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Basic data
| Standard ID | SN/T 5225-2019 (SN/T5225-2019) |
| Description (Translated English) | (Rapid detection method of five diarrhea-causing Escherichia coli in imported and exported foods) |
| Sector / Industry | Commodity Inspection Standard (Recommended) |
| Classification of Chinese Standard | C53 |
| Classification of International Standard | 67.050 |
| Word Count Estimation | 15,169 |
| Date of Issue | 2019 |
| Date of Implementation | 2020-07-01 |
| Issuing agency(ies) | General Administration of Customs |
SN/T 5225-2019: (Rapid detection method of five diarrhea-causing Escherichia coli in imported and exported foods)
---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Rapid detection of five diarrheogenic Escherichia coli in food
for import and export-Multiplex PCR method
The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards
Issued by the General Administration of Customs of the People's Republic of China
2019-12-27 release
2020-07-01 Implementation
Foreword
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
This standard was proposed and managed by the General Administration of Customs of the People's Republic of China.
Drafting organizations of this standard. Zhengzhou Customs of the People’s Republic of China, PLA Center for Disease Control and Prevention, China Animal Health and Epidemiology
Medical Center, Gongbei Customs of the People's Republic of China, Fuzhou Customs of the People's Republic of China.
The main drafters of this standard. Miao Li, Wang Yongliang, Zhang Can, Li Yang, Wang Junjie, Zhang Xiuping, Dang Zhihao, Li Ke, Feng Jiawang,
Zheng Jing.
Rapid detection method of five diarrhoea-causing Escherichia coli in imported and exported foods Multiplex PCR method
1 Scope
This standard specifies enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC,
Also known as Shiga toxin-producing Escherichia coli), enterotoxic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC),
Intestinal-aggregating Escherichia coli (EAEC) five rapid detection methods for diarrhea-causing Escherichia coli.
This standard applies to all kinds of import and export foods and their raw materials, except for those with special inspection methods. Other products can refer to the use of this
standard.
2 Normative references
The following documents are indispensable for the application of this document. For dated reference documents, only the dated version is applicable to this
file. For undated reference documents, the latest version (including all amendments) is applicable to this standard.
GB/T 6682 Analytical laboratory water specifications and test methods
GB 19489 Laboratory Biosafety General Requirements
GB/T 27403 Laboratory Quality Control Specification Food Molecular Biology Testing
3 Terms and definitions
The following terms and definitions apply to this document.
3.1 Multiplex Polymerase Chain Reaction (MPCR) Multiplex Polymerase Chain Reaction
Also known as multiple primer PCR or multiple PCR, it is to add more than two pairs of primers in the same PCR reaction system to amplify multiple primers at the same time.
PCR reaction of a nucleic acid fragment, the reaction principle is that the template gene sequence is first denatured by high temperature to become single-stranded.
Under suitable reaction conditions, the two primers designed according to the template sequence are respectively sent to the corresponding complementary sequence on the two strands of the template DNA.
Are annealed and combined with each other, and then the four kinds of deoxyribonucleic acid (dNTP) are used as substrates under the action of DNA polymerase to make the primers
Extension, and then continue to repeat the cycle of denaturation, annealing and extension, so that the gene fragments to be amplified will be amplified geometrically.
3.2 Diarrheogenic Escherichia coli
Diarrhea-causing Escherichia coli is a type of Escherichia coli that can cause diarrhea in the human body. It is also a kind of
Pathogens that contaminate food and cause human disease. According to virulence factors, pathogenic mechanism, epidemiological characteristics, it will mainly cause diarrhea in the large intestine
There are 5 types of serobacteria. enteropathogenic E.coli (EPEC), enterohemorrhagic Escherichia coli
Enterohemorrhagic E.coli (EHEC, also known as Shiga toxin-producing Escherichia coli), enterotoxic Escherichia coli
(Enteroinvasive E.coli, ETEC), enteroinvasive E.coli (EIEC), intestinal aggregation adhesion
Sexual Escherichia coli (enteroaggregative E.coli, EAEC).
4 Abbreviations
The following abbreviations apply to this document.
DEC Diarrheogenic Escherichia coli
EPEC Enteropathogenic Escherichia coli
EIEC Enteroinvasive Escherichia coli
ETEC Enteroinvasive Escherichia coli
EHEC Enterohemorrhagic Escherichia coli
EAEC Enteroaggregative Escherichia coli
escV protein secretion regulation gene gene encoding LEE-encoded type III secretion system factor
bfpB bundle-forming pilus B gene bundle-forming pilus B
stx1A Shiga toxin I gene Shiga toxin one
stx2A Shiga toxin two gene
elt heat-labile enterotoxin gene heat-labile enterotoxin
invE invasive plasmid regulator
astA Aggregative heat-stable enterotoxin A gene enteroaggregative heat-stable enterotoxin A
estIa heat-stable enterotoxins initially discovered in the isolates from pigs
estIb heat-stable enterotoxins initially discovered in the isolates from human
aggR Aggregative adhesive fimbriae regulator
pic intestinal colonization factor gene protein involved in intestinal colonization
eae gene encoding intimin for Escherichia coli attaching and effacing
ipaH nvasive plasmid antigen H-gene
Polymerase chain reaction
dNTP deoxyribonucleoside triphosphate
5 Method summary
According to the pathogenic mechanism of diarrheal Escherichia coli, this standard detects pathogenic EPEC (bfpB, escV), EIEC (invE),
ETEC (elt, estIa, estIb), EHEC (escV, stx1A, stx2A), EAEC (astA, aggR, pic), using multiple PCR
Technology, according to the size of the band of the PCR amplified product to determine the pathogenic type of diarrheal Escherichia coli.
6 Equipment and materials
6.1 Constant temperature incubator, 36°C ±1°C, 42°C ±1°C.
6.2 Homogenizer.
6.3 Oscillator.
6.4 Sterile homogenization cup or sterile homogenization bag; capacity 500mL.
6.5 Sterile petri dish; diameter 90 mm.
6.6 One-time inoculation loop.
6.7 Pipettes. the ranges are 1 mL, 5 mL and 10 mL respectively, and the scale is 0.1 mL.
6.8 Micropipette. 0.5 µL ～ 2 µL, 2 µL ～ 20 µL, 20 µL ～.200 µL.
6.9 Sterilize PCR reaction tube.
6.10 Electronic balance. Sensitivity 0.1g, 0.01g.
6.11 Constant temperature water bath. 100℃.
6.12 Refrigerated centrifuge. temperature control 4℃～8℃, maximum speed not less than 13 000 r/min.
6.13 Nucleic acid protein analyzer or ultraviolet spectrophotometer.
6.14 PCR thermal cycler.
6.15 Nucleic acid electrophoresis instrument, including power supply, electrophoresis tank, gel preparation tank (length >10 cm) and comb.
6.16 Gel imaging system.
7 Medium and reagents
Except for special instructions, all experimental reagents are of analytical grade. The experimental water meets the requirements of first-grade water in GB/T 6682.all
The reagents are all aliquoted in containers that are not contaminated by DNase.
7.1 Nutrient broth. see A.1.1 in Appendix A.
7.2 Intestinal bacteria enrichment broth. see A.1.2 in Appendix A.
7.3 MacConkey Agar (MAC). See A.1.3 in Appendix A.
7.4 Eosin Meran Agar (EMB). See A.1.4 in Appendix A.
7.5 TE buffer. see A.1.5 in Appendix A.
7.6 10×PCR buffer. see Appendix A in A.1.6.
7.7 50×TAE buffer. see Appendix A in A.1.7.
8 Inspection procedures
The inspection procedures of five diarrheal Escherichia coli in food are shown in Figure 1.
Figure 1 Inspection procedures for five diarrheal Escherichia coli in food
9 Operation steps
9.1 Sample preparation, enrichment culture and separation
Take 25 g (or 25 mL) of the test sample aseptically, add it to a homogenizing cup containing sterilized 225 mL nutrient broth, and use a rotating blade
The homogenizer uses 8 000 r/min to 10 000 r/min to homogenize for 1 min to 2 min; or add it to a homogenizing bag containing 225 mL nutrient broth, and use a pat
Homogenize with a percussion homogenizer for 1 min~2 min. The liquid sample can be shaken and mixed evenly. Incubate at 36 ℃ ± 1 ℃ for 6 h. Take 10 µL and inoculate 30
Culture the intestinal bacteria enrichment broth tube at 42℃±1℃ for 18 hours.
Streak the enrichment solution to inoculate MAC and EMB agar plates, and incubate at 36℃±1℃ for 18 h...
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