Basic requirements for specimen collection
1. The specimens of hospitalized cases are collected by the medical staff of the hospital where they are located, and the specimens of close contacts are collected by the local designated disease control agency and medical institution. When collecting specimens, different sampling areas should be set according to different collection objects. Fever patients go to fever clinics for treatment and sampling. Institutions that do not have fever clinics should set up special sampling areas for fever patients, and sample fever patients and other testing groups to avoid intersection. infection. 2. Asymptomatic infected persons, entry personnel, and close contacts should collect nasopharyngeal swabs for nucleic acid testing during isolation and observation. Two nasopharyngeal swab samples should be collected at the same time when the isolation is released, and different nucleic acid detection reagents should be used for testing respectively. In principle, the secondary testing is carried out by different testing institutions. 3. According to the needs of clinical and laboratory testing, multiple samples can be taken during hospitalization and isolation, and multiple samples such as respiratory tract, blood, and stool can be collected at the same time. Sampling personnel should strictly follow the sampling specifications to collect specimens to ensure that the quality of the collected specimens meets the requirements. At the same time, they should record the information of the examinee in detail, and can use barcode scanning and other information methods to collect relevant information. 4. For population screening, sampling tubes should be determined according to the requirements of the reagents used for nucleic acid extraction and detection, and sampling tubes containing virus inactivating agents (guanidine salts or surfactants, etc.) can be selected. The specimen used for virus isolation should be placed in a sampling tube that does not contain virus inactivation agent. 5. Items and environmental monitoring should be based on monitoring purposes and prevention and control needs to determine the sampling items, location and quantity, sampling should strictly follow the sampling specifications.
(4) Types of specimens collected. Each case must collect acute respiratory tract specimens (including upper respiratory tract specimens or lower respiratory tract specimens). In severe cases, lower respiratory tract specimens shall be collected first; stool specimens, whole blood specimens, serum specimens, and fecal specimens may be collected according to clinical needs. Urine specimen. Articles and environmental specimens are collected according to monitoring requirements.
Specimen types: 1. Upper respiratory tract specimens: including nasopharyngeal swabs, throat swabs, etc. 2. Lower respiratory tract specimens: deep cough sputum, alveolar lavage fluid, bronchial lavage fluid, respiratory tract aspirate, etc. 3. Stool specimens/anal swabs: about 10 grams (the size of peanuts) should be collected. If it is not convenient to keep the stool samples, anal swabs can be collected. 4. Blood specimen: anticoagulant blood, the collection volume is 5ml, it is recommended to use a vacuum blood vessel containing EDTA anticoagulant to collect blood. 5. Serum specimens: try to collect double serum samples during the acute and recovery phases. The first serum should be collected as soon as possible (preferably within 7 days after the onset of illness), and the second serum should be collected in the 3 to 4 weeks after the onset of illness. The collection volume is 5ml, and it is recommended to use a vacuum blood collection tube without anticoagulant. Serum samples are mainly used for the determination of antibodies, and no nucleic acid detection. 6. Urine specimen: collect the mid-morning urine, the collection volume is 2~3ml. 7. Surface specimens: including imported cold-chain food or imported goods on the inner and outer packaging surfaces, as well as transportation and storage tools and other parts that may be contaminated by smear collection. 8. Sewage samples: According to the distribution of the drainage system of the large-scale imported frozen goods processing and treatment sites at the seaport, focus on selecting the sewage outlet, the internal pipe network collection, the downstream of the sewage flow or the connection with the municipal pipe network, etc. 2 to 3 points Bits are sampled.
(4) Specimen collection and processing
1. Nasopharyngeal swab: the sampler gently supports the head of the person to be collected with one hand, and holds the swab with the other. The swab enters the nostril and goes deep along the bottom of the lower nasal passage slowly. Because the nasal passage is curved, it is not acceptable. Excessive force to avoid traumatic bleeding. When the tip of the swab reaches the back wall of the nasopharyngeal cavity, gently rotate it for a week (in case of reflex cough, stay for a while), then slowly take out the swab, and immerse the swab head in 2～3ml virus preservation solution (also can be Use isotonic saline solution, tissue culture fluid or phosphate buffer), discard the tail, and screw the cap tightly.
2. Pharyngeal swab: the person being collected should rinse their mouth with normal saline, and the person sampling shall moisten the swab in sterile saline (it is forbidden to put the swab in the virus preservation solution to avoid antibiotics causing allergies), and the person being collected Lean the head slightly, open the mouth, and make an "ah" sound, exposing the tonsils on both sides, put the swab across the base of the tongue, wipe the tonsils on both sides of the person with a little force for at least 3 times, and then wipe up and down the posterior pharynx for at least 3 times. Next, immerse the swab head in a tube containing 2 to 3 ml of virus preservation solution (isotonic saline solution, tissue culture fluid or phosphate buffer solution can also be used), discard the tail, and screw the tube cap tightly. The throat swab can also be placed in the same tube as the nasopharyngeal swab.
3. Nasopharyngeal extract or respiratory tract extract: Use a collector connected to a negative pressure pump to extract mucus from the nasopharynx or extract respiratory secretions from the trachea. Insert the head of the collector into the nasal cavity or trachea, turn on the negative pressure, rotate the head of the collector and slowly withdraw, collect the extracted mucus, and rinse the collector once with 3ml sampling liquid (you can also use a pediatric catheter connected to a 50ml syringe Alternative collector).
4. Deep cough sputum: After the patient has a deep cough, collect the coughed up sputum in a sampling tube containing 3ml of sampling fluid. If the sputum is not collected in the sampling fluid, 2 to 3ml of the sampling fluid can be added before the test, or an equal volume of sputum digestive juice can be added. The formula of sputum digestive juice storage solution is shown in Table 1.
When using, dilute the storage solution to 50ml with deionized water, mix it with the same volume of sputum, or refer to the reagent instructions for use, or use an equal volume of sputum with 1g/L proteinase K-containing phosphate buffer to convert the sputum .
5. Bronchial lavage fluid: insert the collector head from the nostril or tracheal socket into the trachea (about 30cm deep), inject 5ml of normal saline, turn on the negative pressure, rotate the collector head and slowly withdraw, collect the extracted mucus , And flush the collector with the sampling solution once, or you can use a pediatric catheter connected to a 50ml syringe to replace the collection.
6. Alveolar lavage fluid: After local anesthesia, insert the fiberoptic bronchoscope through the mouth or nose through the pharynx into the bronchus of the right middle lobe or left lung lingual segment, and tap its top into the bronchial branch opening, and slowly add it through the tracheal biopsy hole. Bacterial physiological saline, 30-50ml each time, total 100-250ml, should not exceed 300ml.
7. Stool specimen: Take 1ml of specimen processing solution, pick up the soy bean-sized stool specimen and add it to the tube, gently blow and suck 3 to 5 times, let it stand at room temperature for 10 minutes, centrifuge at 8000 rpm for 5 minutes, and absorb the supernatant. Detection. The fecal specimen treatment solution can be prepared by itself, and the formula is shown in Table 2.
HANK'S solution or other isotonic saline solution, tissue culture fluid or phosphate buffer solution can also be used to dissolve the stool sample to prepare a stool suspension. If the patient has symptoms of diarrhea, take 3~5ml of fecal specimen, gently pipette to mix, centrifuge at 8000rpm for 5 minutes, and absorb the supernatant for use.
8. Anal swab: Use a sterile cotton swab to gently insert it into the anus 3～5cm, then gently rotate it out, immediately put it into a 15ml outer screw cap sampling tube containing 3～5ml virus preservation solution, discard the tail, and rotate it. Tighten the tube cap.
9. Blood specimens: It is recommended to use vacuum blood collection tubes containing EDTA anticoagulant to collect 5ml blood specimens. According to the type of nucleic acid extraction reagents selected, use whole blood or plasma for nucleic acid extraction. To separate plasma, centrifuge the whole blood at 1500-2000 rpm for 10 minutes, and collect the supernatant in a sterile screw-top plastic tube.
10. Serum sample: Collect 5ml of blood sample with vacuum negative pressure blood collection tube, leave it at room temperature for 30 minutes, centrifuge at 1500-2000rpm for 10 minutes, and collect serum in a sterile screw-top plastic tube.
11. Surface specimens: Refer to the method recommended in the "Technical Specifications for Environmental Monitoring of the New Coronavirus in the Agricultural Trade (Market Trade) Market" (WS/T776—2021). After the sampling swab is fully infiltrated with the virus preservation solution, apply it to the surface again and then apply the swab. Put it back into the sampling tube and soak it, take it out and apply the sample again, repeat more than 3 times. Multi-point distributed sampling is performed on objects with large surfaces.
12. Sewage specimens: Refer to the methods recommended in the "Technical Specifications for Environmental Monitoring of New Coronavirus in the Agricultural Trade (Market Trade) Market" (WS/T776—2021). When collecting a swab sample of sewage, immerse the swab in the adsorbed sewage and remove the swab. Put back the sampling tube to soak, take it out and immerse it in the sewage again, repeat more than 3 times, and conduct multi-point distributed sampling for each sewage sampling location. When collecting sewage water samples, use polyethylene plastic bottles to collect 1L～1.5L of sewage. For sewage collection larger than 1.5L, you can use polyethylene plastic buckets or special enrichment equipment for on-site water samples. Before collecting sewage water samples, they should be mixed thoroughly and then sampled; if the sewage is difficult to mix well and stratification occurs, sampling can be done in layers according to the proportion of the water volume of each layer.
13. Other materials: collect according to design requirements and specifications.
(5) Specimen packaging
After the specimens are collected, they are subpackaged in the biological safety cabinet of the second-level biological safety laboratory.
1. All specimens should be placed in a sample collection tube of suitable size with a screw cap and a gasket inside, which is resistant to freezing, and tightened. The sample number, type, name and sampling date are indicated on the outside of the container. 2. Put the sealed specimens into a sealed bag, each bag is limited to one specimen. The sample packaging requirements must meet the corresponding standards of the "Technical Rules for the Safe Transport of Dangerous Goods by Air". 3. When the transportation of external specimens is involved, three-layer packaging shall be carried out according to the type of specimens and the infectious substances of Class A or Class B.
(6) Preservation of specimens
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). Serum samples can be stored at 4°C for 3 days, and can be stored for a long time below -20°C. Special libraries or special counters should be set up to store specimens separately.
(7) Submit specimens for inspection
The specimens should be sent to the laboratory as soon as possible after collection, and the specimens should not be placed at room temperature (25°C) for more than 4 hours after collection. If long-distance transportation is required, it is recommended to use dry ice and other refrigeration methods for storage. Avoid repeated freezing and thawing during specimen transportation.
1. Send specimens
All the original or early cases in the local epidemics discovered in various provinces (autonomous regions and municipalities), key cases related to the epidemiology of the early cases, local cases with unknown sources of infection, imported cases from abroad, imported goods and related environmental positive specimens, etc. At least 2 specimens should be collected in parallel, one is sent to each provincial disease control agency for testing, the other is sent to the China Centers for Disease Control and Prevention for testing and review, and a sample submission form is attached (see Appendix 10-1). New representative strains isolated from various provinces should be promptly sent to the China Centers for Disease Control and Prevention for review and preservation.
2. Transport of specimens and strains
(1) The domestic transportation of new coronavirus strains or other potentially infectious biological materials is classified as Class A, and the corresponding UN number is UN2814. The packaging conforms to the PI620 classification of the ICAO document Doc9284 "Technical Instructions for the Safe Transport of Dangerous Goods by Air" Packaging requirements: The environmental samples belong to category B, and the corresponding UN number is UN3373. The packaging meets the PI650 classification requirements of the ICAO document Doc9284 "Technical Rules for the Safe Transport of Dangerous Goods by Air"; the above standard packaging can be referred to for transportation by other means of transportation. The transportation of new coronavirus strains or other potentially infectious materials should be handled in accordance with the "Administrative Regulations on the Transportation of Highly Pathogenic Microorganisms (Virus) Species or Samples that Can Infect Humans" (formerly Ministry of Health Order No. 45) for the "Transport Permit Certificate" .
(2) International transportation The specimens or strains of the new coronavirus that are transported internationally shall be packaged in a standardized manner, go through the relevant procedures in accordance with the "Regulations on the Administration of Sanitary and Quarantine of Entry and Exit Special Articles", and meet relevant national and international requirements.
(3) Receipt and management of specimens and virus strains. Specimens transported by air arrive at the destination airport and are transported by professional transport vehicles to the receiving unit. Specimens transported by land are transported by professional vehicles. The transporters and receivers shall deal with the specimens Double sign for receipt. The specimens and strains of the new coronavirus should be managed by a dedicated person, accurately record the source, type, quantity, and number registration of the specimens and strains, take effective measures to ensure the safety of the strains and samples, and strictly prevent misuse, malicious use, theft, and theft. Robbery, loss, leakage and other incidents.
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Laboratory testing of the new coronavirus
