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SN/T 5335-2020Technical Specification for Biosafety Operation of African Swine Fever Testing Laboratory (English PDF)

非洲猪瘟检测实验室生物安全操作技术规范

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Issued by

General Administration of Customs

Level / Type

National · Recommended

Issue date

December 30, 2020

Implementation date

July 1, 2021

Scope

SN/T 5335-2020 is the English-translated version of 非洲猪瘟检测实验室生物安全操作技术规范.

SN/T 5335-2020 — This document specifies the basic biological safety requirements of African swine fever testing laboratories, including sample reception and management, sample processing, sample Testing, the harmless treatment of waste and residual samples, the disinfection of facilities and the environment, and the delivery of positive samples for preliminary screening.

This document is applicable to related activities of African swine fever testing laboratories.

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Document preview — SN/T 5335-2020

National Standard of the People's Republic of China

ICS: 65.020.30
Classification: B 41

Issued by: General Administration of Customs

Foreword
1 Scope
2 Normative references
3 Terms and definitions
4 Basic requirements for laboratory biosafety
5 Sample reception and management
6 Sample processing
6.1 Sample preparation
6.2 Sample preparation requirements
6.3 Sample preparation
6.4 Temporary storage of residual samples
7 Sample testing
7.1 Handling method of sample spillage
7.2 How to treat the sample in contact with the skin
7.3 Selection of detection methods
8 Harmless treatment of waste and residual samples

Foreword

This document was drafted in accordance with the rules given in GB/T 1.1-2020.

This document was proposed and managed by the General Administration of Customs of the People's Republic of China.

Drafting organizations of this document. Gongbei Customs of the People's Republic of China, Qingdao Customs of the People's Republic of China, Shanghai of the People's Republic of China Customs, Beijing Customs of the People's Republic of China, Dalian Customs of the People's Republic of China, Changchun Customs of the People's Republic of China, People's Republic of China Shenzhen Customs of the country, Guangzhou Customs of the People's Republic of China, Changsha Customs of the People's Republic of China, and Chongqing Customs of the People's Republic of China.

1 Scope

This document specifies the basic biological safety requirements of African swine fever testing laboratories, including sample reception and management, sample processing, sample Testing, the harmless treatment of waste and residual samples, the disinfection of facilities and the environment, and the delivery of positive samples for preliminary screening.

This document is applicable to related activities of African swine fever testing laboratories.

2 Normative references

The contents of the following documents constitute the indispensable clauses of this document through normative references in the text. Among them, dated quotations Only the version corresponding to that date is applicable to this document; for undated references, the latest version (including all amendments) is applicable Used in this document.

GB/T 18088 Entry and Exit Animal Quarantine Sampling GB/T 18648 African swine fever diagnostic technology GB 19489 Laboratory Biosafety General Requirements GB/T 27401 Laboratory Quality Control Specification Animal Quarantine SN/T 1559 Technical Specification for African Swine Fever Quarantine Regulations on Biosafety Management of Pathogenic Microorganism Laboratories (Order No. 424 of the State Council) Transport and packaging specifications for highly pathogenic animal pathogenic microorganisms (virus) species or samples (Announcement No. 503 of the Ministry of Agriculture) World Organization for Animal Health (OIE) Terrestrial Animal Diagnostic Test and Vaccine Manual (2019 Edition) the Food and Agriculture Organization of the United Nations (FAO) Animal Production and Health Manual (2018 Edition)

3 Terms and definitions

There are no terms and definitions that need to be defined in this document.

4 Basic requirements for laboratory biosafety

4.1 African swine fever virus is a highly pathogenic animal microorganism. For related information, see Appendix A. According to Article 30 of Order No. 424 of the State Council it is determined that those engaged in the isolation of highly pathogenic microorganisms and related experimental activities on animals should be carried out in a biosafety level 3 or above laboratory.

4.2 For laboratories that carry out the detection of African swine fever virus nucleic acid and antibodies, their facilities and equipment should comply with the level of biosafety level 2 in GB 19489.

(BSL-2) and above laboratory requirements. The experimental activities before the sample inactivation should be operated in a biological safety cabinet, waste and tested samples it should be sterilized by high-pressure steam.

4.3 The management, technology, process control and result quality control of laboratories that carry out the detection of African swine fever virus nucleic acid and antibody shall be consistent with Meet the requirements of GB/T 27401.

4.4 For laboratories that carry out the detection of African swine fever virus nucleic acid and antibodies, the types of testing activities they are engaged in should reach the corresponding level of biological Safety laboratory protection requirements, and obtain the approval of the competent authority. Laboratories that have not obtained authorization or testing qualifications should not carry out African swine fever testing.

5 Sample reception and management

5.1 The packaging and transportation of the samples should meet the requirements of the Ministry of Agriculture Announcement No. 503, using sealed three-layer packaging, with a biohazard sticker on the outer packaging.

The risk identification will be delivered to the laboratory by a dedicated person.

5.2 The samples used for African swine fever detection should be refrigerated and transported at 2 °C ~ 8 °C, and delivered to the laboratory as soon as possible (within 24 hours). For viral nucleic acid or the samples for serum antibody testing should be stored temporarily (within 24 hours) at 2 °C ~ 8 °C, and stored at -20 °C or -70 °C for a longer period of time. Need to separate serum of non-anticoagulated blood, the serum was collected after 12 hours to 24 hours at room temperature. The blood used for PCR and other viral nucleic acid detection should be anticoagulated with EDTA.

Organize samples to be collected and submitted for inspection in accordance with the requirements of the Ministry of Agriculture Announcement No. 503.The amount of samples sent should meet the requirements of initial screening test and confirmation of sample delivery.

Meet the requirements of GB/T 18088.

5.3 When the laboratory receives samples, the sampling personnel should check whether the outer packaging is damaged, and confirm that the samples are transported to the laboratory under refrigerated and sealed conditions.

room. If the package is found to be damaged, a sealed package should be added to the outer layer, and the sample delivery personnel should be notified immediately to assess the risk of virus exposure and deal with it.

5.4 After confirming that the outer packaging of the sample is not damaged, open the outer packaging in the sample room and check that the inner packaging of the sample is free of leakage and pollution.

Check that the sample type and quantity are correct, and affix the sample logo.

5.5 A strict management system should be implemented for the African swine fever test samples, and the laboratory should have a special freezer or refrigerator for the African swine fever test samples.

The preservation of products is strictly managed by "double locks". If it is necessary to obtain test samples, it shall be approved by the person in charge of the laboratory, and Sample manager records.

5.6 The sample identification shall include but not limited to the following information. sample number, name, quantity, and test items.

5.7 The samples used for the detection of African swine fever shall not be retained. After the test task is completed, the samples with negative test results should be autoclaved Steam disinfection (121°C, 30 min) or other harmless measures should be used for treatment, and the treatment records should be made. Samples with a positive initial screening result are required If the diagnosis is confirmed, it shall be packaged and sent to the diagnosis laboratory designated by the competent authority.

6 Sample processing

6.1 Sample preparation

The inspector is responsible for processing and testing the samples. Take out the sample, after the sample is thawed, send it to the biosafety laboratory in a sealed state Sample preparation is carried out in the whole cabinet.

6.2 Sample preparation requirements

Before opening the sample package in the biological safety cabinet, an effective disinfectant should be used for surface disinfection, and then aseptic sampling treatment should be carried out.

6.3 Sample preparation

6.3.1 Tissue sample preparation

In the biological safety cabinet, use sterile scissors and tweezers to cut 0.1 g~0.2 g of the sample to be tested, put it into a sterile mortar or centrifuge tube, and add After adding 1 mL~2 mL PBS (0.01 mol/L, pH 7.4, see appendix B for preparation), after thorough grinding and crushing, it is made into 10% uniform tissue.

The slurry was placed in a 1.5 mL sterile centrifuge tube. After centrifugation at 12,000 g for 5 min, the supernatant was taken and inactivated at 60°C for 30 min. As in biosafety Use a tissue grinder outside the cabinet to process samples. Disinfect the surface of the sample tube to be grinded before removing it out of the biological safety cabinet.

After centrifugation, the supernatant was taken in a biological safety cabinet, and inactivated at 60 °C for 30 min for subsequent testing.

6.3.2 Anticoagulation

Directly draw the anticoagulant blood in the biological safety cabinet and aliquot it into a 1.5 mL sterile centrifuge tube for later use. If the blood is sticky and cannot be absorbed, use After making a 10-fold dilution of 0.01 mol/L PBS (pH 7.4), aspirate the anticoagulant blood into a 1.5 mL sterile centrifuge tube. Inactivate at 60 °C for 30 min Then move out of the biological safety cabinet for follow-up testing.

6.3.3 Non-anticoagulated blood

After the non-anticoagulated blood sample is collected, place it at room temperature for 12 to 24 hours for the serum to separate out. Collect the serum in a biological safety cabinet and aliquot it in 1.5 mL sterile centrifuge tube. If it is used for nucleic acid detection, it should be inactivated at 60°C for 30 minutes and then removed from the biological safety cabinet for nucleic acid extraction;

The serum for immunological analysis can be removed from the biological safety cabinet after the surface of the centrifuge tube is disinfected, temporarily stored at 4 °C (within 24 h), and placed at -20 °C or Store at -70°C for a long time. The equipment and facilities that may come into contact with samples during the immunological testing process should be disinfected with effective disinfectants.

6.3.4 Ticks

For the detection of African swine fever virus nucleic acid on ticks, the ticks should be placed in a pre-cooled sterile mortar with sterile tweezers in a biological safety cabinet.

Grind with 1.5 mL of pre-cooled 0.01 mol/L PBS (pH 7.4), transfer the ground homogenate into a 1.5 mL sterile centrifuge tube, 1000 g Centrifuge for 5 min, take the supernatant and inactivate it at 60 °C for 30 min, then remove it out of the biological safety cabinet for virus nucleic acid detection or freeze storage for later use.

6.3.5 Other samples

For other samples of animal products, food, biological products, etc. used for the detection of African swine fever virus nucleic acid, refer to the above-mentioned tissues or liquid samples the processing method of the product, the sample to be tested is inactivated in a biological safety cabinet and then subjected to follow-up testing activities.

6.4 Temporary storage of residual samples

After taking the sample, the remaining samples are sealed and packaged in a biosafety bag, marked, and the surface is disinfected with an effective disinfectant, and then stored Special refrigerator at -20°C or -70°C.

7 Sample testing

7.1 Handling method of sample spillage

When a small amount of liquid sample is spilled during the sample detection process, absorbent paper should be used to absorb it, and then an effective disinfectant should be sprayed on the surface.

deal with. When a large amount of liquid samples are spilled, a towel or paper towel should be used to cover infectious substances and related broken items immediately, and then Spray an effective disinfectant on it for 30 minutes. Clean up absorbent towels or paper towels and broken objects (glass fragments should be cleaned with tweezers) Management), and then spray or wipe the contaminated area with an effective disinfectant. After cleaning, clean utensils and garbage should be autoclaved. test Personnel should wear gloves during the entire operation.

7.2 How to treat the sample in contact with the skin

When the sample is splashed or stained on the skin of hands, face, etc., immediately wash the skin with 75% alcohol or equivalent disinfectant, or Use a cotton ball to soak the above-mentioned disinfectant and wipe the skin thoroughly until it is clean, and then wash the skin with clean water. If the liquid in the sample If it splashes into the eyes, it should be rinsed off with an eyewash immediately, and the water after washing the eyes should be sterilized.

7.3 Selection of detection methods

After the nucleic acid is extracted from the inactivated sample, the laboratory can use the PCR or fluorescent PCR methods recommended in the standard to develop the African swine fever virus Nucleic acid amplification testing. If the separated serum samples are used for immunological testing, attention should be paid to biological safety. For specific operation methods, see OIE "Land Animals Manual of Diagnostic Tests and Vaccines (2019 edition) section 3.8.1, GB/T 18648 or SN/T 1559.

8 Harmless treatment of waste and residual samples

8.1 When the African swine fever test result is negative, the remaining samples should be promptly sterilized by high-pressure steam (121°C, 30 min) and handed over to the designated biosafety waste disposal company shall conduct harmless treatment and keep records.

8.2 Africa...

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This preview omits tables, figures, formulas and parts of the technical clauses. The complete document — 6 pages — is available in the English PDF.

Referenced standards

Normative references

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