SN/T 2641-2010Detection of pathogen in food - PCR-DHPLC method (English PDF)

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°C 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°C 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°C

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.

11.1 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.

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...

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