Since the outbreak of the "new coronavirus COVID-19", companies developing and manufacturing diagnostic reagents have introduced for the first time RT-PCR nucleic acid detection kits to extract and purify viral RNA by qualitative or quantitative collection of oropharyngeal or nasopharyngeal swab samples. coronavirus".
The sample collection and storage container used in this method is a "disposable viral sampling tube" and the following considerations apply to its production and use.
Virus sampling tubes are medical device products. Most domestic manufacturing companies have applied for category 1 products. Few companies have registered 2 types of products.
Recently, many companies have used the "emergency channel" to meet urgent needs in places like Wuhan. "Apply for a type of license. Virus sampling tube consists of a sampling swab, virus preservation solution and outer packaging. Since there is no unified national or industry standard, the products of each manufacturer are very different.
1. Sampling swab: The sampling swab is in direct contact with the sampling site and the material of the sampling head is closely related to the subsequent testing. Sampling swab head should be made of polyester (PE) synthetic fiber or rayon (artificial fiber), calcium alginate sponge or wooden stick swab (including bamboo swabs) should not be used, the swab head should not be made of cotton.
Because cotton fibers have a strong protein adsorption capacity, they are not easily eluted into the subsequent preservation solution. Wooden or bamboo sticks containing calcium alginate and wood components will also absorb proteins when broken and immersed in the preservation solution. It can inhibit subsequent PCR reactions. Synthetic fibers, such as PE fibers, polyester fibers and polypropylene fibers, are recommended for swab head materials.
The use of natural fibers, such as cotton, is not recommended. Nylon fibers are not recommended because nylon fibers (similar to toothbrush heads) can absorb moisture. This leads to insufficient sampling volume and affects the detection rate. The use of calcium alginate sponge sampling swab material is prohibited! There are two types of swab handles: broken type and built-in type.
After sampling, put the separated swab into the preservation tube and break the cap from the position near the sampling head; after sampling, put the built-in swab directly into the preservation tube with the built-in cap. Align the small hole with the top of the handle and tighten the cap. Comparing these two methods, the latter is relatively safe. When a broken swab is used with a smaller storage tube, rupture may result in spilling of liquid from the tube.
Due attention should be paid to the risk of contamination due to improper use. It is recommended that hollow polystyrene (PS) extruded tubing or polypropylene (PP) injection molded indented tubing be used for the material of the swab handle. Whichever material is used, no calcium alginate additives should be added. Wooden or bamboo sticks. In short, sampling swabs should ensure that the volume sampled and released and that the material selected does not contain substances that will affect subsequent testing.
2. Virus Preservation Solution: Two main types of virus preservation solutions are widely used in the market. One is a modified virus maintenance solution based on delivery media and the other is a modified preservation solution for nucleic acid extraction lysates. The main component of the former is Eagle's basic medium (MEM) or Hank's balanced salt, which is supplemented with salts, amino acids, vitamins, glucose and proteins necessary for virus survival. The preservative solution uses phenol red sodium salt as an indicator and the solution is pink at pH 6.6-8.0.
The necessary glucose, L-glutamine and proteins are added to the preservation solution. Protein is provided in the form of fetal bovine serum or bovine serum albumin, which stabilizes the protein shell of the virus. Since this preservation solution is rich in nutrients, it is both beneficial for the survival of the virus and for the growth of the bacteria. If bacteria are contaminated in the preservation solution, they will multiply. The carbon dioxide in the metabolite products will lower the pH of the preserving solution, changing it from pink to yellow.
Therefore, most manufacturers have added antimicrobial ingredients to their formulations. The recommended antimicrobial agents are penicillin, streptomycin, gentamicin and polymyxin B. Sodium azide and 2-methyl are not recommended. inhibitors such as methyl 4-isothiazolin-3-one (MCI) and 5-chloro-2-methyl-4-isothiazolin-3-one (CMCI), as these components can have an effect on the PCR reaction. Since this preservation solution provides samples that are essentially live viruses, the originality of the samples can be maximized and can be used not only for nucleic acid extraction and detection of viruses, but also for virus culture and isolation. It should be noted, however, that when used for detection, nucleic acid extraction and purification must be performed after inactivation.
Another type of preservation solution based on nucleic acid extraction lysates is based on balanced salts, EDTA chelator, guanidine salts (e.g., guanidine isothiocyanate, guanidine hydrochloride, etc.), sodium anionic surfactant (e.g., dodecane) sulfate, cationic surfactant (e.g., tetradecyltrimethylammonium oxalate), phenol, 8-hydroxyquinoline, dithiothreitol (DTT), proteinase K, and several or more other components, and this preservation solution is used to directly lyse the virus to release nucleic acid and eliminate nucleolytic enzymes (RNase). It is more appropriate if used for RT-PCR only, but the lysate can inactivate the virus. This sample should not be used for virus culture and isolation.
EDTA salts (e.g., dipotassium EDTA, disodium EDTA, etc.) are recommended for use as metal ion chelators in virus preservation solutions, and heparin (e.g., sodium heparin, lithium heparin) is not recommended to avoid interfering with PCR assays.
3. Preservation tubes: The material of the preservation tubes should be carefully selected. There are data showing that polypropylene is associated with adsorption of nucleic acids, especially when the high pressure ion concentration is high, and polyethylene plastic (polyethylene) is better than polypropylene (polypropylene). Easy to get hold of DNA/RNA. polytetrafluoroethylene plastic and some specially treated polypropylene plastic containers are more suitable for DNA/RNA storage. In addition, when using fragile swabs, the storage tube should be a container with a height greater than 8 cm to prevent splashing and contamination of the contents when the swab breaks.
4. Water used for the production of preservative solution: Ultrapure water used for the production of preservative solution should be filtered through an ultrafiltration membrane with a molecular weight of 13,000 to ensure removal of bio-derived polymer impurities such as RNase, DNase and endotoxin; plain purified water or distilled water is not recommended.
