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食品安全国家标准 食品微生物学检验 诺如病毒检验
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GB 4789.42-2016
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标准编号: GB 4789.42-2016 (GB4789.42-2016)
中文名称: 食品安全国家标准 食品微生物学检验 诺如病毒检验
英文名称: Detection of norovirus in shellfish -- Conventional RT-PCR and real-time RT-PCR
行业: 国家标准
中标分类: X09
字数估计: 15,118
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): SN/T 1635-2005
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
发布机构: 中华人民共和国国家卫生和计划生育委员会、国家食品药品监督管理总局
GB 4789.42-2016: 食品安全国家标准 食品微生物学检验 诺如病毒检验
GB 4789.42-2016 英文名称: Detection of norovirus in shellfish -- Conventional RT-PCR and real-time RT-PCR
1 范围
本标准规定了食品中诺如病毒(Norovirus)的实时荧光RT-PCR检测方法。
本标准适用于贝类,生食蔬菜,胡萝卜、瓜、坚果等硬质表面食品,草莓、西红柿、葡萄等软质水果等
食品中诺如病毒核酸的检测。
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 实时荧光PCR仪。
2.2 冷冻离心机。
2.3 无菌刀片或等效均质器。
2.4 涡旋仪。
2.5 天平:感量为0.1g。
2.6 振荡器。
2.7 水浴锅。
2.8 离心机。
2.9 高压灭菌锅。
2.10 低温冰箱:-80℃。
2.11 微量移液器。
2.12 pH计或精密pH试纸。
2.13 网状过滤袋:400mL。
2.14 无菌棉拭子。
2.15 无菌贝类剥刀。
2.16 橡胶垫。
2.17 无菌剪刀。
2.18 无菌钳子。
2.19 无菌培养皿。
2.20 无RNase玻璃容器:见E.1.2。
2.21 无RNase离心管、无RNase移液器吸嘴、无RNase药匙、无RNasePCR薄壁管:见E.1.3。
3 试剂
除有特殊说明外,所有实验用试剂均为分析纯;实验用水均为无RNase超纯水:见E.2.1。
3.1 GⅠ、GⅡ基因型诺如病毒的引物、探针:见A.1。
3.2 过程控制病毒的引物、探针:见A.1。
3.3 过程控制病毒:制备见附录C。
3.4 外加扩增控制RNA:制备见附录D。
3.5 Tris/甘氨酸/牛肉膏(TGB E)缓冲液:见E.2.2。
3.6 5×PEG/NaCl溶液(500g/L聚乙二醇PEG8000,1.5mol/LNaCl):见E.2.3。
3.7 磷酸盐缓冲液(PBS):见E.2.4。
3.8 氯仿/正丁醇的混合液:见E.2.5。
3.9 蛋白酶K溶液:见E.2.6。
3.10 75%乙醇:见E.2.7。
3.11 Trizol试剂:见E.2.8。
4 检验程序
诺如病毒检验程序见图1。
5 操作步骤
5.1 病毒提取
注:样品处理一般应在4℃以下的环境中进行运输。实验室接到样品后应尽快进行检测,如果暂时不能检测应将样品保存在-80℃冰箱中,试验前解冻。样品处理和PCR反应应在单独的工作区域或房间进行。每个样品可设置2~3个平行处理。
5.1.1 软质水果和生食蔬菜
5.1.1.1 将25g软质水果或生食蔬菜切成约2.5cm×2.5cm×2.5cm的小块(如水果或蔬菜小于该体
积,可不切)。
5.1.1.2 将样品小块移至带有400mL网状过滤袋的样品袋,加入40mLTGB E溶液(软质水果样品,
需加入30UA.niger果胶酶,或1140UA.aculeatus果胶酶),加入10μL过程控制病毒。
5.1.1.3 室温,60次/min,振荡20min。酸性软质水果需在振荡过程中,每隔10min检测pH,如pH
低于9.0时,使用1mol/LNaOH调pH至9.5,每调整一次pH,延长振荡时间10min。
5.1.1.4 将振荡液转移至离心管,如体积较大,可使用2根离心管。10000r/min,4℃,离心30min。
取上清至干净试管或三角瓶,用1mol/LHCl调pH至7.0。
5.1.1.5 加入0.25倍体积5×PEG/NaCl溶液,使终溶液浓度为100g/LPEG,0.3mol/LNaCl。60s
摇匀,4℃,60次/min,振荡60min。10000r/min,4℃,离心30min,弃上清。10000r/min,4℃,离
心5min紧实沉淀,弃上清。
5.1.1.6 500μLPBS悬浮沉淀。如食品样品为生食蔬菜,可直接将悬浮液转移至干净试管,测定并记
录悬浮液毫升数,用于后续RNA提取。如食品样品为软质水果,将悬浮液转移至耐氯仿试管中。加入
500μL氯仿/丁醇混合液,涡旋混匀,室温静置5min。10000r/min,4℃,离心15min,将液相部分仔
细转移至干净试管,测定并记录悬浮液毫升数,用于后续RNA提取。
5.1.2 硬质表面食品
5.1.2.1 将无菌棉拭子使用PBS湿润后,用力擦拭食品表面(< 100cm2)。记录擦拭面积。将10μL
过程控制病毒添加至该棉拭子。
5.1.2.2 将棉拭子浸入含490μLPBS试管中,紧贴试管一侧挤压出液体。如此重复浸入和挤压3~4次,
确保挤压出最大量的病毒,测定并记录液体毫升数,用于后续RNA提取。硬质食品表面过于粗糙,可
能会损坏棉拭子,可使用多个棉拭子。
5.1.3 贝类
5.1.3.1 戴上防护手套,使用无菌贝类剥刀打开至少10个贝类。
5.1.3.2 使用无菌剪刀、手术钳或其他等效器具在胶垫上解剖出贝类软体组织中的消化腺,置于干净培
养皿中。收集2.0g。
5.1.3.3 使用无菌刀片或等效均质器将消化腺匀浆后,转移至离心管。加入10μL过程控制病毒。加
入2.0mL蛋白酶K溶液,混匀。
5.1.3.4 使用恒温摇床或等效装置,37℃,320次/min,振荡60min。
5.1.3.5 将试管放入水浴或等效装置,60℃,15min。室温,3000r/min,5min离心,将上清液转移至
干净试管,测定并记录上清液mL数,用于后续RNA提取。
5.2 病毒RNA提取和纯化
注:病毒RNA可手工提取和纯化,也可使用商品化病毒RNA提取纯化试剂盒。提取完成后,为延长RNA保存时间可选择性加入RNase抑制剂。操作过程中应佩戴一次性橡胶或乳胶手套,并经常更换。提取出来的RNA立即进行反应,或保存在4℃小于8h。如果长期储存建议-80℃保存。
将病毒提取液加入离心管,加入病毒提取液等体积Trizol试剂,混匀,激烈振荡,室温放置5min,加入
0.2倍体积氯仿,涡旋剧烈混匀30s(不能过于强烈,以免产生乳化层,也可用手颠倒混匀),12000r/min,离
心5min,上层水相移入新离心管中,不能吸出中间层。
5.2.2 病毒RNA提取
离心管中加入等体积异丙醇,颠倒混匀,室温放置5min,12000r/min,离心5min,弃上清,倒置于
吸水纸上,沾干液体(不同样品须在吸水纸不同地方沾干)。
5.2.3 病毒RNA纯化
5.2.3.1 每次加入等体积75%乙醇,颠倒洗涤RNA沉淀2次。
5.2.3.2 于4℃,12000r/min,离心10min,小心弃上清,倒置于吸水纸上,沾干液体(不同样品须在吸
到有沉淀,室温干燥3min,不能过于干燥,以免RNA不溶。
5.2.3.3 加入16μL无RNase超纯水,轻轻混匀,溶解管壁上的RNA,2000r/min,离心5s,冰上保存
备用。
5.3 质量控制
5.3.1 空白对照
以无RNase超纯水作为空白对照(A反应孔)。
5.3.2 阴性对照
以不含有诺如病毒的贝类,提取RNA,作为阴性对照(B反应孔)。
5.3.3 阳性对照
5.3.4 过程控制病毒
5.3.4.1 以食品中过程控制病毒RNA的提取效率表示食品中诺如病毒RNA的提取效率,作为病毒提
取过程控制。
5.3.4.2 将过程控制病毒按5.2步骤提取和纯化RNA。可大量提取,分装为10μL过程控制病毒的
RNA量,-80℃保存,每次检测时取出使用。
5.3.4.3 将10μL过程控制病毒的RNA进行数次10倍梯度稀释(D~G反应孔),加入过程控制病毒
引物、探针,采用与诺如病毒实时荧光RT-PCR反应相同的反应条件确定未稀释和梯度稀释过程病毒
RNA的Ct值。
5.3.4.4 以未稀释和梯度稀释过程控制病毒RNA的浓度lg值为X 轴,以其Ct值为Y 轴,建立标准曲
10-1、10-2、10-3等。
5.3.4.5 将含过程控制病毒食品样品RNA(C反应孔),加入过程控制病毒引物、探针,采用诺如病毒实
时荧光RT-PCR反应相同的反应体系和参数,进行实时荧光RT-PCR反应,确定Ct值,代入标准曲线,
计算经过病毒提取等步骤后的过程控制病毒RNA浓度。
5.3.4.6 计算提取效率,提取效率=经病毒提取等步骤后的过程控制病毒 RNA 浓度×100%,即
(C反应孔)Ct值对应浓度×100%。
5.3.5 外加扩增控制
5.3.5.1 通过外加扩增控制RNA,计算扩增抑制指数,作为扩增控制。
5.3.5.2 外加扩增控制RNA分别加入含过程控制病毒食品样品RNA(H反应孔)、10-1稀释的含过程
录C反应体系和参数,进行实时荧光RT-PCR反应,确定Ct值。
5.3.5.3 计算扩增抑制指数,抑制指数=(含过程控制病毒食品样品RNA+外加扩增控制RNA)Ct值
-(无RNase超纯水+外加扩增控制RNA)Ct值,即抑制指数=(H反应孔)Ct值-(J反应孔)Ct值。
如抑制指数≥2.00,需比较10倍稀释食品样品的抑制指数,即抑制指数=(I反应孔)Ct值-(J反应孔)
Ct值。
5.4 实时荧光RT-PCR
实时荧光RT-PCR反应体系和反应参数详见附录B。反应体系中各试剂的量可根据具体情况或不
同的反应总体积进行适当调整。可采用商业化实时荧光RT-PCR试剂盒。也可增加调整反应孔,实现
一次反应完成GⅠ和GⅡ型诺如病毒的独立检测。将18.5μL实时荧光RT-PCR反应体系添加至反应
GB 4789.42-2016
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Food microbiological examination –
Examination of norovirus
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the PRC;
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 Scope ... 4
2 Equipment and materials ... 4
3 Reagent ... 5
4 Testing procedures ... 6
5 Operating procedures ... 6
6 Results and reports ... 12
Appendix A Real-time fluorescent RT-PCR primers and probes ... 14
Appendix B Real-time fluorescent RT-PCR reaction system and parameters 15
Appendix C Process control virus culture and primers, probes ... 16
Appendix D Preparation of externally added amplification control RNA ) ... 18
Appendix E RNase removal and RNase-free solution preparation ... 20
Foreword
This standard replaces SN/T 1635-2005 “Detection of norovirus in shellfish -
Conventional RT-PCR and real-time RT-PCR”.
As compared with SN/T 1635-2005, the main changes of this standard are as
follows.
- CHANGE the standard name changed into “National food safety standard
- Food microbiological examination - Examination of norovirus”;
- EXTEND the standard test range from “shellfish” to “food”;
- MODIFY the “operational procedures”;
- ADD the “quality control requirements”, as shown in Appendix C;
- DELETE the “normal RT-PCR method”.
National food safety standard -
Food microbiological examination –
Examination of norovirus
1 Scope
This standard specifies real-time fluorescent RT-PCR detection method of
norovirus in food.
This standard applies to the norovirus nucleic acid detection in hard-surfaced
foods such as shellfish, raw vegetables, carrots, melons, nuts and so on AND
such soft foods as the strawberries, tomatoes, grapes and so on.
2 Equipment and materials
In addition to routine sterilization and culture equipment for microbiological
laboratories, other equipment and materials are as follows.
2.1 Real-time fluorescence PCR instrument.
2.2 Freeze centrifuge.
2.3 Sterile blade or equivalent homogenizer.
2.4 Scrollers.
2.5 Balance. sensitivity of 0.1 g.
2.6 Oscillator.
2.7 Water bath.
2.8 Centrifuge.
2.9 Autoclave pot.
2.10 Low temperature refrigerator. -80 °C.
2.11 Micro-pipettes.
2.12 pH meter or precision pH test paper.
5.1.1.2 MOVE the sample piece into a sample bag with a 400 mL mesh filter
bag; ADD 40 mL of TGBE solution (for soft fruit sample, it is needed to add 30
U of A.niger pectinase or 1140 U of A.aculeatus pectinase); ADD 10 μL of
process control virus.
5.1.1.3 At room temperature, MAKE it subject to oscillation for 20 min at the
speed of 60 times/min. As for the acid soft fruit, it shall detect the pH value at
the interval of 10 min during oscillation; if the pH is less than 9.0, USE the 1
mol/L NaOH to adjust the pH to 9.5; EXTEND the oscillation duration for 10
min every time the pH is adjusted.
volume is large, it may use two centrifuge tubes. CONTRIFUGE it at 4 °C for
30 min at the speed of 10000 r/min. TAKE the supernatant; PLACE it into a
clean test tube or conical flask; USE 1 mol/L HCl to adjust the pH to 7.0.
5.1.1.5 ADD 0.25 times volumes of 5 x PEG/NaCl solution so that the final
solution concentration is 100 g/L PEG and 0.3 mol/L NaCl. SHAKE it uniformly
within 60 s; MAKE it subject to oscillation at 4 °C for 60 min at the speed of 60
times/min. CENTRIFUGE it at 4 °C for 30 min at the speed of 10000 r/min;
DISCARD the supernatant; CENTRIFUGE it at 4 °C for 5 min at the speed of
10000 r/min to solidify the precipitation; DISCARD the supernatant.
vegetable, the suspension can be transferred directly to a clean test tube, and
the millimeters of suspension is determined and recorded, for subsequent RNA
extraction. If the food sample is soft fruit, the suspension is transferred into a
chloroform-resistant test tube. ADD 500 μL of chloroform/butanol mixed
solution; MIX it uniformly in vortex; LET it stand at room temperature for 5 min.
CENTRIFUGE it at 4 °C for 15 min at the speed of 10000 r/min; carefully
TRANSFER the liquid phase part into a clean test tube; DETERMINE the
RECORD the millimeters of suspension, for the subsequent RNA extraction.
5.1.2 Hard surfaced food
cm2) with force; RECORD the wipe area. ADD 10 μL of the process control
virus to this swab.
5.1.2.2 IMMERSE the cotton swab in a 490 μL PBS test tube; PRESS it onto
the tube wall to extrude the solution out. REPEAT the immersion and extrusion
for 3 ~ 4 times, to ensure extruding out the maximum amount of virus;
DETERMINE and RECORD the millimeters of solution, for the subsequent
RNA extraction. Since the hard-surfaced food surface may be too rough AND it
may damage the cotton swab, so it may use several cotton swabs.
ADD the same volume of isopropyl alcohol into the centrifuge tube; MAKE it
CENTRIFUGE it for 5 min at the speed of 12000 r/min; DISCARD the
supernatant; PLACE it upside down onto the absorbent paper to make it dry
(different samples must be dried at different positions of the absorbent paper).
5.2.3 Virus RNA purification
5.2.3.1 ADD the equal volume of 75% ethanol each time; MAKE it upside down
to rinse the RNA precipitation twice.
5.2.3.2 CENTRIFUGE it at 4 °C for 10 min at the speed of 12000 r/min;
carefully DISCARD the supernatant; PLACE it upside down on the absorbent
paper to make it dry (different samples must be dried at different positions of
micro-sampler to make it dry; USE one absorption tip for one sample; DO not
let the absorption tip touch the precipitation; DRY it at room temperature for 3
min; AND it shall not be over-dried to avoid RNA insoluble.
5.2.3.3 ADD 16 μL of RNase-free ultrapure water; MIX it uniformly and gently;
DISSOLVE the RNA on the tube wall; CENTRIFUGE it for 5 s at the speed of
2000 r/min; PRESERVE it on ice to prepare for use.
5.3 Quality control
5.3.1 Blank control
USE the RNase-free ultrapure water as a blank control (A reaction hole).
EXTRACT the RNA from the shellfish containing no norovirus as a negative
control (B reaction hole).
5.3.3 Positive control
USE the externally added amplification control RNA as a positive control (J
reaction hole).
5.3.4 Process control viruses
5.3.4.1 USE the extraction efficiency of the process control virus RNA in the
food to indicate the extraction efficiency of the norovirus RNA in the food, as
the virus extraction process control.
accordance with the procedures in 5.2. It may extract it in large amount;
added amplification control RNA) Ct value, that is, the inhibition index = (H
reaction hole) Ct value - (J reaction hole) Ct value. If the inhibition index is ≥
2.00, it shall compare the inhibition index of the 10-fold diluted food sample,
that is, the inhibition index = (I reaction hole) Ct value - (J reaction hole) Ct
value.
5.4 Real-time fluorescent RT-PCR
Real-time RT-PCR reaction system and reaction parameters are described in
Appendix B. The amount of each reagent in the reaction system can be
total reaction volume. A commercially available real-time fluorescent RT-PCR
kit can be used. It can also increase and adjust the reaction hole to realize the
independent detection of the GI type and GII type norovirus in one time. After
adding the 18.5 μL of real-time fluorescent RT-PCR reaction system into the
reaction hole, ADD the following substances into different reaction holes to
detect the GI or GII genotype norovirus.
A reaction hole. blank control, ADD 5 µL of RNase-free ultrapure water + 1.5
µL of GI or GII type primer probe;
B reaction hole. negative control, ADD 5 µL of negative extraction control RNA
C reaction hole. virus extraction process control 1, ADD 5 μL of process control
virus contained food sample RNA + 1.5 μL of process control virus primer
probe;
D reaction hole. virus extraction process control 2, ADD 5 μL of process control
virus RNA + 1.5 μL of process control virus primer probe;
E reaction hole. virus extraction process control 3, ADD 5 μL of 10-1 fold
dilution process control virus RNA + 1.5 μL of process control virus primer
probe;
F reaction hole. virus extraction process control 4, ADD 5μL of 10-2 fold dilution
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