Vietnam: What are the requirements for proliferation and treatment of bacterial samples in food and animal feeding stuffs under National Standard TCVN 11925: 2017?

Main Content

• What are the requirements for proliferation and treatment of bacterial samples in food and animal feeding stuffs in Vietnam under National Standard TCVN 11925: 2017?
• What are the requirements for nucleic acid extraction under National Standard TCVN 11925:2017 on microbiology of food and animal feeding stuffs in Vietnam?
• What are the standards related to the proliferation of microbiology in Vietnam?

What are the requirements for proliferation and treatment of bacterial samples in food and animal feeding stuffs in Vietnam under National Standard TCVN 11925: 2017?

In subsection 6.1 Section 6 of National Standard TCVN 11925:2017, the requirements for proliferation and treatment of bacterial samples in food and animal feeding stuffs in Vietnam are:

Food samples must be proliferated according to the corresponding standards or other appropriate standards. Other proliferation environments that are preferable to PCR can be used, if used, these environments need to be validated, so that the performance is at least equivalent to that of the environment specified in specific standards.

Some proliferation media recommended in standards contain fewer PCR inhibitors than others, so careful consideration should be given when selecting a sample preparation method.

For some products, special care should be paid to prevent the growth of competitive microbiology (for example by the addition of chemicals or selective antibiotics).

The least harmful methods, such as dilution, centrifugation, protein decomposition, filtration, gravity centrifugation, immune magnetic separation, etc. can be done. In the absence of a PCR response, more precise methods such as boiling, using complex agents or harsh chemicals such as chloroform and ethanol, or test kits with similar effects. Simple physical methods can be used to reduce the fat content of high-fat samples. Complexes can be used to reduce the high calcium content of dairy products that may cause inhibition.

What are the requirements for nucleic acid extraction under National Standard TCVN 11925:2017 on microbiology of food and animal feeding stuffs in Vietnam?

In subsection 6.2 Section 6 of National Standard TCVN 11925:2017 on microbiology of food and animal feeding stuffs, the requirements for nucleic acid extraction are as follows:

* DNA extraction

- DNA release and purification

Several principles of DNA extraction can be combined. For example, the following steps can be taken:

+ Decomposite proteins in cell extracts by proteases (e.g. proteinase K) and RNA by ribonucleases.

+ Precipitate peptides formed by organic solvents (e.g. mixture of phenol and chloroform) leaving DNA in the aqueous phase.

+ Purify DNA solution and thicken it by precipitation in ethanol in the presence of hexavalent cations.

+ Obtain precipitated DNA by centrifugation.

+ Wash DNA with ethanol and mix it in buffer solution [e.g. tris(hydroxymethyl) aminomethane/EDTA buffer (Tris-EDTA buffer) or Tris.

DNA co-precipitation is glycogen, polyethylene glycol (PEG), or transport RNA (t-RNA) which can be used to improve DNA acquisition during precipitation steps; use only precipitates that do not have any nuclease activity, do not have PCR inhibitors/competitions, and do not have any sequence similarities with the potential PCR targets studied.

Note: Use of vacuum freeze dryers to dry DNA pellets obtained after the precipitation step may cause cross-contamination.

DNA can be released when the heat cell breaks down (e.g. by boiling for 10 min). After boiling, centrifugal samples are cooled and the upper pot is used to run PCR. Before boiling, to facilitate cell breakdown, enzymatic treatment (e.g., lysozyme, mutanolysine can be applied to Gram-positive bacteria) followed by incubation of protease/proteinase. Other methods such as vigorous agitation of particles may be needed when organisms have cell walls that are difficult to break down (e.g., Mycobacterium spp.).

Any other method, including commercial test kits, can be used to extract nucleic acids if comparable results are given.

- Quantity and quality of DNA

The quantity and quality of extracted DNA using the given method on a given sample substrate is required for good repeatability and reproducibility during amplification by PCR, providing sufficient DNA is present in the sample substrate. In particular, the method used must yield recovery of DNA fragments of medium size equal to or larger than those of the PCR products studied.

The concentration and purity of the isolated DNA can be estimated by fluorescence or gel electrophoresis methods. Purified DNA can be quantified by spectroscopic methods.

A quick way to assess DNA quality and quantity is electrophoresis on agarose gel, followed by ethylium bromide staining and fluorescence measurement under ultraviolet (UV) light (see Reference [1]).

For some methods of sample preparation (e.g. boiling), it is necessary to apply the nucleic acid solution directly after preparation when the nucleic acids released are unstable.

In general, repeated coagulation and defrosting of nucleic acid solutions should be avoided.

Use appropriate plastic utensils to preserve nucleic acids with low copy counts.

Note: Some test tube materials can bind nucleic acids.

What are the standards related to the proliferation of microbiology in Vietnam?

In Appendix A promulgated together with National Standard TCVN 11925:2017 on microbiology of food and animal feeding stuffs, standards related to the proliferation of microbiology in Vietnam are required as follows:

Table A.1 - Specific standards for microbiology (bacteria)

Accordingly, the standards listed in Table A.1 include information regarding the proliferation of microbiology (bacteria).