SN/T 5192-2020Technical specifications for dermatophilia quarantine (English PDF)

5.1 Equipment and disinfectants

5.1.1 Equipment

Work clothes, latex gloves, line gloves, protective masks, masks, goggles, mirrors, flashlights, boiling sterilizers, washbasins, Ear clips.

5.1.2 Disinfecting drugs

0.1% mercury liter water, 2.5% Lysur, 0.1% Promethazine or 75% alcohol cotton ball, etc.

5.2 Inspection method

5.2.1 The examiner and assistant should wear work clothes, latex gloves, masks, goggles and protective masks, choose an appropriate position, Good animals are checked.

5.2.2 Check the head (especially the lips), neck, back, chest and abdomen, limbs, perineum, and tail for superficial exudation and localization.

Limited keratinous rash or hard scab.

5.2.3 At the end of the inspection, take off the gloves. The hands of the inspector and assistants need to use 0.1% Mercury Liquor or 2.5% Lysuror or 0.1% Promethazine or 75% alcohol cotton balls are thoroughly disinfected. Work clothes and used equipment are soaked in 2.5% Lysur for 1 hour, or boiled for 10 minutes. Check Use 10% lime milk, 2% hot alkaline water or 10%-20% bleaching powder solution to disinfect the epidemic site.

5.3 Clinical features

The appearance of characteristic clinical symptoms, such as exudative dermatitis, rash, or scab on the skin or mucous membrane of the body, can be preliminarily determined.

5.4 Judgment

A preliminary diagnosis can be made based on the clinical characteristics (see 5.3). For further diagnosis, disease materials or serum samples should be collected for laboratory examination.

6.1 Material preparation

6.1.1 Equipment

Tissue masher, constant temperature water bath, constant temperature biochemical incubator, ordinary refrigerator and low temperature refrigerator (4 °C, -20 °C), centrifuge and separation Heart tube, ordinary biological microscope, fluorescence microscope, lens cleaning paper, scalpel, tweezers, micropipette, plastic dropper, glass slide, cover Slides, inoculation loops, alcohol lamps.

6.1.2 Medium and reagents

Formaldehyde, polymyxin B, PBS (0.1 mol/L pH7.2), see Appendix A.

Sheep blood agar medium (plate) or serum broth medium (test tube), Gram staining solution, Giemsa staining solution, press GB/T 14926.43 stipulates preparation.

6.2 Sample collection and delivery

6.2.1 Sample collection

Collect disease materials such as crust and subcutaneous exudate from the lesion aseptically.

6.2.2 Sample delivery

Under low temperature conditions (2 °C~8 °C), the sample should be sent to the laboratory as soon as possible (within 24 h~48 h).

6.3 Smear or microscopic examination of diseased material

Freshly peeled fresh and moist crust can be directly imprinted on a glass slide to make a pressed sheet; hardened crust or air-dried crust is immersed in PBS Soak it overnight to make it fully wet, and then press it tightly on the glass slide to make a pressed tablet. Exudate from the subcutaneous scab, use an inoculation loop to hook the Coat the glass slide to make a smear.

6.4 Pathogen isolation and culture

Take the prepared sheep blood agar medium (plate) or serum broth medium (test tube), and use the collected samples aseptically Use the inoculation loop to hook the exudate from the skin of the scab, or cut out a small piece of scab (1 cm×1 cm), rinse with PBS for 5 times, then use a tissue masher Emulsify to make a 10% suspension, inoculate it in the culture medium, inoculate 2 plates or test tubes for each sample. If the suspension is filtered through a 0.45 µm filter membrane, can fully reduce or eliminate pollution, or add 1 000 U/mL polymyxin B to the culture medium to inhibit the growth of contaminating bacteria.

Place the inoculated plate or test tube at 37°C and incubate at a constant temperature. Check whether there is contamination by bacteria in the first 24 hours. Observe every day after 24 hours, Cultivate for 4-7 days.

6.5 Pathogen identification

6.5.1 Colony identification

Observe that there are undoubtedly characteristic colonies on the medium. The broth medium forms a biofilm. After culturing on the plate for 24 h~48 h, after culturing Hemolytic, yellow-gray, rough colonies (about 1 mm in diameter) formed on the base. Faint yellow smooth colonies often appeared after 72 h. Crude Rough colonies are formed by branched hyphae, and smooth colonies are formed by cocci.

6.5.2 Gram stain identification

6.5.2.1 Gram staining identification shall be performed in accordance with the provisions of GB/T 14926.43.

6.5.2.2 Result judgment. This bacteria is a gram-positive bacteria with a purple color. Microscopic examination to look for branched and multi-septate hyphae or parallel arrangement of gram Yang Sex cocci-like cell clusters.

6.5.3 Giemsa staining identification

6.5.3.1 Giemsa staining identification shall be operated in accordance with the provisions of GB/T 14926.43.

6.5.3.2 Result judgment. the bacterial cell is divided into branch-like long hyphae, and divides horizontally into multirow coccus or ovale coccus, Congo Dermatophiles have a darker stain, so the darkly stained Dermatophagoides contrasts with the light or pink counterstained background of keratinocytes or neutrophils.

Poor, it is easier to distinguish Dermatophilus Congo in thick smears. For suspected bacteria, the next PCR test or immunofluorescence test is required Make a diagnosis.

6.5.4 Physiological and biochemical identification

6.5.4.1 Physiological and biochemical identification shall be operated in accordance with the provisions of GB/T 14926.43.

6.5.4.2 Physiological and biochemical identification results, see Appendix B.

7.1 Materials and reagents

7.1.1 Apparatus and equipment

PCR detector, high-speed bench-top refrigerated centrifuge (centrifugal speed above 12 000 r/min), bench-top centrifuge (centrifugal speed 2 000 r/min), Mixer, refrigerator (2 °C~8 °C and -20 °C), micro adjustable pipette (10 µL, 100 µL, 1000 µL) and matching belt filter Wicking tip, eppendorf centrifuge tube (1.5 mL plastic centrifuge tube with lid), voltage stabilization, steady flow electrophoresis apparatus and horizontal electrophoresis tank, electrophoresis gel Image system (or UV analyzer).

7.1.2 Primer

Add sterile distilled water to prepare 20 µmol/L working solution. The PCR primer sequence is shown in Table 1.

7.1.3 Reagents

Unless otherwise specified, the reagents used in this document are of analytical grade.

Proteinase K, 10% SDS solution, phenol, chloroform, isopropanol (pre-cooled at -20°C), 3 mol/L NaAc, 75% ethanol, Taq enzyme (5 U/µL), 10×PCR buffer, dNTPs (containing dATP, dTTP, dCTP, dGTP each 10 mmol/L), electrophoresis Buffer (0.5×TBE buffer), EB, electrophoresis loading buffer, 0.1 mol/L PBS (pH 8.0), see Appendix C.

7.2 Preparation of DNA template

7.2.1 Take n sterilized 1.5 mL centrifuge tubes, where n is the sum of the tested sample, positive control and negative control (positive control, negative control).

The control has been marked in the kit) and numbered.

7.2.2 Add 1 mL of pH 7.0 PBS to each tube, pick a single suspected characteristic colony from the culture medium after separation, and mix it in PBS.

Centrifuge at 4 °C 15 000 r/min for 1 min, and remove the supernatant.

7.2.3 The precipitate was resuspended in 50 µL distilled water, boiled for 10 min, centrifuged at 4 °C 15,000 r/min for 3 min, the precipitate was removed, and the supernatant was transferred to in a new centrifuge tube.

7.2.4 Add proteinase K to each tube to a final concentration of 100 µg/mL and SDS to a final concentration of 10 mg/mL, in a water bath at 55°C for 2 h.

7.2.5 Add phenol,. chloroform. isoamyl alcohol mixture (volume ratio 25.24.1) and chloroform respectively to each tube for extraction Once, transfer the water phase to a new centrifuge tube.

7.2.6 Add 1/10 volume of 3 mol/L sodium acetate and 2.5 times volume of anhydrous ethanol to each tube, mix gently and set it to settle at -20 °C for 1 h.

Centrifuge at 12 000 r/min for 15 min at 4 °C, and discard the supernatant.

7.2.7 Each tube was washed with 75% ethanol, centrifuged at 12 000 r/min for 5 min at 4 °C, dried, and suspended in sterilized 20 µL ultra in pure water, use an appropriate amount as a PCR template. For long-term storage, it should be placed in a -70 °C refrigerator.

7.3 PCR reaction

Extend at 72 °C for 60 s, 35 cycles, then extend at 72 °C for 10 min, and finally hold at 4 °C.

7.4 Control establishment

From the beginning of DNA extraction, a positive control and a negative control are set up. Contains the known Congo Dermophila NCTC 11184 strain (U.S.

ATCC) bacterial suspension was used as a positive control, and distilled water was used as a negative control.

7.5 Agarose electrophoresis of PCR products

Prepare 1% agarose (containing 0.5 µg/mL EB) plate with TBE running buffer. Put the plate into the horizontal electrophoresis tank to make electrophoresis the buffer just flooded the glue surface. Mix 8 µL sample and 2 µL sample buffer and add to the sample well. Set DNA standards during electrophoresis the molecular weight is used as a control. 5 V/cm electrophoresis for about 30 minutes, when the bromophenol blue reaches the bottom, it stops. Observe the results under UV light.

7.6 Judgment of results

The positive control will show a 500 bp DNA fragment, and the negative control does not have the nucleic acid band, the test is valid. If the sample comes out A DNA fragment of 500 bp is judged to be positive, otherwise it is judged to be negative.

When a positive amplified fragment appears, it is necessary to sequence the amplified product.

8.1 Fluorescent antibody test

8.1.1 Test equipment

Constant temperature incubator, fluorescence microscope, glass slide, cover glass, glass pencil, inoculation loop, tweezers, staining tank.

8.1.2 Test reagent

PBS-T (0.1 mol/L pH7.2), glycerol buffer, acetone. ethanol (mixed by volume ratio 1.1, pre-cooled at -20 °C), physiological brine Standard antigens, standard positive serum, standard negative serum, fluorescein isothiocyanate (FITC)-labeled anti-antibodies, etc. are regulated by the country Provided by the designated unit and used according to the instructions.

8.1.3 Operating procedures

8.1.3.1 Prepare the antigen slide, use a glass pencil to draw an area (0.5 cm in diameter) on the slide, and directly smear or smear the diseased material according to 5.2 and 5.3.

Press the tablet, or hook a single colony on the agar medium with an inoculating loop, spread evenly, make a thin smear, and air dry.

8.1.3.2 Add pre-cooled acetone and ethanol dropwise, fix the cells for 5 min to 10 min; wash 3 times with PBS-T, 5 min each time, and spin dry.

8.1.3.3 Add 100 µL of blocking solution to cover the entire specimen, incubate at 37 °C for 30 min, spin dry; wash 3 times with PBS-T, each time Spin dry for 5 minutes.

8.1.3.4 Add 50 µL of standard serum, incubate overnight at 4 °C in a humidified box; wash 3 times with PBS-T, 5...