Measures taken by PCR laboratories to remove contamination from nucleic acid amplification products have been repeatedly studied and practiced in various ways over the past few years. Today, we're going to talk about how they work.
1
Chlorine-containing disinfectants
Disinfectants that are soluble in water and produce hypochlorous acid are collectively referred to as chlorine-containing disinfectants.
The exact mechanism of action of hypochlorous acid is not well understood, but its oxidation is thought to denature proteins to achieve the effect of effectively killing bacteria, fungi and viruses.
The effective chlorine in the usual chlorine-containing disinfectant does not refer to the content of chlorine, but the oxidation capacity of the disinfectant, which is equivalent to the oxidation capacity of how much chlorine.
The characteristics are broad spectrum, high efficiency, low toxicity, strong irritating odor, corrosive, bleaching effect, because of the great influence of organic matter, so unstable, need to be prepared on the same day to use.
Most of the chlorine-containing disinfectants commonly used in PCR laboratories are formulated using 84 disinfectant solution, chlorine-containing disinfection tablets, and analytical pure sodium hypochlorite solutions.
(1) 84 disinfectant: the main ingredient is sodium hypochlorite. The effective chlorine content of the 84 disinfectants on the market varies slightly from manufacturer to manufacturer, usually 2%-10%.
(2) Chlorine-containing disinfection tablets: the main active ingredient is trichloroisocyanuric acid (is an organic compound, is a very strong oxidant and chlorination agent), the available chlorine content is 450-550mg / tablet.
(3) Analysis of pure sodium hypochlorite solution: it is a highly efficient chlorine-containing disinfectant. Hypochlorous acid is generally considered to be an active ingredient for the sterilization of sodium hypochlorite.
10% sodium hypochlorite solution: Prepared with an analytical pure hypochlorous acid solution with pure water. The available chlorine concentration is 0.5%.
Principle of sodium hypochlorite to remove PCR amplification product contamination:
Sodium hypochlorite can create a wide "gap" in the PCR amplification product (DNA double strand), hindering the extension of nucleic acid polymerase, so it can be used for the control of PCR product contamination.
IT is generally recommended that PCR laboratories do not use 84 disinfectant directly or use its formulation because its exact concentration is unknown.
2
Alcohol disinfectants
Ethanol solution has low cost, fast disinfection effect, volatile, generally no damage to people and goods, and is generally disinfected by microorganisms such as viruses, bacteria and fungi.
The mechanism of action of ethanol, it is generally believed that ethanol has a strong permeability when mixed with water, which can destroy the cell membrane or viral envelope of pathogenic microorganisms, resulting in their lysis, protein degeneration, death or inactivation.
Ethanol solutions commonly used in PCR laboratories are 70% ethanol and 75% ethanol.
(1) 75% ethanol (alcohol) disinfectant: commercialized alcohol disinfectant.
(2) 70% ethanol solution: commercially available analytical pure absolute ethanol is prepared with pure water.
The effective sterilization concentration of ethanol ranges from 60%-90%, the concentration is less than 50% or the disinfection effect of absolute ethanol is significantly weakened, and some studies believe that 70% concentration is the most effective.
3
Nucleic acid/nuclease remover
In the regulatory requirements for nucleic acid detection of the new crown virus at home and abroad, it is mentioned that residual nucleic acid can be removed by reagents such as nucleic acid scavengers.
The advantage of this type of nucleic acid/nuclease remover is that it uses biodegradation or chemical degradation to effectively remove DNA/RNA and DNA/RNA enzymes from the surface of the object, which is non-toxic to the human body, does not contain corrosive acids and alkalis, and does not damage metals.
4
Ultraviolet (UV) irradiation
The use of ultraviolet lamps to irradiate experimental tables or related instruments and equipment after each detection operation is an indispensable method for clinical PCR laboratories to eliminate or reduce amplification pollution.
The main mechanism by which ultraviolet irradiation removes nucleic acid contamination is base-based oxidation, which breaks the single and double chains and forms a cyclobutane ring in the adjacent pyrimidine base certificate, which forms an intra-chain pyrimidine dimer that inhibits polymerase-mediated chain extension.
The distance, intensity and timing of Irradiation during ultraviolet light are critical to the effect of removing nucleic acid contamination.
5
Conclusion
Details determine success or failure, especially for the experimental environment and highly demanding PCR laboratories, any one link is not done well may affect the entire experimental results. Therefore, the inspector should always bear in mind the concept of "no nucleic acid" and "no gene", run the anti-pollution idea through the entire experimental process, strictly abide by the operating procedures, and ensure the quality of testing.
【References】
Real-Time Fluorescence PCR Technology
Clinical Detection Technology for Novel Coronavirus Infection
Source: Evening class time of Primary 6
Editor: Ren Mileage Reviewer: Xiao Ran