On February 21, the National Health and Wellness Commission released the Novel Coronavirus Pneumonia Prevention and Control Program (5th edition), along with the Technical Guidelines for Laboratory Testing of Novel Coronavirus Infection in Pneumonia (5th edition) as an annex.
This technical guide was developed to guide disease control departments at all levels and other relevant institutions in the laboratory testing of novel coronavirus pneumonia.
I. Specimen collection
(A) the collection of objects
Suspected and aggregated cases of novel coronavirus pneumonia, those requiring diagnosis or differential diagnosis of novel coronavirus infection, or other environmental or biological materials requiring further screening tests.
(B) specimen collection requirements
1. Technical personnel engaged in specimen collection for novel coronavirus testing should have undergone biosafety training (training qualified) and have the appropriate experimental skills. Personal Protective Equipment (PPE) requirements for sampling personnel: N95 and above protective masks, goggles, one-piece protective clothing, double-layer latex gloves, waterproof boot covers; if contact with patient blood, body fluids, secretions or excretions, the outer latex gloves should be replaced in a timely manner.
2. Specimens of hospitalized cases are collected by the medical and nursing staff of the hospital.
3 close contacts specimens are collected by the local designated disease control agencies, medical institutions.
4, according to the needs of laboratory testing, can be combined with the disease process multiple sampling.
(C) the type of specimens collected
Each case must collect acute respiratory specimens (including upper respiratory specimens or lower respiratory specimens), with priority given to lower respiratory specimens in severe cases; stool specimens, whole blood specimens, serum specimens can be retained according to clinical needs.
Specimen types.
1. Upper respiratory specimens: including nasopharyngeal swabs, pharyngeal swabs, etc.
2. Lower respiratory specimens: deep cough sputum, alveolar lavage fluid, bronchial lavage fluid, respiratory aspirates, etc.
3. Stool specimens: Retain about 10 grams of stool specimens (peanut size), if it is not convenient to retain stool specimens, anal swabs can be collected.
4. Blood specimens: Try to collect as much anticoagulated blood as possible within 7 days after the onset of the disease, with a collection volume of 5 ml.
5. Serum specimens: Try to collect both acute and recovery serum. The first serum should be collected as soon as possible (preferably within 7 days after the onset of the disease), and the second serum should be collected in the third to fourth week after the onset of the disease. A volume of 5 ml should be collected, and the use of a non-anticoagulant vacuum blood tube is recommended. The serum specimen is mainly used for antibody determination and no nucleic acid testing is performed.
(iv) Specimen collection and processing
1) Nasopharyngeal swab: The sampler should hold the head of the person being collected with one hand and hold the swab with the other hand. The swab should be inserted against the nostril and slowly penetrate backward along the bottom of the lower nasal passage. When the tip of the swab reaches the posterior wall of the nasopharyngeal cavity, gently rotate it for a week (in case of reflex coughing, it should be left for a moment), then slowly remove the swab and dip the swab head into a tube containing 2-3 ml of virus preservation solution (isotonic salt solution, tissue culture solution or phosphate buffer can also be used), discard the tail part and screw the cap tightly.
2. Pharyngeal swab: the collected person first rinses his mouth with saline, the sampling personnel puts the swab into sterile saline and moistens it (it is forbidden to put the swab into virus preservation solution to avoid allergy caused by antibiotics), the collected person tilts his head slightly, opens his mouth wide and makes an "ah" sound to reveal the pharyngeal tonsils on both sides, crosses the swab over the root of the tongue and puts the swab in the pharyngeal tonsils on both sides of the collected person. The swab is then dipped into a tube containing 2 to 3 ml of virus preservation solution (isotonic salt solution, tissue culture solution or phosphate buffer can also be used), the end is discarded and the cap is tightened. A pharyngeal swab can also be placed in the same tube as a nasopharyngeal swab.
3. Nasopharyngeal aspirate or respiratory aspirate: A collector connected to a negative pressure pump is used to aspirate mucus from the nasopharynx or respiratory secretions from the trachea. Insert the head of the collector into the nasopharynx or trachea, connect the negative pressure, rotate the head of the collector and withdraw it slowly to collect the extracted mucus, and rinse the collector once with 3 ml of sampling fluid (a pediatric catheter can also be connected to a 50 ml syringe to replace the collector).
4. Deep coughing of sputum: After asking the patient to cough deeply, collect the coughing sputum in a 50 ml screw-top plastic tube containing 3 ml of sampling solution. If sputum is not collected in the sampling solution, 2 to 3 ml of sampling solution can be added before the test, or sputum digestion solution of equal volume of sputum can be added.
Sputum digest solution storage solution formulation.
Dilute the storage solution with deionized water to 100 ml before use. sputum can also be liquefied with an equal volume of sputum containing 1 g/L proteinase K in phosphate buffer.
5. Bronchial lavage solution: Insert the collector head into the trachea (about 30 cm deep) through the nostril or tracheal socket, inject 5 ml of saline, connect the negative pressure, rotate the collector head and withdraw it slowly. Collect the extracted mucus and rinse the collector once with the sampling fluid (a pediatric catheter connected to a 50 ml syringe can also be used to collect instead).
6. Alveolar lavage fluid: After local anesthesia, insert the fiberoptic bronchoscope through the mouth or nose through the pharynx into the bronchus of the middle lobe of the right lung or the lingual segment of the left lung, fit its tip into the opening of the bronchial branches, and slowly add sterilized saline through the tracheal biopsy hole, 30-50 ml each time, totaling 100-250 ml, which should not exceed 300 ml.
7. Stool specimen: take 1ml of specimen processing solution, pick a soybean-sized stool specimen and add it to the tube, blow gently 3-5 times, let it stand at room temperature for 10 minutes, centrifuge at 8000rpm for 5 minutes, and aspirate the supernatant for testing.
The stool specimen treatment solution can be prepared by the patient: 1.211 g Tris, 8.5 g NaCl, 1.1 g anhydrous calcium chloride or 1.47 g calcium chloride with crystal water, dissolved in 800 ml deionized water, pH adjusted to 7.5 with concentrated hydrochloric acid, and supplemented with deionized water to 1000 ml.
Stool suspensions can also be prepared by dissolving stool specimens in HANK'S solution or other isotonic salt solutions, tissue culture fluid or phosphate buffer solution. If the patient shows symptoms of diarrhea, keep 3-5 ml of stool specimen, gently blow and mix, then centrifuge at 8000 rpm for 5 minutes and aspirate the supernatant for use.
8、Anal swab: Gently insert a sterile cotton swab into the anus for 3~5cm, then gently rotate and pull out, and immediately put it into a 15ml outer screw cap sampling tube containing 3~5ml of virus preservation solution, discard the tail, and screw the cap tightly.
9. Blood specimens: It is recommended that 5 ml of blood specimens be collected using a vacuum blood tube containing EDTA anticoagulant, and that whole blood or plasma be used for nucleic acid extraction depending on the type of nucleic acid extraction reagent chosen. If plasma separation is required, whole blood is centrifuged at 1500-2000 rpm for 10 minutes and the supernatant is collected in sterile screw-top plastic tubes.
10. Serum specimen: Collect 5 ml of blood specimen by vacuum negative pressure blood collection tube, leave it at room temperature for 30 minutes, centrifuge it at 1500-2000 rpm for 10 minutes, and collect the serum in a sterile screw-top plastic tube.
Other materials: collected according to the design requirements specification.
(E) Specimen packaging
After collection, the specimens are divided and packed in biosafety cabinets in biosafety level II laboratories.
1. All specimens should be placed in a suitable sized sample collection tube with a screw cap with a gasket inside and freezing resistance, tightened. The specimen number, type, name and date of sampling should be indicated on the outside of the container.
2. Seal the specimens in sealed bags, and limit one specimen per bag. The specimen packaging requirements should be in accordance with the corresponding standards of the Technical Rules for the Safe Transport of Dangerous Goods by Air.
3. For the transport of external specimens, the specimens should be packed in three layers according to the type of specimens, in accordance with Class A or Class B infectious substances.
(F) specimen preservation
Specimens used for virus isolation and nucleic acid detection should be tested as soon as possible, and specimens that can be tested within 24 hours can be stored at 4 ℃; specimens that cannot be tested within 24 hours should be stored at -70 ℃ or below (if there is no -70 ℃ storage conditions, then stored temporarily in the -20 ℃ refrigerator). Serum can be stored at 4℃ for 3 days, and below -20℃ for long term storage. Specimens should be stored separately in special storage or special cabinets. Repeated freezing and thawing of specimens should be avoided during transport.
(G) Specimen delivery
Specimens should be sent to the laboratory as soon as possible after collection, and if long-distance transportation is required, it is recommended to use dry ice and other refrigeration methods for storage.
1、Sending specimens
Specimens of aggregated cases from each province (autonomous region, municipality directly under the Central Government) should be sent to the Institute for Prevention and Control of Viral Diseases of the Chinese Center for Disease Control and Prevention for testing and review, with the sample delivery form (see attached).
2. Transportation of pathogens and specimens
2.1 Domestic transportation
The transport packaging classification of novel coronavirus strains or other potentially infectious biological materials belongs to Class A, corresponding to UN2814, and the packaging conforms to the PI602 classification packaging requirements of ICAO Doc9284 "Technical Rules for the Safe Transport of Dangerous Goods by Air"; environmental samples belong to Class B, corresponding to UN3373, and the packaging conforms to ICAO Doc9284 "Dangerous Goods by Air". Doc9284 "Technical Rules for the Safe Transport of Dangerous Goods by Air" PI650 classification packaging requirements; transported by other means of transport can refer to the above standard packaging.
New coronavirus strains or other potentially infectious materials should be transported in accordance with the "Regulations for the transport of highly pathogenic microorganisms (viruses) or samples that can infect humans" (former Ministry of Health Order No. 45) for the "Certificate of Carriage".
2.2 International transport of new coronavirus strains or samples in the international transport, should be standardized packaging, in accordance with the "Entry-Exit Special Articles Health and Quarantine Management Regulations" for the relevant procedures, and meet the relevant national and international requirements.
2.3 strain and sample management new coronavirus strains and their samples should be managed by dedicated personnel, accurate records of the source, type and number of strains and samples, number registration, take effective measures to ensure the safety of strains and samples, and prevent misuse, malicious use, theft, robbery, loss, leakage and other events.
Second, the laboratory detection of novel coronavirus
The conventional detection method for novel coronavirus infection is real-time fluorescence RT-PCR, and any detection of novel coronavirus must be performed in a qualified laboratory by personnel trained in relevant technical safety. The nucleic acid assays in this guideline focus on the openreading frame1ab (ORF1ab) and nucleocapsidprotein (N) of the novel coronavirus genome.
Laboratory confirmation of positive cases requires one of the following two conditions.
1. 2 targets of the novel coronavirus (ORF1ab, N) are positive by real-time fluorescence RT-PCR in the same specimen. If there is a positive result for a single target, the test needs to be resampled and retested. If it is still positive for a single target, it is judged as positive.
2. Two specimens with a single positive target by real-time fluorescence RT-PCR at the same time, or the same type of specimen with a single positive target in both sampling and testing, can be judged as positive.
A negative nucleic acid test result does not exclude novel coronavirus infection. Factors that may produce a false negative need to be excluded, including: poor sample quality, such as respiratory samples from the oropharynx and other sites; samples collected too early or too late; failure to properly preserve, transport, and handle samples; and the presence of the technology itself, such as viral mutation and PCR inhibition.
