GB/T 27982-2026Diagnostic techniques for peste des petits ruminants (English PDF)

6.1 Epidemiology

6.1.1 Ruminants are generally susceptible, with no obvious seasonality or age-related differences. Goats and sheep are typical hosts, with goats generally being more susceptible than sheep. Susceptible. Among wild animals, bovines are the most susceptible. Camels, pigs, and many other animals are atypical hosts.

6.1.2 The main sources of infection are sick and dead goats, sheep, and wild ruminants.

6.1.3 The main route of infection is through the respiratory tract, spreading over short distances via aerosols produced by sneezing and coughing; infection can also occur through direct contact. The virus can be transmitted through animals or their secretions and excrement, or through contact with bedding, feed, etc., contaminated with the virus.

6.1.4 High morbidity and mortality rates, with morbidity and mortality rates reaching over 90%; some patients die acutely without obvious symptoms.

6.2 Clinical Symptoms

6.2.1 Sudden onset of fever, with body temperature reaching 40°C~42°C on the 2nd to 3rd day. The fever lasts for about 3 days. In acute cases, the fever begins at the onset of fever. Death occurs within 4 to 6 days. Subclinical cases have milder symptoms and may recover within 10 to 14 days after onset.

6.2.2 Diarrhea begins 2-3 days after the onset of fever, accompanied by severe dehydration, weight loss, and collapse. Pregnant ewes may experience abortion.

6.2.3 A large amount of discharge comes from the eyes and nose, initially watery, increasing in volume as the disease progresses, becoming purulent, then drying out, and emitting a foul odor. It has a foul odor. Symptoms usually include sneezing and coughing, shortness of breath, and difficulty breathing.

6.2.4 The oral mucosa is congested, and numerous necrotic spots and ulcers appear on the oral epithelium and nasal mucosa. After the necrotic tissue sloughs off, it forms well-defined lesions. Superficial erosions. Epithelial damage appears on the surface of the lips, gums, dental lamina, tongue, and vulva of the ewe, with excessive salivation in the oral cavity.

6.3 Necropsy lesions

6.3.1 Severe congestion was observed in the upper respiratory tract mucosa, accompanied by ulcers in the nasal cavity and tracheal mucosa.

6.3.2 Bronchopneumonia and apical pneumonia appeared in the lungs.

6.3.3 Severe congestion and ulceration of the digestive tract mucosa. Intestinal congestion is most commonly limited to the duodenum, ileum, cecum, and upper colon, and occasionally... Diffuse congestion was observed throughout the intestines; hemorrhage at the ileocecal valve was common; severe hemorrhage occasionally occurred at the top of the longitudinal folds of the large intestine, forming a zebra-like appearance. stripe.

6.3.4 Mildly enlarged and edematous mesenteric lymph nodes, swollen and edematous pulmonary lymph nodes, and swollen spleen.

6.4 Clinical assessment Meeting the epidemiological characteristics outlined in 6.1, exhibiting any of the clinical symptoms described in

6.2 or any of the necropsy lesions described in 6.3, and not yet diagnosed with any other disease. This can be preliminarily identified as a suspected case of peste des petits ruminants (PPR).

7.1 Sample Collection

7.1.1 Collect one swab each from the eyes, nose, mouth, and anus of suspected diseased animals, and preserve them in 1 mL of sterile

0.01 mol/L PBS or similar solution. Store in liquid. Aseptically collect 5-10 mL of whole blood and separate the serum.

7.1.2 Select infected animals that have just been culled or have died within 24 hours to collect tissue samples. Collect mesenteric lymph nodes aseptically from each infected animal. Three to four portions each of the tracheal and bronchial lymph nodes, and approximately 10 to 20 grams each of diseased tissues from the spleen, intestines, and lungs, were placed in sample preservation tubes (bags).

7.2 Sample Preservation and Transportation

7.2.1 Sample collection and transportation shall comply with the provisions of NY/T 541.Sample packaging shall be leak-proof, seepage-proof, and spill-proof, and puncture-proof shall be avoided. damage.

7.2.2 Samples should be stored at 4°C for no more than 24 hours. If long-term storage is required, they should be stored at -70°C or below, and the freeze-thaw cycle should not exceed 3 times.

8.1 Instruments and Equipment

8.1.1 Biosafety cabinet.

8.1.2 Cell incubator.

8.1.3 Inverted microscope.

8.1.4 Benchtop low-temperature high-speed centrifuge.

8.1.5 Grinding machine.

8.1.6 Cell culture flasks.

8.2 Reagents and Materials

8.2.1 Vero-derived cells expressing canine SLAM, African green monkey kidney cells, or primary lamb kidney/lung cells. 8.2.2

0.01 mol/L PBS. Prepared according to A.1 in Appendix A.

8.2.3 DMEM cell culture medium. Prepared according to A.2.

8.2.4 DMEM cell maintenance medium. Prepared according to A.3.

8.3 Operating Procedures

8.3.1 Tissue Sample Processing. Cut approximately 1g of tissue material from lymph nodes, spleen, lungs, etc., and add 10mL of solution containing penicillin (100IU/mL) and streptomycin. The 100 µg/mL styraxe was thoroughly ground in PBS buffer, centrifuged at 3000 rpm for 10 min, and the supernatant was filtered through a 0.45 µm filter membrane. spare. Swab sample preparation. Twist the cotton swab thoroughly in the sample collection tube, wring it dry, and discard the swab. Filter the sample through a 0.45µm filter membrane for later use.

8.3.2 Wash cells once with

0.01 mol/L PBS. Inoculate cells with 1 mL of the pre-treated sample, incubate at 37°C for 60 min, then add... The sample was inoculated into DMEM growth medium and cultured in a 37°C, 5% (v/v) CO2 incubator. A blank control without any inoculation was also set up.

8.3.3 Observe cytopathic effects daily. Within 5 days of inoculation, cells should exhibit cytopathic effects (CPE), manifested as cell fusion and formation of... Syncytosis occurs, and inclusion bodies are formed in the nucleus and cytoplasm. If no cytopathic effect occurs 5-7 days after inoculation, the cells should be blindly passaged three times.

8.3.4 Repeat the freeze-thaw cycle three times for the cell suspension exhibiting cytopathic effects, centrifuge at 3000 r/min for 10 min, collect the supernatant, and store it. Pending inspection.

8.4 Result Determination

8.4.1 The blank control Vero cell monolayer was intact, the cells were regularly elliptical, relatively dense, and had few floating dead cells (see Appendix). (Figure B.1) The experiment was successful.

8.4.2 Under the established experimental conditions, if the cells in the inoculated sample exhibit cell aggregation, shrinkage, or even severe cell detachment and fragmentation, The image (see Figure B.2) was identified as a suspected positive result for peste des petits ruminants virus isolation. If it is confirmed as positive by Chapter 9 or Chapter 10, it is determined to be a true case of peste des petits ruminants. Positive for veterinary virus isolation. Cell cultures were blindly passaged three times without cytopathic effects, and were negative as determined by Chapter 9 or Chapter 10, thus classified as small... The ruminant plague virus isolation was negative. Cell cultures were blindly passaged three times without cytopathic effects, and were therefore identified as positive according to Chapter 9 or Chapter 10. The test result was positive for peste des petits ruminants virus (PPR) nucleic acid. 9.RT-PCR Method

9.1 Instruments and Equipment

9.1.1 PCR amplification instrument.

9.1.2 Benchtop low-temperature high-speed centrifuge.

9.1.3 Electrophoresis apparatus and horizontal electrophoresis tank.

9.1.4 Gel imaging device.

9.1.5 Mortar and pestle (or tissue homogenizer).

9.1.6 Micro-adjustable pipettes (2.5µL, 10µL, 100µL,.200µL, 1000µL and other sizes).

9.1.7 RNase-free centrifuge tubes.

9.1.8 PCR amplification tube.

9.2 Reagents and Materials

9.2.1 DEPC-treated water shall be prepared in accordance with C.1 in Appendix C. 9.2.2 5×TAE buffer, prepared according to C.2. 9.2.3 1.5% agarose gel, prepared according to C.3. 9.2.4 2× one-step RT-PCR reaction buffer.

9.2.5 Enzyme mixture (containing reverse transcriptase, Taq enzyme, and RNase inhibitors, etc.).

9.2.6 Nucleic acid electrophoresis loading buffer.

9.2.7 Primers, sequence information is shown in Appendix D, D.1.

9.2.8 Positive control for nucleic acid extraction. Small ruminant virus (vaccine strain) cell culture, etc.

9.2.9 Positive control. Small ruminant virus (vaccine strain) nucleic acid or equivalent positive plasmid, plasmid sequence see D.2.

9.2.10 Nucleic acid extraction negative control. Anticoagulated blood from small ruminant disease-negative animals.

9.2.11 Negative control. DEPC-treated water or empty plasmid.

9.3.1 Nucleic Acid Extraction The swabs and tissue samples collected in

7.1 or the supernatant obtained in

8.3.4 were extracted using a viral nucleic acid extraction kit or an automated nucleic acid extractor. Viral nucleic acid in the liquid. A positive control and a negative control should be set up for each nucleic acid extraction.

9.3.2 Preparation of the reaction system Prepare a 25 µL reaction system according to the following composition.

---12.5 µL 2× one-step RT-PCR reaction buffer;

---1.0 µL forward primer (10 µmol/L);

---1.0 µL reverse primer (10 µmol/L);

---1.0 µL enzyme mixture;

---5.0µL viral nucleic acid;

---4.5µL DEPC treated water. Mix well, centrifuge briefly, and then place in a PCR amplification instrument for amplification. A positive control and a negative control should be included in each test. The reaction system should be varied depending on the kit used.

9.3.3 Reaction Program Setup Reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 3 min; denaturation at 94°C for 20 s, annealing at 56°C for 30 s, extension at 72°C for 35 s, 40 cycles. Ring; 72°C for 5 min extension. The reaction procedure may vary depending on the kit.

9.3.4 Electrophoresis of RT-PCR amplification products Take 5 µL of RT-PCR product, add loading buffer according to the ratio, mix well, add to a 1% agarose gel, and electrophoresis for 30 min. 40 min. After electrophoresis, place the agarose gel in a gel imaging system to observe the results.

9.4 Result Determination

9.4.1 The positive control should show a specific amplification band of 351 bp (N gene), and the negative control should show no specific amplification band. Established.

9.4.2 Under the established experimental conditions, the tested sample showed a 351bp (N gene) specific amplification band, which was different from the positive control band. If the sample's mass and size match, it is determined to be positive for peste des petits ruminants virus (PPR) nucleic acid; if the tested sample shows no specific amplification band, it is determined to be PPR. The ruminant disease virus nucleic acid test was negative.

10.1 Instruments and Equipment

10.1.1 Fluorescent PCR amplification instrument.

10.1.2 Benchtop low-temperature high-speed centrifuge.

10.1.3 Mortar and pestle (or automatic homogenizer).

10.1.4 Micro-adjustable pipettes (2.5µL, 10µL, 100µL,.200µL, 1000µL and other sizes).

10.1.5 RNase-free centrifuge tubes.

10.1.6 PCR tubes or plates.

10.2 Reagents and Materials

10.2.1 Primers and probes. General sequence information is shown in D.3, and differential sequence information is shown in D.4.

10.2.2 One-step RT-qPCRMix kit (commercially available).

10.2.3 Commercial nucleic acid extraction kits.

10.2.4 Positive control for nucleic acid extraction. Inactivated small ruminant virus (vaccine strain) cell culture, etc., were used.

10.2.5 Nucleic acid extraction negative control. Vero cell cultures, etc.

10.2.6 Positive control. Universal type using small ruminant virus (vaccine strain) nucleic acid or equivalent positive plasmid (sequence shown in D.5); Differential type The plasmid of the small ruminant disease virus vaccine strain (sequence shown in D.6) and the plasmid of the small ruminant disease virus wild-type strain (sequence shown in D.7) were used.

10.2.7 Negative control. DEPC-treated water or empty plasmid.

10.3 Nucleic Acid Extraction Same as 9.3.1.

10.4 Universal Fluorescent RT-PCR Procedure

10.4.1 Reaction System The reagents required to prepare a 25 µL reaction system are as follows:

---12.5 µL 2× one-step Real-time RT-PCR reaction buffer;

---1.0 µL upstream primer (10 µmol/L);

---1.0 µL of downstream primer (10 µmol/L);

---0.5 µL probe (10 µmol/L);

---1.0 µL enzyme mixture;

---5.0µL viral nucleic acid;

---4.0µL DEPC treated water. Tightly cap the PCR tube or seal it with a protective film. After brief centrifugation, amplify the tube in a fluorescence PCR instrument. A positive control and a negative control should be included in each assay. Negative control. The reaction system may vary depending on the kit used.

10.4.2 Reaction Conditions Reverse transcription at 50°C for 10 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 45 s, 40 cycles, at each FAM fluorescence signals were collected at 60°C during cycling. Reaction conditions may vary depending on the kit used.

10.5 Procedure for Differentiating Fluorescent RT-PCR

10.5.1 Reaction System The reagents required to prepare a 25 µL reaction system are as follows:

---12.5 µL 2× one-step Real-time RT-PCR reaction buffer;

---1.0 µL upstream primer (10 µmol/L);

---1.0 µL of downstream primer (10 µmol/L);

---0.5 µL vaccine strain probe (10 µmol/L);

---0.5 µL wild-type virus probe (10 µmol/L);

---1.0 µL enzyme mixture;

---5.0µL viral nucleic acid;

---3.5µL DEPC treated water. Tightly cap the PCR tube or seal it with a protective film. After brief centrifugation, amplify the tube in a fluorescence PCR instrument. A positive control and a negative control should be included in each assay. Negative control. The reaction system may vary depending on the kit used.

10.5.2 Reaction Conditions Reverse transcription at 50°C for 15 min; pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min, 40 cycles, extension... Both FAM and VIC fluorescence signals are collected simultaneously during the process. Reaction conditions may vary depending on the kit used.

10.6 Result Determination

10.6.1 The Ct values of both the positive control and the reaction positive control should be < 30 and a specific amplification curve should appear. The negative control and reaction positive control should also be extracted. Negative controls showed no Ct values and no specific amplification curves, thus the experiment was valid.

10.6.2 Under the established experimental conditions, if the Ct value of the tested sample in the general detection method is < 37 and a specific amplification curve appears, it is judged as a small reverse reaction. Positive for peste des petits ruminants virus (PPR) nucleic acid. A Ct value >=37 or no Ct value indicates a negative PPR nucleic acid result.

10.6.3 Under the established experimental conditions, the Ct value of the test sample that collects the VIC signal by the discriminative detection method is < 37 and shows specific amplification. The increasing curve indicated a positive result for the small ruminant virus vaccine strain nucleic acid. Samples with collected FAM signals had a Ct value < 37 and exhibited specificity. A positive amplification curve indicates a positive result for wild-type peste des petits ruminants virus (PPR) nucleic acid. A Ct value >=37 or no Ct value indicates a positive result for PPR nucleic acid. Acid-negative.

11.1 Instruments and Equipment

11.1.1 Microplate reader.

11.1.2 Constant temperature incubator.

11.1.3 Bottle or plate washing machine.

11.1.4 Micro-adjustable pipettes (2.5µL, 10µL,.200µL and other sizes).

11.2 Reagents and Materials

11.2.1 Antigen plates coated with recombinant petr ruminant virus N protein, preparation method is given in Appendix E.

11.2.2 Control serum. positive serum and negative serum.

11.2.3 Enzyme-labeled antibody. HRP-labeled monoclonal antibody against the N protein of peste des petits ruminants virus. The preparation method is given in Appendix F.

11.2.4 Sample dilution solution.

11.2.5 Washing buffer.

11.2.6 Enzyme-labeled monoclonal antibody dilution solution.

11.2.7 Substrate solution.

11.2.8 Termination solution.

11.3 Test Procedure

11.3.1 Add 25 µL of sample diluent to each well of the serum to be tested, followed by 25 µL of the serum to be tested. Include a positive serum control well. Two control wells, each containing 50 µL of positive serum; two negative serum control wells, each containing 50 µL of negative serum; and two blank control wells, each containing 50 µL of negative serum. Add 50 µL of diluent to each well. Incubate at 37°C for 60 min.

11.3.2 Add 300 µL of working concentration washing solution to each well, discard the washing solution, repeat the washing process 4 times, and then pat dry.

11.3.3 Add 50 µL of working concentration of enzyme-labeled antibody to each well and incubate at 37°C for 30 min.

11.3.4 Add 300 µL of working concentration washing solution to each well, discard the washing solution, repeat washing 4 times, and then pat dry.

11.3.5 Add 50 µL of substrate chromogenic solution to each well and incubate at room temperature (20°C~25°C) in the dark for 10 min~15 min.

11.3.6 Add 50 µL of stop solution to each well to terminate the colorimetric reaction.

11.3.7 Use an ELISA reader to read the OD value of each well at a wavelength of 450 nm.

11.4 Calculation of Blocking Rate The sample blocking rate is calculated according to formula (1).

11.5 Result Determination If the positive control blocking rate is >60% and the negative control blocking rate is < 40%, the test is valid; otherwise, the test should be repeated. A sample with a blocking rate >50% is considered positive for peste des petits ruminants (PPR) antibodies; a sample with a blocking rate <=50% is considered positive for PPR. Antibody negative.