GB/T 18644-2020Diagnostic techniques for porcine cysticercosis (English PDF)

Foreword

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

This standard replaces GB/T 18644-2002 "Cysticercus cellulosae diagnosis technology", compared with GB/T 18644-2002, except for editorial revision In addition, the main technical changes are as follows:

---The scope part adds PCR and Dot-ABC-ELISA technology (see Chapter 1);

--- Added abbreviations (see Chapter 3);

---Added PCR detection method for pathogens (see Chapter 5);

---Modified the detection antigen and detection steps of the enzyme-linked immunosorbent assay, and directly performed antibody detection on serum samples (see Chapter 6, Chapter 3 of the 2002 edition);

---Added Dot-ABC-ELISA method for detecting circulating antigens (see Chapter 7);

--- Added comprehensive judgment (see Chapter 8);

---Added pictures of normal saline and cysticercus sacrum (see Appendix A), PCR primer positions, solution preparation and electrophoresis pictures (see attached Record B), ELISA reagents and their preparations (see Appendix C, Appendix A of the 2002 edition), the recombinant protein of Cysticercus cellulosae TS-CC18 Preparation (see Appendix D), sample addition example for indirect enzyme-linked immunosorbent assay (see Appendix E), Cysticercus cellulosae TS-CC18 and enolization Enzyme monoclonal antibody preparation (see Appendix F), Dot-ABC-ELISA related reagent preparation and result determination (see Appendix G).

Please note that certain contents of this document may involve patents. The issuing agency of this document is not responsible for identifying these patents.

Introduction

Porcine cysticercosis (porcinecysticercosis) is a serious damage caused by the larva of Taeniasolium Zoonotic parasitic diseases. The World Animal Health Organization lists the disease as one of the diseases to be declared. Currently, the disease is widespread in development The number of cases in countries and developed countries is also increasing year by year, which not only affects the development of the pig industry and causes huge economic losses, but also A serious threat to human health.

The diagnosis of swine cysticercosis includes pathogenic diagnosis and serological diagnosis. The purpose of pathogenic diagnosis is to determine the worms in clinical necropsy samples The morphological characteristics of the body can be judged as Cysticercus cellulosae by observing under a microscope, there are two neatly arranged small hooks on the top of the head segment. Cysticercus It is mainly parasitic in the liver, so it should be distinguished from the cysticercus of Taenia asiatica. At present, the pre-mortem diagnosis of the disease mainly uses serological methods.

The antigens used include worm antigens, cyst fluid antigens, or secreted metabolic antigens. Although these antigens have high sensitivity, they have poor specificity.

And the source of antigen is very limited. In view of the above reasons, this revision of GB/T 18644-2002 "Cysticercus cellulosae Diagnosis Technology", except for some The antigen used in ELISA was changed to a recombinant protein of Cysticercus cellulosae stage specific 18ku gene (TS-CC18), and serum circulating antigen was added (CAg) detection methods and pathogen PCR detection methods to meet the epidemiological investigation of cysticercosis, drug treatment effect evaluation and animal Different needs such as circulation risk assessment.

The issuing authority of this document draws attention to the fact that when a declaration conforms to this document, Chapter 6 Indirect ELISA may be involved. (ELISA) related patent use.

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 microscopic examination, polymerase chain reaction method (PCR method), indirect enzyme-linked immunosorbent test (intermediate enzyme-linked immunosorbent assay) of swine cysticercosis Connected with ELISA), dot biotin-avidin complex enzyme-linked immunosorbent test (Dot-ABC-ELISA) diagnostic technology and comprehensive judgment.

This standard applies to the diagnosis of cysticercosis in pigs and 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 document. For undated references, the latest version (including all amendments) applies to this document.

CNAS-GL029:2018 Guidelines for Accreditation of Testing Laboratories in the Field of Gene Amplification

3 Abbreviations

The following abbreviations apply to this document.

Avidin-HRP. Avidin-Horseradish Peroxidase (Avidin-Horseradish Peroxidase)

DAB. Diaminobenzidine (3,3'-Diaminobenzidine)

Dot-ABC-ELISA. dot biotin-avidin complex enzyme-linked immunosorbent assay (Dot-Avidin-Biotin-Complex- ELISA)

ELISA. Enzyme-LinkedImmunosorbentAssay

HRP. Horseradish Peroxidase (HorseRadish Peroxidase)

IPTG. Isopropyl beta-D-Thiogalactoside (Isopropyl beta-D-Thiogalactoside)

NC. Nicrocelulose

OPD. O-phenylenediamine

PBS. Phosphate Buffered Saline (PhosphateBufferedSaline)

PBST. Washing solution (PBScontaining0.5% Tween-20)

PCR. Polymerase Chain Reaction (PolymeraseChain Reaction)

TMB. TetramethylBenzidine (3,3',5,5'-TetramethylBenzidine)

4 Microscopic examination