SN/T 1162-2020 English PDFUS$279.00 · In stock
Delivery: <= 3 days. True-PDF full-copy in English will be manually translated and delivered via email. SN/T 1162-2020: (Technical specifications for quarantine of infectious pancreatic necrosis) Status: Valid SN/T 1162: Historical versions
Basic dataStandard ID: SN/T 1162-2020 (SN/T1162-2020)Description (Translated English): (Technical specifications for quarantine of infectious pancreatic necrosis) Sector / Industry: Commodity Inspection Standard (Recommended) Classification of Chinese Standard: B41 Classification of International Standard: 65.020.30 Word Count Estimation: 13,123 Date of Issue: 2020-12-30 Date of Implementation: 2021-07-01 Older Standard (superseded by this standard): SN/T 1162-2002 Regulation (derived from): General Administration of Customs Announcement No. 136 [2020] Issuing agency(ies): General Administration of Customs SN/T 1162-2020: (Technical specifications for quarantine of infectious pancreatic necrosis)---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.Quarantine protocol for infectious pancreatic necrosis The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards Replace SN/T 1162-2002 Technical specifications for quarantine of infectious pancreatic necrosis Issued by the General Administration of Customs of the People's Republic of China 2020-12-30 release 2021-07-01 implementation ForewordThis document is drafted in accordance with GB/T 1.1-2020. This document replaces SN/T 1162-2002 "Infectious Pancreatic Necrosis Virus (IPNV) Enzyme Chain Immunosorbent Test (ELISA) Diagnosis method". Compared with SN/T 1162-2002, the main technical changes of this document are as follows. --Added the description of clinical symptoms; --- Added SYBR Green fluorescent RT-PCR method; - Added RT-PCR method; - Modified the comprehensive judgment. This document was proposed and managed by the General Administration of Customs of the People's Republic of China. Drafting organizations of this document. Shenzhen Customs of the People's Republic of China, Shenzhen Institute of Inspection and Quarantine. The main drafters of this document. Liu Hong, Zheng Xiaocong, Liu Ying, Wang Jinjin, Jia Peng, Wen Zhiqing, He Junqiang, Yu Li, Shi Xiujie, Lan Wensheng, Wang Hongying, Ye Yiyou. The previous versions of the documents replaced by this document are as follows. - SN/T 1162-2002. Technical specifications for quarantine of infectious pancreatic necrosis1 ScopeThis document specifies the isolation of infectious pancreatic necrosis virus, SYBR Green real-time fluorescent reverse transcription polymerase chain reaction, reverse Record the detection methods of polymerase chain reaction and enzyme-linked immunosorbent assay. This document is applicable to the epidemiological surveillance, detection and quarantine of infectious pancreatic necrosis.2 Normative referencesThe following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. For undated reference documents, the latest version (including all amendments) is applicable to this document. GB/T 6682 Analytical laboratory water specifications and test methods GB/T 18088 Entry and Exit Animal Quarantine Sampling 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. BSA. bovine serum albumin (bovine serum albumin); BF-2.blue fin fish cell line; CHSE-214.Chinook salmon embryo cell line (chinook salmon embryo cell line); Ct. cycle threshold (cycle threshold); DEPC. Diethyl pyrocarbonate (diethy pyrocarbonate); dNTP. deoxynucleoside triphosphate; ELISA. enzyme-linked immunosorbent assay; HRP. horseradish peroxidase (horseradish peroxidase); OD value. optical density value (optic density value); RNA. deoxyribonucleic acid (ribonucleic acid); RTG-2.rainbow trout gonadal cell line; RT-PCR. reverse transcription polymerase chain reaction (reverse transcription polymerase chain reaction).4 overviewInfectious pancreatic necrosis (IPN) is caused by infectious pancreatic necrosis virus (infectious pancreatic necrosis, IPN). Pancreatic necrosis virus (IPNV) caused a very serious infectious and viral fish disease, mainly in salmon Fry and juvenile fish of the family fish (see Appendix A).5 Reagents and materials5.1 Water. Comply with the first grade water specification in GB/T 6682. 5.2 DEPC water. For the preparation method, see B.1 in Appendix B or purchase commercial reagents. 5.3 IPNV reference strain. 5.4 Cell line. CHSE-214, RTG-2 or BF-2 cell line. 5.5 HEPES. 4-hydroxyethylpiperazine ethanesulfonic acid. 5.6 Rabbit anti-IPNV reference antiserum. 5.7 Goat anti-IPNV antibody. 5.8 Enzyme-labeled goat anti-rabbit IgG. use according to the instructions. 5.9 Primer. Use DEPC water to prepare each primer into a 100 μmol/L storage solution, and store it below -20 ℃; when using, take an appropriate amount. Prepare 20 μmol/L working solution to avoid multiple freezing and thawing. The specific sequence is as follows. IPNV F. 5'-ACTACGAACCCCCAGGACAAA-3'; IPNV R. 5'-GTCTCGTCCTCTAGGCGGACGTA-3'. Used for SYBR Green real-time fluorescent RT-PCR to amplify the 122 bp fragment of the IPNV VP2 gene. IPNV PrD1.5'-AAAGCCATAGCCGCCCATGAAC-3'; IPNV PrD2.5'-TCTCATCAGCTGGCCCAGGTAC-3'. Used for RT-PCR to amplify the 174 bp fragment between the IPNV NS and VP3 genes.6 Equipment and equipment6.1 Microplate reader. 6.2 Inverted microscope. 6.3 Constant temperature incubator. 6.4 PCR thermal cycler. 6.5 Gel imager.7 Isolation and identification of IPNV7.1 Sampling The sampling quantity shall be implemented in accordance with GB/T 18088.For fish with clinical symptoms, take the whole fish fry less than or equal to 4 cm in length, if The yolk sac should be removed for fish with yolk sac; fish fry with a body length of 4 cm to 6 cm should be taken from internal organs (including kidneys); fish with a body length of more than 6 cm should be taken from liver, Kidney and spleen. Liver, kidney and spleen are taken from fish without clinical symptoms; ovarian fluid is also required for mature female fish. The egg shell is taken from fish eggs. 7.2 Sample processing Should be kept below 10 ℃ for processing. First homogenize the sample with a tissue grinder, and then suspend it in the fine particles at a final dilution of 1.10. Cell culture medium (see B.2 in Appendix B). If the sample has not been treated with antibiotics before homogenization, it must be added to the cell culture medium 1 000 IU/mL penicillin and 1 000 μg/mL streptomycin. Incubate at 15 ℃ for 2 h ~ 4 h or at 4 ℃ for 6 h ~ 24 h. 7 000 r/min Centrifuge for 15 min and collect the supernatant. The ovarian fluid does not need to be homogenized, it should be diluted more than 2 times. Centrifuge samples of ovarian fluid in the same way and later The supernatant was used directly in the step. 7.3 Virus isolation Dilute the 1.10 tissue homogenate supernatant twice with cell culture medium by 10 times, and then dilute 1.10, 1.100, 1.1 000 3 The diluted supernatant was inoculated into CHSE-214, RTG-2 or CHSE-214, RTG-2 or In the BF-2 cell monolayer, each well (2 cm2) of the cell monolayer is inoculated with a maximum of 100 μL of diluent. After adsorption at 15 ℃ ~20 ℃ for 0.5 h ~1 h, Add cell culture medium. Incubate at 18 ℃ ± 2 ℃. After the positive control group and the test sample were inoculated with cells, they were inspected with an inverted microscope every day for 7 days. The blank control cells should be normal. If, except for the positive control cells, there is no cytopathic (CPE) in the cell culture of the tested homogenate supernatant diluent, then culture 7 After d, it is necessary to subculture with sensitive cells. During subculture, freeze-thaw and collect the cell monolayer culture inoculated with the supernatant diluent of the tissue homogenate. The nutrient was centrifuged at 7 000 r/min 4 ℃ for 15 min, and the supernatant was collected. Inoculate the supernatant into a fresh cell monolayer and culture for 7 days, every day Microscopic examination. If there is no CPE after blind transmission, the result is judged as negative. If CPE appears in the cell culture inoculated with the supernatant diluent of the tested homogenate or the cell culture after blind transmission, it should be applied immediately SYBR Green real-time fluorescent RT-PCR, conventional RT-PCR or ELISA methods for IPNV identification. CPE appears as a local area The domain cells begin to lyse, the surrounding cells shrink and become round, gradually develop, and finally all the cells fall off. If there is no CPE in the positive control, the test is invalid. You must switch to sensitive cells and a new batch of tissue samples according to the above method. Method for virological examination. 7.4 SYBR Green real-time fluorescent RT-PCR detection of IPNV 7.4.1 Setting up a comparison Blank control, negative control and positive control should be set for each reaction. For blank control, DEPC water can be used instead of sample; negative control It is a tissue or cell culture that does not contain IPNV; a positive control is a positive tissue or cell culture that contains IPNV or contains a fragment of interest RNA. 7.4.2 RNA extraction Take.200 μL of the tissue supernatant of the sample to be tested or the cell culture of the sample to be tested, add 1 mL of Trizol reagent, and use a pipette Fully pipette 10 to 20 times, and place at room temperature for 5 minutes; add.200 μL of chloroform, vortex for 30 s to mix, and place at room temperature 15 min; centrifuge at 12 000 r/min for 10 min; take the upper water phase to a new centrifuge tube, add an equal volume of isopropanol, and turn upside down. Mix it again and place it at -20 ℃ for 20 min; centrifuge at 12,000 r/min for 10 min; discard the supernatant, and wash the pellet with 1 mL of 75% ethanol; Centrifuge at 8 000 r/min for 10 min, discard the supernatant, and dry the pellet at room temperature for 5 min; add 20 μL of DEPC water to dissolve the RNA pellet. 4 ℃ ice Keep in the box for later use. The RNA solution should avoid repeated freezing and thawing and be used for testing as soon as possible. RNA extraction can also use equivalent commercial RNA extraction kits instead of the above methods. 7.4.3 Reaction system In a 0.2 mL PCR reaction tube, prepare the reaction system according to Table 1, and add samples according to the blank control, negative control, sample to be tested, positive Sexual control... ......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of SN/T 1162-2020_English be delivered?Answer: Upon your order, we will start to translate SN/T 1162-2020_English as soon as possible, and keep you informed of the progress. The lead time is typically 1 ~ 3 working days. The lengthier the document the longer the lead time.Question 2: Can I share the purchased PDF of SN/T 1162-2020_English with my colleagues?Answer: Yes. 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