How to prevent and handle nucleic acid contamination in PCR testing laboratories?

Article Source: Dr. Yuan Takes You for Tests

It is impossible for nucleic acid testing laboratories to completely eliminate nucleic acid contamination. Managers and laboratory personnel of nucleic acid testing laboratories must recognize that nucleic acid contamination is inevitable, but the frequency and probability of contamination are controllable. What we need to do is to always remain vigilant, do our utmost to minimize the probability of pollution, and be able to detect pollution at the earliest possible time and deal with it promptly.

Let's first talk about the most significant laboratory nucleic acid contamination incident in history:

On April 18, 2020 local time, The Washington Post broke a major piece of news. The COVID-19 test kits produced by the US CDC in laboratories were contaminated, causing the nucleic acid testing across the United States to be delayed by at least one month. On January 21st, the CDC had "confirmed the development" of a detection method for the novel coronavirus and used it to confirm the first case of COVID-19 in the United States. In late January, the US CDC distributed novel coronavirus test kits to 26 public health laboratories, and false positive reactions occurred in 24 of them.

The CDC urgently recalled diagnostic reagents. It was not until March 2nd that the CDC's COVID-19 reagents, produced by Integrated DNA Technologies (IDT), were put back into use. The price of this laboratory contamination was that the United States missed the golden window of opportunity for epidemic prevention and control, with tens of thousands or even hundreds of thousands of ordinary people dying. Every laboratory should keep this lesson in mind and constantly remind itself that nucleic acid contamination can occur at any time, regardless of the level of the laboratory.

Preventive measures for nucleic acid contamination

1. Laboratory layout

The layout of nucleic acid testing laboratories is fundamental to preventing contamination. A reasonable layout can significantly reduce the probability of contamination. Generally, it is divided into four areas: reagent preparation area, specimen preparation area, nucleic acid amplification area and product analysis area. For fluorescence quantitative PCR that does not require electrophoresis, the nucleic acid amplification area and the product analysis area can be combined. Each area should be independently ventilated and the direction of air flow can be controlled.

Some nucleic acid testing laboratories were built without taking into account the need for nucleic acid amplification and were constructed as ordinary laboratories. In this case, I personally suggest that the amplification and product analysis area should be far away from the reagent preparation area and the specimen preparation area, and try not to be on the same floor or in the same area. The most undesirable situation is when several laboratories are right next to each other, or even door to door.

2. Each area has its own dedicated items

Each of the four separated working areas must be equipped with dedicated instruments and equipment. Each area must be clearly marked to prevent equipment and items such as pipettes or reagents from being removed from their respective areas, which could cause confusion of equipment and items between different working areas. Work clothes of different colors or with distinct distinguishing marks should be used in different work areas for easy identification.

3. People and goods flow in one direction only

When entering each working area, one must strictly follow a single-direction sequence, that is, only from the reagent storage and preparation area, specimen preparation area, amplification reaction mixture preparation and amplification area (referred to as the amplification area) to the product analysis area to avoid cross-contamination.

4. Personnel training management
No matter how good the laboratory design is, if the laboratory personnel do not follow the procedures, everything will be in vain. Strengthening the training and management of laboratory personnel is of vital importance.

5. Quality Control 

Negative quality control: The risk of nucleic acid contamination in the laboratory is positively correlated with the volume of tests. Therefore, when testing activities are frequent, more negative quality control should be added, including negative samples, negative extraction controls, negative amplification controls, template-free controls, and even reaction systems, primers, probes, and extraction reagents can be used as controls to test for contamination of components in the testing process and reaction system. Negative controls can use several randomly inserted test samples. Ensure that pollution can be detected at the first moment.

Positive quality control: Avoid or minimize the use of overly strong plasmid positive controls.

6. Processing of PCR products
Some standards suggest that PCR products be soaked in 1mol/L hydrochloric acid. I think this can easily lead to the exposure and spread of PCR products, posing a very high risk. Some laboratories, from a biosafety perspective, apply high pressure to all experimental waste. I do not recommend this either, as PCR products do not pose a biosafety risk. High pressure can cause the PCR products to spread, increasing the risk of contamination in the laboratory. My personal suggestion is that for PCR products, surface disinfection should be carried out and they can be treated as ordinary medical waste.

Measures for handling nucleic acid contamination

The first step in detecting contamination is to suspend the experiment and assess the impact of false positives on previous test results. Thoroughly clean and tidy up the experiment, replace the relevant reagents and consumables as much as possible, and clean and remove nucleic acid from the instruments and equipment. All personnel go home to take a bath and change clothes.

1. Chemical treatment method

Use commercialized DNA removers

1 mol/l hydrochloric acid and various chemical substances are used to degrade DNA.