Thoughts on the production and use of virus sampling tube

Virus blood collection belongs to medical device products. Most domestic production enterprises are filed according to class I products, and few enterprises are registered according to class II products. In order to meet the emergency needs of Wuhan and other places, many enterprises apply for class I filing license through the "emergency channel". The virus sampling tube is composed of sampling swab, virus preservation solution and outer packaging. Since there is no unified national standard or industrial standard, the products of various manufacturers vary greatly.

1. Sampling swab: the sampling swab directly contacts the sampling part, and the material of its sampling head is closely related to subsequent detection. The sampling swab head shall be made of polyester (PE) synthetic fiber or rayon (man-made fiber). Calcium alginate sponge or wooden swab (including bamboo swab) shall not be used. The material of the swab head shall not be cotton products, because the cotton fiber has strong adsorption on protein and is not easy to elute into the subsequent preservation solution; The broken wooden stick or bamboo stick containing calcium alginate and wooden components will also adsorb protein and even inhibit the subsequent PCR reaction. Synthetic fibers such as PE fiber, polyester fiber and polypropylene fiber are recommended as the materials for manufacturing the swab head. Natural fibers such as cotton and nylon fibers are not recommended, because nylon fibers (similar to toothbrush head) have poor water absorption, resulting in insufficient sampling and affecting the detection rate. Calcium alginate sponge is prohibited for sampling swab materials! The swab handle has two types: broken type and built-in type. The broken swab is put into the preservation tube after sampling, and the tube cover can be tightened after breaking from the part close to the sampling head; Built in swab after sampling, directly put the sampling swab into the preservation tube, align the built-in small hole of the preservation tube cover with the handle dingduan, and tighten the tube cover. Compared with the two methods, the latter is relatively safe. When the broken swab is used together with the storage tube with small size, the liquid in the tube may splash out when it is broken. Full attention shall be paid to the possible pollution risk caused by improper use of the product. Hollow polystyrene (PS) extrusion tube or polypropylene (PP) injection molding indentation tube is recommended as the material for manufacturing the swab handle, and calcium alginate additives cannot be added no matter what material is used; Wooden sticks or bamboo sticks are not recommended. In short, the sampling swab shall ensure the sampling amount and release amount, and the selected materials shall not have substances that affect the subsequent detection.

2. Virus preservation solution: there are two kinds of virus preservation solutions widely used in the market, one is the improved virus maintenance solution based on the delivery of culture medium, and the other is the improved preservation solution of nucleic acid extraction lysate.

The main component of the former is eagle's basic culture medium (MEM) or Hank's equilibrium salt, which is added with salts, amino acids, vitamins, glucose and proteins required for virus survival. This preservation solution uses phenol red sodium salt as an indicator. When the pH value of the solution is 6.6-8.0, the solution is pink. The preservation solution is added with necessary glucose, l-glutamine and protein. The protein is provided in the form of fetal bovine serum or bovine serum albumin, which can stabilize the protein shell of the virus. Because the preservation solution is rich in nutrients, it is conducive to the survival of viruses, but also conducive to the growth of bacteria. If bacteria are polluted in the preservation solution, they will multiply in large numbers, and the carbon dioxide in its metabolites will reduce the pH value of the preservation solution from pink to yellow. Therefore, most manufacturers have added antibacterial agents to the formula. The recommended antibacterial agents are penicillin, streptomycin, gentamicin and polymyxin B. sodium azide, 2-methyl-4-isothiazolin-3-one (MCI) and 5-chloro-2-methyl-4-isothiazolin-3-one (CMCI) are not recommended, because these components have an impact on the PCR reaction. Because the sample provided by this preservation solution is basically a live virus, it can maintain the originality of the sample to a large extent. In the follow-up, it can not only be used for nucleic acid extraction and detection of the virus, but also for virus culture and separation. However, it should be noted that nucleic acid extraction and purification must be carried out after inactivation.

Another preservation solution is prepared based on nucleic acid extraction and lysis solution. The main components are equilibrium salts, EDTA chelating agent, guanidine salt (such as guanidine isothiocyanate, guanidine hydrochloride, etc.), anionic surfactant (such as sodium dodecyl sulfate), cationic surfactant (such as ammonium tetradecyl trimethyloxalate), phenol, 8-hydroxyquinoline, dithiothreitol (DTT) Protease K and other components. This preservation solution directly cleaves the virus, releases nucleic acid and eliminates nucleic acid decomposing enzyme (RNase). If it is only used for RT-PCR, it is more appropriate, but the lysate can inactivate the virus. This sample cannot be used for virus culture and separation.

EDTA salts (such as dipotassium ethylenediamine tetraacetate, disodium ethylenediamine tetraacetate, etc.) are recommended as metal ion chelators used in virus preservation solution, and heparins (such as heparin sodium and heparin lithium) are not recommended to avoid affecting PCR detection.

3. Preservation tube: the material of preservation tube shall be selected carefully. Some data suggest that polypropylene is related to the adsorption of nucleic acid. Especially at high tension ion concentration, polyethylene plastic is easier to grasp DNA / RNA than polypropylene. Polyethylene propylene plastic and some specially treated polypropylene plastic containers are more suitable for DNA / RNA storage. In addition, when using a breakable swab, a container with a height greater than 8 cm shall be selected as far as possible to prevent the contents from splashing pollution when the swab is broken.

4. Water for the production of preservation solution: the ultrapure water used for the production of preservation solution shall be filtered through an ultrafiltration membrane with a molecular weight of 13000 to ensure the removal of polymer impurities from biological sources, such as RNase, DNase and endotoxin. It is not recommended to use ordinary purified water or distilled water.