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食品安全国家标准 食品微生物学检验 大肠埃希氏菌O157:H7/NM检验
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GB 4789.36-2016
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标准编号: GB 4789.36-2016 (GB4789.36-2016)
中文名称: 食品安全国家标准 食品微生物学检验 大肠埃希氏菌O157:H7/NM检验
英文名称: National Food Safety Standard -- Food Microbiological Examination -- Examination of Escherichia Coli O157:H7/MN
行业: 国家标准
中标分类: X09
字数估计: 13,177
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB/T 4789.36-2008
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
GB 4789.36-2016: 食品安全国家标准 食品微生物学检验 大肠埃希氏菌O157:H7/NM检验
GB 4789.36-2016 英文名称: National Food Safety Standard -- Food Microbiological Examination -- Examination of Escherichia Coli O157:H7/MN
1 范围
本标准适用于食品中大肠埃希氏菌O157:H7/NM的检验。
2 设备和材料
除微生物实验室常规灭菌及培养设备外,其他设备和材料如下:
2.1 恒温培养箱:36℃±1℃。
2.2 冰箱:2℃~5℃。
2.3 恒温水浴箱:46℃±1℃。
2.4 天平:感量0.1g、0.01g。
2.5 均质器。
2.6 显微镜:10倍~100倍。
2.7 无菌吸管:1mL(具0.01mL刻度)、10mL(具0.1mL刻度)或移液器及吸头。
2.8 无菌均质杯或无菌均质袋:容量500mL。
2.9 无菌培养皿:直径90mm。
2.10 pH计或精密pH试纸。
2.11 长波紫外光灯:365nm,功率≤6W。
2.12 微量离心管:1.5mL或2.0mL。
2.13 磁板、磁板架、样品混合器。
2.14 微生物鉴定系统。
3 培养基和试剂
3.1 改良EC肉汤(mEC+n):见A.1。
3.2 改良山梨醇麦康凯琼脂(CT-SMAC):见A.2。
3.3 三糖铁琼脂(TSI):见A.3。
3.4 营养琼脂:见A.4。
3.5 半固体琼脂:见A.5。
3.6 月桂基硫酸盐胰蛋白胨肉汤-MUG(MUG-LST):见A.6。
3.7 氧化酶试剂:见A.7。
3.8 革兰氏染色液:见A.8。
3.9 PBS-Tween20洗液:见A.9。
3.10 亚碲酸钾(AR级)。
3.11 头孢克肟(Cefixime)。
3.12 大肠埃希氏菌O157显色培养基。
3.13 大肠埃希氏菌O157和H7诊断血清或O157乳胶凝集试剂。
3.14 鉴定试剂盒。
3.15 抗-E.coliO157免疫磁珠。
第一法 常规培养法
4 检验程序
大肠埃希氏菌O157:H7/NM常规培养法检验程序见图1。
图1 大肠埃希氏菌O157:H7/NM常规培养法检验程序
5 操作步骤
5.1 增菌
以无菌操作取检样25g(或25mL)加入到含有225mLmEC+n肉汤的均质袋中,在拍击式均质
器上连续均质1min~2min;或放入盛有225 mL mEC+n肉汤的均质杯中,8000r/min~
10000r/min均质1min~2min。36℃±1℃培养18h~24h。
5.2 分离
取增菌后的 mEC+n肉汤,划线接种于CT-SMAC平板和大肠埃希氏菌O157显色琼脂平板上,
36℃±1℃培养18h~24h,观察菌落形态。在CT-SMAC平板上,典型菌落为圆形、光滑、较小的无色
菌落,中心呈现较暗的灰褐色;在大肠埃希氏菌 O157显色琼脂平板上的菌落特征按产品说明书进行
判定。
5.3 初步生化试验
在CT-SMAC和大肠埃希氏菌 O157显色琼脂平板上分别挑取5个~10个可疑菌落,分别接种
TSI琼脂,同时接种 MUG-LST肉汤,并用大肠埃希氏菌株(ATCC25922或等效标准菌株)做阳性对照
和大肠埃希氏菌 O157:H7(NCTC12900或等效标准菌株)做阴性对照,于36℃±1℃培养18h~
24h。必要时进行氧化酶试验和革兰氏染色。在TSI琼脂中,典型菌株为斜面与底层均呈黄色,产气或
不产气,不产生硫化氢(H2S)。置 MUG-LST肉汤管于长波紫外灯下观察,MUG阳性的大肠埃希氏菌
株应有荧光产生,MUG阴性的应无荧光产生,大肠埃希氏菌 O157:H7/NM 为 MUG试验阴性,无荧
光。挑取可疑菌落,在营养琼脂平板上分纯,于36℃±1℃培养18h~24h,并进行下列鉴定。
5.4 鉴定
5.4.1 血清学试验
在营养琼脂平板上挑取分纯的菌落,用O157和H7诊断血清或O157乳胶凝集试剂作玻片凝集试
验。对于H7因子血清不凝集者,应穿刺接种半固体琼脂,检查动力,经连续传代3次,动力试验均阴
性,确定为无动力株。如使用不同公司生产的诊断血清或乳胶凝集试剂,应按照产品说明书进行。
5.4.2 生化试验
5.4.2.1 自营养琼脂平板上挑取菌落,进行生化试验。大肠埃希氏菌O157:H7/NM 生化反应特征见
表1。
5.4.2.2 如选择生化鉴定试剂盒或微生物鉴定系统,应从营养琼脂平板上挑取菌落,用稀释液制备成浊
度适当的菌悬液,使用生化鉴定试剂盒或微生物鉴定系统进行鉴定。
5.4.3 毒力基因测定(可选项目)
样品中检出大肠埃希氏菌O157:H7或O157:NM时,如需要进一步检测Vero细胞毒素基因的存
在,可通过接种Vero细胞或HeLa细胞,观察细胞病变进行判定;也可使用基因探针检测或聚合酶链反
应(PCR)方法进行志贺毒素基因(stx1、stx2)、eae、hly等基因的检测。如使用试剂盒检测上述基因,应按照产品的说明书进行。
6 结果报告
综合生化和血清学试验结果,报告25g(或25mL)样品中检出或未检出大肠埃希氏菌 O157:H7
或大肠埃希氏菌O157:NM。
第二法 免疫磁珠捕获法
7 检验程序
大肠埃希氏菌O157:H7/NM免疫磁珠捕获法检验程序见图2。
图2 大肠埃希氏菌O157:H7/NM免疫磁珠捕获法检验程序
8 操作步骤
同5.1。
8.2 免疫磁珠捕获与分离
8.2.1 应按照生产商提供的使用说明进行免疫磁珠捕获与分离。当生产商的使用说明与下面的描述
可能有偏差时,按生产商提供的使用说明进行。
8.2.2 将微量离心管按样品和质控菌株进行编号,每个样品使用1只微量离心管,然后插入到磁板架
上。在漩涡混合器上轻轻振荡E.coliO157免疫磁珠混悬液后,用开盖器打开每个微量离心管的盖子,
每管加入20μLE.coliO157免疫磁珠悬液。
8.2.3 取mEC+n肉汤增菌培养物1mL,加入到微量离心管中,盖上盖子,然后轻微振荡10s。每个
样品更换1只加样吸头,质控菌株必须与样品分开进行,避免交叉污染。
转动10min,使E.coliO157与免疫磁珠充分接触。
8.2.5 捕获:将磁板插入到磁板架中浓缩磁珠。在3min内不断地倾斜磁板架,确保悬液中与盖子上的
免疫磁珠全部被收集起来。此时,在微量离心管壁中间明显可见圆形或椭圆形棕色聚集物。
8.2.6 吸取上清液:取1支无菌加长吸管,从免疫磁珠聚集物对侧深入液面,轻轻吸走上清液。当吸到
液面通过免疫磁珠聚集物时,应放慢速度,以确保免疫磁珠不被吸走。如吸取的上清液内含有磁珠,则
应将其放回到微量离心管中,并重复8.2.5步骤。每个样品换用1支无菌加长吸管。
免疫磁珠的滑落:某些样品特别是那些富含脂肪的样品,其磁珠聚集物易于滑落到管底。在吸取上
清液时,很难做到不丢失磁珠,在这种情况下,可保留50μL~100μL上清液于微量离心管中。如果在
后续的洗涤过程中也这样做的话,脂肪的影响将减小,也可达到充分捕获的目的。
器上转动或用手轻微转动3min,洗涤免疫磁珠混合物。重复上述步骤8.2.5~8.2.7。
8.2.8 重复上述步骤8.2.5~8.2.6。
8.2.9 免疫磁珠悬浮:移走磁板,将免疫磁珠重新悬浮在100μLPBS-Tween20洗液中。
8.2.10 涂布平板:将免疫磁珠混匀,各取50μL免疫磁珠悬液分别转移至CT-SMAC平板和大肠埃希
氏菌O157显色琼脂平板一侧,然后用无菌涂布棒将免疫磁珠涂布平板的一半,再用接种环划线接种平
板的另一半。待琼脂表面水分完全吸收后,翻转平板,于36℃±1℃培养18h~24h。
注:若CT-SMAC平板和大肠埃希氏菌O157显色琼脂平板表面水分过多时,应在36℃±1℃下干燥10min~
20min,涂布时避免将免疫磁珠涂布到平板的边缘。
GB 4789.36-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Microbiological Examination of Food Hygiene
Examination of Escherichia Coli O157.H7/NM
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the
People’s Republic of China;
China Food and Drug Administration.
3. No action is required - Full-copy of this standard will be automatically &
immediately delivered to your EMAIL address in 0~60 minutes.
Table of Contents
Foreword ... 3
1 Application Scope ... 4
2 Apparatus and Materials ... 4
3 Culture Media and Reagents ... 5
4 Test Procedures ... 5
5 Operating Procedures ... 6
6 Result Reporting ... 8
7 Test Procedures ... 8
8 Operating Procedures ... 9
9 Result Reporting ... 11
Annex A Culture Media and Reagents ... 12
Foreword
This Standard replaces GB/T 4789.36-2008, Microbiological Examination of Food
Hygiene – Examination of Escherichia Coli O157.H7/NM.
Compared with GB/T 4789.36-2008, the major changes of this Standard are as follows.
-- the standard name is modified into “National Food Safety Standard –
Microbiological Examination of Food Hygiene – Examination of Escherichia Coli
O157.H7/NM.”;
-- the application scope of the standard is modified;
-- the apparatus and materials are modified;
-- the text descriptions of culture media and biochemical reactions are modified;
-- “Method II The Principle of the Immunomagnetic Beads Capture Method” is
deleted;
-- “Method III Full-automatic Enzyme-linked Fluorescence Immunoassay
Instrument Screening Method” is deleted;
-- “Method IV Full-automatic Pathogenic Bacteria Detecting System Screening
Method” is deleted.
National Food Safety Standard -
Microbiological Examination of Food Hygiene
Examination of Escherichia Coli O157.H7/NM
1 Application Scope
This Standard specifies the test method of Escherichia coli o157.h7/nm in foods.
This Standard applies to the test of Escherichia coli o157.h7/nm in foods.
2 Apparatus and Materials
In addition to the conventional sterilization and culture equipment in the microbiological
laboratory, other apparatus and materials are as follows.
2.1 Thermostatic incubator. 36°C ± 1°C.
2.2 Refrigerator. 2°C ~ 5°C.
2.3 Thermostatic water bath. 46°C ± 1°C.
2.4 Balance. sensitivities 0.1 g and 0.01 g.
2.5 Homogenizer.
2.6 Microscope. 10X ~ 100X.
2.7 Sterile pipettes. 1 ml (having graduates of 0.01 ml), 10 ml (having graduates of
0.1 ml) or pipettes and tips.
2.8 Sterile homogeneous cups or sterile homogeneous bags. capacity 500 ml.
2.9 Sterile culture dishes. diameter 90 mm.
2.10 pH meters or precise pH test paper.
2.11 Long-wave UV lamps. 365 nm, power ≤ 6 W.
2.12 Microcentrifuge tubes. 1.5 ml or 2.0 ml.
2.13 Magnetic plate, magnetic plate frame, specimen mixer.
2.14 Microbiological identification system.
3 Culture Media and Reagents
3.1 Modified EC broth (mEC + n). see A.1.
3.2 Modified Sorbitol-MacConkey agar (CT-SMAC). see A.2.
3.3 Trisaccharide iron agar (TSI). see A.3.
3.5 Semi-solid agar. see A.5.
3.6 Lauryl sulphate peptone broth – MUG (MUG-LST). see A.6.
3.7 Oxidase reagent. see A.7.
3.8 Gram stain solution. see A.8.
3.9 PBS-Tween 20 lotion. see A.9.
3.10 Potassium tellurite (analytical reagent).
3.11 Cefixime.
3.12 Chromogenic medium for Escherichia coli O157.
3.13 Diagnostic serum for Escherichia coli O157 and H7 or latex agglutination
3.14 Identification kit.
3.15 Anti-E. coli O157 immunomagnetic beads.
Method I The Routine Culture Method
4 Test Procedures
See Figure 1 for the test procedures of the routine culture method of Escherichia coli
O157. H7/NM.
Simmons citrate Negative
Lysine decarboxylase Positive (purple)
Ornithine decarboxylase Positive (purple)
Raffinose fermentation Positive
MUG test Negative (no fluorescence)
Dynamic test Dynamic or non-dynamic
5.4.2.2 If the biochemical identification kit or microbiological identification system is
selected, pick colonies from the nutrient agar plate, use the diluent to make the
bacterial suspension of appropriate turbidity and use the biochemical identification kit
or microbiological identification system for identification.
5.4.3 Virulence genes test (optional item)
When Escherichia coli O157. H7 or O157. NM is detected in the specimen, it may be
existence of Vero cytotoxin requires further tests; it may also be tested by using the
gene probe tests or polymerase chain reaction (PCR) method for Shiga toxin genes
(stx1 and stx2), eae, hly and so on. If kits are used for the test of the above-mentioned
genes, the product manuals shall be followed.
6 Result Reporting
Summarize the biochemical and serological test results and report whether
Escherichia coli O157.H7 or Escherichia coli O157.NM is detected or not detected in
the 25 g (or 25 ml) of specimen.
Method II The Immunomagnetic Beads Capture
7 Test Procedures
See Figure 2 for the test procedures of the immunomagnetic beads capture method of
Escherichia coli O157.H7/NM.
8.2.2 Number the microcentrifuge tubes in accordance with the specimens and
quality-control strains, use one microcentrifuge tube for each specimen, and then
insert into the magnetic plate frame. Vibrate the Escherichia coli O157
immunomagnetic beads suspension gently on the vortex mixer, use an opener to open
the cap of each microcentrifuge tube, and add 20 μL of Escherichia coli O157
immunomagnetic beads suspension in each tube.
put on the cap, and then vibrate gently for 10 s. Replace one specimen adding tip for
each specimen, and the quality-control strains shall be separated from the specimen
to prevent cross-contamination.
8.2.4 Binding. at 18°C ~ 30°C, rotate the above-mentioned microcentrifuge tube
together with the magnetic plate frame on the specimen mixer or use hands to rotate
gently for 10 min to make Escherichia coli O157 fully contact with the immunomagnetic
beads.
8.2.5 Capture. insert the magnetic plate into the concentrated magnetic beads in the
magnetic plate frame. Tilt the magnetic plate frame within 3 min to ensure the
rounded or oval brown accumulation becomes visible in the middle of the wall of the
microcentrifuge tubes.
8.2.6 Absorption of supernatant. take one sterile elongated pipette and absorb gently
the supernatant by inserting deep into the liquid from the immunomagnetic beads
accumulation opposite to the liquid level. When the liquid is absorbed until the liquid
level passes through the immunomagnetic accumulation, slow down to ensure no
immunomagnetic beads are absorbed away. If the supernatant absorbed contains
magnetic beads, place them back in the microcentrifuge tube, and repeat the
procedures in 8.2.5. Replace one sterile elongated tube for each specimen.
especially those specimens containing much fat is liable to fall to the bottom. When
absorbing the supernatant, it is hard to prevent the loss of magnetic beads; and under
such circumstances, 50 μL ~ 100 μL of supernatant may be reserved in the
microcentrifuge tube. If it is operated like this in the later washing process, the effects
of fat will become smaller and the purpose of full capture may also be achieved.
8.2.7 Washing. move away the magnetic plate from the frame, add 1 ml of PBS-
Tween 20 lotion in each microcentrifuge tube, and rotate on a specimen mixer or use
hand to rotate gently for 3 min to wash the immunomagnetic beads mixture. Repeat
the above-mentioned procedures 8.2.5 ~ 8.2.7.
8.2.9 Immunomagnetic beads suspension. move away the magnetic plate and make
the immunomagnetic beads suspend in 100 μL of PBS-Tween 20 lotion once again.
Annex A
Culture Media and Reagents
A.1 Modified EC broth (mEC+n)
A.1.1 Ingredients
Tryptone 20.0 g
Bile salt No.3 1.12 g
Lactose 5.0 g
KH2PO4 1.5 g
NaCl 5.0 g
Novobiocin sodium salt solution (20 mg/ml) 1.0 ml
Distilled water 1 000 ml
A.1.2 Preparation
Except novobiocin, dissolve all ingredients in the water, heat to boiling, calibrate the
pH to 6.9 ± 0.1 at 20°C ~ 25°C, and load separately. Conduct autoclaved sterilization
for 15 min at 121°C as standby. Prepare novobiocin stock solution of concentration 20
mg/ml and conduct disinfection by filtering. Wait until the temperature ...
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