SN/T 1632.2-2013Determinetion of Enterobacter sakazakii (Cronobacter spp.)from dehydrated powdered milk for export. Part 2: PCR method (English PDF)

Foreword

SN/T 1632 "Enterobacter sakazakii in milk powder exports (Craunot sp) test methods" is divided into three parts.

--- Part 1. Separation and counting;

Part --- Article 2. PCR method;

--- Part 3. Fluorescent PCR method.

This section SN/T 1632 Part 2.

This section drafted in accordance with GB/T 1.1-2009 given rules.

The partial replacement of SN/T 1632.2-2005 "Enterobacter sakazakii in milk powder test methods - Part 2. PCR testing methods." this section compared with the SN/T 1632.2-2005, the main technical changes are as follows.

--- The standard according to GB/T 1.1-2009 specification text format;

--- Modify the standard English name;

--- Deleted the definitions, terms and abbreviations;

--- Remove the separation medium crystal violet neutral red bile dextrose agar (VRBGA).

Please note that some of the content of this document may involve patents. Release mechanism of the present document does not assume responsibility for the identification of these patents.

This part by the National Certification and Accreditation Administration of People's Republic of China and focal points.

1 Scope

SN/T 1632 provisions of this part of the milk powder Enterobacter sakazakii (Craunot sp) of the PCR detection method.

This section applies to milk Enterobacter sakazakii (Craunot sp) the rapid qualitative detection of other foods may refer to.

2 Normative references

The following documents for the application of this document is essential. For dated references, only the dated version suitable for use herein Member. For undated references, the latest edition (including any amendments) applies to this document.

Laboratory use specifications and test methods GB/T 6682 Analysis GB/T 27403-2008 food quality control laboratory of molecular biology standardized testing SN/T 1632.1 milk Enterobacter sakazakii (Craunot sp) - Part 1. Separation and Counting

3 Equipment and materials

3.1 Balance. Range 2kg, a sense of the amount of 0.1g.

3.2 water bath. to maintain the water temperature 44.5 °C ± 0.2 °C.

3.3 Incubator. 35 °C ~ 37 °C and 43 °C ~ 45 °C.

3.4 PCR instrument.

3.5 centrifuge.

3.6 UV gel imager.

3.7 electrophoresis.

3.8 pH meter.

3.9 Pipette. 2µL ~ 10µL, 10µL ~ 100µL, 20µL ~ 200µL, 100µL ~ 1000µL.

4 Media and reagents

Test water should comply with GB/T 6682 stipulated a water.

4.1 Modified lauryl sulfate tryptone broth - vancomycin (mLST-Vm) (see Appendix A, A.1).

4.2 brain heart infusion (BHI) (see A.2).

4.3 nutrient broth (NB) (see A.3).

4.4 Primer SAKAF. 5'-GGGTTGTCTGCGAAAGCGAA-3 '; SAKAR. 5'GTCTTCGTGCTGCGAGTTTG-3'.

4.5 TaqDNA polymerase.

4.6 dNTP. dATP, dTTP, dCTP, dGTP.

4.7 10 × PCR buffer.

4.8 Enterobacter sakazakii control strains. ATCC29544, or equivalent strains.

4.9 DNA extraction kit. bacterial genome DNA extraction kit.