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Article(id=1153433694023963045, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433686872679135, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241030001, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1730217600000, receivedDateStr=2024-10-30, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752929622416, onlineDateStr=2025-07-19, pubDate=1744646400000, pubDateStr=2025-04-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752929622416, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752929622416, creator=13701087609, updateTime=1752929622416, updator=13701087609, issue=Issue{id=1153433686872679135, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='7', pageStart='1', pageEnd='322', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752929620712, creator=13701087609, updateTime=1757656380159, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1173259152974561742, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433686872679135, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1173259152978756047, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153433686872679135, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=12, endPage=18, ext={EN=ArticleExt(id=1153433694833463739, articleId=1153433694023963045, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Identification of Tricholoma matsutake and its counterfeits based on TaqMan real-time fluorescence polymerase chain reaction technology and DNA barcoding technology, columnId=1153433689657692523, journalTitle=Journal of Food Safety & Quality, columnName=Highlight: Technology Centre of Dalian Customs, runingTitle=null, highlight=null, articleAbstract=

Objective To establish a method for qualitative determination of Tricholoma matsutake ingredients by a TaqMan real-time fluorescence polymerase chain reaction (PCR), and identify the conformity of Tricholoma matsutake and its products with their labels by DNA barcoding technology. Methods Taking the pol gene of Tricholoma matsutake as the target gene, specific primers and probes were designed to study and analyze their specificity, sensitivity and repeatability. The established TaqMan real-time PCR method of Tricholoma matsutake was used to detect different types of commercially available samples and identify their authenticity. Results The established method was used for real-time PCR detection, and there was no cross-reactivity between Tricholoma matsutake and 32 kinds of other edible fungi, animals and plants, indicading strong specificity of the method. The sensitivity was 0.01% Tricholoma matsutake and 0.01 ng/μL Tricholoma matsutake genomic DNA. The authenticity test of 160 commercially available Tricholoma matsutake and their products showed that there was counterfeiting of dried Tricholoma matsutake. The test results did not match the labels at a rate of 36.70%. DNA barcoding technology was used to further identify the species of counterfeit matsutake mushrooms, with species attributes of Stropharia rugosoannulata, Tricholoma giganteum and Tricholoma bakamatsutake. The non-compliance rates with labels in the authenticity testing resultes of other collected matsutake products were 23.70%-60.00%, respectively. Conclusion This method has high sensitivity and strong specificity, which can quickly and accurately identify the authenticity and compliance of Tricholoma matsutake ingredients.

, correspAuthors=Ai-Fu YANG, Jing XU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Ai-Fu YANG, Chao WAN, Xin QI, Xue-Hua LIU, Li JIANG, Jing XU), CN=ArticleExt(id=1153433721928667363, articleId=1153433694023963045, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=基于TaqMan实时荧光聚合酶链式反应和DNA条形码技术鉴别松茸及其伪品, columnId=1153433689825464685, journalTitle=食品安全质量检测学报, columnName=本期重点：大连海关, runingTitle=null, highlight=null, articleAbstract=

目的 建立松茸(Tricholoma matsutake)成分的TaqMan实时荧光聚合酶链反应(polymerase chain reaction, PCR)定性检测方法, 并结合DNA条形码技术鉴定松茸及其制品成分与其标签的符合性。方法 以松茸的pol基因为靶基因, 设计特异性引物及探针, 对其特异性、灵敏度、重复性开展研究分析, 并将建立的松茸成分TaqMan实时荧光PCR检测方法对不同种类市售松茸样品开展检测, 鉴别其真实性。结果 采用建立的TaqMan实时荧光PCR方法进行检测, 松茸与其他32种食用菌和动植物样品均无交叉反应, 方法特异性强; 灵敏度为质量分数0.01%的松茸和0.01 ng/μL松茸基因组DNA; 对市售160份松茸及其制品开展真实性检测, 研究表明松茸干品存在假冒现象, 检测结果与标签不符合率为36.70%, 采用DNA条形码技术进一步对假冒松茸的物种进行鉴定, 物种属性为皱环球盖菇(Stropharia rugosoannulata)、巨大口蘑(Tricholoma giganteum)和栎松口蘑(Tricholoma bakamatsutake), 其他收集的松茸制品成分检测结果中与标签不符合率分别为23.70%~60.00%。结论 该方法灵敏度高, 特异性强, 可快速精准地对松茸成分进行真实性和符合性鉴别。

, correspAuthors=杨爱馥, 徐静, authorNote=null, correspAuthorsNote=
* 杨爱馥(1978—), 女, 博士, 正高级工程师, 主要研究方向为食品安全检验。E-mail:
* 徐静(1982—), 女, 博士, 研究员, 主要研究方向为食品安全检验。E-mail:
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Food Science and Technology, 2024, 49(5): 342-348., articleTitle=Establishment of visual nucleic acid test strip for rapid identification of Tricholoma Matsutake and its products, refAbstract=null)], funds=[Fund(id=1173278635315311013, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, awardId=2023HK134, language=CN, fundingSource=海关总署科研项目(2023HK134), fundOrder=null, country=null), Fund(id=1173278635369836966, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, awardId=2023DK08, language=CN, fundingSource=大连海关科研项目(2023DK08), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1173278628411486559, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, xref=null, ext=[AuthorCompanyExt(id=1173278628419875168, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, companyId=1173278628411486559, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Technology Center of Dalian Customs District, Dalian 116001, China), AuthorCompanyExt(id=1173278628428263777, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, companyId=1173278628411486559, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=大连海关技术中心, 大连 116001)])], figs=[ArticleFig(id=1173278631724986763, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=EN, label=Fig.1, caption=Detection results of 18S rRNA for Tricholoma matsutake and other samples, figureFileSmall=ZgQKwSEyRW339vR3ADxK2w==, figureFileBig=jyWScHXU+cY2ayHIFLePHA==, tableContent=null), ArticleFig(id=1173278631834038668, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=CN, label=图1, caption=松茸及其他试验样品18S rRNA检测结果

注: 0.04线条为荧光阈值, 下图同。

, figureFileSmall=ZgQKwSEyRW339vR3ADxK2w==, figureFileBig=jyWScHXU+cY2ayHIFLePHA==, tableContent=null), ArticleFig(id=1173278631909536141, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=EN, label=Fig.2, caption=Specific detection results of Tricholoma matsutake by TaqMan real-time fluorescence PCR, figureFileSmall=hgLQZLLVhtGQ1zEA1xWVRg==, figureFileBig=dHE8NiwgkRWnfeiRTDrDmw==, tableContent=null), ArticleFig(id=1173278632006005134, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=CN, label=图2, caption=松茸成分TaqMan实时荧光PCR特异性检测结果

注: 阴性对照: 27种其他食用菌样品和5种动、植物样品; 空白对照: ddH2O。

, figureFileSmall=hgLQZLLVhtGQ1zEA1xWVRg==, figureFileBig=dHE8NiwgkRWnfeiRTDrDmw==, tableContent=null), ArticleFig(id=1173278632094085519, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=EN, label=Fig.3, caption=Sensitivity test results of simulated samples, figureFileSmall=qSaRuIXOvjue4Ao88hZ0tg==, figureFileBig=MeexPUzhWxIzbjUWF1CZCQ==, tableContent=null), ArticleFig(id=1173278632169582993, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=CN, label=图3, caption=模拟样品灵敏度检测结果

注: 1. 100.00%松茸; 2. 10.00%松茸; 3. 1.00%松茸; 4. 0.10%松茸; 5. 0.01%松茸; 6. 空白对照ddH2O。

, figureFileSmall=qSaRuIXOvjue4Ao88hZ0tg==, figureFileBig=MeexPUzhWxIzbjUWF1CZCQ==, tableContent=null), ArticleFig(id=1173278632240886162, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=EN, label=Fig.4, caption=Sensitivity detection results of different genomic DNA mass concentrations, figureFileSmall=0zzuRcm1M135dBeE2zBVFA==, figureFileBig=xdBujw7d/rrJRrMv8owX6A==, tableContent=null), ArticleFig(id=1173278632312189332, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=CN, label=图4, caption=不同基因组DNA质量浓度灵敏度检测结果

注: 1. 100.00 ng/μL; 2. 10.00 ng/μL; 3. 1.00 ng/μL; 4. 0.10 ng/μL; 5. 0.01% ng/μL; 6.空白对照ddH2O。

, figureFileSmall=0zzuRcm1M135dBeE2zBVFA==, figureFileBig=xdBujw7d/rrJRrMv8owX6A==, tableContent=null), ArticleFig(id=1173278632433824150, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=EN, label=Table 1, caption=

Primer and probe sequence information by TaqMan real-time fluorescence PCR

, figureFileSmall=null, figureFileBig=null, tableContent=
类别 引物名称 引物序列(5'-3')
上游引物 Pol-F CAGATGCAGCGTCACCTATCC
下游引物 Pol-R GGCTGAACGGTTCTCAATCAC
探针 Pol-P FAM-AGGCAGACGTGATGGATGG AATACCTGTCAAG-TAMRA
), ArticleFig(id=1173278632513515928, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=CN, label=表1, caption=

TaqMan实时荧光PCR法引物探针序列信息

, figureFileSmall=null, figureFileBig=null, tableContent=
类别 引物名称 引物序列(5'-3')
上游引物 Pol-F CAGATGCAGCGTCACCTATCC
下游引物 Pol-R GGCTGAACGGTTCTCAATCAC
探针 Pol-P FAM-AGGCAGACGTGATGGATGG AATACCTGTCAAG-TAMRA
), ArticleFig(id=1173278632609984922, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=EN, label=Table 2, caption=

Species attribute identification names of Tricholoma matsutake and other edible mushrooms

, figureFileSmall=null, figureFileBig=null, tableContent=
样品编号 样品名称 鉴定结果
1 松茸(吉林延边) Tricholoma matsutake
2 松茸(四川甘孜) Tricholoma matsutake
3 松茸(云南香格里拉) Tricholoma matsutake
4 姬松茸 Agaricus blazei
5 赤松茸 Stropharia rugosoannulata
6 白松茸 Tricholoma giganteum
7 黑松露 Tuber indicum
8 白松露 Tuber huidongense
9 金针菇 Flammulina yunnanensis
10 褐盖牛肝菌 Boletus umbrinipileus
11 网纹牛肝菌 Boletus reticulatus
12 黄牛肝菌 Leccinum aurantiacum
13 白牛肝菌 Boletus bainiugan
14 皱盖牛肝菌 Rugiboletus extremiorientalis
15 黑牛肝菌 Neoboletus obscureumbrinus
16 羊肚菌 Morchella sextelata
17 元蘑 Hohenbuehelia serotina
18 白口蘑 Tricholoma album
19 栎松口蘑 Tricholoma bakamatsutake
20 黑虎掌菌 Sarcodon aspratus
21 杏鲍菇 Pleurotus eryngii
22 猴头菌 Hericium erinaceus
23 黑木耳 Auricularia auricula
24 鸡油菌 Cantharellus yunnanensis
25 香菇 Lentinula edodes
26 黑皮鸡枞 Oudemansiella raphanipies
27 银耳 Tremella fuciformis
28 茶树菇 Agrocybe salicaciola
29 红菇 Russula vinosa
30 竹荪 Dictyophora indusiata
), ArticleFig(id=1173278632740008347, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=CN, label=表2, caption=

松茸和其他食用菌物种属性鉴定名称

, figureFileSmall=null, figureFileBig=null, tableContent=
样品编号 样品名称 鉴定结果
1 松茸(吉林延边) Tricholoma matsutake
2 松茸(四川甘孜) Tricholoma matsutake
3 松茸(云南香格里拉) Tricholoma matsutake
4 姬松茸 Agaricus blazei
5 赤松茸 Stropharia rugosoannulata
6 白松茸 Tricholoma giganteum
7 黑松露 Tuber indicum
8 白松露 Tuber huidongense
9 金针菇 Flammulina yunnanensis
10 褐盖牛肝菌 Boletus umbrinipileus
11 网纹牛肝菌 Boletus reticulatus
12 黄牛肝菌 Leccinum aurantiacum
13 白牛肝菌 Boletus bainiugan
14 皱盖牛肝菌 Rugiboletus extremiorientalis
15 黑牛肝菌 Neoboletus obscureumbrinus
16 羊肚菌 Morchella sextelata
17 元蘑 Hohenbuehelia serotina
18 白口蘑 Tricholoma album
19 栎松口蘑 Tricholoma bakamatsutake
20 黑虎掌菌 Sarcodon aspratus
21 杏鲍菇 Pleurotus eryngii
22 猴头菌 Hericium erinaceus
23 黑木耳 Auricularia auricula
24 鸡油菌 Cantharellus yunnanensis
25 香菇 Lentinula edodes
26 黑皮鸡枞 Oudemansiella raphanipies
27 银耳 Tremella fuciformis
28 茶树菇 Agrocybe salicaciola
29 红菇 Russula vinosa
30 竹荪 Dictyophora indusiata
), ArticleFig(id=1173278632857448860, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=EN, label=Table 3, caption=

Repeatability and stability results of TaqMan real-time fluorescence PCR for Tricholoma matsutake detection (n=3)

, figureFileSmall=null, figureFileBig=null, tableContent=
项目 DNA质量浓度/(ng/μL) Ct值 变异系数/%
批内重复 100.00 22.58±0.10 0.46
10.00 25.23±0.15 0.61
1.00 28.64±0.11 0.40
批间重复 100.00 22.94±0.31 1.35
10.00 25.48±0.38 1.49
1.00 29.00±0.31 1.07
), ArticleFig(id=1173278633054581149, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=CN, label=表3, caption=

松茸成分TaqMan实时荧光PCR方法重复性和稳定性试验结果(n=3)

, figureFileSmall=null, figureFileBig=null, tableContent=
项目 DNA质量浓度/(ng/μL) Ct值 变异系数/%
批内重复 100.00 22.58±0.10 0.46
10.00 25.23±0.15 0.61
1.00 28.64±0.11 0.40
批间重复 100.00 22.94±0.31 1.35
10.00 25.48±0.38 1.49
1.00 29.00±0.31 1.07
), ArticleFig(id=1173278633172021662, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=EN, label=Table 4, caption=

Label compliance test resultsof commercial Tricholoma matsutake

, figureFileSmall=null, figureFileBig=null, tableContent=
样品名称 样品数量
/份		 松茸成分阳性样品数量
/份		 松茸成分阳性比例
/%		 检测结果与标签不符比例
/%
松茸鲜品 15 15 100.00 -
松茸冻品 15 15 100.00 -
松茸干品 30 19 63.30 36.70
松茸酱 20 13 65.00 35.00
松茸调味粉 20 9 45.00 55.00
松茸饼干
(米粉)		 15 10 66.60 23.70
杂菌包干货(标签含松茸) 15 9 60.00 40.00
杂菌碎干货(标签含松茸) 10 4 40.00 60.00
杂菌包干货
(标签不含松茸)		 10 0 - -
杂菌碎干货(标签不含松茸) 10 0 - -
), ArticleFig(id=1173278635126567327, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153433694023963045, language=CN, label=表4, caption=

市售松茸商品标签符合性检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
样品名称 样品数量
/份		 松茸成分阳性样品数量
/份		 松茸成分阳性比例
/%		 检测结果与标签不符比例
/%
松茸鲜品 15 15 100.00 -
松茸冻品 15 15 100.00 -
松茸干品 30 19 63.30 36.70
松茸酱 20 13 65.00 35.00
松茸调味粉 20 9 45.00 55.00
松茸饼干
(米粉)		 15 10 66.60 23.70
杂菌包干货(标签含松茸) 15 9 60.00 40.00
杂菌碎干货(标签含松茸) 10 4 40.00 60.00
杂菌包干货
(标签不含松茸)		 10 0 - -
杂菌碎干货(标签不含松茸) 10 0 - -
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基于TaqMan实时荧光聚合酶链式反应和DNA条形码技术鉴别松茸及其伪品
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杨爱馥 * , 万超 , 齐欣 , 刘雪华 , 姜丽 , 徐静 *
食品安全质量检测学报 | 本期重点：大连海关 2025,16(7): 12-18
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食品安全质量检测学报 | 本期重点：大连海关 2025, 16(7): 12-18
基于TaqMan实时荧光聚合酶链式反应和DNA条形码技术鉴别松茸及其伪品
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杨爱馥* , 万超, 齐欣, 刘雪华, 姜丽, 徐静*
作者信息
  • 大连海关技术中心, 大连 116001

通讯作者:

* 杨爱馥(1978—), 女, 博士, 正高级工程师, 主要研究方向为食品安全检验。E-mail:
* 徐静(1982—), 女, 博士, 研究员, 主要研究方向为食品安全检验。E-mail:
Identification of Tricholoma matsutake and its counterfeits based on TaqMan real-time fluorescence polymerase chain reaction technology and DNA barcoding technology
Ai-Fu YANG* , Chao WAN, Xin QI, Xue-Hua LIU, Li JIANG, Jing XU*
Affiliations
  • Technology Center of Dalian Customs District, Dalian 116001, China
出版时间: 2025-04-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241030001
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摘要
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目的 建立松茸(Tricholoma matsutake)成分的TaqMan实时荧光聚合酶链反应(polymerase chain reaction, PCR)定性检测方法, 并结合DNA条形码技术鉴定松茸及其制品成分与其标签的符合性。方法 以松茸的pol基因为靶基因, 设计特异性引物及探针, 对其特异性、灵敏度、重复性开展研究分析, 并将建立的松茸成分TaqMan实时荧光PCR检测方法对不同种类市售松茸样品开展检测, 鉴别其真实性。结果 采用建立的TaqMan实时荧光PCR方法进行检测, 松茸与其他32种食用菌和动植物样品均无交叉反应, 方法特异性强; 灵敏度为质量分数0.01%的松茸和0.01 ng/μL松茸基因组DNA; 对市售160份松茸及其制品开展真实性检测, 研究表明松茸干品存在假冒现象, 检测结果与标签不符合率为36.70%, 采用DNA条形码技术进一步对假冒松茸的物种进行鉴定, 物种属性为皱环球盖菇(Stropharia rugosoannulata)、巨大口蘑(Tricholoma giganteum)和栎松口蘑(Tricholoma bakamatsutake), 其他收集的松茸制品成分检测结果中与标签不符合率分别为23.70%~60.00%。结论 该方法灵敏度高, 特异性强, 可快速精准地对松茸成分进行真实性和符合性鉴别。

松茸  /  TaqMan实时荧光聚合酶链式反应技术  /  DNA条形码技术  /  快速检测  /  真实性鉴别

Objective To establish a method for qualitative determination of Tricholoma matsutake ingredients by a TaqMan real-time fluorescence polymerase chain reaction (PCR), and identify the conformity of Tricholoma matsutake and its products with their labels by DNA barcoding technology. Methods Taking the pol gene of Tricholoma matsutake as the target gene, specific primers and probes were designed to study and analyze their specificity, sensitivity and repeatability. The established TaqMan real-time PCR method of Tricholoma matsutake was used to detect different types of commercially available samples and identify their authenticity. Results The established method was used for real-time PCR detection, and there was no cross-reactivity between Tricholoma matsutake and 32 kinds of other edible fungi, animals and plants, indicading strong specificity of the method. The sensitivity was 0.01% Tricholoma matsutake and 0.01 ng/μL Tricholoma matsutake genomic DNA. The authenticity test of 160 commercially available Tricholoma matsutake and their products showed that there was counterfeiting of dried Tricholoma matsutake. The test results did not match the labels at a rate of 36.70%. DNA barcoding technology was used to further identify the species of counterfeit matsutake mushrooms, with species attributes of Stropharia rugosoannulata, Tricholoma giganteum and Tricholoma bakamatsutake. The non-compliance rates with labels in the authenticity testing resultes of other collected matsutake products were 23.70%-60.00%, respectively. Conclusion This method has high sensitivity and strong specificity, which can quickly and accurately identify the authenticity and compliance of Tricholoma matsutake ingredients.

Tricholoma matsutake  /  TaqMan real-time fluorescence polymerase chain reaction  /  DNA barcoding technology  /  rapid detection  /  authenticity identification
杨爱馥, 万超, 齐欣, 刘雪华, 姜丽, 徐静. 基于TaqMan实时荧光聚合酶链式反应和DNA条形码技术鉴别松茸及其伪品. 食品安全质量检测学报, 2025 , 16 (7) : 12 -18 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241030001
Ai-Fu YANG, Chao WAN, Xin QI, Xue-Hua LIU, Li JIANG, Jing XU. Identification of Tricholoma matsutake and its counterfeits based on TaqMan real-time fluorescence polymerase chain reaction technology and DNA barcoding technology[J]. Journal of Food Safety & Quality, 2025 , 16 (7) : 12 -18 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241030001
正文
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0 引言
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松茸(Tricholoma matsutake)又称松口蘑, 属于世界上珍贵的药食同源性天然野生菌类[1-3]。因含有松茸醇、松茸多糖及松茸多肽等活性物质, 营养和商业价值极高, 更有“菌中之王”的美誉[4-7]。
我国作为松茸生产和出口的主要国家之一, 每年有大量优质的松茸出口至欧洲和日本。由于松茸是我国国家二级重点保护野生植物, 属于国家限制出口货物; 同时因松茸现无人工栽培种植技术且自然产量极其有限, 供不应求的市场关系导致其经济价值急剧上升, 市场价格更是居高不下[8-9]。为了获取更高利润, 一些商家将名字中含有“松茸”二字的姬松茸、白松茸、赤松茸等其他廉价菌类按照松茸价格进行售卖, 对消费者造成误导, 更有不法商家销售假冒或掺假的松茸产品[10]。
目前, 松茸的鉴定方法主要包含形态特征识别[11-12]及理化检测方法[13-14], 但受形态完整性和加工工艺等因素的影响, 难以实现松茸的精准和快速鉴别[15]。TaqMan实时荧光聚合酶链式反应(polymerase chain reaction, PCR)技术及DNA条形码技术因操作简便、特异性强、稳定性高且不受样品基质状态影响, 已成为物种鉴定及食品真实性检测公认的主流手段[16-18], 并在深加工食品鉴别[19-20]、微生物检测[21-22]、动植物源性成分鉴定[23-25]等领域广泛应用。其中, 核糖体内转录间隔区(internal transcribed space, ITS)是真菌公认的通用DNA条形码。AOKI等[26]采用DNA条形码技术中ITS分子标记验证了来自8个国别松茸样品的系统进化树可聚成一个松茸群。MURATA等[27]基于PCR方法将3类反转录转座子和单拷贝基因进行比较, 通过分析它们之间的基因组拷贝数以阐明松茸的系统发育树, 更有效地区分与松茸相近的其他物种。周亭亭等[28]采用DNA指纹鉴定技术将松茸及其制品与其他7种易伪品进行鉴别, 研究表明方法可有效区别松茸部分伪品。但面对目前我国没有松茸真伪鉴别检验标准的现状, 急需建立一种简便、准确、高效的松茸鉴伪方法, 为海关执法及市场监管提供技术保障[29]。
本研究靶向松茸的Pol特异性基因序列, 建立基于TaqMan实时荧光PCR检测技术的松茸成分快速、精准的检测方法, 适用于原料及其制品中松茸成分的真伪鉴别, 旨在为保护消费者权益、营造良好营商环境、促进我国松茸产业健康稳定发展提供技术保障。
1 材料与方法
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1.1 材料与试剂
收集产自吉林延边、四川甘孜和云南香格里拉地区的松茸样品各10份; 松茸易伪品食用菌3种, 包括姬松茸、赤松茸、白松茸; 其他食用菌24种, 包括黑松露、白松露、金针菇、褐盖牛肝菌、网纹牛肝菌、黄牛肝菌、白牛肝菌、皱盖牛肝菌、黑牛肝菌、羊肚菌、元蘑、白口蘑、栎松口蘑、黑虎掌菌、杏鲍菇、猴头菌、黑木耳、鸡油菌、香菇、黑皮鸡枞、银耳、茶树菇、红菇、竹荪; 其他异源性动、植物样品共计5种, 包括猪肉、鳕鱼、鸡肉、牛肉、大豆。所收集样品均采购于吉林、四川、云南和大连地区各农贸市场及超市。
植物基因组DNA提取试剂盒(货号DP305, 天根生化科技北京有限公司); PrimeSTAR® Max DNA Polymerase (货号R045Q)、Premix Ex TaqTM Probe qPCR(货号RR390 A)[宝生物工程(大连)有限公司]; 引物、探针及扩增产物均委托华大基因合成和测序。
1.2 仪器与设备
QuantStudio6Flex实时荧光定量PCR仪(美国ABI公司); 5415R小型高速冷冻离心机(德国Eppendorf公司); DK-8D电热恒温水浴锅(上海一恒科学仪器有限公司); Epoch酶标仪(美国Biotek公司)。
1.3 试验方法
1.3.1 样品的处理及DNA的提取
将收集的样品经研磨处理后, 按照植物基因组DNA提取试剂盒操作说明进行所有样品的DNA提取, 并使用酶标仪测定提取基因组DNA的纯度和浓度。
1.3.2 物种属性鉴定
依据行业标准SN/T 4625—2016《DNA条形码筛选与质量要求》中ITS引物序列(ITS5/ITS4), 对收集的松茸及其他食用菌样品基因组DNA进行PCR扩增反应, 并将扩增产物进行测序, 扩增片段序列在美国国家生物技术信息中心(National Center of Biotechnology Information, NCBI)进行BLAST同源性比对, 以确认各试验样品的物种属性。
1.3.3 TaqMan实时荧光PCR法引物及探针的设计
在NCBI数据库中下载松茸Pol基因扩增靶标参考序列(GenBank: AB016926.1), 使用Mega 4软件对序列进行比对和分析, 再按照引物探针的差异性原则, 采用Primer Premier 5.0软件设计目的基因的引物和探针。具体引物探针基因序列如表1所示。
1.3.4 TaqMan实时荧光PCR法反应体系及反应条件
反应体系: Premix Ex TaqTM 12.5 μL, 上下游引物(浓度均为10 μmol/L)各1.0 μL, TaqMan探针(浓度为10 μmol/L) 0.5 μL, 模板DNA 2.0 μL, ddH2O 8 μL, 总反应体系共计25 μL。
反应条件: 95 ℃ 30 s, 1个反应循环; 95 ℃ 5 s, 60 ℃ 30 s, 40个反应循环。荧光阈值为实时荧光定量PCR仪默认值。
1.3.5 TaqMan实时荧光PCR法特异性试验
按照1.3.1节提取松茸及其他食用菌和其他异源性动、植物样品的基因组DNA, 并测定其纯度和浓度后进行TaqMan实时荧光PCR扩增反应, 同时以ddH2O水作为空白对照, 测试本试验的特异性。
1.3.6 TaqMan实时荧光PCR法灵敏度试验
松茸成分质量分数灵敏度试验: 将赤松茸、白松茸、白口蘑和栎松口蘑4种易混食用菌等量混匀后, 按照100.00%、10.00%、1.00%、0.10%和0.01%的质量比例与待测物松茸样品混合制成模拟试验研究基质, 分别提取基因组DNA后进行TaqMan实时荧光PCR反应, 以确定松茸成分质量分数的检测灵敏度。
松茸成分质量浓度灵敏度试验: 将提取的松茸基因组DNA按照10倍系列稀释, 稀释后各质量浓度分别为100.00、10.00、1.00、0.10和0.01 ng/μL, 以此分别为DNA反应模板进行TaqMan实时荧光PCR反应, 以确定松茸成分质量浓度的检测灵敏度。
1.3.7 重复性和稳定性试验
将质量浓度为100.00、10.00和1.00 ng/μL的松茸基因组DNA分别作为模板进行TaqMan实时荧光PCR反应, 通过检测结果的Ct值计算变异系数。
1.3.8 实际市售样品松茸成分的检测试验
采购松茸及其制品共计160份, 包括松茸鲜品15份、松茸冻品15份、松茸干品30份、松茸酱20份、松茸调味粉20份、松茸饼干(米粉)15份、标签含松茸成分的杂菌包干货15份、标签含松茸成分的杂菌碎干货10份以及标签不含松茸成分的杂菌包干货10份、标签不含松茸成分的杂菌包碎干货10份, 检测其是否含有松茸成分。
1.4 数据处理
试验数据采用Microsoft Excel 2016对检测结果的标准偏差进行分析。
2 结果与分析
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2.1 样品属性符合性鉴定
基于DNA条形码技术对收集的松茸及其近源食用菌样品的属性进行符合性鉴定, 经BLAST同源性比对后, 各样品的物种属性鉴定结果如表2所示, 从鉴定结果可知3份松茸样品及27种其他食用菌样品的属性均与商品标识相一致。
2.2 真核生物18S rRNA内参基因检测结果
参照行业标准SN/T 5637—2023《6种常见黑松露成分定性检测方法实时荧光PCR法》中真核生物18S rRNA通用引物序列, 对收集的35种样品基因组DNA进行TaqMan实时荧光PCR扩增反应。检测结果如图1所示, 包括松茸在内的35种样品18S rRNA内参基因检测结果均为阳性, 以双蒸水为DNA模板的空白对照未出现典型性扩增曲线, 所有样品的Ct值在14.16~18.78范围之间。测试结果表明当本研究采用TaqMan实时荧光PCR方法对松茸进行特异性成分检测时, 所提取的样品DNA溶液均适用于扩增反应。
2.3 松茸成分TaqMan实时荧光PCR特异性检测结果
采用本研究收集的松茸以及27种食用菌样品和其他5种异源性动、植物样品进行松茸成分TaqMan实时荧光PCR方法的特异性研究。检测结果如图2所示, 产自吉林延边、四川甘孜和云南香格里拉地区的松茸样品检测结果为阳性, 均出现典型性扩增曲线; 姬松茸、赤松茸、白松茸、黑松露、白松露等其他27种食用菌样品和猪肉等5种异源性动、植物样品及双蒸水阴性对照检测结果均为阴性。结果显示, 无论松茸近源物种还是异源物种与其之间均不存在交叉反应, 说明本研究建立的松茸成分TaqMan实时荧光PCR检测方法特异性良好, 适用于松茸成分的精准检测。
2.4 松茸成分TaqMan实时荧光PCR灵敏度检测结果
2.4.1 松茸成分质量分数灵敏度试验结果
将4种易和松茸混淆的食用菌等量混匀后, 按照不同质量比例再与松茸样品进行混合制成模拟基质, 分别提取其基因组DNA, 以此为模板采用TaqMan实时荧光PCR方法检测松茸成分质量分数的灵敏度。结果如图3所示, 松茸成分TaqMan实时荧光PCR方法的质量分数检测灵敏度可达0.01%。
2.4.2 松茸成分质量浓度灵敏度试验结果
以10倍系列稀释后制备的5个不同质量浓度松茸样品基因组DNA为模板, 采用TaqMan实时荧光PCR方法检测松茸成分。检测结果如图4所示, 当松茸成分质量浓度为0.01 ng/μL时, 松茸成分检测结果为阳性, 仍有典型性扩增曲线的出现。确定松茸成分TaqMan实时荧光PCR方法的基因组DNA质量浓度检测灵敏度为0.01 ng/μL。
2.5 重复性和稳定性试验结果
选取质量浓度为100.00、10.00和1.00 ng/μL的松茸基因组DNA溶液, 以此为模板进行TaqMan实时荧光PCR反应, 研究设立每个浓度进行3次平行试验, 每次平行进行3次重复试验, 通过检测结果Ct值分析方法的重复性和稳定性。统计分析表明, 批内变异系数介于0.40%~0.61%之间, 批间变异系数介于1.07%~1.49%之间, 批内和批间变异系数均小于2%(表3)。研究表明本试验建立的松茸成分TaqMan实时荧光PCR检测方法重复性好且稳定性强。
2.6 市售样品松茸成分TaqMan实时荧光PCR检测结果
市售松茸鲜品、冷冻品、干品、松茸酱、松茸调味粉、松茸饼干(米粉)、杂菌包以及杂菌碎样品的松茸成分检测结果如表4所示。其中松茸鲜品和松茸冻品均出现典型性扩增曲线, 检测结果均为阳性; 松茸干品中存在假冒现象, 检测结果与标签不符比例为36.70%, 进一步采用DNA条形码技术对假冒松茸进行物种鉴定, 基因序列比对显示假冒松茸物种属性为皱环球盖菇(Stropharia rugosoannulata)、巨大口蘑(Tricholoma giganteum)和栎松口蘑(Tricholoma bakamatsutake); 松茸制品包括松茸酱、松茸调味粉、松茸饼干(米粉)检测结果中与标签不符的比例分别为35.00%、55.00%和23.70%; 标签含松茸成分的杂菌包干货、杂菌碎干货样品检测结果中与标签不符的比例分别为40.00%和60.00%; 标签不含松茸成分的杂菌包干货、杂菌碎干货样品均未出现典型性扩增曲线, 检测结果为阴性。检测结果表明, 本研究建立的松茸成分检测方法可准确有效地鉴别市售商品中是否含有松茸成分, 具有较强的应用性及广泛的适用性。
3 讨论与结论
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随着分子生物检测技术的飞速发展, 为食品鉴伪技术的发展提供了新的思路和方法, 确保高值食品市场的稳定及安全, 维护正常的进出口贸易及国内市场秩序, 保护消费者的合法权益。本研究采用真菌通用DNA条形码ITS分子标记对收集的28种食用菌进行物种属性鉴定, 经BLAST同源性比对后样品属性均与商品标识一致。这与郑梦迪等[30]验证ITS2标记的DNA条形码技术可有效区分可食用真菌和有毒菌类结果相一致。进一步说明DNA条形码技术在高值真菌类产品物种鉴定中的适用性。本研究建立了基于TaqMan实时荧光PCR技术的松茸及其制品鉴伪方法, 并从特异性、灵敏度、重复性方面分析本方法的可行性。研究表明, 该方法检测灵敏度可达到0.01 ng/μL松茸基因组DNA, 高于朱建宇等[31]建立的PCR扩增结合可视化核酸试纸条检测方法中模板DNA最低检出量0.1 ng/μL。TaqMan实时荧光PCR法的检测灵敏度高于普通PCR, 并且TaqMan实时荧光PCR还具有特异性高、全封闭检测模式、安全无污染等优势, 更适用于实验室和检测机构对松茸及其制品进行精准快速检测。
通过对160份市售松茸及其制品进行松茸成分检测, 发现市售松茸鲜品和冻品因产品形态完整度高, 物种属性鉴定也是Tricholoma matsutake; 但松茸干品存在假冒现象, 检测结果与标签不符比例达36.70%, 经DNA条形码序列分析显示假冒松茸物种的属性为皱环球盖菇、巨大口蘑和栎松口蘑, 伪品价值均远远低于松茸成本; 其他收集松茸制品检测结果中与标签不符的比例达23.70%~60.00%, 验证酱类、粉末等市售深加工松茸制品掺伪现象更是较为普遍。TaqMan实时荧光PCR技术检测松茸及其制品真伪方法的建立, 可规范松茸及其相关产品的市场, 为减少或避免口岸贸易和市场流通中松茸虚假贸易的发生提供了有力的技术支撑, 以促进我国松茸产业的健康稳定发展。
基金
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  • 海关总署科研项目(2023HK134)
  • 大连海关科研项目(2023DK08)
文献
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2025年第16卷第7期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241030001
  • 接收时间：2024-10-30
  • 首发时间：2025-07-19
  • 出版时间：2025-04-15
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  • 收稿日期：2024-10-30
基金
海关总署科研项目(2023HK134)
大连海关科研项目(2023DK08)
作者信息
    大连海关技术中心, 大连 116001

通讯作者:

* 杨爱馥(1978—), 女, 博士, 正高级工程师, 主要研究方向为食品安全检验。E-mail:
* 徐静(1982—), 女, 博士, 研究员, 主要研究方向为食品安全检验。E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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