SN/T 5225-2019Rapid detection method of five diarrhea-causing Escherichia coli in imported and exported foods (English PDF)

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.

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.

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°C.

6.12 Refrigerated centrifuge. temperature control 4°C~8°C, 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