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WS/T 571-2017 English PDF

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WS/T 571-2017: Detection of diphyllobothroid larvae
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Basic data

Standard ID WS/T 571-2017 (WS/T571-2017)
Description (Translated English) Detection of diphyllobothroid larvae
Sector / Industry Health Industry Standard (Recommended)
Classification of Chinese Standard C62
Word Count Estimation 12,163
Date of Issue 2017-08-01
Date of Implementation 2018-02-01
Regulation (derived from) State-Health-Communication (2017) 11
Issuing agency(ies) National Health and Family Planning Commission of the People's Republic of China

WS/T 571-2017: Detection of diphyllobothroid larvae

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Detection of diphyllobothroid larvae ICS 11.020 C 62 WS People's Republic of China Health Industry Standard Split head tapeworm larva detection 2017-08-01 released 2018-02-01 implementation Issued by the National Health and Family Planning Commission of the People's Republic of China

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009. Drafting organizations of this standard. Chinese Center for Disease Control and Prevention, Institute of Parasitic Disease Control, Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai Municipal Diseases Disease Prevention and Control Center. Drafters of this standard. Chen Shaohong, Li Shuqing, Zhang Xiaoping, Xu Xuenian, Lu Yan, Cai Yuchun, Zhang Yongnian, Li Hao, Irene, Zheng Bin. Split head tapeworm larva detection

1 Scope

This standard specifies the operating procedures for the detection of larvae of the larvae. This standard applies to disease prevention and control institutions at all levels, medical institutions, and food testing institutions for the inspection of larvae of Schistocephalus in fish, frogs and snakes. Measurement.

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. GB/T 18088 Entry and Exit Animal Quarantine Sampling Standard GB/T 6682 Analytical laboratory water specifications and test methods

3 Terms and definitions

The following terms and definitions apply to this document. 3.1 Sparganosis The larvae of Schizocephalus are the third stage larvae of Pseudophylla and Schizocephae The general term for insects (see Appendix A). 3.2 Sparganosis The third-stage larva of Schizocephalus parasitic in fish. 3.3 Sparganosis mansoni The third-stage larva of Dimonia mansoni parasitic in frogs, snakes, or humans.

4 Equipment

4.1 Biological microscope. 4.2 Stereo microscope. 4.3 PCR thermal cycler. 4.4 Electrophoresis instrument. 4.5 Gel imaging system. 4.6 Ultra-clean workbench. 4.7 High-speed centrifuge. 4.8 Surgical instruments for dissection.

5 Reagent materials

5.1 Reagents Pepsin digestion solution; Tris-acetic acid electrophoresis buffer (TAE); agarose gel; 6× loading buffer; 100～2000 bp DNA marker; Sterilized double distilled water (ddH2O); 70% ethanol (see Appendix B, B.1 for preparation). 5.2 Primer Amplification of the primer sequence of the schistocyst ribosomal DNA transcription spacer (ITS); amplification of the primer sequence of the cytochrome C oxidase (Cox1) of the schistocyst Columns; the sequence of the specific primers of S. mansoni and the sequence of the specific primers of S. mansoni (see Appendix B, B.2). 5.3 Positive control The positive control is the positive clone plasmid of the corresponding gene fragment of Schizocephalus or the whole genome DNA of Schizocephala and Schizocephalus.

6 Detection steps

6.1 Sample preparation 6.1.1 Types of samples. marine fish, freshwater fish, frogs and snakes. 6.1.2 Sample collection. Sampling shall be carried out in accordance with GB/T 18088. 6.2 Sample testing 6.2.1 Tablet inspection method Use surgical scissors to dissect the examined fish, frogs and snakes one by one. Take out the internal organs, scrape off the inner lining of the abdominal cavity and observe the surface of the abdominal cavity If there are suspicious white spots on the surface, use surgical scissors or a scalpel to separate the skin and flesh, use two small tweezers to tear open the muscles, and take out the white spots. The tissues were pressed with glass slides, and the results were determined by microscopic examination with a biological microscope. 6.2.2 Protease digestion method Weigh 250 g of the sample and cut it into small pieces, add the digestion solution at a ratio of 1.5 between the sample and the pepsin digestion solution, and digest at 37°C until there is no naked eye Visible fleshy tissue. After digestion, filter with a 0.8mm×0.8mm (10 mesh) mesh sieve, and place the filtrate in a sharp-bottomed graduated cylinder and add water (with water According to GB/T 66828) to the maximum scale, the sediment is washed until the water is clear, all the sediment is placed in a flat dish, and the result is determined by a stereo microscope. 6.2.3 Nucleic acid detection method 6.2.3.1 Sampling Pick the worms under a stereo microscope and use them for preliminary identification. Set up a positive control for PCR reaction, the control is the corresponding gene sheet of Schizocephalus The positive cloning plasmid or the whole genomic DNA of schistocephala and schistocephala. Sterile water was used as a blank control. 6.2.3.2 DNA extraction (see Appendix B, B.3) 6.2.3.3 Reaction system The total volume is 25 L, template DNA 2 L, upstream and downstream primers (10 mol/L) each 0.5 L, dNTPs 2 L, MgCl2 2.5 L, 10×Buffer 2.5 L, Taq enzyme (5 U/µL) 0.2 L, supplement with ddH2O to 25 L. 6.2.3.4 PCR reaction program Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 7 min. 6.2.3.5 Electrophoresis Take 10 L of product and 2 L of 6× loading buffer to mix, add sample to 1.5% agarose gel containing ethidium bromide. In 1×TAE In the buffer, electrophoresis at 3 V/cm～4 V/cm for about 30 minutes. When the bromophenol blue reaches the bottom, the electrophoresis is stopped, and the gel imaging system is used for analysis. 6.3 Results judgment 6.3.1 Schizocephaly identification If the size of the larva detected from the fish is 2mm~20 mm long, 2mm~3 mm wide, milky white, the head is spoon-shaped, and its dorsal and ventral surfaces are different There is a narrow and deep concave suction groove, the front end of the body is concave and slightly larger, the body is not segmented but has horizontal folds, and the tail is thin, stick-like, with and The cephaloceles similar to worms can be preliminarily identified as schistocephala of the genus Schizocephalus (see Appendix C). 6.3.2 Determination of the morphology of Sparganosis If the size of the larva detected from a frog or snake is 0.5cm~80cm long and 0.3cm~1cm wide, it has a long band shape, milky white or light yellow, There is no suction groove at the front end of the body, and a hole in the center of the top end is recessed into a tunnel shape, and extends backward to form a blind tube. The body is not divided into sections and has irregularities. The folds can be preliminarily judged as sparganosis of the genus Diomong (see Appendix C). 6.3.3 Judgment of nucleic acid amplification results 6.3.3.1 There are bands in the amplified fragments of the target gene but no bands in the blank control, the experimental results are valid. 6.3.3.2 Amplification with specific primers of Schizocephalus broad-segmented, a characteristic band of 428 bp appeared, and the species was preliminarily determined as Schizocephalys broad-segmented. 6.3.3.3 Sparganosis mansoni specific primers were amplified, and a characteristic band of 156 bp appeared. It was preliminarily determined that the insect species was sparganosis mansoni. 6.3.3.4 If you want to further identify the larvae, you need to sequence the primer amplification product and compare its sequence with the sequence on GenBank After planting. AA

Appendix A

(Informative appendix) Etiology information A.1 Form A.1.1 The morphology of the schistocephala Broad-segment sparganosis is 2 mm~20 mm long, 2mm~3 mm wide, milky white, with a spoon-shaped head segment, with a narrow and deep concave on each dorsal and ventral surface The sucking trough is concave and slightly larger at the front end of the body, the body is not segmented but has horizontal folds, and the tail is thin, stick-like, and has a head section similar to that of an adult. The surface of the sparganosis cortex is covered with microhairs, about 1.5 µm in length. A.1.2 Sparganosis morphology of Diagonus Sparganosis mansoni is 0.5cm~80 cm long, 0.3cm~1 cm wide, long ribbon shape, milky white or light yellow, no suction groove at the tip of the worm, and the top There is a hole in the center that is recessed inward to form a tunnel, and extends backward to form a blind tube. The body of the worm is not segmented and has irregular folds. A.2 Life history Adults of Schizocephalus latifolia live in the small intestines of humans, dogs, cats, pigs and other animals. After the eggs are discharged with the host feces, at 15~25℃ After 7d~15d of development in the water, larvae hatched. When the hooked larvae are swallowed by Cyclops, they spend 2 to 3 weeks in their blood cavity. Breed the original cercariae. When the infected Cyclops is swallowed by small fish or juvenile fish, protocercaria can develop into sparganosis in the muscles, gonads, and eggs of the fish. Sparganosis can be excreted with fish eggs. When the big fish swallows small fish or fish eggs containing schizocephaly, the schistocephala can invade the muscle tissues of the big fish. Continue to survive until the final host eats fish with sparganosis, sparganosis can develop into adults in their intestines for 5 to 6 weeks. Adults in the end The main body can live for 5 to 13 years. Adults of D. mansoni live in the small intestines of cats, dogs and carnivorous wild animals, and their eggs are excreted with the host’s feces. At a suitable temperature, after 2 to 5 weeks of development, hexacoccid larvae will develop into protocercaria after being swallowed by Cyclops, which can be swallowed by frogs. It develops into sparganosis, and human ingestion of a second intermediate host containing sparganosis can cause sparganosis mansoni. The second intermediate that can infect sparganosis mansoni The host is frogs; birds, snakes, and pigs can be used as transfer hosts. BB

Appendix B

(Normative appendix) Technical methods related to detection B.1 Reagent preparation B.1.1 Pepsin digestion solution Pepsin 2 g, concentrated hydrochloric acid 0.7 mL, add distilled water to 100 mL, ready to use. B.1.2 Tris-acetic acid electrophoresis buffer (TAE) Tris (Tris base) 242 g, glacial acetic acid 57.1 mL, pH 8.0 0.5 mol/L EDTA solution 100 mL, add The volume of distilled water is adjusted to 1000 mL to make 50×TAE buffer solution, and it is mixed and stored at 4°C for later use. Dilute 50 times before use. B.1.3 1×TAE liquid 50× TAE 20 mL, add distilled water to make the volume to 1000 mL, and mix well for use. B.1.4 10 mg/mL ethidium bromide solution 1 g of ethidium bromide, add distilled water to make the volume to 100 mL, magnetically stir until completely dissolved, and store at room temperature in the dark. B.1.5 1.5% agarose gel 1.5 g agarose, add 1×TAE to make the volume to 100 mL, after completely melted, cool the solution to 60℃, add 10 mg/mL ethidium bromide 5 µL (Final concentration 0.5 µg/mL), mix gently to prepare a gel. B.1.6 6×Sampling buffer Bromophenol blue 0.25 g, sucrose 40 g, add distilled water to 100 mL. Store at 4°C for later use. B.1.7 Commercial kit Taq enzyme, dNTP and other nucleic acid extraction and PCR reagents can choose commercial reagents and kits. B.2 Primer template See Table B.2 for the primers related to the larvae of Split head tapeworm B.3 DNA extraction B.3.1 Pick a single insect body to be identified and put it into a sterile centrifuge tube, add sterile distilled water, change the water every half an hour, repeat the washing 3 times, wash After completion, discard the distilled water in the centrifuge tube, crush the worms in the centrifuge tube with a grinding rod, add 180 µl Buffer ATL, 20 µl proteinase K, After mixing, incubate at 56°C for 1~3 h until digestion is complete, shaking occasionally during the period. B.3.2 After the digestion is complete, vortex for 15 s, add.200 µl Buffer AL, vortex and mix immediately, and then add.200 µl ethanol (9 6～100%), vortex and mix well. B.3.3 Place the spin column on the collection tube, suck the mixture from the previous step into the spin column, centrifuge at 8000 r/min for 1 min, and discard the collection tube. B.3.4 Place the spin column in a new collection tube, add 500 µl Buffer AW1, centrifuge at 8000 r/min for 1 min, and discard the collection tube. B.3.5 Put the spin column in a new collection tube, add 500 µl Buffer AW2, centrifuge at 14000 r/min for 3 minutes, and discard the collection tube. B.3.6 Place the spin column in a sterile 1.5 ml centrifuge tube, add.200 µl Buffer AE, incubate for 1 min at room temperature, and centrifuge at 8000 r/min For 1 min, the DNA obtained by centrifugation is stored in a refrigerator at -20°C for later use (repeat this step to increase the DNA recovery rate). B.4 Explanation of experimental reagent codes (included in the kit) CC

Appendix C

(Informative appendix) Physical map of sparganosis C.1 The broad-segmented schistocephala in the muscle of white fish is shown in Figure C.1 Figure C.1 C.2 Schizocephala in trout muscle is shown in Figure C.2 Figure C.2 C.3 Sparganosis mansoni in a frog is shown in Figure C.3 Figure C.3 C.4 Sparganosis mansoni of snake body is shown in Figure C.4

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