1. About the manufacture of virus sampling tubes
Virus sampling tubes belong to medical device products. Most domestic manufacturers are registered according to the first-class products, and few companies are registered according to the second-class products. Recently, in order to meet the emergency needs of Wuhan and other places, many companies have taken the “emergency channel” “Apply for the first-class record permission. The virus sampling tube is composed of a sampling swab, virus preservation solution and outer packaging. Since there is no unified national standard or industry standard, the products of various manufacturers vary greatly.
1. Sampling swab: The sampling swab directly contacts the sampling site, and the material of the sampling head is closely related to the subsequent detection. The sampling swab head should be made of Polyester (PE) synthetic fiber or Rayon (man-made fiber). Calcium alginate sponge or wooden stick swabs (including bamboo sticks) cannot be used, and the material of the swab head cannot be cotton products. Because cotton fiber has a strong adsorption of protein, it is not easy to elute into the subsequent storage solution; and when a wooden stick or bamboo stick containing calcium alginate and wooden components is broken, soaking in the storage solution will also adsorb protein, and even will It can inhibit the subsequent PCR reaction. It is recommended to use synthetic fibers such as PE fiber, polyester fiber and polypropylene fiber for the material of the swab head. Natural fibers such as cotton are not recommended. Nylon fibers are also not recommended because nylon fibers (similar to toothbrush heads) absorb water. Poor, resulting in insufficient sampling volume, affecting the detection rate. Calcium alginate sponge is forbidden for sampling swab material! Swab handle has two types: broken and built-in. The broken swab is placed in the storage tube after sampling, and the tube cap is broken after being broken from the position near the sampling head; the built-in swab directly puts the sampling swab into the storage tube after sampling, and the storage tube tube cover is built in Align the small hole with the top of the handle and tighten the tube cover. Comparing the two methods, the latter is relatively safe. When the broken swab is used in conjunction with a smaller size storage tube, it may cause liquid splashing in the tube when broken, and full attention should be paid to the risk of contamination caused by improper use of the product. It is recommended to use hollow polystyrene (PS) extruded tube or polypropylene (PP) injection creasing tube for the material of the swab handle. No matter what material is used, calcium alginate additives cannot be added; wooden sticks or Bamboo sticks. In short, the sampling swab should ensure the amount of sampling and the amount of release, and the selected materials must not have substances that affect subsequent testing.
2. Virus preservation solution: There are two kinds of virus preservation solutions widely used in the market, one is a virus maintenance solution modified based on the transport medium, and the other is a modified solution for nucleic acid extraction lysate.
The main component of the former is Eagle’s basic culture medium (MEM) or Hank’s balanced salt, which is added with the salts, amino acids, vitamins, glucose and protein necessary for virus survival. This storage solution uses phenol red sodium salt as an indicator and solution. When the pH value is 6.6-8.0, the solution is pink. The necessary glucose, L-glutamine and protein are added to the preservation solution. 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 the virus but also beneficial to the growth of bacteria. If the preservation solution is contaminated with bacteria, it will multiply in large quantities. The carbon dioxide in its metabolites will cause the preservation solution pH to fall from pink Turns yellow. Therefore, most manufacturers have added antibacterial ingredients to their formulations. The recommended antibacterial agents are penicillin, streptomycin, gentamicin and polymyxin B. Sodium azide and 2-methyl are not recommended Inhibitors such as 4-methyl-4-isothiazolin-3-one (MCI) and 5-chloro-2-methyl-4-isothiazolin-3-one (CMCI) because these components have an effect on the PCR reaction. Since the sample provided by this preservation solution is basically a live virus, the originality of the sample can be kept to the greatest extent, and it can be used not only for the extraction and detection of virus nucleic acids, but also for the cultivation and isolation of viruses. However, it should be noted that when used for detection, nucleic acid extraction and purification must be performed after inactivation.
Another kind of preservation solution prepared based on nucleic acid extraction lysate, the main components are balanced salts, EDTA chelating agent, guanidine salt (such as guanidine isothiocyanate, guanidine hydrochloride, etc.), anionic surfactant (such as dodecane Sodium sulfate), cationic surfactants (such as tetradecyltrimethylammonium oxalate), phenol, 8-hydroxyquinoline, dithiothreitol (DTT), proteinase K and other components, This storage solution is to directly cleave the virus to release the nucleic acid and eliminate the RNase. If only used for RT-PCR, it is more suitable, but the lysate can inactivate the virus. This kind of sample cannot be used for virus culture separation.
The metal ion chelating agent used in the virus preservation solution is recommended to use EDTA salts (such as dipotassium ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, etc.), and it is not recommended to use heparin (such as sodium heparin, lithium heparin), so as not to affect PCR detection.
3. Preservation tube: The material of the preservation tube should be selected carefully. There are data suggesting that polypropylene (Polypropylene) is related to the adsorption of nucleic acid, especially at high tension ion concentration, polyethylene (Polyethylene) is more preferred than polypropylene (Polypropylene) Easy to grasp DNA/RNA. Polyethylene-propylene polymer (Polyallomer) plastic and some specially processed polypropylene (Polypropylene) plastic containers are more suitable for DNA/RNA storage. In addition, when using a breakable swab, the storage tube should try to select a container with a height greater than 8 cm to prevent the contents from being splashed and contaminated when the swab is broken.
4. Water for production preservation solution: The ultrapure water used for production preservation solution should be filtered through an ultrafiltration membrane with a molecular weight of 13,000 to ensure the removal of polymer impurities from biological sources, such as RNase, DNase, and endotoxin, and ordinary purification is not recommended. Water or distilled water.
2. Use of virus sampling tubes
Sampling using the virus sampling tube is mainly divided into oropharyngeal sampling and nasopharyngeal sampling:
1. Oropharyngeal sampling: First press the tongue with the tongue depressor, then extend the head of the sampling swab into the throat to wipe the bilateral pharyngeal tonsils and posterior pharyngeal wall, and wipe the posterior pharyngeal wall with light force, avoid touching the tongue unit.
2. Nasopharyngeal sampling: measure the distance from the tip of the nose to the ear lobe with a swab and mark with a finger, insert the sampling swab into the nasal cavity in the direction of the vertical nose (face), the swab should extend at least half the length of the ear lobe to the tip of the nose, Leave the swab in the nose for 15-30 seconds, gently rotate 3-5 times, and withdraw the swab.
It is not difficult to see from the method of use, whether it is an oropharyngeal swab or a nasopharyngeal swab, sampling is a technical task, which is difficult and contaminated. The quality of the collected sample is directly related to the subsequent detection. If the collected sample has a viral load Low, easy to cause false negatives, difficult to confirm the diagnosis.
Post time: Jun-21-2020