Chinese National Standards

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Surveillance for Allergen and GMO - IP Controls

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鑑証和物種鑑定

Authentication and Species

Identification

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DNA Authentication and Species Identification

DNA 鑑証和物種鑑定 • Species ID 生物物種鑑定: Mammals, birds, reptiles, amphibians and fishes

– Beef, Pork, Chicken, Fish, etc

– Allergens : seafood, nuts, etc

– Particular species

• Meat in vegetarian products 素食產品內有否含肉類成分

• Substitution of low cost / fake product for expensive product 偽劣食品辯證

– Pork to substitute beef

– Oil fish vs. cod fish

• Chinese Medicinal Material and Tonic Products Authentication 中藥材及保健食品鑑証

– Bird nest vs. artificial jelly

– Chinese medicine and herbs e.g. Ginseng （人参）

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Example : Authentication of Chinese Medicinal Material

中藥材DNA鑑証

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Authentication Methodology

鑑定方法

Reference: Authentication of Chinese Medicinal Material by DNA Sequencing. Pang-Chui Shaw,

Department of Biochemistry & Institute of Chinese Medicine, The Chinese University of Hong Kong

Common Authentication Method

一般鑑定方法

PHYSICAL APPEARANCE

物理方法

CHEMICAL TECHNOLOGY

化學方法

Principle Morphological & organoleptic appearance

Qualitative & quantitative assessment of chemical composition

Advantages

Fast, inexpensive Applicable on crude extracts; various chromatographic & spectroscopic methods available

Disadvantages Subjective, experience-based, low accuracy, hard to identify processed materials

May contain hundreds or thousands of different chemicals: vary according to growth & storage conditions

利用DNA分子技術作鑒定 42

Chinese Pharmacopoeia 中國藥典 2005 冬虫夏草 Cordyceps Sinensis

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Cordyceps sinensis 冬蟲夏草 : Adenosine, Cordycepin

Cordyceps militaris 蛹蟲草

Active ingredient or chemical component

can be obtained /purchased easily

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鉴别试验 1、显微鉴别 2、理化鉴别 3、薄层色谱 4、液相色谱鉴别 5、气相色谱鉴别 6、DNA分子标记鉴别 DNA分子标记鉴别是指通过比较药材间DNA 分子遗传多样性差异来鉴别药材基源、确定学名的方法。适用于采用性状、显微、理化以及色谱鉴别等方法难以鉴定的样品的鉴别，如同属多基源物种、动物药等的鉴别。 （1）DNA提取、纯化方法的考察 通过多种方法的优化，建立切实可行的DNA提取、纯化方法，确定最佳条件，获取高质量的药材总DNA，并提供研究数据。 （2）DNA分子标记方法的确定 通过多种方法对多样品的比较，确定适于目的物鉴别的分子标记方法，优化各种条件、参数，并提供研究数据。 （3）PCR反应条件的确定 通过实验，优化PCR反应条件、参数，并提供研究数据。 （4）电泳检查：通过实验，优化琼脂糖凝胶电泳条件、参数，并提供研究数据。 （5）实验过程要防止外源DNA的污染。 7、中药指纹图谱技术

摘自 2009《中国药典》中药质量标准研究制定技术要求

Chinese Pharmacopoeia 中國藥典 2010

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Chinese Pharmacopoeia 中國藥典 2010

PCR methods are documented

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美国食品及藥品管理局 USFDA OTC, IND/NDA

Guidance for Industry : Botanical Drug Products (USFDA)

• Description of Botanical Used : The following information should be provided for each

of the botanical raw materials ingredients in a botanical drug product : … Name of

variety species, genus and family, …

Botanical Raw Materials :

• “… It is important for the safe conduct of clinical trials to ensure the proper identity of

botanical raw materials used in the trials. Since there is no history of U.S. experience

for botanical raw materials marketed only outside the United States, a certificate of

authenticity of the plant and plant parts should be provided for such materials…”

• “… If more than one variety of a given species is used, each should be specified. A

sample of plant, plant parts, or other botanical materials should be retained and

stored under appropriate conditions…These samples will be used for verification of

identify, if needed.

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中药管理国家法规

《中华人民共和国药品管理法》（主席令第45号）

第五条 国务院药品监督管理部门主管全国药品监督管理工作。国务院有关部门在各自的职责范围内负责与药品有关的监督管理工作。

省、自治区、直辖市人民政府药品监督管理部门负责本行政区域内的药品监督管理工作。省、自治区、直辖市人民政府有关部门在各自的职责范围内负责与药品有关的监督管理工作。

国务院药品监督管理部门应当配合国务院经济综合主管部门，执行国家制定的药品行业发展规划和产业政策。

第六条 药品监督管理部门设置或者确定的药品检验机构，承担依法实施药品审批和药品质量监督检查所需的药品检验工作。

第六十四条 药品监督管理部门有权按照法律、行政法规的规定对报经其审批的药品研制和药品的生产、经营以及医疗机构使用药品的事项进行监督检查，有关单位和个人不得拒绝和隐瞒。

药品监督管理部门进行监督检查时，必须出示证明文件，对监督检查中知悉的被检查人的技术秘密和业务秘密应当保密。

第六十五条 药品监督管理部门根据监督检查的需要，可以对药品质量进行抽查检验。抽查检验应当按照规定抽样，并不得收取任何费用。所需费用按照国务院规定列支。

药品监督管理部门对有证据证明可能危害人体健康的药品及其有关材料可以采取查封、扣押的行政强制措施，。。。

《中华人民共和国药品管理法实施条例》

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第八章 药品监督

第五十六条 药品监督管理部门（含省级人民政府药品监督管理部门依法设立的药品监督管理机构，下同）依法对药品的研制、生产、经营、使用实施监督检查。

第五十七条 药品抽样必须由两名以上药品监督检查人员实施，并按照国务院药品监督管理部门的规定进行抽样；被抽检方应当提供抽检样品，不得拒绝。

药品被抽检单位没有正当理由，拒绝抽查检验的，国务院药品监督管理部门和被抽检单位所在地省、自治区、直辖市人民政府药品监督管理部门可以宣布停止该单位拒绝抽检的药品上市销售和使用。

第五十八条 对有掺杂、掺假嫌疑的药品，在国家药品标准规定的检验方法和检验项目不能检验时，药品检验机构可以补充检验方法和检验项目进行药品检验；经国务院药品监督管理部门批准后，使用补充检验方法和检验项目所得出的检验结果，可以作为药品监督管理部门认定药品质量的依据。

第五十九条 国务院和省、自治区、直辖市人民政府的药品监督管理部门应当根据药品质量抽查检验结果，定期发布药品质量公告。药品质量公告应当包括抽验药品的品名、检品来源、生产企业、生产批号、药品规格、检验机构、检验依据、检验结果、不合格项目等内容。药品质量公告不当的，发布部门应当自确认公告不当之日起5日内，在原公告范围内予以更正。

《中药品种保护条例》（国务院令第106号）-1993

《中华人民共和国药品注册法》

《中华人民共和国海关法》

中药国家法规

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Example I : Authentication of Cordyceps sinensis

(Ophiocordyceps sinensis) 冬蟲夏草鑑証 • Definition (Chinese Pharmacopoeia 2010) : 真菌冬蟲夏草寄生於蝙蝠蛾幼蟲體上的子座與幼蟲屍

體的複合物 (Cordyceps sinensis + Hepialus spp.)

• Detection using real time PCR using species specific primers and probes developed by HKLife

/TFSL

• Verified by DNA sequencing on different regions on both fungus and the moth

• Standards obtained from various reputable sources including National Institute for the Control of

Pharmaceutical and Biological Products (中國食品藥品檢定研究院)

• Cordyceps spp : Cordyceps sinensis 冬蟲夏草, Cordyceps militaris 北冬蟲夏草, 蛹蟲草,

Cordyceps hawkseii 亞香棒蟲草, Cordyceps gunii 古尼蟲草, Cordyceps liangshanensis 涼山蟲草,

Cordyceps gracillis 新疆蟲草, Cordyceps ophioglossoids大團囊蟲草, C. sobolifer, etc.

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Analysis on products obtained from market

Product Type Cordyceps spp.

Product 1 丸(HKX-XXXX) C. sobolifer

Product 2 膠囊(HKXX-XXXX) C. militaris

Product 3 膠囊 (HKXX-XXXX) C. sobolifer

Product 4 膠囊 (HKXX-XXXX) C. militaris

Product 5 菌絲體膠囊 C. militaris

Product 6 C. militaris

Product 7 C. militaris

Product 8 N/A

Product 9 : 原條冬蟲夏草 C. sinensis

Product 10 : 冬蟲夏草 C. sinensis

Product 11 : Raw powder C. militaris

Product 12 : Raw powder C. militaris

Product 13 : Pure Cordyceps

Extract C. militaris

Product 14 : Pure Cordyceps

Extract C. militaris

All claimed Cordyceps sinensis

powder / tablets contain other Cordceps

species and C. militaris is the most

commonly used substitutes

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Example II: Edible Bird Nest 燕窩

• Salivary secretion of Aerodramus species such

as A. fuciphagus爪哇金絲燕 and A. maximus大金絲燕, or other Apus species.

• Valuable functional food

• Counterfeit edible bird nest: substituted by

porcine skin, fungus, seaweed, etc.

• Specific primers and probes were developed for

the real-time PCR / PCR detection

• Sequencing approaches to differentiate different

species

Drawback :

No habitat considerations

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Aerodramus maximus sample

Aerodramus fuciphagus sample

Multiplex real-time PCR detection for Aerodramus maximus and Aerodramus fuciphagus

Negative control and

negative sample

Deposition of gene sequences of various Aerodramus species to genebank

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Selected Nucleic Acid / DNA Test Conducted at HKLife / TFSL

HK Hasma DNA Sequencing + Species Identification

HK Isolated colonies for Bacteria ID DNA Sequencing + Species Identification

CN 硫酸軟骨素 Cow, Porcine, Shark Species Identification

CN Vegetarian soup powder Animal Screening

HK 冬蟲夏草膠囊 Cordyceps sinensis (real time PCR + Sequencing

HK Ginseng 人蔘粉 DNA Sequencing Qualitative Test

HK Cinnamon 肉桂 DNA Authentication

HK Beef / Minced beef牛柳/免治牛肉 Porcine test by real time PCR

HK Porcine bone soup 豬骨湯底 Porcine test by real time PCR

Thailand Samples for virus detectoin DNA Sequencing + species ID

HK Edible Bird Nest 印尼白燕 Manihot Esculeuta

HK Coix lacryma-jobi(薏米) DNA Authentication

HK Pure Water Chestnut Powder DNA Authentication

HK Fish Maw Fish

HK Deer tendon DNA Authentication of species of deer tendon

HK Sausage Test Sequencing (black spot)

HK Sea Cucumber Sequencing (Sea Cucumber)

Chile Blueberry for yeast & mold ID Sequencing (yeast & mold)

HK Caviar (魚子醬) Huso Huso identification

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Applications of Molecular NAT on Food Safety and Quality Control

分子核酸檢測(DNA檢測)方法在食品安全及品管的應用

• Screening for pathogens 致病原檢測

– E.g. Bacteria and virus such as Salmonella(沙門氏菌)and Norovirus(諾沃克病毒)

• Detection of GMOs / Allergens 基因改造及致敏原檢測

– E.g. Qualitative & Quantitative Detection of GM DNA in food

• Authentication 鑑証

– E.g. Bird nest, Traditional Chinese Medicine, wine (grapes, rice)

• Species-specific identification 物種鑑定

– E.g. Detection of meat adulteration in vegetarian food, species identification

• Monitoring of catering and manufacturing facility 衛生監測

– E.g. Bacterial contamination in workplace

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1. Avoidance of introduction /

Early detection

2. Segregation / Isolation

3. Equipment cleaning /

Elimination

4. Labelling if needed

The methods that used for risk identification

are very critical

Identify the Risks

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Food Hazard • Biological

– Bacteria and viral pathogens

– E.Coli, Listeria, Samonella

– Norovirus

– V.C. / V.P.

– S. suis

– Avian influenza virus

– Allergen

• Chemical – Contaminants : Dioxin, Mercury,

Sudan III/IV

– Antibiotics

– Pesticides

• Physical

Identify the Risks

The World Health Organization estimates that about 70% of the approximately 1.5 billion episodes of diarrhea occurring globally each year have been caused by biologically contaminated food (www.ifpro.org)

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Example : New Emerging Disease

Streptococcus Suis

(豬鏈球菌) • Gram-positive, facultative

anaerobic coccus

• Total of 35 serotypes (1-34, ½ )

• Domesticated pigs as the major host (found in many parts of the world)

• Human infection was first described in the 1960s and is

increasingly recognized

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Bird Flu 禽流感 Found in Frozen Duck Meat HANOI, Vietnam -- The bird flu virus was found in processed frozen duck meat shipped for human consumption from China to Japan two years ago - another reminder of how easily the disease can cross borders and how hard it is to kill, health officials said Wednesday.

In a recent study published online in the journal Virology, a team of Japanese researchers reported a form of the H5N1 virus was found in duck meat exported from China's Shandong province into Japan in 2003.

The World Health Organization said this was not the first time the virus has been found in processed meat, but it stressed the findings illustrate the virus' strength and presence in Asian poultry stocks.

Example : New Emerging Disease

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Example : Pathogen Identification

Pork Tapeworm (T. Solium) specific testing by PCR

S S2 S3 Pos Pork Neg M

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Major Technical Consideration

主要技術考慮 • DNA degradation DNA 降解

– Highly processed / fermented matrices

– Require a high performance DNA extraction method

– HKLife has long been providing extraction kits for highly processed food and fermented products (oriental food matrices) such as soy sauce, oils, etc

• Adulteration攙雜 – Presence of small amount of true product in fake product

– Quantitative analysis is needed

• Requirement of reference materials / CRM 標準物質/有證標準物質 – reference / voucher sequences, specific biomarkers, etc.

Detection of GMO on same sample using different extraction methods

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Conclusion 總結 • Molecular (nucleic acid) based testing has wide application on food sector

分子 (核酸測試) 在食品領域上有很廣泛的應用

• Regulatory agencies including China SFDA and AQSIQ recognized the

needs for the development DNA based methods for such purposes 全球各檢驗部門都認可

• For industry, good and reliable approaches as provided by DNA based

methods could help quality control and assurances 對工業方面,可帮助品質管理控制

• Help protecting industry and market : manufacturer, trader and customer帮助保障生產商,貿易商以及消費者,從而保障整個市場

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Conclusion 總結 Opportunity to Hong Kong 對香港的機遇:

• DNA based test / authentication methods

available in Hong Kong and could provide

service based analysis or in form of test kits

香港有這方面的技術:服務,試劑

• Help developing branding for good quality

products : TCM products, tonic products,

good for trade and manufacturing

development 建立優良產品品牌:促進貿易生產

• New opportunity on testing and certification

on food and TCM as encouraged by Hong

Kong Government掌握發展新技術,開拓測試認證機遇

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Thank you

多謝各位

www.haikanglife.com

www.foodsafetylabs.com

info@haikanglife.com

terence.lau@haikanglife.com

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