Translated English of Chinese Standard: SN/T2641-2010 Translated by: www.chinesestandard.net Wayne Zheng et al. Email: Sales@ChineseStandard.net ICS 65.020.30 B 40 INDUSTRY STANDARD OF THE PEOPLE S REPUBLIC OF CHINA Detection of pathogen in food PCR-DHPLC method 食品中常见致病菌检测 PCR-DHPLC 法 SN/T 2641 2010 Issued on: November 01, 2010 Implemented on: May 01, 2011 Issued by: General Administration of Quality Supervision, Inspection and Quarantine of the People s Republic of China www.chinesestandard.net 1
Table of Contents Foreword... 1 1 Scope... 1 2 Normative References... 1 3 Abbreviations... 2 4 Biosecurity measures... 2 5 The measures for waste disposal and pollution prevention... 2 6 Principles... 2 7 Reagents and materials... 3 8 Main instruments and equipments... 4 9 Methods summary and detection procedures... 4 9.1 Method summary... 4 9.2 Inspection process... 4 10 Operating steps... 6 10.1 Sample preparation, enrichment culture and separation... 6 10.2 Preparation of template DNA... 6 10.3 PCR amplification... 6 10.4 DHPLC detection... 7 10.5 The contrast settings of quality control... 8 11 Results and determination... 8 11.1 Quality control standard:... 8 11.2 Results determination and report... 8 Annex A (Normative) Sequences of primers used in the PCR-DHPLC detection of common-seen-pathogen in food... 9 Annex B (Informative) List of Standards... 11 Annex C (Informative) Examples of DHPLC detection map of pathogen in food... 13 References and Original Chinese Documents... 14 www.chinesestandard.net 2
Foreword This Standard is prepared according to the rules specified in GB/T 1.1-2009. This Standard is under the jurisdiction of Certification and Accreditation Administration of the People's Republic of China. The responsible drafting organizations are Liaoning Entry-Exit Inspection and Quarantine Bureau of P.R. China, Fujian Entry-Exit Inspection and Quarantine Bureau of P.R. China, Jiangxi Entry-Exit Inspection and Quarantine Bureau of P.R. China, Jilin Entry-Exit Inspection and Quarantine Bureau of P.R. China and Beijing Yingjiusi Technology Development Co., Ltd. The chief drafting staffs of this Standard include Cao Jijuan, Zheng Qiuyue, Xu Junyi, Huang Xiaorong, Geng Limei, Sun Zheping, Wang Xu, Yu Chang, Shao Biying, Zheng Jing, Yang Chunhua, Wang Zhenguo and Gao Xiaobo. www.chinesestandard.net 1
Detection of pathogen in food PCR-DHPLC method 1 Scope This Standard specifies the detection method of salmonella and other 30 kinds of pathogen in food-pcr-dhplc method. This Standard applies to the rapid detection of salmonella and other 30 kinds of pathogen in food. Note: 30 kinds of common pathogen include: Salmonella, shigella, staphylococcus aureus, yersinia enterocolitica, listeria monocytogenes, campylobacter jejuni, bending thermophilic bacteria, enterohemorrhagic Escherichia coli O157:H7, enterotoxigenic escherichia coli, intestinal pathogenic escherichia coli, intestinal invasive escherichia coli, enterobacter sakazakii, vibrio parahaemolyticus, vibrio cholerae, vibrio vulnificus, vibrio alginolyticus, vibrio mimicus, vibrio fluvialis, vibrio metschnikovi, aeromonas hydrophila, clostridium botulinum, clostridium perfringens, bacillus cereus, hemolytic streptococcus, brucella, lactic acid bacteria, pseudomonas aeruginosa, klebsiella pneumoniae, bacillus proteus vulgaris, and proteus mirabilis. 2 Normative References The articles contained in the following documents have become this Standard when they are quoted herein. For the dated documents so quoted, all the modifications (including all corresponding corrections) or revisions made thereafter shall be applicable to this Standard. GB/T 6682 GB 19489 Water for analytical laboratory use - Specification and test methods Laboratories - General requirements for biosafety GB/T 27403 Criterion on quality control of laboratories - Molecular biological testing of food www.chinesestandard.net 1
10 Operating steps 10.1 Sample preparation, enrichment culture and separation Conduct the enrichment and separation culture according to the relevant national standards, industry standards or international authoritative standard methods in Annex B. 10.2 Preparation of template DNA 10.2.1 Preparation of enrichment broth template DNA TAKE 1.5mL corresponding pathogen enrichment broth cultured in 10.1 (if the secondary culture is required, take a secondary enrichment broth), and PLACE into 1.5mL sterile centrifuge tube, 13,000g-CENTRIFUGATE for 1min; ABANDON the supernatant and TAKE the precipitation, ADD 567μL TE solution (ph8.0), SUSPEND, ADD 30μL 10% SDS and 3μL proteinase K (20mg/mL), MIX evenly, 37 WARM-BATH for 1h; ADD 100μL sodium chloride (5mol/L), MIX evenly, ADD 80μL CTAB/NaCl solution (10% CTAB and 0.7mol/L NaCl), MIX evenly, 65 WARM-BATH for 10min; ADD an equal volume of chloroform/isoamyl alcohol (with volume ratio of 24:1), MIX evenly, 13,000g-CENTRIFUGATE for 10min; TAKE the supernatant, ADD an equal volume of phenol/trichloromethane/isoamylol (with the volume ratio of 25:24:1), MIX evenly, 13,000g-CENTRIFUGATE for 10min; TAKE the supernatant, ADD 0.6 times the volume of isopropanol, MIX evenly, 13,000g-CENTRIFUGATE for 10min; TAKE the precipitation, WASH with 70% ethyl alcohol for two times, DRY it, Add 100μL TE solution (ph8.0) to dissolve -- this is the DNA solution. If it can not be detected immediately, STORE at -20 for spare use. PREPARE enrichment broth template DNA of positive control bacterial strain and a negative control bacterial strain in the same way. May also use commercial DNA extraction kit and PREPARE the template DNA according to the instructions. 10.2.2 Preparation of suspicious bacterial colony template DNA PICK suspicious bacterial colony or thallus obtained from separation in 10.1, or 1.5mL of its passage nutrient solution, PREPARE DNA template according to steps in 10.2.1 for detection. May also use commercial DNA extraction kit and PREPARE the template DNA according to the instructions. 10.3 PCR amplification www.chinesestandard.net 6
10.4.1, set up detection procedure and run. 10.5 The contrast settings of quality control Positive contrast and the negative contrast shall be set up in process of detection respectively. The positive contrast is standard strain of the target pathogen and the negative contrast is standard strain of non-target pathogen. 11 Results and determination 11.1 Quality control standard: 11.1.1 Negative contrast: No absorption peak appears. 11.1.2 Positive contrast: The typical absorption peak of PCR product appears, and the peak absorption value is greater than 3mV. 11.1.3 For those that may not meet the above contrast quality control standard, it is deemed to be invalid. 11.2 Results determination and report For example of salmonella, the example of DHPLC detection patterns refers to Annex C: a) If there is no amplification absorption peak in detected samples, the results can be determined as negative and directly report that the XXX pathogen can not be detected; b) If the typical absorption peak of PCR product appears in detected samples and the absorption peak is more than 3mV, the sample XXX pathogen can be determined as suspicious positive; c) If the typical absorption peak of PCR product occurs in detected samples but the absorption peak is less than 3mV, then propose to adjust the amplification parameters of the PCR to redetect. If the absorption peak of the redo-results is still less than 3mV, the XXX pathogen is negative; otherwise the XXX pathogen can be determined as suspicious positive; d) For the suspicious positive results of XXX pathogen, further biochemical identification and report shall be carried out according to the relevant classical detection methods of Annex B. www.chinesestandard.net 8
Annex B (Informative) List of Standards GB 4789.4 National food safety standard Food microbiological examination: Salmonella GB/T 4789.5 Microbiological examination of food hygiene-examination of shigella GB/T 4789.6 Microbiological examination of food hygiene-examination of diarrheogenic Escherichia coli GB/T 4789.7 Microbiological examination of food hygiene - Examination of Vibrio parahaemolyticus GB/T 4789.8 Microbiological examination of food hygiene - Examination of yersinia enterocolitica GB/T 4789.9 Microbiological examination of food hygiene - Examination of campylobacter jejuni GB 4789.10 Food microbiological examination: Staphylococcus aureus GB/T 4789.11 Microbiological examination of food hygiene-examination of streptococcus hemolyticus GB/T 4789.12 Microbiological examination of food hygiene-examination of Clostridium botulinum and botulinus toxi GB/T 4789.13 Microbiological examination of food hygiene-examination of Clostridium perfringens GB/T 4789.14 Microbiological examination of food hygiene-examination of bacillus cereus GB 4789.30 National food safety standard Food microbiological examination: Listeria monocytogenes GB 4789.35 National food safety standard Food microbiological examination: Lactic acid bacteria GB/T 4789.36 Microbiological examination of food hygiene - Examination of Escherichia coli O157:H7/NM GB 4789.40 National food safety standard Food microbiological examination: www.chinesestandard.net 11
Enterobacter sakazakii SN 0175 Method for detection of campylobacter species in food for export SN/T 0184.1 Detection of Listeria monocytogenes in foods for import and export SN/T 0751 SN/T 1022 SN/T 1962 Method for determination of Aeromonas hydrophila in food for export Detection of Vibrio cholerae in food for import and export Detection of Klebsiella in food NMKL No.156 Pathogenic Vibrio species. Detection and enumeration in foods www.chinesestandard.net 12
References and Original Chinese Documents [1] SN/T 2641-2010 Common food pathogen detection by PCR-DHPLC. http://www.chinesestandard.net/default.aspx?pdf-english-id=sn/t%202641-2010 www.chinesestandard.net 14