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食品安全国家标准 食品微生物学检验 创伤弧菌检验
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标准编号: GB 4789.44-2020 (GB4789.44-2020)
中文名称: 食品安全国家标准 食品微生物学检验 创伤弧菌检验
英文名称: National food safety standard - Food microbiological examination - Vibrio vulnificus
行业: 国家标准
中标分类: X09
字数估计: 17,170
发布日期: 2020-09-11
实施日期: 2021-03-11
标准依据: 国家卫生健康委员会 国家市场监督管理总局公告2020年第7号
GB 4789.44-2020
National food safety standard - Food microbiological examination - Vibrio vulnificus
中华人民共和国国家标准
食品安全国家标准
食品微生物学检验 创伤弧菌检验
2020-09-11发布
2021-03-11实施
中华人民共和国国家卫生健康委员会
国 家 市 场 监 督 管 理 总 局 发 布
食品安全国家标准
食品微生物学检验 创伤弧菌检验
1 范围
本标准适用于鱼、虾、蟹、贝类等水产品中创伤弧菌的检验。
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℃、7℃~10℃。
2.3 恒温水浴锅。
2.4 均质器或无菌研钵。
2.5 天平:感量0.1g。
2.6 PCR仪。
2.7 电泳仪或毛细管电泳仪。
2.8 凝胶电泳成像系统或紫外检测仪。
2.9 生物安全柜。
2.10 高速离心机(最大转速至少15000r/min)。
2.11 涡旋振荡器。
2.12 微量可调移液器(量程2.5μL、10μL、100μL、1000μL)及配套吸头。
2.13 精密pH试纸或pH计。
2.14 无菌试管:规格18mm×180mm和15mm×100mm。
2.15 无菌吸管:规格1mL(具0.01mL刻度)和10mL(具0.1mL刻度)。
2.16 无菌锥形瓶:容量250mL、500mL和1000mL。
2.17 无菌培养皿:直径90mm。
2.18 无菌手术剪、镊子、钳子等。
2.19 PCR反应管。
3 培养基和试剂
3.1 蛋白胨-氯化钠-纤维二糖-多黏菌素E(PNCC)增菌液:见A.1。
3.2 纤维二糖-多黏菌素E(CC)琼脂培养基:见A.2。
3.3 改良纤维二糖-多黏菌素B-多黏菌素E(mCPC)琼脂培养基:见A.3。
3.4 3%氯化钠胰蛋白胨大豆琼脂培养基:见A.4。
3.5 磷酸盐缓冲液(PBS):见A.5。
3.6 3%氯化钠三糖铁琼脂斜面:见A.6。
5 操作步骤
5.1 样品制备
5.1.1 新鲜样品采集后应于3h内完成检验,若不能在规定时间内完成,则将样品置于7℃~10℃条
件下保存(因创伤弧菌在4℃条件下极易形成活而不可培养的状态,因此样品勿放4℃条件下),并尽可
能在24h内完成检验;冷冻样品应在不超过45℃的温热条件下解冻,解冻时间不超过15min。
5.1.2 取样:鱼类和头足类取其表面组织、肠和鳃。贝类则取全部内容物(包括贝肉和体液)。甲壳类
取整个动物或其中心部分(包括肠和鳃)。如为带壳贝类或硬壳甲壳类,则应先用流动自来水冲洗外壳
并用滤纸吸干表面水分,然后无菌操作打开外壳,按上述要求取相应部分。
5.1.3 以无菌操作取上述处理后的样品25g,加入PNCC增菌液225mL,用旋转刀片式均质器以
8000r/min均质1min,或用拍击式均质器均质2min,充分混匀制备成1∶10的样品匀液。如无均质
器,则将样品放入无菌乳钵中充分研磨,取磨碎后的样品25g转入500mL无菌锥形瓶中,加入PNCC
增菌液225mL,充分振荡混匀,制备成1∶10的样品匀液。
5.2 增菌
将5.1.3制备的1∶10样品匀液于36℃±1℃培养18h±1h。
5.3 PCR检测
PCR实验环境条件和过程控制应参照GB/T 27403《实验室质量控制规范 食品分子生物学检
测》规定执行,下同。
5.3.1 DNA模板的制备
在距离PNCC增菌液的液面下1cm处吸取1mL置于1.5mLEP管中,9000r/min离心3min,
弃去上清液。向沉淀中加入1mLPBS将其悬浮并充分清洗后,9000r/min离心3min,弃去上清液,
按此步骤反复清洗沉淀2次~3次,弃去最后一遍上清液,加入1mL无菌超纯水,100℃煮沸10min,
继而12000r/min离心5min,上清液用于PCR分析。若不能及时分析则于-20℃保存备用。
也可以用商品化的DNA提取试剂盒按其说明书要求提取制备DNA模板。
5.3.2 PCR扩增
5.3.2.1 引物
信息见表1。
5.3.2.2 对照设置
每次PCR反应使用创伤弧菌标准菌株作为阳性对照,同时,使用除创伤弧菌之外的其他弧菌的标
准菌株作为阴性对照,以灭菌去离子水作为空白对照。
5.3.2.3 PCR反应体系
见表2。
5.3.2.4 PCR反应程序
预变性:94℃、5min;变性:94℃、1min;退火:62℃、1min;延伸:72℃、1min;循环数:30;终延
伸:72℃、10min;电泳检测。若不能立即检测则4℃保存备用。
5.3.3 电泳
凝胶电泳检测PCR扩增产物。用0.5×TBE缓冲液配制1.5%的琼脂糖凝胶(含EB0.5μg/mL或
Goldview5μL/100mL或Gelred5μL/50mL等DNA染料),取5μLPCR扩增产物与1μL6×核酸
电泳上样缓冲液混合后,点样,同时有一孔加入DNA分子量标准(范围为100bp~1000bp)。根据以
下公式:电泳槽正负极的距离(cm)×5V/cm计算并设置电压,使用0.5×TBE缓冲液恒压电泳,根据
5.3.4 结果判定
质控系统:阴性对照和空白对照均未出现扩增条带,阳性对照出现预期大小(519bp)的扩增条带,
则检测系统正常。否则,任一种对照如果出现非上述正常结果,应重做实验,同时排除污染因素。
阳性结果:在质控系统正常的情况下,待测样品出现预期大小(519bp)的扩增条带,判定PCR结果
为阳性。
阴性结果:在质控系统正常的情况下,待测样品未出现预期大小(519bp)的扩增条带,判定PCR结
果为阴性。
5.4 分离
用10μL接种环在距离PNCC增菌液的液面下1cm 沾取一环增菌液,分别划线接种于CC和
圆形、扁平,光照下呈透明或中心不透明但边缘透明的黄色至橘黄色菌落,菌落直径1mm~2mm,菌
落周围可出现(或不出现)黄色晕圈。
5.5 分纯培养
从CC平板和mCPC平板上各挑取至少5个创伤弧菌可疑菌落(少于5个时全选),分别接种于
3%氯化钠胰蛋白胨大豆琼脂平板,36℃±1℃培养18h±1h后用于后续鉴定。创伤弧菌在3%氯化
钠胰蛋白胨大豆琼脂平板上的菌落形态为圆形、乳白色、湿润、隆起、直径1mm~2mm。
5.6 鉴定
创伤弧菌可采用菌落特征结合生化特性或PCR方法进行鉴定。
5.6.1 菌落特征及生化特性
该步骤用于创伤弧菌的初步鉴定,进行以下4项鉴定实验时,挑取的菌落应来自分纯后的同一个单
菌落。
a) 革兰氏染色镜检:从5.5中3%氯化钠胰蛋白胨大豆琼脂平板上挑取创伤弧菌疑似菌落进行革
兰氏染色并镜检。创伤弧菌为革兰氏阴性,显微镜下菌体为棒状、弧状、卵圆状等多种形态,无
芽胞。
b) 氧化酶试验:用接种环从5.5中3%氯化钠胰蛋白胨大豆琼脂平板上挑取创伤弧菌疑似菌落适
量,涂布在用氧化酶试剂润湿(如无菌滤纸)或滴有氧化酶试剂的白色或无色载体上(如载玻
片)。如果涂菌部位在10s之内变紫色(偶有蓝紫色),即为氧化酶试验阳性,不变色为氧化酶
试验阴性。创伤弧菌为氧化酶试验阳性。
量,转种于3%氯化钠三糖铁琼脂斜面并穿刺底层(注意接种针不要触及试管底部),36℃±
1℃培养24h观察结果。创伤弧菌在3%氯化钠三糖铁琼脂斜面中生长时试管底层变黄,无
气泡,斜面颜色不变黄(偶有斜面颜色变黄现象)。
d) 嗜盐性试验:用接种针从5.5中3%氯化钠胰蛋白胨大豆琼脂平板上挑取创伤弧菌疑似菌落,
分别接种于含0%、3%、6%、8%和10%氯化钠的胰蛋白胨水中,36℃±1℃培养24h,观察液
体的混浊情况。创伤弧菌在含0%、8%和10%氯化钠的胰蛋白胨水中不生长或微弱生长,胰
蛋白胨水澄清透亮或稍混浊,而在含3%和6%氯化钠的胰蛋白胨水中生长旺盛,胰蛋白胨水
混浊。
5.6.1.2 确证实验
18h±1h后,取纯培养物分别接种于含3%氯化钠的赖氨酸脱羧酶试验培养基和 MR-VP培养基中,
36℃±1℃培养24h~48h后观察结果;另取少许纯培养物接种于3%氯化钠三糖铁琼脂斜面,
36℃±1℃培养18h后用于ONPG试验。也可选择生化鉴定试剂盒进行鉴定。
创伤弧菌生化特性见表3,与其他弧菌的鉴别见表4。
5.6.2 PCR鉴定
本部分试验可替代5.6.1用于创伤弧菌的快速鉴定。
5.6.2.1 DNA模板的制备
从5.5中3%氯化钠胰蛋白胨大豆琼脂平板上挑取创伤弧菌疑似菌落,转至500μL无菌超纯水中,
充分混匀后制成肉眼可见的混浊菌悬液,煮沸10min,12000r/min离心5min,取上清液用于PCR分
也可以用商业化的DNA提取试剂盒按其说明提取制备DNA模板。
5.6.2.2 PCR扩增和电泳
同5.3.2和5.3.3。
5.6.2.3 结果判定
质控系统:阴性对照和空白对照均未出现扩增条带,阳性对照出现预期大小(519bp)的扩增条带,
则检测系统正常。否则,任一种对照如果出现非上述正常结果,应重做实验,同时排除污染因素。
阳性结果:在质控系统正常的情况下,待测样品出现预期大小(519bp)的扩增条带,判定PCR结果
为阳性,该菌株是创伤弧菌。
阴性结果:在质控系统正常的情况下,待测样品未出现预期大小(519bp)的扩增条带,判定PCR结
5.7 创伤弧菌分型(选做)
5.7.1 DNA模板的制备
取鉴定结果为创伤弧菌的菌株制备DNA模板,制备方法同5.6.2.1。
5.7.2 PCR扩增
5.7.2.1 引物
PCR分型用引物信息见表5。
5.7.2.2 对照设置
每次PCR反应使用含有目的基因的创伤弧菌标准菌株作为阳性对照,同时,使用除创伤弧菌之外
的其他革兰氏阴性菌标准菌株作为阴性对照,以灭菌去离子水作为空白对照。
同表2。
5.7.2.4 PCR反应程序
预变性:94℃、5min;变性:94℃、1min;退火:55℃(vcg 基因)、58℃ (16SrRNA基因)、64℃
(Bt2基因及SerE基因)1min;延伸:72℃、1min;循环数:30个循环(vcg 基因及16SrRNA基因)、
35个循环(Bt2基因及SerE基因);终延伸:72℃、5min;电泳。若不能及时电泳检测,则将扩增产物于
4℃短期(1d~2d)储存。
5.7.3 电泳
同5.3.3。
5.7.4 结果判定
统正常。否则,任一种对照如果出现非上述正常结果,应重做实验,同时排除污染因素。
在质控系统正常的情况下,如果待测菌株出现97bp大小的条带,则判定该株创伤弧菌为vcgC
型;如果出现199bp大小的条带,则判定该株创伤弧菌为vcgE型;
如果待测菌株出现285bp大小的条带,则判定该株创伤弧菌为16SrRNAA型,如果出现839bp
大小的条带,则判定该株创伤弧菌为16SrRNAB型,如果同时出现285bp大小和839bp大小的两条
带,则判定该株创伤弧菌为16SrRNAA/B型;
如果待测菌株出现665bp大小的条带,则判定该株创伤弧菌为血清E型;如果待测菌株出现
344bp大小的条带,则判定该株创伤弧菌为生物Ⅱ型。
6 结果与报告
GB 4789.44-2020
National food safety standard-Food microbiological examination-Vibrio vulnificus
National Standards of People's Republic of China
National Food Safety Standard
Food microbiology inspection Vibrio vulnificus inspection
2020-09-11 released
2021-03-11 implementation
National Health Commission of the People's Republic of China
Issued by the State Administration for Market Regulation
National Food Safety Standard
Food microbiology inspection Vibrio vulnificus inspection
1 Scope
This standard applies to the inspection of Vibrio vulnificus in fish, shrimp, crab, shellfish and other aquatic products.
2 Equipment and materials
In addition to the routine sterilization and culture equipment in the microbiology laboratory, other equipment and materials are as follows.
2.1 Constant temperature incubator. 36℃±1℃.
2.2 Refrigerator. 2℃~5℃, 7℃~10℃.
2.3 Constant temperature water bath.
2.4 Homogenizer or sterile mortar.
2.5 Balance. Sensitivity 0.1g.
2.6 PCR machine.
2.7 Electrophoresis apparatus or capillary electrophoresis apparatus.
2.8 Gel electrophoresis imaging system or UV detector.
2.9 Biological safety cabinet.
2.10 High-speed centrifuge (maximum speed is at least 15000r/min).
2.11 Vortex oscillator.
2.12 Micro adjustable pipette (range 2.5μL, 10μL, 100μL, 1000μL) and matching tips.
2.13 Precision pH test paper or pH meter.
2.14 Sterile test tube. specifications 18mm×180mm and 15mm×100mm.
2.15 Sterile straws. specifications 1mL (with a 0.01mL scale) and 10mL (with a 0.1mL scale).
2.16 Sterile Erlenmeyer Flask. Capacity 250mL, 500mL and 1000mL.
2.17 Sterile petri dish. 90mm in diameter.
2.18 Sterile surgical scissors, tweezers, forceps, etc.
2.19 PCR reaction tube.
3 Medium and reagents
3.1 Peptone-sodium chloride-cellobiose-polymyxin E (PNCC) enrichment solution. see A.1.
3.2 Cellobiose-polymyxin E (CC) agar medium. see A.2.
3.3 Improved cellobiose-polymyxin B-polymyxin E (mCPC) agar medium. see A.3.
3.4 3% Sodium Chloride Tryptone Soy Agar Medium. See A.4.
3.5 Phosphate buffered saline (PBS). see A.5.
3.6 3% sodium chloride trisaccharide iron agar slope. see A.6.
5 Operation steps
5.1 Sample preparation
5.1.1 The inspection should be completed within 3 hours after the fresh sample is collected. If it cannot be completed within the specified time, the sample should be placed at 7℃~10℃.
Store under the sample (because Vibrio vulnificus is very easy to form a live and uncultivated state at 4°C, so do not put the sample at 4°C), and as much as possible
The inspection can be completed within 24h; frozen samples should be thawed under warm conditions not exceeding 45℃, and the thawing time should not exceed 15min.
5.1.2 Sampling. Take the surface tissues, intestines and gills of fish and cephalopods. Shellfish take the entire contents (including shellfish and body fluids). Crustaceans
Take the whole animal or its central part (including intestines and gills). For shellfish or hard-shelled crustaceans, rinse the shell with running tap water first
And use filter paper to absorb the surface moisture, then aseptically open the shell, take the corresponding part according to the above requirements.
5.1.3 Take 25g of the above-treated sample by aseptic operation, add 225mL of PNCC enrichment solution, and use a rotating blade homogenizer to
Homogenize at 8000r/min for 1 min, or homogenize with a flapping homogenizer for 2 min, and mix thoroughly to prepare a sample homogenate of 1.10.If there is no homogeneity
Then put the sample into a sterile mortar and grind it thoroughly, take 25g of the ground sample and transfer it into a 500mL sterile conical flask, add PNCC
225 mL of the enrichment solution, shake and mix thoroughly, and prepare a sample homogenate of 1.10.
5.2 Bacteria enhancement
Incubate the 1.10 sample homogenate prepared in 5.1.3 at 36°C±1°C for 18h±1h.
5.3 PCR detection
The environmental conditions and process control of PCR experiments should refer to GB/T 27403 "Laboratory Quality Control Regulations for Food Molecular Biology Inspection"
The provisions of "Test" shall be implemented, the same below.
5.3.1 Preparation of DNA template
At a distance of 1cm below the surface of the PNCC enrichment solution, draw 1mL into a 1.5mL EP tube, centrifuge at 9000r/min for 3min,
Discard the supernatant. Add 1mL PBS to the pellet to suspend it and wash it thoroughly, centrifuge at 9000r/min for 3min, and discard the supernatant.
Repeat this step to wash the precipitate 2 to 3 times, discard the last supernatant, add 1 mL sterile ultrapure water, and boil at 100°C for 10 min.
Then centrifuged at 12000r/min for 5min, and the supernatant was used for PCR analysis. If it cannot be analyzed in time, store it at -20℃ for later use.
Commercial DNA extraction kits can also be used to extract and prepare DNA templates according to their instructions.
5.3.2 PCR amplification
5.3.2.1 Primer
The information is shown in Table 1.
5.3.2.2 Control settings
Each PCR reaction uses the standard strain of Vibrio vulnificus as a positive control, and at the same time, uses the standard of Vibrio vulnificus.
Quasi-strains were used as negative control, and sterilized deionized water was used as blank control.
See Table 2.
5.3.2.4 PCR reaction program
Pre-denaturation. 94℃, 5min; Denaturation. 94℃, 1min; Annealing. 62℃, 1min; Extension. 72℃, 1min; Cycle number. 30; Final extension
Stretching. 72℃, 10min; electrophoresis detection. If it cannot be detected immediately, store at 4°C for later use.
5.3.3 Electrophoresis
Gel electrophoresis to detect PCR products. Prepare 1.5% agarose gel with 0.5×TBE buffer (containing EB0.5μg/mL or
DNA dyes such as Goldview 5μL/100mL or Gelred5μL/50mL), take 5μL PCR amplification product and 1μL 6× nucleic acid
After the electrophoresis loading buffer is mixed, spot the sample, and add DNA molecular weight standard (range 100bp~1000bp) to a hole at the same time. According to
The following formula. the distance between the positive and negative electrodes of the electrophoresis tank (cm)×5V/cm calculate and set the voltage, use 0.5×TBE buffer for constant pressure electrophoresis, according to
5.3.4 Results judgment
Quality control system. Negative control and blank control showed no amplified bands, and positive control showed amplified bands of expected size (519bp).
The detection system is normal. Otherwise, if any kind of control has abnormal results as mentioned above, the experiment should be repeated and the pollution factors should be excluded.
Positive result. Under the normal condition of the quality control system, the sample to be tested has an amplified band of the expected size (519bp), and the PCR result is judged
Is positive.
Negative result. When the quality control system is normal, the sample to be tested does not show the expected size (519bp) amplified band, and the PCR result is determined
The fruit is negative.
5.4 Separation
Use a 10μL inoculation loop to dip a loop of enrichment solution 1cm below the surface of the PNCC enrichment solution, and inoculate it in CC and
Round, flat, yellow to orange colonies with transparent or opaque center but transparent edges under light, colony diameter 1mm~2mm, bacteria
A yellow halo may appear (or not appear) around the area.
5.5 fractional culture
Pick at least 5 suspicious Vibrio vulnificus colonies from each of the CC plate and mCPC plate (select all if less than 5) and inoculate them respectively
3% Sodium Chloride Tryptone Soy Agar Plate, cultured at 36℃±1℃ for 18h±1h for subsequent identification. Vibrio vulnificus is chlorinated at 3%
The colony on the Sodium Tryptone Soy Agar plate is round, milky white, moist, raised, and 1mm~2mm in diameter.
5.6 Identification
Vibrio vulnificus can be identified by colony characteristics combined with biochemical characteristics or PCR methods.
5.6.1 Colony characteristics and biochemical characteristics
This step is used for the preliminary identification of Vibrio vulnificus. When performing the following 4 identification experiments, the selected colonies should come from the same single after purification.
Colony.
a) Gram staining microscopic examination. pick out suspected colonies of Vibrio vulnificus from 5.5 3% sodium chloride tryptone soy agar plates for leather
Ran's staining and microscopic examination. Vibrio vulnificus is gram-negative, and the bacteria under the microscope are rod-shaped, arc-shaped, oval-shaped, etc.
Spore.
b) Oxidase test. Use an inoculating loop to pick out the suspected colony of Vibrio vulnificus from 5.5 3% sodium chloride tryptone soy agar plate.
Amount, spread on a white or colorless carrier moistened with oxidase reagent (such as sterile filter paper) or dripped with oxidase reagent (such as glass
sheet). If the smeared part turns purple (occasionally blue-purple) within 10s, it means the oxidase test is positive, and the color does not change as oxidase
The test was negative. Vibrio vulnificus is positive for the oxidase test.
Transfer to the 3% sodium chloride trisaccharide iron agar slope and puncture the bottom layer (be careful not to touch the bottom of the test tube), 36℃±
Observe the results after incubating at 1℃ for 24h. When Vibrio vulnificus grows on the slope of 3% sodium chloride triose iron agar, the bottom of the test tube turns yellow.
Bubbles, the color of the slope does not change to yellow (sometimes the color of the slope turns yellow).
d) Halophilicity test. Use an inoculation needle to pick out suspected colonies of Vibrio vulnificus from 5.5 3% sodium chloride tryptone soy agar plates.
Inoculate respectively in tryptone water containing 0%, 3%, 6%, 8% and 10% sodium chloride, incubate at 36°C±1°C for 24h, observe the solution
The turbidity of the body. Vibrio vulnificus does not grow or grows weakly in tryptone water containing 0%, 8% and 10% sodium chloride.
Peptone water is clear and bright or slightly turbid, while it grows vigorously in tryptone water containing 3% and 6% sodium chloride, tryptone water
turbid.
5.6.1.2 Confirmation experiment
After 18h±1h, the pure culture was inoculated into Lysine Decarboxylase Test Medium and MR-VP Medium Containing 3% Sodium Chloride.
Observe the results after culturing at 36°C±1°C for 24h~48h; another small amount of pure culture was inoculated on the 3% sodium chloride triose iron agar slope,
After incubating at 36℃±1℃ for 18h, it was used for ONPG test. A biochemical identification kit can also be selected for identification.
The biochemical characteristics of Vibrio vulnificus are shown in Table 3, and the identification of Vibrio vulnificus is shown in Table 4.
5.6.2 PCR identification
This part of the test can replace 5.6.1 for the rapid identification of Vibrio vulnificus.
5.6.2.1 Preparation of DNA template
Pick the suspected colony of Vibrio vulnificus from the 3% sodium chloride tryptone soy agar plate in 5.5 and transfer it to 500 μL sterile ultrapure water.
After mixing thoroughly, make a turbid bacterial suspension visible to the naked eye, boil it for 10 minutes, centrifuge at 12000r/min for 5 minutes, and take the supernatant for PCR analysis.
Commercial DNA extraction kits can also be used to extract and prepare DNA templates according to their instructions.
5.6.2.2 PCR amplification and electrophoresis
Same as 5.3.2 and 5.3.3.
5.6.2.3 Judgment of results
Quality control system. Negative control and blank control showed no amplified bands, and positive control showed amplified bands of expected size (519bp).
The detection system is normal. Otherwise, if any kind of control has abnormal results as mentioned above, the experiment should be repeated and the pollution factors should be excluded.
Positive result. Under the normal condition of the quality control system, the sample to be tested has an amplified band of the expected size (519bp), and the PCR result is judged
Is positive, the strain is Vibrio vulnificus.
Negative result. When the quality control system is normal, the sample to be tested does not show the expected size (519bp) amplified band, and the PCR result is determined
5.7 Typing of Vibrio vulnificus (optional)
5.7.1 Preparation of DNA template
The DNA template was prepared from the strain with the identification result of Vibrio vulnificus, and the preparation method was the same as 5.6.2.1.
5.7.2 PCR amplification
5.7.2.1 Primer
See Table 5 for primer information for PCR typing.
5.7.2.2 Control settings
Each PCR reaction uses the standard strain of Vibrio vulnificus containing the target gene as a positive control, and at the same time, uses other than Vibrio vulnificus
Other standard strains of gram-negative bacteria were used as negative controls, and sterilized deionized water was used as a blank control.
Same as Table 2.
5.7.2.4 PCR reaction program
Pre-denaturation. 94℃, 5min; Denaturation. 94℃, 1min; Annealing. 55℃ (vcg gene), 58℃ (16SrRNA gene), 64℃
(Bt2 gene and SerE gene) 1min; extension. 72℃, 1min; cycle number. 30 cycles (vcg gene and 16SrRNA gene),
35 cycles (Bt2 gene and SerE gene); final extension. 72℃, 5min; electrophoresis. If electrophoresis detection cannot be performed in time,
Short-term (1d~2d) storage at 4℃.
5.7.3 Electrophoresis
Same as 5.3.3.
5.7.4 Results judgment
The system is normal. Otherwise, if any kind of control has abnormal results as mentioned above, the experiment should be repeated and the pollution factors should be excluded.
Under the normal condition of the quality control system, if the tested strain has a band with a size of 97bp, it is judged that the strain of Vibrio vulnificus is vcgC
Type; if a band of.199bp size appears, the strain of Vibrio vulnificus is judged to be of type vcgE;
If a band of 285bp size appears in the tested strain, it is determined that the strain of Vibrio vulnificus is of the 16S rRNAA type, if it appears 839bp
The size of the band, the strain of Vibrio vulnificus is determined to be of the 16SrRNAB type, if two 285bp size and 839bp size appear at the same time
Belt, it is determined that the strain of Vibrio vulnificus is 16SrRNAA/B type;
If the test strain has a 665bp band, it is judged that the strain of Vibrio vulnificus is serotype E; if the test strain appears
A band with a size of 344bp indicates that the strain of Vibrio vulnificus is biological type II.
6 Results and reports
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