GB/T 16551-2020Diagnostic techniques for classical swine fever (English PDF)

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009.

This standard replaces GB/T 16551-2008 "Swine Fever Diagnostic Technology". Compared with GB 16551-2008, the main technical changes are as follows:

--- Modify the "scope" (see Chapter 1, Chapter 1 of the 2008 edition);

--- Added "Normative Reference Documents" (see Chapter 2);

---Added "abbreviations" (see Chapter 3);

--- Revised the "Clinical and Pathological Diagnosis" section and renamed it "Clinical Symptoms and Pathological Changes" (see Chapter 4, 2008 edition chapter 2);

--- Added "Sample Collection, Storage, Transportation and Processing" section (see Chapter 5);

--- Modified the "Etiological Diagnosis" section and renamed it "Laboratory Etiological Diagnosis Methods" (see Chapter 6, Chapter 3 of the 2008 edition);

--- Deleted the "Rabbit Body Interactive Immunity Test" part (see 3.1 in the 2008 edition);

---Modified the "Immunoenzyme Staining Test" and "Direct Immunofluorescence Antibody Test" and renamed "Immunofluorescence Antibody Test" (FAT)" and "Immune Peroxidase Test (IPT)" (see 6.1, 6.2, and 3.2, 3.4 of the 2008 edition);

---Modified the "virus isolation and identification test" part, renamed "swine fever virus isolation and identification" (see 6.3, 2008 edition 3.3);

--- Deleted "Swine fever virus reverse transcription polymerase chain reaction (RT-PCR)" (see 3.5 in 2008 edition);

---Added "Swine fever virus RT-nPCR detection method" and "Swine fever virus real-time fluorescent RT-PCR detection method" (see 6.4, 6.5);

---Modified the "serological diagnosis" part and renamed it "laboratory antibody detection method" (see Chapter 7, Chapter 4 of the 2008 edition);

---Modified the "fluorescent antibody virus neutralization test" and renamed it "swine fever virus neutralization test" (see 7.1, 2008 edition 4.1);

--- Deleted "Swine Fever monoclonal antibody enzyme-linked immunosorbent assay" (see 4.2 in the 2008 edition);

---Added "swine fever virus blocking ELISA antibody detection method", "swine fever antibody indirect ELISA detection method" and "swine fever virus Chemiluminescence antibody detection method" (see 7.2, 7.3, 7.4).

Introduction

Swine fever (Classicalswinefever, CSF) is caused by swine fever virus (Classicalswinefevervirus, CSFV) infection A highly contact fatal infectious disease that can cause huge economic losses and social impact. Recognized by the World Organization for Animal Health (OIE) Listed as a disease that must be reported, my country stipulates that CSF is a type of animal disease.

Pigs, including wild boars, are the only natural host of this disease. Diseased pigs and infected pigs are the source of infection of this disease. Pigs of different ages, genders, and breeds are all Susceptibility, can occur all year round. Infected pigs can excrete toxins through secretions and excrement before they become ill, and continue the entire course of the disease. With infected pigs straight Contact is the main mode of transmission of the disease. The virus can also be transmitted indirectly through semen, embryos, pork, and swill. Humans and other animals such as Rodents, insects, and utensils can all become important media.

CSFV is a member of the Flaviviridae (Flaviviridae) Pestivirus. It has only 1 serotype and 3 genotypes.

There are 10 genotypes, and there are good antigen cross-reactions among different genotypes. In recent years, due to clinical persistent infection of CSFV The chronic and recessive forms of infection without typical clinical symptoms and pathological changes caused by the phenomenon, as well as the phenomenon of mixed infection of multiple diseases, the clinical symptoms of CSF Bed diagnosis and pathological diagnosis can only be used as the basis for preliminary diagnosis. Laboratory testing of CSF pathogens is the main method to determine viral infection.

Law requires diagnostic techniques and standards to be more specific and sensitive. Due to the need for diagnosis and the development of diagnostic technology, GB/T 16551-2008 has After failing to meet the requirements. In view of the common characteristics of CSF infection with low viral load and short viremia time, it is most suitable for the examination of live animals.

The test sample is tonsils first, followed by anticoagulated whole blood; if it is a dead animal, tonsils, spleen, kidney, pancreas, ileum, ileocecal valve, and Mesenteric lymph nodes and submandibular lymph nodes and other organs with high toxic content are tested. Using CSFV antigen immunoassay [immunofluorescence anti Body test (Fluorescent antibody test, FAT)/Immunoperoxidase test (Immunoperoxidase test, IPT)] can be detected Viral antigens in infected tissues and cells are used for diagnosis; if the result is suspicious or the sample is insufficient, it can be confirmed by nucleic acid detection technology.

If the sample size is large, the real-time fluorescent RT-PCR detection method of classical swine fever virus can be used for preliminary screening, and the positive ones use classical swine fever virus RT-nPCR Detection methods and sequence determinations are used for diagnosis and genotyping; samples that cannot be diagnosed above are diagnosed with classic CSFV isolation techniques.

The isolated virus can be used for further research. The above three types of methods are all recommended by OIE.

At present, the implementation of comprehensive immunization with CSF vaccine is an important means for our national defense to control CSF. CSFV antibody detection is mainly used for CSF vaccine Evaluation of immunization effects, and for some countries and regions that do not implement immunization CSF vaccine, CSF antibody testing can be used as an unimmunized pig The basis for the diagnosis of CSF infection in pigs with pestilence vaccine. In my country, the ELISA method has become a method of monitoring the CSFV antibody protection level and evaluation after immunization.

The most important means to determine the immune effect of pigs, according to different testing purposes, different methods can be selected. For my country's CSF vaccine In the large-scale census carried out after the immunization, the indirect ELISA antibody detection method can more truly reflect the level of neutralizing antibodies, which is Program formulation and antibody level monitoring provide reliable data support; blocking ELISA antibody detection method can be used for introduction due to its strong specificity The detection of breeding pigs; the chemiluminescence antibody detection method is a new enzyme-linked immunoassay method developed in recent years.

Objects increase the sensitivity of detection, while increasing the linear range of detection. The CSFV chemiluminescent antibody detection method in this standard adopts CSFV antibody calibrator, and draw a calibration curve in the test, can make the antibody test results relatively quantitative. In addition, use this method to Line detection can greatly shorten the detection time and is suitable for field promotion. The final antibody confirmation can use the "gold standard" method for virus neutralization Test (Virusneutralizationtest, VNT). Both the ELISA method and the VNT method are recommended by OIE and are also international Trade designated test.

This standard involves 5 kinds of CSF pathogens and 4 kinds of CSFV antibody laboratory detection methods, which can be used for CSF pathogens and antibodies.

Qualitative testing. Users can choose the appropriate method according to their own experimental conditions, ability level and the actual situation of the sample to be tested.

The issuing agency of this document draws attention to the fact that when the declaration conforms to this document, it may involve 7.2 and 7.3 and CSFVE2 protein expression and purification Use of related patents.

The issuing agency of this document has no position on the authenticity, validity and scope of the patent.

The patent holder has assured the issuing organization of this document that he is willing to work with any applicant under reasonable and non-discriminatory terms and conditions.

Negotiations on patent licensing. The statement of the patent holder has been filed with the issuing agency of this document.

Please note that in addition to the above patents, certain contents of this document may still involve patents. The issuing agency of this document is not responsible for identifying these Li's responsibility.

1 Scope

This standard specifies the clinical symptoms and pathological changes of swine fever, sample collection, preservation, transportation and processing, and laboratory pathogenic diagnosis methods And laboratory antibody detection methods.

This standard applies to the diagnosis of swine fever in pigs (domestic pigs, wild boars).

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this file. For undated references, the latest version (including all amendments) applies to this document.

GB 19489 Laboratory Biosafety General Requirements

GB/T 27540 Real-time fluorescent RT-PCR detection method for swine fever virus

GB/T 34729-2017 Swine fever virus blocking ELISA antibody detection method

GB/T 35906 Indirect ELISA detection method for swine fever antibodies

GB/T 36875 RT-nPCR detection method for swine fever virus

NY/T 541 Technical Specification for Collection, Storage and Transportation of Veterinary Diagnostic Samples

3 Abbreviations

The following abbreviations apply to this document.

CSF. Classicalswinefever

CSFV. Classicalswinefevervirus

EDTA. Ethylenediaminetetraacetic acid

ELISA. Enzymelinked immunosorbent assay (Enzymelinkedimmunosorbentassay)

FAT. Immunofluorescent antibody test (Fluorescentantibodytest)

FITC. Fluoresceinisothiocyanate

HRP. Horseradish peroxidase (Horseradishperoxidase)

IPT. Immunoperoxidase test (Immunoperoxidasetest)

MEM. Basic Eagle's medium (Minimumeagle'smedium)

NCU. National clinical unit (Nationalclinicalunit)

NIF. Neutralization-immunofluorescence

NPLA. Peroxidase-linked neutralization test (Neutralizationperoxidase-linked antibody)

PBS. Phosphate buffered saline (Phosphatebufferedsaline)

PK-15. Porcine kidney passage cell line (Pigkidney-15celline)

RT-nPCR. Nested PCR technology (Reversetranscriptionnestpolymerasechainreaction)

RT-PCR. Reverse transcription polymerase chain reaction (Reversetranscription polymerase chain reaction)

TCID50. half of tissue infection dose (50% tissuecultureinfectivedose)