SN/T 5196-2020Technical specification for quarantine of porcine rotavirus infection (English PDF)

Foreword

This document is drafted in accordance with 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. Fuzhou Customs of the People's Republic of China, Nanjing Customs of the People's Republic of China, Nanning of the People's Republic of China Customs, Shenzhen Customs of the People's Republic of China, Haikou Customs of the People's Republic of China, Xiamen Rui Deli Calibration and Testing Technology Co., Ltd.

1 Scope

This document specifies the enzyme-linked immunosorbent assay for porcine rotavirus infection, reverse transcription-polymerase chain reaction and real-time fluorescent reverse transcription-Polymerase chain reaction detection technology.

This document is suitable for antibody detection and pathogenic nucleic acid identification of porcine rotavirus infection.

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 6682 Analytical laboratory water specifications and test methods GB/T 18088 Entry and Exit Animal Quarantine Sampling GB 19489 Laboratory Biosafety General Requirements

3 Terms, definitions and abbreviations

There are no terms and definitions that need to be defined in this document. The following acronyms apply to this document.

4 Apparatus and equipment

4.1 Class II biological safety cabinet.

4.2 Autoclave.

4.3 96-well microtiter plate.

4.4 Constant temperature incubator.

4.5 Plate washer.

4.6 Microplate reader.

4.7 Refrigerated centrifuge.

4.8 Single-channel adjustable micropipette (2 µL~20 µL, 20 µL~200 µL, 100 µL~1000 µL).

4.9 Multi-channel adjustable micropipette (10 µL~100 µL).

4.10 Thermal cycler.

4.11 Horizontal electrophoresis apparatus.

4.12 Gel imager.

4.13 Fluorescence PCR machine.

4.14 Refrigerator (2 °C~8 °C and -20 °C).

5 Reagents and materials

5.1 Water. Comply with the first grade water specification in GB/T 6682.

5.2 Porcine rotavirus VP6 protein.

5.3 Standard positive serum. preparation of piglet immunized with porcine rotavirus vaccine, and it is positive by virus neutralization test.

5.4 Standard Negative Serum. The serum of piglets without maternal antibodies and not immunized with porcine rotavirus vaccine is negative by the virus neutralization test.

5.5 Sterilized normal saline. See A.1 in Appendix A for the preparation.

5.6 Coating liquid. For preparation, see Appendix A, A.2.

5.7 Phosphate buffered saline (PBS). For preparation, refer to Appendix A, A.3.

5.8 Washing solution. See A.4 in Appendix A for the preparation.

5.9 Sealing solution. See A.5 in Appendix A for the preparation.

5.10 Sample diluent. For preparation, see Appendix A in A.6.

5.11 Anti-pig PRV enzyme-labeled antibody.

5.12 Substrate color developing solution. For preparation, see Appendix A in A.7.

5.13 Stop solution. See A.8 in Appendix A for the preparation.

5.14 Diethyl pyrocarbonate treated water (DEPC water). See Appendix A in A.9 for the preparation.

5.15 Trizol.

5.16 Trichloromethane.

5.17 Isopropanol.

5.18 Absolute ethanol.

5.19 Reverse transcription kit.

5.20 Taq DNA polymerase.

5.21 dNTPs (including dCTP, dGTP, dATP, dTTP).

5.22 10×PCR buffer.

5.23 Agarose.

5.24 Molecular weight standard (100 bp ~ 600 bp).

5.25 TBE electrophoresis buffer, see Appendix A in A.10 for the preparation.

5.26 Loading buffer, see Appendix A in A.11 for preparation.

5.27 EB (10 mg/mL), see A.12 in Appendix A for the preparation.

5.28 DNA purification and recovery kit.

5.29 Quality control substances. the positive control is the positive tissue containing PRV or the virus culture medium containing PRV; the negative control is the PRV-free organization.

5.30 Sequence of primer F1 and R1 for RT-PCR.

Amplify a 309 bp fragment in the conservative sequence of each PRV strain (see Appendix B), and after the primer is dissolved to 10 µmol/L with ddH2O, Store at -20 °C for later use.

5.31 Primer F2, R2 and probe sequence for real-time fluorescent RT-PCR.

The primers and probes were dissolved to 10 µmol/L with ddH2O, and then stored at -20 °C for later use.

6 Sampling and sample preparation

6.1 Determine the number of animals to be sampled according to GB/T 18088; the detection of pathogens involved should be carried out in the secondary biosafety laboratory.

Disposal should be carried out in accordance with the requirements of GB 19489.

6.2 Serum samples. blood is collected aseptically by the anterior vena cava blood collection method, and after it has spontaneously coagulated, it is centrifuged at 4 000 r/min for 10 min.

6.3 Stool or anal swab sample. collect a stool sample, add an equal volume of sterilized physiological saline to grind the homogenate, and centrifuge at 4 °C 5 000 r/min 15 minutes, collect the supernatant for testing. Collect anal swab samples, infiltrate it with a small amount of sterile physiological saline, and take the supernatant for testing.

6.4 Tissue samples. Collect tissue samples such as intestines and stomach, add an equal volume of sterilized physiological saline to grind the homogenate, and centrifuge at 4 °C 5 000 r/min 15 minutes, collect the supernatant for testing.

7 ELISA detection method