Principles for prevention and treatment of PCR contamination in nucleic acid detection

First, the scope of application:

This principle applies to laboratories involved in PCR methods for food safety testing.

This principle applies to the prevention and treatment of contamination in PCR laboratories.

Second, the source of pollution:

(1) Cross-contamination between samples: Sample contamination is mainly caused by contamination caused by improper handling of samples during transportation, storage, and placement. Sample nucleic acid templates can cause contamination between samples during the extraction process due to improper handling.

(2) Contamination of reagents: contamination of related reagents during nucleic acid extraction and amplification due to improper operation.

(3) PCR amplification product contamination: This is the most common and most common contamination in the PCR reaction. Very small amounts of PCR product contamination can cause false positives. The most likely form of contamination of the PCR product is aerosol contamination. When the reaction tube is shaken vigorously, the cover is opened, and the sample is repeatedly sucked and sucked, an aerosol can be formed to cause contamination.

(4) Contamination of cloning plasmids: When a cloning plasmid was used as a positive control, contamination of the cloning plasmid sometimes occurred.

(5) Contamination of laboratory equipment: such as contamination of pipettes.

Third, the method of preventing pollution:

(1) Reasonable laboratory partitioning. Laboratory supplies, including pipettes, should be used exclusively for each work area in the laboratory. If possible, PCR reagents should be added to the laminar flow table (such as biosafety cabinet, ultra-clean workbench) equipped with UV lamps. Micro-centrifuges for PCR and disposable gloves should be placed in the workbench. Pipettes and other essential items for each range.

(2) Correct experimental operation:

a. The operation of adding PCR reagents and template should be carried out in strict accordance with the requirements. The sample should be slow and try to be done in one time. Avoid multiple suctions to avoid cross-contamination or aerosol pollution.

b. The gloves should be changed frequently during the experiment. After entering or leaving different areas or performing template operation, the gloves should be replaced in time.

c. The Microcentrifuge Tube containing the PCR reagent should be centrifuged (about 10 s) before opening to remove the liquid from the tube wall and the tube cover to the bottom of the tube, thus reducing the chance of contaminating the glove or the sampler. The opening action should be light to prevent the liquid in the tube from splashing or forming an aerosol, causing pollution.

d. When performing multiple PCR reactions at the same time, the main reaction mixture should be prepared, then dispensed into the PCR reaction tube, and finally the sample nucleic acid template is added to reduce the pollution caused by the cumbersome single addition operation.

(3) Experimental instruments and reagents:

a. The laboratory's own reagents, such as deionized water, buffer, etc., should be autoclaved or filtered to remove bacteria before use.

b. Packing PCR reagents: All PCR reagents should be dispensed in small amounts to reduce the number of repeated samplings and stored at -20 ° C. When appropriate, it is best to use it exclusively. In addition, the PCR reagent and the PCR reaction solution should be stored separately from the sample and the PCR product, and should not be placed in the same ice box or refrigerator.

c. All tips, reaction tubes, etc. should be used at one time, and should be subjected to high pressure treatment before use. If conditions permit, it is best to use a tip with a filter. Pipettes in each work area shall be marked and shall be used for fixed use and shall not be used interchangeably.

(4) The experimental design should follow the relevant regulations of the internal quality control of the laboratory. At least the experiment should:

a. Set the target DNA positive control reaction. Appropriate dilution of the target sequence should be performed at a different location in the laboratory prior to the experiment to prevent the concentrated solution of the target DNA from being brought to the laboratory for PCR.

b. Set the PCR reagent negative control and the target DNA negative control reaction.

Fourth, analyze the cause of pollution:

(1) Reagent contamination: The result of detecting a positive positive control reaction. If the result of the negative control reaction is positive, it indicates that one or several reagents in the PCR reaction system are contaminated. If the positive control reaction is negative, it indicates that the inhibitory substance is present in the PCR reaction system, or one to several PCR reaction reagents are invalid.

(2) Environmental pollution: After the possibility of contamination of the reagent is excluded, if the pollution still exists, it may be considered as environmental pollution. Common sources of contamination may be contamination of various experimental surfaces, including bench tops, instrumentation surfaces, various switches or handles, etc. For these contaminations, a wiping experiment can be used to find suspected sources of contamination. The steps are as follows:

a. Wipe the suspected source of contamination with a sterile cotton swab soaked in sterile water;

b. 0.1mL deionized water soak;

c. Take 5 μL for PCR experiment;

d. Electrophoresis test results.

If the exact source of contamination is not found after the above tracking experiments, the contamination may be caused by an aerosol of the PCR product in the air.

V. Pollution treatment:

(1) Environmental pollution:

a. Dilute acid treatment method: The apparatus can be wiped or soaked with 1 mol/L hydrochloric acid to dissociate the residual DNA.

b. Ultraviolet irradiation (UV) method: ultraviolet wavelength (nm) is generally selected 254nm and 300nm, irradiation for 30min. When UV is selected as the elimination of residual PCR product contamination, the length of the PCR product and the distribution of bases in the product sequence should be considered. UV irradiation is only effective for long fragments of 500 bp or more, and has little effect on short fragments.

c. If it is likely to be aerosol contamination, the test site should be replaced. If the conditions are not allowed, redesign the new primer (no correlation with the original primer).

(2) Reagent contamination:

If the control shows that the contamination caused by the reagent, the reagent should be replaced immediately.