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SN/T 2641-2010: Detection of pathogen in food - PCR-DHPLC method
SN/T 2641-2010
ICS 65.020.30
B 40
INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
SN/T 2641–2010
Detection of pathogen in food—
PCR-DHPLC method
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
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
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.
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 Water for analytical laboratory use - Specification and test methods
GB 19489 Laboratories - General requirements for biosafety
GB/T 27403 Criterion on quality control of laboratories - Molecular biological testing of
food
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
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 b...