Laboratory testing begins with sample collection, and sample collection is the easiest to overlook. The most important thing for sample collection is to select the correct sample type, use appropriate sampling tools, and carry out reasonable transportation and processing.

I.Sample type

Common sample types are as follows:

The sampling method can refer to the technical specifications for environmental monitoring of the new coronavirus in the agricultural trade (bazaar) market”:

1. Swab samples from throat swabs, hands, clothes and other objects of practitioners: it is required to use the virus preservation solution in the virus sampling tube and fully infiltrate the sampling cotton swabs. The surface of the ground) is repeatedly applied and rinsed for more than 3 times. At the same time, it is necessary to perform multi-point distributed sampling on the surface of the sampled object.

2. Food surface swab samples: Food samples cannot be collected directly. The food to be collected should be separated carefully and stored in a clean sampling bag before collecting swab samples. It is required to use the virus preservation solution in the virus sampling tube, after fully soaking the sampling cotton swab, to repeatedly smear and rinse the surface of the food sample to be collected more than 3 times. At the same time, it is necessary to perform multi-point distributed sampling on the sample surface.

3. Sewage samples: According to the distribution of the drainage system in the market, select 2-3 sewage sampling locations, focusing on the internal pipe network collection, the downstream of the water flow direction, or the connection with the municipal pipe network. To collect a swab sample, immerse it in the sewage with a sampling cotton swab to make it adsorb the sewage and rinse it in the sampling tube more than 3 times. To collect sewage samples, use polyethylene plastic bottles to collect 30-500 mL sewage water samples; for sewage collection with a volume greater than 500 mL, polyethylene plastic buckets or on-site water sample special enrichment equipment can be used. At the same time, it is necessary to conduct multi-point distributed sampling for the sewage sampling location.

4. Animal samples: For live animals, sampling swabs can be used to collect body surface swabs, oropharyngeal swabs and anal swabs, and excrement or secretion samples can also be collected and recorded accordingly on the record sheet. For animal samples that have undergone skinning, etc., use cotton swabs to collect their body surface and body cavity swab samples. It is required to use the virus preservation solution in the virus sampling tube to fully infiltrate the sampling cotton swab, and then repeat the application of the surface of the food sample to be collected. Shabu more than 3 times. At the same time, it is necessary to perform multi-point distributed sampling on the sample surface.

5. Other appliances: containers for transporting and breeding animals such as cages or fish tanks. First, observe or understand the specific types of animals stored and bred in the container, and collect swab samples on the inner wall of the container or liquid samples of the contents.

6.Collect aerosol samples in areas where people gather, poorly ventilated local trading areas, offices, and rest rooms.

II.Sampling tools
1. Blood collection tube
If you need to do nucleic acid testing and anticoagulant samples later, it is recommended to use a vacuum blood vessel containing EDTA anticoagulant to collect blood; for serum samples, it is recommended to use a vacuum blood vessel without anticoagulant.

2. Sampling swab

It is recommended to use polypropylene swabs instead of calcium alginate swabs or swabs with wooden poles, because they may contain substances that inactivate certain viruses and inhibit PCR testing. Swabs with cotton heads cannot be used, because cotton fibers have a strong adsorption of protein and are not easily eluted into the subsequent preservation solution.

In the “Technical Specifications for 10-in-1 Mixed Collection and Detection of New Coronavirus Nucleic Acids”, it is clearly stipulated that “the sampling swab should be made of polyester, nylon and other non-cotton and non-calcium alginate materials, and the handle should be made of non-wood material. The breaking point is about 3cm from the top of the swab head, which is easy to break.”

The CDC in the United States also mentioned “Use only synthetic fiber swabs with plastic or wire shafts. Do not use calcium alginate swabs orswabs with wooden shafts, as they may contain substances that inactivate some viruses and inhibit PCR testing.

Currently widely used is a disposable sterile nylon flocking swab combined with a plastic rod. The flocking swab has better water absorption and release rate. According to different sampling sites, the swab head also has different sizes and fibers. The thickness. ”

3. Sample transportation preservation solution

Virus inactivation preservation solution: an appropriate amount of guanidine salt concentration and a variety of virus lysis components can directly lyse the virus to release nucleic acid, inactivate RNase and DNase, and ensure the integrity of nucleic acid, which can be used for subsequent qPCR detection. It cannot be used for virus cultivation and isolation.

Virus transport and preservation solution: contains salts, amino acids, vitamins, glucose and protein needed for the survival of the virus, which can maintain the activity of the virus. It can be used for the nucleic acid extraction and detection of the virus in the future, and it can also be used for the cultivation and separation of the virus.

4. Sample preservation tube and sample bag

The sample storage tube should be a sterile plastic tube with a screw cap that does not adsorb nucleic acid. Each sample tube should be covered with a sample bag to prevent leakage and contamination of the sample.

III. Interfering substances in the sample

Interfering substances in PCR experiments

1. endogenous interferent

Substances present in the body sample can interfere with the detection of other substances.

Blood: heme (>1mg/ml), hemin (>0.1ng/μl), triglycerides, IgG, etc.;

Urine: Urea (20mM);

Stool: plant polysaccharides, cholate;

Tissue: protease, collagen;

Milk: protease, calcium ion;

Drugs: antibiotics, antiviral drugs, hormones, etc.

other.

2. exogenous interferent

It comes from substances that can interfere with the detection of other substances in vitro. Sample additives and substances that can be contacted during sample collection and processing:

Test supplies: serum separation gel, sample collection container and rubber stopper, catheter, catheter flushing fluid, glove powder, etc.

Anticoagulant: heparin (>0.1 U/mL), EDTA (>0.5 mM), etc.;

Organic solvents: isopropanol (>0.1 IU/ml), betaine, dimethyl sulfoxide, formamide, glycerin, PEG, etc.;

Disinfectants: alcohols (ethanol>1%), aldehydes, phenols (phenol>0.2%), peroxides, strong acids (HCL), strong alkalis (NaOH), etc.;

Detergent: SDS (>0.005%);

Soil: Humus

Dyestuff: Indigo

other.

Under actual outdoor sampling conditions, such as collecting environmental samples, the samples may contain a variety of interfering substances, and the composition of interfering substances in each location is different, which requires PCR interference experiments to be carried out according to local conditions.

IV. Sample storage and transportation conditions

Freshly collected clinical specimens should be sent to the laboratory within 2 to 4 hours after collection at 2°C to 8°C. Store at 4℃ within 24h. Specimens used for virus isolation and nucleic acid detection should be tested as soon as possible. Specimens that can be tested within 24 hours can be stored at 4°C; specimens that cannot be detected within 24 hours should be stored at -70°C or below (if not- Store at 70°C in a refrigerator at -20°C).

Summary: The standardized collection, transportation and storage of samples are essential for subsequent testing. The false negatives of many laboratory tests are caused by improper collection, transportation and storage of samples that lead to degradation of viral nucleic acid. However, many laboratories still ignore the importance of sampling. I hope this article can awaken everyone’s attention to sampling and make more scientific and reasonable sampling!

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