. edodes in deeply processed products. Establishing a molecular biological detection method for L. edodes-derived components is therefore of great importance. In this study， a real-time fluorescent PCR detection method for L. edodes-derived components in food was developed， utilizing specific primers and probes designed based on the sequence of the mitochondrial NADH dehydrogenase subunit 1 （ND1） gene of L. edodes. This method specifically amplified DNA from various L. edodes varieties （including winter shiitake， thick-cap shiitake， thin-cap shiitake， and wild L. edodes）， while showing no cross-reactivity with 28 other common edible fungi. The detection limits of the method were determined to be 100 copies·μL?1 and 0.05% （mass fraction）， with excellent repeatability and verified accuracy in practical applications. This highly specific， sensitive， stable， and practical method effectively meets the requirements for detecting L. edodes-derived components in food products.

Key words: Lentinus edodes, NADH dehydrogenase subunit 1, real-time fluorescent PCR, component detection

中图分类号:

Q523

苏丽娜, 李凤新, 吴杰, 曾宪东, 郑剑. 食品香菇源性成分荧光PCR检测方法研究[J]. 生物技术进展, 2026, 16(1): 149-156.

Lina SU, Fengxin LI, Jie WU, Xiandong ZENG, Jian ZHENG. Fluorescence PCR Detection Method for Shiitake-derived Components in Food[J]. Current Biotechnology, 2026, 16(1): 149-156.

图/表 10

表1 PCR扩增引物和探针

Table 1 Primers and Probes of PCR amplification

引物和探针	序列(5'→3')	扩增大小/bp
L. edodes F4	5'-TTTACTGTCTCCCAACCTGCC-3'	87
L. edodes R4	5'-GCCCTATCCCAGCCTAAAGGA-3'	87
L. edodes P4	5'-TGCCTAGACTCCCTTGCCACCCCAGCCT-3'	-

图1 重组质粒酶切图谱注：1—酶切前质粒；2—酶切后质粒条带；M—DL2000 Plus分子量标准。

Fig. 1 Restriction enzyme digestion analysis of recombinant plasmid

图2 香菇ND1基因的系统发育分析

Fig. 2 Phylogenetic analysis based on ND1 gene of Lentinula edodes

图3 香菇源性成分实时荧光PCR特异性检测结果

Fig. 3 Results of specific detection for Lentinus edodes-derived components by real-time PCR

图4 阳性重组质粒实时荧光PCR标准扩增曲线

Fig. 4 Standard amplification curves of real-time PCR of positive recombinant plasmid

图5 标准曲线

Fig. 5 Standard curves

图6 香菇源性成分实时荧光PCR灵敏性试验结果

Fig. 6 Results of sensitivity test for Lentinus edodes-derived components by real-time PCR

图7 香菇源性成分实时荧光PCR重复性试验结果

Fig. 7 Results of repeatability test for Lentinus edodes-derived components by real-time PCR

表2 香菇源性成分实时荧光PCR重复性验证结果

Table 2 Repeatability verification results of Lentinus edodes-derived components by real-time PCR

香菇质量含量	平均C_t 值	标准偏差	变异系数
0.05%	33.38	1.28%	1.28%
0.50%	28.67	1.17%	1.17%
5%	23.68	1.19%	1.19%
0%	-	-	-

图8 香菇源性成分实时荧光PCR实际应用试验结果

Fig. 8 Results of practical application for Lentinus edodes-derived components by real-time PCR

参考文献 26

[1]	张光亚.中国常见食用菌图鉴[M].昆明:云南科技出版社,1999.
[2]	佩格勒D N,杨T W K,姚一建.香菇的学名及其在当代真菌分类中的位置[J].真菌学报,1993,12(3):226-231.
	PEGLER D N, UK T W K, YAO Y J. Nomenclature of shii-take and its taxonomic position[J]. Mycosystema, 1993, 12(3): 226-231.
[3]	王健,图力古尔,李玉.香菇属(Lentinus)真菌的研究进展兼论中国香菇属的种类资源[J].吉林农业大学学报,2001,23(2):41-45.
	WANG J, TU L, LI Y. Studies on Lentinus and the species of Lentinus in China: a review[J]. J. Jilin Gricultural Univ., 2001, 23(2): 41-45.
[4]	中国科学院生物多样性委员会.中国生物物种名录2024版[M]. 北京:科学出版社,2024.
[5]	卯晓岚.中国香菇属的种类及香菇的自然分布[J].中国食用菌,1996,15(3):34-36.
	MAO X L. Thespicies of Lentinus and distribution of Lentinus edodes in China[J]. Edible Fungi China, 1996, 15(3): 34-36.
[6]	董浩然,姜宁,陆欢,等.香菇多糖结构与功能研究进展[J].生物技术进展,2024,14(6):911-919.
	DONG H R, JIANG N, LU H, et al.. Research progress on structure and function of lentinan[J]. Curr. Biotechnol., 2024, 14(6): 911-919.
[7]	RUKHSAR S, USMAN M, YOUSAF N, et al.. Mushrooms in modern cosmetics: unlocking anti-aging, antioxidant, and therapeutic potential[J/OL]. Arch. Dermatol. Res., 2025, 317(1): 542[2025-10-15]. .
[8]	XIANG Q, LI J, QIN P, et al.. Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes [J/OL]. PLoS One, 2018, 13(1): e0190226[2025-10-15]. .
[9]	谭琦,潘迎捷,黄为一.中国香菇育种的发展历程[J].食用菌学报,2000,7(4):48-52.
	TAN Q, PAN Y J, HUANG W Y. The development process of Lentinula edodes breeding in China[J]. Acta Edulis Fungi, 2000, 7(4): 48-52.
[10]	中国农村网. 我国香菇市场与产业调查分析报告[EB/OL]. [2025-10-15]. .
[11]	中国食用菌协会. 中国食用菌协会公共服务平台[EB/OL]. [2025-10-15]. .
[12]	中国食用菌协会.2023年度全国食用菌统计调查结果分析[J].中国食用菌,2025,44(1):120-129.
	China Edible Fungi Association. Analysis on the results of the national statistical survey of edible fungi in 2023[J]. Edible Fungi China, 2025, 44(1): 120-129.
[13]	ROYSE D J, MAY B. Identification of shiitake genotypes by multilocus enzyme electrophoresis: catalog of lines[J]. Biochem. Genet., 1987, 25(9-10): 705-716.
[14]	FUKUDA M, NAKAI Y F, HIBBETT D S, et al.. Mitochondrial DNA restriction fragment length polymorphisms in natural populations of Lentinula edodes [J]. Mycol. Res., 1994, 98(2): 169-175.
[15]	WU X, LI H, ZHAO W, et al.. SCAR makers and multiplex PCR-based rapid molecular typing of Lentinula edodes strains[J]. Curr. Microbiol., 2010, 61(5): 381-389.
[16]	ZHANG Y, MOLINA F I. Strain typing of Lentinula edodes by random amplified polymorphic DNA assay[J]. FEMS Microbiol. Lett., 1995, 131(1): 17-20.
[17]	张美彦,宋春艳,于海龙,等.基于SNP分型的香菇交配型AS-PCR鉴定[J].食用菌学报,2019,26(2):1-9.
	ZHANG M Y, SONG C Y, YU H L, et al.. Mating-type identification of Lentinula edodes based on SNP genotyping by AS-PCR[J]. Acta Edulis Fungi, 2019, 26(2): 1-9.
[18]	郑梦迪,于佳琦,王明珞.基于DNA条形码的食用菌与其易混有毒真菌的鉴定[J].食品安全质量检测学报,2024,15(1):102-109.
	ZHENG M D, YU J Q, WANG M L. Identification of edible fungi and their easily mixed wild toxic fungi based on DNA barcodes[J]. J. Food Saf. Qual., 2024, 15(1): 102-109.
[19]	魏传正,王朦,张鹏,等.基于二代测序技术的MNP标记鉴别刺芹侧耳菌株[J].食用菌学报,2023,30(1):1-9.
	WEI C Z, WANG M, ZHANG P, et al.. Identification of Pleurotus eryngii strains by MNP makers based on next-generation sequencing[J]. Acta Edulis Fungi, 2023, 30(1): 1-9.
[20]	弓志青,张永琥,沈小刚,等.基于近红外光谱技术快速定量检测香菇面制品中香菇含量的研究[J].中国果菜,2025,45(1):8-12.
	GONG Z Q, ZHANG Y H, SHEN X G, et al.. Research on rapid and quantitative detection of mushroom content in mushroom flour products based on near infrared spectroscopy technology[J]. China Fruit Veg., 2025, 45(1): 8-12.
[21]	孙坤秀.基于显微和化学指纹特征的香菇粉掺假检测方法研究[D].无锡:江南大学,2022.
[22]	王迪.PCR技术在食品微生物检测中的应用研究[J].食品安全导刊,2024(13):187-189.
	WANG D. Study on the application of PCR technique in food microbiological detection[J]. China Food Saf. Mag., 2024(13): 187-189.
[23]	付理文,张宇,依含,等.Taqman多重实时荧光PCR同步定量检测6种动物源性成分方法的建立[J].中国生物工程杂志,2017,37(9):48-59.
	FU L W, ZHANG Y, YI H, et al.. Establishment and application of multiplex fluorescent real-time PCR for detecting six kinds of animal derived materials[J]. China Biotechnol., 2017, 37(9): 48-59.
[24]	JIANG T, LUO S, CHEN Q, et al.. Effect of integrated application of gamma irradiation and modified atmosphere packaging on physicochemical and microbiological properties of shiitake mushroom (Lentinus edodes)[J]. Food Chem., 2010, 122(3): 761-767.
[25]	SUGUI M M, ALVES DE LIMA P L, DELMANTO R D, et al.. Antimutagenic effect of Lentinula edodes (BERK.) Pegler mushroom and possible variation among lineages[J]. Food Chem. Toxicol., 2003, 41(4): 555-560.
[26]	余卉茹,纪艺,彭城,等.肉类掺假检测技术研究进展[J].生物技术进展,2022,12(2):213-221.
	YU H R, JI Y, PENG C, et al.. Research progress on detection technology of meat adulteration[J]. Curr. Biotechnol., 2022, 12(2): 213-221.

食品香菇源性成分荧光PCR检测方法研究

Fluorescence PCR Detection Method for Shiitake-derived Components in Food

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