A. About the production and manufacture of virus sampling tubes
Virus sampling tube belongs to the medical device products, domestic manufacturers are mostly in accordance with a class of products for the record, according to the second class product registration of enterprises are very few, recently in order to meet the urgent needs of Wuhan and other places, there are many enterprises are to take the "emergency channel" to apply for a record permit. Virus sampling tube consists of a sampling swab, virus preservation solution and packaging. Since there is no uniform national standard or industry standard, the products of various manufacturers vary greatly.
1, sampling swab: sampling swab direct contact with the sampling site, the material of the sampling head and the subsequent detection of close relationship. Sampling swab head should be made of Polyester (PE) synthetic fiber or Rayon (artificial fiber), can not use calcium alginate sponge or wooden stick swab (including bamboo swabs), swab head material can not be cotton products, because the cotton fiber adsorption of protein is stronger, not easy to elute to the subsequent preservation solution; and containing calcium alginate and wooden components of the wooden stick or bamboo swab after fracture Immersion in the preservation solution will also adsorb proteins and even inhibit the subsequent PCR reaction. The material used to manufacture the swab head is recommended to use PE fiber, polyester fiber, polypropylene fiber and other synthetic fibers, not recommended natural fibers such as cotton, and nylon type fibers, because nylon type fibers (similar to toothbrush brush head) poor water absorption, resulting in insufficient sampling volume, affecting the detection rate. Sampling swab material prohibited calcium alginate sponge! There are two types of swab handles, fracture type and built-in type. Fracture type swab in the sampling into the preservation tube from the sampling head near the part of the break and tighten the cap; built-in swab in the sampling directly after the sampling swab into the preservation tube, the preservation tube cap built-in small hole aligned with the top of the handle tighten the cap can be. The latter is relatively safe when comparing the two methods. When using the fracture swab with a smaller size preservation tube, the fracture may cause the liquid inside the tube to spill out, and due attention should be paid to the risk of contamination that may be caused by improper use of the product. The material used to manufacture the swab handle is recommended to use hollow polystyrene (PS) extrusion tube or polypropylene (PP) injection indentation tube, regardless of the material used can not add calcium alginate type additives; the use of wooden sticks or bamboo sticks is not recommended. In short, the sampling swab should ensure that the sampling volume and release, the selected material can not have a substance that affects the subsequent detection.
2, virus preservation solution: There are two main types of virus preservation solutions widely used in the market, one is the virus maintenance solution improved based on the transport medium, and the other is the preservation solution improved by nucleic acid extraction lysate.
The main component of the former is Eagle's basic culture solution (MEM) or Hank's balanced salt with added salts, amino acids, vitamins, glucose, and proteins necessary for virus survival. This preservation solution uses phenol red sodium salt as an indicator, and the solution is pink at a solution pH of 6.6-8.0. The preserving solution is supplemented with essential glucose, L-glutamine, and protein, and protein is provided in the form of fetal bovine serum or bovine serum albumin, which stabilizes the protein shell of the virus. Due to the rich nutrient content of the preservation fluid, it is conducive to virus viability but also to bacterial growth. If the preservation fluid is contaminated with bacteria, it will multiply and the carbon dioxide in its metabolites will cause the pH of the preservation fluid to drop and change from pink to yellow. Thus, most manufacturers have added bacteriostatic components to the formulation. The recommended inhibitors are penicillin, streptomycin, gentamicin and polymyxin B. Inhibitors such as sodium azide, 2-methyl-4-isothiazolin-3-one (MCI) and 5-chloro-2-methyl-4-isothiazolin-3-one (CMCI) are not recommended because of the effect of these components on the PCR reaction. Since the samples provided by this preservation solution are basically live viruses, the originality of the samples can be maintained to the maximum extent, and they can be subsequently used not only for nucleic acid extraction detection of viruses, but also for virus culture and isolation. However, it should be noted that the sample must be inactivated and then purified by nucleic acid extraction.
Another preservation solution based on nucleic acid extraction lysate is formulated with several or more components such as balanced salts, EDTA chelator, guanidine salts (e.g. guanidine isothiocyanate, guanidine hydrochloride, etc.), anionic surfactants (e.g. sodium dodecyl sulfate), cationic surfactants (e.g. tetradecyltrimethylammonium oxalate), phenol, 8-hydroxyquinoline, dithiothreitol (DTT), proteinase K, etc. This preservation solution is a direct lysis of the virus to release nucleic acid and eliminate nucleolytic enzymes (RNase), which is more appropriate if used only for RT-PCR, but the lysate can inactivate the virus, and such samples cannot be used for virus culture isolation.
Virus preservation solution using metal ion chelator recommended to use EDTA salts (such as ethylenediaminetetraacetic acid dipotassium, ethylenediaminetetraacetic acid disodium, etc.), not recommended to use heparin class (such as heparin sodium, heparin lithium), so as not to affect the PCR detection.
3, preservation tube: manufacturing preservation tube material should pay attention to the choice, there are data suggesting that polypropylene (Polypropylene) and nucleic acid adsorption, especially in high tension ion concentration, polyethylene plastic (Polyethylene) than polypropylene (Polypropylene) easier to catch DNA / RNA. polyethylene - propylene polymer ( Polyallomer) plastic and some specially treated polypropylene plastic containers are more suitable for DNA/RNA storage use. In addition, when using foldable swabs, the preservation tube should try to choose a container with a height greater than 8 cm to prevent contamination of the contents from splashing out when the swab is folded.
4. Water for the production of preservation solution: Ultra-pure water used for the production of preservation solution should be filtered through an ultrafiltration membrane with a molecular weight of 13,000 to ensure the removal of polymeric impurities of biological origin, such as RNase, DNase and endotoxin, and the use of ordinary purified water or distilled water is not recommended.
Second, about the use of virus sampling tubes
The use of virus sampling tube sampling is mainly divided into oropharyngeal sampling and nasopharyngeal sampling.
1, oropharyngeal sampling: first press the tongue with a tongue depressor, then extend the head of the sampling swab into the pharynx to wipe the bilateral pharyngeal tonsils and the posterior pharyngeal wall, wipe the posterior pharyngeal wall with mild force, avoid touching the tongue.
2、Nasopharynx sampling: measure the distance from the tip of the nose to the earlobe with a swab and mark it with your finger, insert the sampling swab into the nasal cavity in the direction of vertical nose (face), the swab should reach at least half of the length from the earlobe to the tip of the nose, make the swab stay in the nose for 15-30 seconds, gently rotate it 3-5 times and withdraw the swab.
It is easy to see from the use of the method, whether oropharyngeal swabs or nasopharyngeal swabs, sampling is a technical task, difficult and easy to contaminate, the quality of the collected samples is directly related to the subsequent detection, if the collected samples have a low viral load, it is easy to cause false negatives and difficult to confirm the diagnosis.
Currently, the recommended samples for the kits sold on the market are mostly oropharyngeal swabs or nasopharyngeal swabs and alveolar lavage fluid. If venous blood samples, especially special nucleic acid detection tubes, are used to collect blood and extract purified RNA for testing, this can greatly reduce the work difficulty of sampling personnel. After all, the difficulty of collecting venous blood samples is not high, and like the detection of hepatitis C RNA, the extracted and purified RNA from about 5 ml of EDTA-anticoagulated blood sample separated plasma can fully meet the needs of PCR testing.
It is expected that the diagnostic reagent R&D enterprises and medical institutions and research institutions will verify the correlation between the test results of oropharyngeal (nasopharyngeal) swab samples and those of venous blood samples as soon as possible, and launch highly sensitive kits that can adapt to a variety of sample tests as soon as possible.
