SN/T 5191-2020Technical specifications for quarantine of avian nephritis (English PDF)

4.1 Introduction to Avian Nephritis

Refer to Appendix A for an introduction to avian nephritis.

4.2 Clinical symptoms

This disease can significantly inhibit the growth and weight gain of chicks within 1 month of age, and other symptoms are not obvious. Generally presents a transient recessive infection, this disease After artificial inoculation of chicks without special pathogenic microorganisms, the concentration of urate in plasma was 2-10 times higher than the normal concentration, and returned to normal 21 days later.

4.3 Pathological changes

The pathological changes of this disease are mainly manifested in the kidneys, and other organs are generally unchanged. As the age increases, the degree of disease is lessened, 4 days old the chicks inside are the most severely diseased and may even die. Necropsy of dead chicks, kidney, spleen, peritoneal surface and legs are seen A large amount of urate deposits around the tendons, and only mild nephritis lesions occurred at 2 months of age.

A preliminary diagnosis of avian nephritis can be made based on epidemiology, clinical symptoms and pathological changes, and further diagnosis depends on laboratory diagnosis.

5.1 Biosecurity measures

See GB 19489 for test environment and protection requirements.

5.2 Pathogen isolation and identification

5.2.1 Materials and reagents

5.2.1.1 Equipment

Biological safety cabinets, centrifuges, carbon dioxide incubators, refrigerated high-speed centrifuges, grinders, inverted microscopes, etc.

5.2.1.2 Water

It meets the first-level water requirements of GB/T 6682.

5.2.1.3 Reagents

Trypsin digestion solution, cell growth solution, cell maintenance solution, double antibody solution, please refer to Appendix B.1~B.4 for specific preparation.

5.2.1.4 Cell

Chicken kidney cells (CKC).

5.2.2 Virus isolation and culture

5.2.2.1 Sample collection and preparation

5.2.2.1.1 When there are no clinical symptoms, live poultry shall aseptically collect cloacal swabs of sick chickens (with visible feces), and young poultry shall collect fresh feces;

After the animal died, the contents of the kidney and rectum were aseptically taken as disease feed.

5.2.2.1.2 The sample is placed in isotonic phosphate buffered saline (PBS) at pH 7.0 containing antibiotics.

5.2.2.1.3 Stool and crushed tissues are made into 10%-20% (mass concentration) suspension with PBS containing antibiotics, and the swab is immersed in 3 mL Shake well in PBS containing antibiotics. Incubate at 4 °C for 4 h or overnight, or at room temperature for 2 h. The swab was discarded after repeated squeezing.

5.2.2.1.4 Centrifuge at 4 °C 3 000 r/min for 10 min, take the supernatant and filter and sterilize it through a 0.22 µm filter membrane.

5.2.2.2 Sample transportation and storage conditions

Samples for immediate laboratory testing should be stored at 4 °C or placed in a refrigerator for fast delivery, samples that cannot be tested immediately Store frozen at -70°C.

5.2.2.3 Sample inoculation

Inoculate the prepared samples in a monolayer of CKC cells cultured in a 24-well cell culture plate, inoculate at least 4 wells for each sample, set the correct for habitual cell control, at least 4 wells, 0.1 mL~0.2 mL per well, adsorbed at 37°C for 1 h, decanted excess liquid, added 1 mL of maintenance solution, and placed Incubate in a 5% carbon dioxide constant temperature incubator at 37°C for 3 to 5 days.

5.2.2.4 Observation of results

24 hours after the sample is inoculated, observe the cell status daily under an inverted microscope and make corresponding records. If the cell control well is normal, and when cells become rounded, clustered or plaques are formed due to cell shedding in the seeding sample wells, transfer to the sample after freezing and thawing three times If the culture plate is newly overgrown with a single layer of cells, if the observation still shows similar cytopathic changes and is becoming more obvious, the cell culture can be harvested and placed Put it into the refrigerator at -70 °C for identification. If there is no change between the control cells and the inoculated sample cells, pass them blindly for two more passages. If there is a fine According to the above principles, harvest the cell culture and put it in the refrigerator at -70 °C; if there is no cytopathic disease, it is avian nephritis virus.

The result is negative.

5.2.2.5 Culture identification

Since avian enteroviruses can cause the same CPE, the harvested culture should be identified. Reverse transcription-polymerization can be used Enzyme chain reaction (RT-PCR), real-time fluorescent RT-PCR and enzyme-linked immunosorbent assay (ELISA) methods to identify whether it is avian kidney Inflammatory virus infection.

5.3 Reverse transcription-polymerase chain reaction (RT-PCR)

5.3.1 Instruments and equipment

5.3.1.1 PCR thermal cycler.

5.3.1.2 Electrophoresis instrument.

5.3.1.3 UV gel imager.

5.3.1.4 High-speed desktop refrigerated centrifuge.

5.3.1.5 Biological safety cabinet.

5.3.1.6 Adjustable pipettes. 1 µL~10 µL, 20 µL~100 µL, 100 µL~1000 µL.

5.3.2 Reagents and materials

5.3.2.1 0.01 mol/L PBS buffer. pH 7.2.

5.3.2.2 Lysis Solution Trizol. Store at 4°C.

5.3.2.3 Trichloromethane.

5.3.2.4 Isopropanol.

5.3.2.5 DEPC water.

5.3.2.6 Magnesium chloride. 25 mmol/L.

5.3.2.7 dNTPS. contains 10 mmol/L of dCTP, dGTP, dATP, and dTTP each.

5.3.2.8 5× reverse transcriptase buffer and 10×Taq enzyme buffer.

5.3.2.9 Primer. 10 µmol/L. Designed according to the ORF1 b gene sequence, the length of the amplified product is 450 bp.

5.3.2.10 Taq enzyme. 5 U/µL.

5.3.2.11 Reverse transcriptase AMV. 10 U/µL.

5.3.2.12 75% ethanol.

5.3.2.13 Positive control. cell culture inoculated with ANV virus.

5.3.2.14 Negative control. normal chicken kidney cells (CKC).

5.3.3 Sample collection and preparation

See 5.2.2.1.

5.3.4 Sample transportation and storage conditions

See 5.2.2.2.

5.3.5 Test procedure

5.3.5.1 Sample RNA extraction

5.3.5.1.1 In the sample processing area, take 150 µL of the fecal supernatant sample and place it in a 1.5 mL sterile centrifuge tube after processing, mark it, and set it at the same time.

Establish positive sample control and negative sample control, add 3 times volume of Trizol, shake and mix for 20 s, and let stand for 5 min.

5.3.5.1.2 Add.200 µL of chloroform, shake and mix for 20 s, let stand for 5 min; centrifuge at 4 °C 12 000 r/min for 15 min, and take the supernatant Place another labeled 1.5 mL sterile centrifuge tube, add an equal volume of isopropanol, and let stand at -20°C for 15 min.

5.3.5.1.3 Centrifuge at 12 000 r/min at 4°C for 15 minutes, gently discard the supernatant, and add 75% ethanol prepared with 1 mL of DEPC water.

5.3.5.1.4 Centrifuge at 4 °C 12 000 r/min for 15 min, gently discard the ethanol, invert the filter paper, dry for 10 min, add 20 µL DEPC the RNA is dissolved in water, and the extracted RNA must be reverse transcribed within 2 hours. If it needs to be stored, it must be placed in a refrigerator at -70 °C.

5.3.5.2 Reverse transcription synthesis of cDNA

Add 10 µL reverse transcription template (sample RNA), 1 µL downstream primer PANV-R, 4 ul 5 times reverse transcriptase concentration to the PCR tube Shrink buffer, 2 µL dNTP, 0.5 ul RNase inhibitor (20 U/µL), 1 µL reverse transcriptase AMV (10 U/µL) and 1.5 µL water, the total volume is 20 µL. Mix thoroughly and centrifuge briefly to allow the liquid to settle to the bottom of the PCR tube. Reverse transcription at 42 °C for 45 min, immediately Ice bath, used as PCR template.

5.3.5.3 PCR amplified DNA

5.3.5.3.1 50 µL PCR reaction system composition