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SN/T 4656.7-2020 English PDF

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SN/T 4656.7-2020: (Import and export textiles biosafety inspection method - Part 7: Pseudomonas aeruginosa)
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Standard similar to SN/T 4656.7-2020

GB/T 32614   GB 31701   GB 18401   SN/T 4656.10   SN/T 4656.9   SN/T 4656.8   

Basic data

Standard ID SN/T 4656.7-2020 (SN/T4656.7-2020)
Description (Translated English) (Import and export textiles biosafety inspection method - Part 7: Pseudomonas aeruginosa)
Sector / Industry Commodity Inspection Standard (Recommended)
Classification of Chinese Standard W09
Classification of International Standard 59.080.99
Word Count Estimation 18,157
Date of Issue 2020-08-27
Date of Implementation 2021-03-01
Regulation (derived from) General Administration of Customs Announcement No. 98 [2020]
Issuing agency(ies) General Administration of Customs

SN/T 4656.7-2020: (Import and export textiles biosafety inspection method - Part 7: Pseudomonas aeruginosa)


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Biosafety testing methods of the textiles for import and export- Part 7.Pseudomonas aeruginosa The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards 2020-08-27 release 2021-03-01 implementation ICS 59.080.99 W 09 Issued by the General Administration of Customs of the People's Republic of China

Foreword

SN/T 4656 "Methods for Biosafety Inspection of Imported and Exported Textiles" is divided into the following parts. --Part 1.Candida albicans; --Part 2.Escherichia coli; --Part 3.Coliform flora; --Part 4.Staphylococcus aureus; --Part 5.Total number of colonies; --Part 6.Salmonella; --Part 7.Pseudomonas aeruginosa; --Part 8.General Rules This part is part 7 of SN/T 4656. This part was drafted in accordance with the rules given in GB/T 1.1-2009. Please note that some of the contents of this document may involve patents. The issuing agency of this document is not responsible for identifying these patents. This part is proposed and managed by the General Administration of Customs of the People's Republic of China. Drafting organizations of this section. Zhengzhou Customs of the People's Republic of China, Shijiazhuang Customs of the People's Republic of China. The main drafters of this section. Li Ke, Guo Huiqing, Yu Jianying, Guo Hualin, Lian Sumei, Xu Chao, Qiao Qing. Import and export textile biosafety inspection methods Part 7.Pseudomonas aeruginosa

1 Scope

This part of SN/T 4656 specifies the inspection method for Pseudomonas aeruginosa in import and export textiles. This part is applicable to the inspection of Pseudomonas aeruginosa of import and export textiles.

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 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 8170 Numerical rounding rules and the expression and determination of limit values SN/T 1538.1 Guidelines for the Preparation of Culture Medium Part 1.General Rules for Quality Assurance of Laboratory Culture Medium Preparation SN/T 1538.2 Medium Preparation Guide Part 2.Practical Guide for Medium Performance Testing SN/T 4656.8 Biosafety Inspection Method for Import and Export Textiles Part 8.General Rules

3 Terms and definitions

The following terms and definitions apply to this document. 3.1 Biosafety of textiles The harmful organisms themselves or their metabolites carried in textiles pose potential threats to the ecological environment and human health, and their effects A series of effective prevention and control measures taken. 3.2 Pseudomonas aeruginosa Also known as Pseudomonas aeruginosa, straight or slightly curved gram-negative, aerobic, non-bacillus bacillus, can use acetamide as a carbon source and decompose Acetamide produces alkali; can reduce nitrate, liquefy gelatin, grow well at 42 ℃, has high lethality, strong hemolysis, and drug resistance Strong sex and other characteristics. This fungus is widely distributed in nature, the main growth condition is humid environment, other conditions are not demanding, and it can temporarily parasitize It is on the skin, so the rate of contact body surface infection is quite high. 3.3 Loop-mediated isothermal amplification; LAMP Two pairs of special inner and outer primers designed according to the conserved genes of the target sequence unique to the target bacteria, specifically recognize the six on the target sequence In an independent area, use Bst DNA polymerase to start the cyclic chain displacement reaction, and keep it for 30 min to 60 min under isothermal conditions. Complete nucleic acid amplification efficiently and quickly. The pyrophosphate ions precipitated from dNTPs combine with Mg2 in the reaction solution to produce by-products (coke Magnesium phosphate) to form a milky white precipitate, add the color-developing solution, and the result can be determined by the color change observation.

4 Materials and equipment

4.1 Sterile scissors and tweezers. 4.2 Electronic balance. with a sense of 0.01 g. 4.3 Sterile homogeneous bag. 4.4 Slap type homogenizer. 400 mL. 4.5 Sterile specification plate. 20 cm2. 4.6 Sterile dry cotton swabs. 4.7 Micropipette. measuring range 0.5 μL ～ 20 μL, measuring range 100 μL ～ 1 000 μL, measuring range 1 mL ～ 10 mL. 4.8 Sterile small triangular flask. 25 mL. 4.9 Sterile glass plates or disposable sterile plastic plates. 15 mm × 90 mm. 4.10 Sterile L-shaped glass holder. 4.11 Constant temperature incubator. 36 ℃ ±1 ℃, 42 ℃ ±1 ℃. 4.12 Inoculation loop. 4.13 Microscope. 10×～100×. 4.14 Fully automatic microbial biochemical identification system. 4.15 Sterilized centrifuge tube. 1.5 mL. 4.16 High-speed refrigerated centrifuge. 2 000 r/min ~ 13 000 r/min. 4.17 LAMP reaction tube. 4.18 Water bath. 65 ℃ ±1 ℃. 4.19 LAMP real-time turbidity meter.

5 Medium and reagents

Except for special instructions, all experimental reagents are analytically pure; the experimental water used for molecular detection meets the requirements of Grade 1 water in GB/T 6682 Claim. 5.1 Sterile normal saline. see A.2. 5.2 SCDLP liquid medium. see A.3. 5.3 Acetamide agar. see A.4. 5.4 Gram staining solution. see A.5. 5.5 Oxidase reagent. see A.6. 5.6 Aqueous nitrate peptone medium. see A.7. 5.7 Nutrient agar. see A.8. 5.8 Sterilized double distilled water. 5.9 10×LAMP amplification buffer. containing.200 mmol/L Tris-HCl (pH 8.8), 100 mmol/L ammonium sulfate, 500 mmol/L Potassium chloride, 20 mmol/L magnesium sulfate, 1% Tween20. 5.10 Primers. According to the conservative sequence of the ETA gene of Pseudomonas aeruginosa, design a set of LAMP-specific primers. 5.11 100 mmol/L MgSO4. 5.12 10 mmol/L deoxynucleoside triphosphate (dNTP) mixture. the concentration of each nucleotide is 10 mmol/L. 5.13 Bst DNA polymerase. 8 U/μL. 5.14 Chromogenic solution. SYBR Green I dye, 1 000×. 5.15 Standard strain of Pseudomonas aeruginosa.

6 Sample preparation

6.1 Weighing method Open the submitted sample aseptically, cut the sample evenly with sterile scissors (4.1), and accurately weigh the cut out on the electronic balance (4.2) 25 g of the sample, cut into pieces, add it to a sterile homogenization bag (4.3) containing 225 mL of sterile diluent, and beat with a flapping homogenizer (4.4) For 1 min to 2 min, mix thoroughly to obtain a 1.10 sample homogenate. 6.2 Multi-point sampling elution method Open the sample to be tested in a sterile method, arrange 5 sampling points evenly around and in the middle of the sample, and use a sterile specification plate (4.5) according to each The sampling points are cut according to the area of 4 cm×5 cm (20 cm2), and the sampling area of every 20 cm2 is 1 test sample, and a total of 5 samples are collected for each sample. For sample inspection, the sampling area is 100 cm2.Put the 5 samples collected above into a sterile homogenization bag (4.3) containing.200 mL of sterile diluent, Use a flapping homogenizer (4.4) to beat for 1 min to 2 min, mix thoroughly, and use the sample homogenate as the stock solution. 6.3 Cotton swab smearing method Aseptically open the sample to be tested, moisten a sterile dry cotton swab (4.6) with sterile diluent, and distribute the control evenly around and in the middle of the sample 5 sampling points, use a sterile specification plate (4.5) to smear evenly in the area of 20 cm2 for each sampling point, and use 1 for each sampling point Sterile dry cotton swab (4.6), spread evenly in the area of 4 cm×5 cm (20 cm2), immediately cut off the cotton with sterile scissors (4.1) Swab the hand contact part, put the smeared part into a sterile homogenization bag (4.3) containing 50 mL of sterile diluent and mix well to make 1.10 Sample homogenate. 6.4 Selection of sample preparation method 6.4.1 The sample preparation is generally based on method 6.1 as the reference method. 6.4.2 When the sample is larger or thicker or porous, method 6.2 should be used for sample preparation. When using method 6.2, if the sample is tested If the area is too large or too small, the sampling points can be increased or decreased proportionally. 6.4.3 When the sample has a dense texture and is not easy to cut and prepare; or the sample is expensive and the customer requires non-destructive testing, the sample preparation should be used in 6.3 method. 6.4.4 When using methods 6.1 and 6.2, if the test sample absorbs a large amount of water and cannot absorb enough sample for conversion, the diluent may be suitable. When increased until enough to suck out the conversion. 7 Method 1 Quantitative Test Method for Pseudomonas aeruginosa Plate Count Method 7.1 Principle Pseudomonas aeruginosa has acetamidase, which can use acetamide as a carbon source and decompose acetamide to produce alkali. Sample inoculated with acetamide After the base, the target bacteria can make the acetamide medium red; the combined Pseudomonas aeruginosa is oxidase-positive, can reduce nitrate to produce gas and in The characteristics of growth at 42 ℃ can accurately determine Pseudomonas aeruginosa. 7.2 Inspection procedure 7.3 Operation steps 7.3.1 Dilution of samples Use a micropipette (4.7) to pipette 1 mL of the prepared sample homogenate, and slowly inject it into the sample containing 9 mL of sterile In the small triangular flask (4.8) (note that the tip of the tip does not touch the liquid surface), shake the small triangular flask or use a sterile tip to repeatedly pipette The mixture is evenly mixed to prepare a homogeneous sample solution of the next dilution. According to the above operating procedures, prepare a 10-fold serial dilution sample homogenate. Every pass Increase and dilute once, and replace with a sterile tip. 7.3.2 Sample inoculation According to the actual contamination of the sample, select 2 to 3 suitable dilution sample homogenates (including the original solution), and each dilution is divided into Do not pipet 1 000 μL of sample homogenate to inoculate two dry acetamide agar (5.3) plates with 500 μL and 500 μL inoculum respectively. Then use a sterile L-shaped glass holder (4.10) to evenly coat the entire plate, taking care not to touch the edge of the plate. If the sample liquid is not easy to absorb, you can The plate is placed in a constant temperature incubator (4.11) at 36 ℃ ± 1 ℃ for 1 h. After the sample is absorbed, the plate is turned over and placed upside down in the incubator. Raise for 24 h ± 2 h. 7.3.3 Count of typical or suspicious colonies Colony morphology of Pseudomonas aeruginosa on acetamide agar (5.3) plate. milky white flat colonies, moist, neat edges, colonies The surrounding medium turns red. Select the typical or suspicious Pseudomonas aeruginosa colonies, and the two plates with the same dilution are all typical or suspicious For the plates with the total number of suspected colonies in the range of 20 CFU to.200 CFU, count the number of typical or suspicious colonies. 7.3.4 Confirmation test 7.3.4.1 Overview Pick at least 5 (all less than 5) typical or suspicious colonies from the counted acetamide agar (5.3) plate, and do The following confirmatory test. 7.3.4.2 Staining microscopic examination Pick a single colony on the acetamide agar (5.3) plate, smear it, stain with Gram stain (5.4), and under the microscope (4.13) Observe its colony morphology. Pseudomonas aeruginosa is a gram-negative bacterium under the microscope. It is slender and varies in length, sometimes in the shape of a ball or thread. Arranged in pairs or short chains. 7.3.4.3 Oxidase test Take a piece of clean filter paper and place it in a sterile glass plate (4.9), use a micropipette (4.7) to suck up the oxidase reagent (5.5) and add it dropwise Put one drop on the filter paper, only make the filter paper wet, not too wet, use the inoculation loop (4.12) to pick a single colony on the acetamide agar (5.3) plate Spread it on the filter paper, at 10...

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