How to detect the trace of mycoplasma

2017-09-15 14:22:26 GMT+0800


Mycoplasma, a prokaryotic microorganism that is similar to bacteria but does not have cell walls, was found in Nocard in 1898. It can grow and reproduce on an inanimate artificial medium with a diameter of 50-300nm and can be passed through a bacterial filter.

Mycoplasma is known in the past as the pleuropneumonia (pleuropneumonia -like organisms, PPLO), which was officially named mycoplasma in 1967.

Mycoplasma (mycoplasma) : also known as the mold, for the smallest of the simplest prokaryotes found at present, the number of genes is 480.

The only cell that is visible in mycoplasma is the ribosome (mycoplasma is a prokaryotic cell, and the prokaryotic cell organelles are only ribosomes).

Mycoplasma structure is also relatively simple, most of which are spherical, without cell wall, only the membrane of the three-layer structure, so it has great variability.

Mycoplasma, the tiny microbe, is a big headache in cell culture.

Mycoplasma pollution may be derived from culture medium, serum or experimental operator, which can affect cell growth rate, cell morphology, gene expression, cell metabolism and cell vitality.

Mycoplasma infection is not easy to detect, so it is very important to test the mycoplasma regularly.

There are eight main types of mycoplasma in the cultured cells of the contaminated laboratory, but there is no single test method to detect these eight mycoplasma.

Mycoplasma is very tenacious, and the vast majority of antibiotics used in cell culture are ineffective.

For example, penicillin mainly ACTS on bacterial cell walls, but mycoplasma has no cell walls and therefore is not affected.

Impeccable cell culture technology is always the best way to prevent mycoplasma infection, timely find out other cultures by mycoplasma infection is very important also, so you can quickly take effective measures before the infection spread.

Here are some common methods to detect mycoplasma in cultured cells.

The scanning electron microscopy of the mycoplasma surface of human fibroblasts was observed

Separation culture

The separation culture method is the gold standard method for the detection of mycoplasma, with the highest accuracy.

This method is sampled from a suspicious cell culture system and is inoculated into an AGAR plate most suitable for mycoplasma growth.

If the samples contain mycoplasma, they will grow wild on this AGAR plate and eventually form a visible characteristic colony.

There is no false negative result in the separation culture method, so it is regarded as the gold standard of mycoplasma.

However, there are two disadvantages to the separation culture method. One is that the detection time is too long and it will take at least 4 weeks for the mycoplasma to produce a clear clone.

The second is that although the majority of mycoplasma species can be detected, the separation and culture methods are also less powerful, such as mycoplasma m. hyorhinis.

If you really don't want to wait that long, or hope to get the preliminary results in the process of separation and culture, you can try DNA, PCR, or enzyme testing.

But it is important to note that the above method can separate all types of mycoplasma detection and sensitivity is not high culture method, so it's best to combine the two methods to obtain the most reliable test results.

DNA testing

DNA testing requires a combination of suspicious samples and instructed cells, so it usually takes a few days.

Instructions for the DNA tests for cells is usually larger Vero cell cytoplasm area, if mycoplasma is contained in the original sample, so when the cell DNA stained by fluorescent dyes (e.g., Hoechst dye), can be observed around the instructions cell nuclear fluorescence spots or fluorescent particles.

The isolation culture method is used to detect the mycoplasma m. hyorhinis strain, which is not reliable, and the DNA method can accurately detect it.

The PCR method

The PCR method can also detect m. hyorhinis strain, and the PCR method detects mycoplasma in just a few hours, which is the fastest but most insensitive mycoplasma detection method.

This method USES PCR to detect suspicious samples using the primer of mycoplasma DNA, where PCR primers usually target the 16S rRNA gene of mycoplasma.

In the process of gel electrophoresis, mycoplasma DNA is shown as a specific stripe to indicate the presence of mycoplasma.

PCR can detect most mycoplasma, but it is prudent to use another method to verify this.

Enzyme testing and ELISA

In addition, there are some other mycoplasma detection methods.

Such as enzymatic detection is suspicious samples are added to the specific substrate, within the system of mycoplasma ADP can be transformed to ATP enzyme, then can use glowing luciferase ATP enzyme can indicate the presence of mycoplasma.

ELISA can also be used to mycoplasma detection, based on the method of ELISA mycoplasma detection in general use for mycoplasma 16 s rRNA genes tagged probe or antibodies, to detect whether they contain mycoplasma cultures.

Regular testing

Mycoplasma infection causes the cells to wither, so it is important to train the cells to perform mycoplasma.

In general, mycoplasma should be tested every 1 to 3 months.

It is the key to the cell culture lab to deal with mycoplasma infection.

Suggestions on prevention of mycoplasma pollution:

In cell culture, it mainly prevents the pollution of mycoplasma from the following aspects:

Control of environmental pollution;

Rigorous experimental operation;

Cell culture medium and apparatus should be sterile;

Appropriate antibiotics are added to the cell culture medium.

When mycoplasma is contaminated with cells, especially important cell lines, it is necessary to remove mycoplasma, which is commonly treated with antibiotics, antiserum treatment, antibiotics plus antiserum and complement.

The most prominent structural feature of mycoplasma is that there is no cell wall. In general, antibiotics such as endoamide and vancomycin are not sensitive to the biosynthesis of the cell wall.

General resistance to polymycin, rifampicin and sulfonamide drugs.

The most inhibitory activity of mycoplasma and the antibiotics commonly used in mycoplasma infection are tetracycline, macrocyclic ester and some fluoroquinolone.

Other classes of antibiotics such as aminoglycoside and chloramphenicol have a small inhibitory effect on mycoplasma, so it is often not used as a chemical treatment agent for mycoplasma infection.

Mycoplasma contamination in cell culture is actually a relatively common, according to data, can reach 30% or higher. Mycoplasma contamination when cells grow well in expression, nor death, at the same time, the medium is clear. Time is up to a week. Then basically can determine is the mycoplasma contamination, willing to check test, is not willing to directly use reagent treatment.

Avoid pollution cells, can begin from prevention, super clean bench items placed to reasonable, enter cells room for shoes and wear a lab coat, experimenter after wearing gloves to use alcohol disinfection, avoid cells cross contamination, and so on.

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