CN106521031A - Method for detecting norovirus in food based on negatively charged membrane concentration - Google Patents
Method for detecting norovirus in food based on negatively charged membrane concentration Download PDFInfo
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Abstract
The invention discloses a method for detecting norovirus in a food based on negatively charged membrane concentration. The method comprises the following steps: immersing a sample to be detected in an eluant, carrying out oscillation elution at room temperature, and centrifuging the above obtained solution to obtain a supernatant; adjusting the pH value of the supernatant to 2-4, and carrying out suction filtration by using a negatively charged filter membrane with the aperture of 0.45 [mu]m to obtain suction-filtered filter paper; and shearing the suction-filtered filter paper, extracting RNA, carrying out a real-time quantitative PCR to obtain the Ct value of the sample to be detected, and finally obtaining the content of the norovirus in the sample to be detected according to a norovirus NoV GI standard curve and a norovirus NoV GII standard curve. Compared with existing local standard (DBS13/001-2015), the method has the advantages of time saving, high efficiency, and increase of the virus enrichment rate by 5-10%.
Description
(1) technical field
The present invention relates to norovirus detection method in a kind of food, and in particular to three kinds of food norovirus wash-outs and suction
Attached method, while and using real-time fluorescence quantitative PCR detection concentrating virus amount (PCRU/mL), to improve disease in detection food
The sensitivity of poison pollution.
(2) background technology
In recent years, the frequent generation of global food origin disease and pernicious food pollution event, makes food security become one
World health problem that is individual serious and constantly expanding, and it is increasingly becoming the focus of global concern.With International Trade, the world
The rapid growth of tourist industry, the globalization of food products market have caused the high risk of food, and food origin disease is had in the whole world
The trend for spreading.As the No.1 problem of food security, caused by virus more than half in the generation of food origin disease.Promise
If viral (noroviruse, NoV) is to cause one of most common food-borne virus of acute human gastroenteritis in world wide, should
Virus is main
Route of transmission is wanted to eat contaminated food, water and life contact, wherein it is important to eat contaminated food
One of approach.
As developed country is increasingly deep with research to the growing interest of norovirus, recent year is to norovirus
Research have started to pay attention to.Norovirus (Norovirus, NoV) belong to the Cécile Nowak of Caliciviridae (Caliciviruses)
Tobamovirus, is to be found with the method for Electronic Speculum by scholars such as Kapikian first for 1972.Norovirus genome molecules size
For 7.3-7.6Kb, generally according to NV viral rna polymerases code area nucleotides or the difference of coat protein region amino acid sequence,
Can be 5 different genotype (GGI, GGII, GGIII, GGIV and GGV) by NV point, wherein GGI and GGII is common causing a disease
Type.Norovirus resistance to external world is stronger, can generally cause the alimentary infection disease of self limiting, light moderate, be characterized in
High incidence, low pathogenic dosage.The master that norovirus are presently believed to be world-wide prevalence, non-bacterial gastroenteritis is broken out
Want reason.The ground such as in November, 2006, Japan, Singapore, Italy there occurs the norovirus collection body-sensing relevant with food in succession
Less than two month to dates, dye event, particularly Japan, there occurs that 35.76 ten thousand people have infected norovirus.China was reported from nineteen ninety-five
After accusing the first norovirus cases of infection, norovirus infectivity acute gastroenteritis (epidemic situation master is repeatedly broken out in domestic many areas
Guangdong to be concentrated on and Zhejiang Province).Norovirus have been increasingly becoming important public health problem.
In daily life, there is the distribution of various virus on the surface or inside of many foods.For a long time, by
The factor such as low, detection technique backwardness of the content of virus in food, people fail to obtain due heavy for viral contaminated food
Depending on.Breaking out for food-borne promise such as disease is most relevant with edible contaminated water and food.In relevant food most at present
The research of poisonous and harmful substance focuses primarily upon the neurotoxin that heavy metal, pathogenic bacteria, the residual beast of agriculture be residual and its food itself is produced
Deng the research to the especially norovirus detection technique of the food-borne virus with which as carrier is also few.China and big in the world
The detection of virus in food of the majority state in addition to shellfish does not have more ripe technical standard perform.Therefore, compel
It is essential and will develops in effective shellfish the method for quick and tracing technology of virus, ensures food safety and common people's health.
The virus detection procedure of food mainly includes sample pretreatment, elution of virus, viral concentration, nucleic acid extraction, PCR inspections
Survey etc..In view of food composition is complicated, various PCR inhibitors may be rich in, while in practical operation, food samples amount is sick greatly
Malicious content is low, therefore elution of virus and concentration are the committed steps of norovirus detection in food.Elution of virus and concentration are exactly
Virion is eluted from foodstuff surface by appropriate buffer solution for a kind of method for further concentrating.Wash-out solution
Acid-base value is generally very big to the wash-out influential effect of virion.In most of the cases, with the alkaline buffer of pH9 to 10.5
Liquid is viral to elute food surface.Because alkaline environment can be such that virion separates from food substrate.However, in acyclic acidic
In border, virion is attached to the ability of food surface and can then be strengthened.So sour environment can affect the effect of elution of virus
Power, so as to the sensitivity for reducing detecting.As in many foods (especially fruit or vegetable type food), acid ingredient content is higher, so
Alkaline buffer based on Tris- is usually used to be eluted.In addition, the use of other kinds of elution buffer
Also have been reported that.As sodium bicarbonate buffer liquid is used for the acquisition of the poliovirus in berry and cold vegetable dish in sauce.Phosphoric acid buffer
Liquid (pH7.6) is used for wash-out of hepatitis A virus in romaine lettuce and fresh strawberry etc..After elution of virus, can in concentration step
Using method it is then very many, they include polyethylene glycol (PEG) precipitate, ultracentrifugation, ultrafiltration, immuno-concentration and filter membrane absorption etc.
Method.Polyethylene glycol (PEG) precipitation method, ultracentrifugation, ultrafiltration are the more methods of viral concentration employing in current food,
But they equally exist time-consuming, high cost and the shortcomings of operation has high demands.Filter membrane absorption was usually used in virus in water body in the past
Concentration, also have document report in the wash-out and concentration process to food after appropriate process in recent years, using filter membrane
The method of absorption can also reach preferable concentrated effect, while the method also has and saves time, efficiently and detection sensitivity height etc. is excellent
Point.
Due to virus in the food content of pollution often relatively low, and viremic individuals less (diameter is about 50-300nm), it is difficult
In direct detection, conventional nucleic acid PCR and median tissue cell infection amount (TCID50) are that virus predominantly detects method in food.
With the development of Protocols in Molecular Biology, for conventional nucleic acid PCR, emerging fluorescent quantitative PCR technique makes to fall ill
The advantages of whole detection process of poison is provided with real-time detection, quantitatively accurate, sensitivity is high and reproducible.At present, due to promise
As virus can't be cultivated completely in laboratory conditions, fluorescent quantitative PCR technique has become the main of norovirus
Detection method.Developed country from shellfish food develops into other food to norovirus detection research using the technology, and I
State starts late to norovirus detection technique research, and current fragmentary report is also only limitted in the shellfishes such as oyster.Fluorescent quantitation
The introducing of round pcr, will further enhance scope, high efficiency and the sensitivity of norovirus detection in food.
In sum, although the current existing not understatement of method in the world with regard to norovirus concentration detection in different food products
Road, but due in various method for concentration material it is different with the physicochemical property of material, absorption-elution requirement, operating process, cause not
Also differ greatly with method for concentration result, various countries do not have comparativity yet.Therefore, in food norovirus it is whole concentrated
Journey is optimized, and improves the thickening efficiency of virus, China set up it is a kind of to food in norovirus efficiently, quickly concentration and
Detection method reduces norovirus infection for ensuring food safety, and ensures people's life health, and the inspection to norovirus
Survey and control with important theory and practice meaning.
(3) content of the invention
It is an object of the present invention to provide it is a kind of efficiently, efficiently in food norovirus method for concentration, it is with instant food
The norovirus (NoV GI and NoV GII) polluted in product are object, utilize easier method for concentration, realize instant food
The concentration of norovirus in product, be in instant food norovirus efficiently quickly detection and food-borne diarrhoea case are traced back
Source provides technical support.
The technical solution used in the present invention is：
The present invention provides a kind of method for concentrating norovirus in detection food based on negatively charged membrane, and methods described is：(1)
Elution of virus：Testing sample is soaked with eluent, shaken at room temperature wash-out (preferred 10-30min) is centrifuged (preferred 8000-
10000rpm is centrifuged 15-30min), obtain supernatant；The testing sample is veterinary antibiotics, salad or cooked meat, if treating
Test sample product are fruit, then addition pectase and cellulase in eluent；The eluent is one of following：Eluent BE,
Eluent ALK, eluent SPE or eluent GE；The eluent BE is consisted of：Trishydroxymethylaminomethane 12.11g/L is sweet
Propylhomoserin 3.75g/L, beef extract 10-30g/L, solvent are ddH2O, pH=9.5；The eluent GE is consisted of：Glycine
3.75g/L, NaCl 8.775g/L, solvent are ddH2O, pH=9.5；The eluent ALK is consisted of：KH2PO46.8g/L,
NaCl 58.5g/L, Trinton X-100 1mL/L, solvent is ddH2O, pH=9.2；The eluent SPE is consisted of：
NaHCO384g/L, soybean protein 10-20g/L, solvent are ddH2O, pH=9.3；(2) viral concentration：Take supernatant and adjust pH value
To 2～4, aperture is adopted for 0.45 μm of negative electrical charge filter membrane suction filtration (preferred Milliflex PLUS vavuum pump S-Pak, 0.45 μ m
47mm, Millipore, Germany), obtain the filter paper after suction filtration；(3) virus concentration detection：Filter paper after suction filtration is shredded,
Extracting RNA carries out real-time fluorescence quantitative PCR reaction, obtains testing sample Ct values, according to norovirus NoV GI calibration curves with
Norovirus NoV GII calibration curves, it is final to obtain norovirus content in testing sample；The norovirus NoV GI standards
Curve is with 1.27～1.27 × 105PCRU/mL norovirus standard concentration is ordinate, with the Ct of real-time fluorescence quantitative PCR
It is worth and is made for abscissa, the norovirus NoV GII calibration curves is with 0.86～0.86 × 108PCRU/mL promises such as disease
Malicious NoV GII standard concentrations are ordinate, are made with the Ct values of real-time fluorescence quantitative PCR as abscissa.
Further, when step (1) testing sample is fresh vegetables or fruit, the eluent is eluent BE；Institute
State eluent BE to consist of：Trishydroxymethylaminomethane (Tris) 12.11g/L, glycine (Glycine) 3.75g/L, beef leaching
Cream (Beef extract) 10g/L, solvent is ddH2O, pH=9.5；When the testing sample is salad, the eluent is to wash
De- liquid GE, the eluent GE are consisted of：Glycine 3.75g, NaCl 8.775g, ddH2O 1L, pH=9.5.
Further, the pectase addition is calculated as 1-18U/mL with effluent volume, the cellulase addition with
Effluent volume is calculated as 1-5.6U/mL.
Further, when the testing sample is salad, the supernatant that shaken at room temperature wash-out is obtained is with volume ratio 2：1 addition examination
Agent Vertrel XF, oscillation extraction (preferably vibrate 1-5min, stand 15min), and collecting water is mutually used for viral concentration.
Further, step (1) testing sample is cooked meat, and the eluent is eluent ALK or eluent
SPE；The eluent SPE is consisted of：NaHCO384g/L, soybean protein 10g/L, solvent are ddH2O, pH=9.3.
Further, the testing sample is cooked meat, if the supernatant after eluent vibration wash-out is floated containing grease-like
Float, then by supernatant volume ratio 1:(supernatant is 3 with mixeding liquid volume ratio for 1 chloroform and n-butanol mixed liquor:1) extract
Take, collecting water is mutually used for viral concentration.
Further, step (2) supernatant adjusts pH value to 3-3.5.
Further, after step (2) suction filtration, the secondary suction filtration of PBS for adding 0.1mol/L, pH=3.5 (should
The RNA inhibitor that step is adsorbed in being effectively reduced filter membrane), obtain the filter paper after suction filtration.
Further, AVL lysates (Qiagen, Germany) are added after step (3) filter paper is shredded, and (step is saved
Secondary wash-out and concentration process after conventional film concentrating virus), acutely vibration 5-8min or high-speed striking on turbula shaker
After 2-3min, RNA is extracted.
Enrichment facility of the present invention includes Milliflex PLUS vavuum pumps, aseptic filtration funnel and filter membrane, and wherein filter membrane is put
It is placed at the filter holder of Milliflex PLUS vavuum pumps (an equal amount of filter paper under filter membrane, can be padded), it is fixed well
After filter support and funnel, activate switch can be filtered.
The each eluent Jing autoclave sterilizations of the present invention process (115 DEG C, 15min), need not carry out centrifugation and go the removal of impurity, can
4 DEG C are deposited within one week, preserving also can be frozen in -20 DEG C such as to need long-term (3-6 month).
Norovirus standard curve making method of the present invention is：1 part of NoV GI standard items is taken, by QIAamp Viral
RNA Mini Kit specifications extract RNA, based on One step RT-PCR technology (primer and probe are shown in Table 1), by standard sample
This RNA carries out 1:As detection template after 10 gradient dilutions, by judging the PCRU using lowest detection dilution factor as virus,
And according to virus stock solution used difference dilution factor (i.e. virus concentration 1.27～1.27 × 105PCRU/mL) corresponding PCRU (i.e. Ct
Value) between relation set up NoV GI calibration curves；Same method makes the calibration curve of NoV GII standard items.
The food collection container used by all steps, viral concentration sterilizing containers sterilization treatment in the present invention.
Compared with prior art, beneficial effect of the present invention is mainly reflected in：
(1) technology of virus in alkaline eluant and negatively charged membrane reserve ration, alkaline eluant wash-out virus are employed
Absorption of the virus to food substrate is effectively reduced, negatively charged membrane concentration can reduce PCR inhibitor during Viral extraction, prominent behaviour
The advantages of making letter, fast separating rate, favorable repeatability, low cost and not needing Large expensive instrument to be adapted to field operation.
(2) pectase and cellulase is added to substantially increase virus during fruit-vegetable food (instant) wash-out of the present invention is processed
Elution efficiency；(supernatant by a certain percentage in cooked meat (instant) eluent supernatant：Mixed liquor=3：1) add chloroform-just
Butanol (chloroform：N-butanol=1:1) mixed liquor, can effectively reduce the compositions such as lipid and albumen, improve the detection spirit of virus
Sensitivity.Pectase and cellulase are added in the process of salad food (instant) wash-out, (filter by a certain percentage in filtrate after wash-out
Liquid：Vertrel XF=2：1) cleaning agent Vertrel XF are added, makes elution of virus rate and verification and measurement ratio all increase.
(3) present invention is 0.45 μm of aperture negative electrical charge S-PAK filter membranes, film surface and disease using the miillpore filter of concentrating virus
Malicious Percentage bound is high, is easy to the concentration absorption of virus, volume concentration to greatly enhance.Aperture is moderate simultaneously, can be with filtering food
PCR suppresses composition, is easy to nucleic acid extraction, improves the sensitivity of detection.
(4) the negative electrical charge filter membrane viral adsorption that this invention is adopted, is directly added into AVL lysates and virus is extracted on filter membrane
Nucleic acid, eliminates the secondary concentration for adding the secondary wash-out of eluent virus and adding flocculant precipitate virus.
(5), based on above eluent and the advantage of method for concentration, norovirus NoV GII are respectively in romaine lettuce, indigo plant for the present invention
Detection in the certain kind of berries, salad and ham is limited to 6.5,65,650 and 650PCRU/15g, average thickening efficiency is up to 69.54,22.43,
13.64 with 7.76%；And norovirus NoV GI detection limits respectively in romaine lettuce, blueberry, salad and meat instantaneity food
For 7.8,78,780 and 780PCRU/15g, average thickening efficiency is up to 57.23,28.35,11.87% and 4.56%.The present invention
Not only save time compared with existing provincial standard (DBS13/001-2015), efficiently；And viral concentration rate can also improve 5-10%.
(4) illustrate
The calibration curve of Fig. 1 norovirus (NoV GI and GII) concentration and Ct values, A are norovirus GII type；B be promise such as
Viral GI types.
Viral extraction flow chart in the instant food of Fig. 2.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：Norovirus concentration and the foundation of detection method in romaine lettuce sample
Before virus in concentration romaine lettuce sample, first to the disinfection (autoclave sterilization such as viral concentration container
Process).
1. using the norovirus in BE elution romaine lettuce samples
Every part of 15g packing of fresh vegetables (romaine lettuce).140 μ L norovirus positive excrement supernatant, point are added in every part of vegetables
Shape coats (5 μ l are per point) in dish leaf, is placed in 30min in two stage biological safety cabinet.After air-drying, dish leaf is shredded, load 50ml
In centrifuge tube, 30ml eluent BE (tris 12.11g, glycine 3.75g, beef extract 10g, ddH are added2O
1L, pH=9.5), vortex oscillation 30min under room temperature, 10000rpm centrifugation 15min, supernatant are transferred to another 50ml centrifuge tube
In.
2. using norovirus in negatively charged membrane concentrate eluant
Supernatant is adjusted with the hydrochloric acid of 1mol/L makes pH=3.5, and the supernatant after adjustment is moved in ultrafiltration cup, using 0.45 μm
(S-Pak, 0.45 μ m 47mm, Millipore, Germany, is purchased from moral to the Milliflex PLUS vavuum pumps of negative electrical charge filter membrane
MILLIPORE companies of state) negative pressure leaching, again toward ultrafiltration cup add 500mL PBS (0.1mol/L, pH=3.5) buffer solution
Filter membrane (step can reach the purpose for removing RNA inhibitor in Partial Food) is run through, filter membrane is taken out, after shredding, is put into
In the EP pipes of 1.5ml, next step nucleic acid extraction is treated.
3. the detection of norovirus copy number
3.1.RNA extract
Norovirus RNA on negative electrical charge filter membrane is extracted using QIAamp Viral RNA Mini Kit kits, first
560 μ L AVL lysates (Qiagen, Germany) are added in the EP pipes equipped with filter membrane fragment, is put in oscillator after mixing
Acutely vibration 30min, takes supernatant into another EP pipe after 8000 × g centrifugations, and remaining steps carry out RNA's according to its specification
Extract, extract the viral RNA for finishing and save backup in -20 DEG C.
3.2. one-step method quantitative fluorescent PCR
NoV GI (JJV1F/JJV1R, JJV1P) and NoV GII (JJV2F/JJV2R, JJV2P) primers and probe are by Shanghai
Sheng Gong biotech firms synthesize (table 1).Viral RNA template adopts One Step PrimescriptTMRT-PCR Kit(TaKaRa)
Reverse transcription amplification (RT-PCR) is carried out, reaction is carried out on quantitative real time PCR Instrument (ABI 7500Real Time, American AB I),
And to result fluoroscopic examination.Amplification system (25 μ L)：2 × buffer, 12.5 μ L, reverse transcriptase and each 0.5 μ L of Taq enzyme, it is upper and lower
Trip primer (20 μm of ol/L) each 0.6 μ L, probe (20 μm of ol/L) 0.3 μ L, 5 μ L of RNA templates, ddH2O 5μL.Amplification condition：42
DEG C reverse transcription reaction 30min；95 DEG C of denaturations 2min；95 DEG C of denaturation 5s, 55 DEG C of annealing, extension 35s, 39 circulations.
3.3. make calibration curve
Draw calibration curve：1 part of NoV GI standard items and NoV GII standard items are taken respectively, by QIAamp Viral RNA
Mini Kit specifications extract RNA, based on One step RT-PCR technology (primer and probe are shown in Table 1), by master sample
RNA carries out 1:As detection template after 10 gradient dilutions, by judging the PCRU using lowest detection dilution factor as virus, and root
According to virus stock solution used difference dilution factor (i.e. NoV GI virus concentrations 1.27～1.27 × 105PCRU/mL, NoV GII virus concentrations
0.86～0.86 × 108PCRU/mL) relation between corresponding PCRU (i.e. Ct values) sets up calibration curve (Fig. 1), NoV
GI calibration curve Y=-0.44X+14.46, NoV GII calibration curve Y=-0.35X+14.47.
3.4. the amount (PCRU/mL) of fluorescence quantitative PCR detection concentrating virus
Experiment above repeats three parts, and during sample detection, every part of sample detects initial viral stoste, eluent and concentration simultaneously
Liquid.During every part of sample detection comprising 1 part of negative control (sample is not added with during nucleic acid extraction, only plus elution buffer), 1 part it is blank
Control is (with ddH in real-time fluorescence RT-PCR system2O replaces RNA).Ct values<40 are judged to detect NoV GI and NoV GII.
The calculating of detection limit：1 part of NoV GI and NoV GII standard items is taken respectively, by 1:10 carry out gradient series dilution
(100、10-1、10-2、10-3、10-4、10-5... .) after, respectively take 140 μ l and be applied on 15g Chinese leafs, each of the above dilution factor sample
Product repeat six parts, and per part elutes according to the method described above, concentrates and detect virus, with six parts of detection samples head in each dilution factor
Secondary when there is being all feminine gender, the viral level corresponding to a upper dilution factor is detection limit of this method in romaine lettuce.
4. the calculating of norovirus wash-out and thickening efficiency
Formula is as follows：
5. conclusion
According to the result of quantitative fluorescent PCR, positive control be initially added in romaine lettuce sample norovirus NoV GII and
The amount of NoV GI is 6.5 × 10 respectively10PCRU/15g and 7.8 × 108Promise such as disease after PCRU/15g, romaine lettuce sample elution and concentration
The result of calculation of the eluting rate, enrichment factor and detection limit of poison concentrates promise in romaine lettuce sample using the method as shown in table 2 and table 3
Eluting rate, enrichment factor and detection limit such as virus N oV GII is respectively 74.75%, 69.54% and 6.5 PCRU/15g；NoV
GI eluting rates, enrichment factor and detection limit are respectively 73.32%, 57.23% and 7.8 PCRU/15g.
Embodiment 2：Norovirus concentration and the foundation of detection method in blueberry sample
Before virus in concentration blueberry sample, first to the disinfection (autoclave sterilization such as viral concentration container
Process).
1st, using the norovirus in BE elution blueberry samples
Every part of 15g packing of fruit (blueberry).Add 140 μ l norovirus NoV GI and NoV GII positive in every part of blueberry
Excrement supernatant, point-like coat blueberry surface (5 μ l are per point), are placed in 30-60min in two stage biological safety cabinet.After air-drying, dress
Enter in 50ml centrifuge tubes, addition 30ml eluent BE (Tris 12.11g, Glycine 3.75g, Beef extract 20g,
ddH2O 1L, pH=9.5), add pectase (30000U/g, 0.02g/mL) and cellulase (10000U/g, 0.01g/mL)
Each 150 μ l, vortex oscillation 30min under room temperature, 10000rpm centrifugation 15min, supernatant are transferred in another 50ml centrifuge tube.
2nd, using norovirus in negatively charged membrane concentrate eluant
Supernatant is adjusted with the hydrochloric acid of 1mol/L makes pH=3.5, and the supernatant after adjustment is moved in ultrafiltration cup, adopt aperture for
The Milliflex PLUS vavuum pump negative pressure leachings of 0.45 μm of negative electrical charge filter membrane, again toward ultrafiltration cup add 500mL PBS
(0.1mol/L, pH=3.5) buffer solution is run through filter membrane, and (step can reach the mesh for removing RNA inhibitor in Partial Food
), filter membrane is taken out, after shredding, is put in the EP pipes of 1.5ml, is treated next step nucleic acid extraction.
3rd, the detection of norovirus copy number
3.1.RNA extract
Norovirus RNA on negative electrical charge filter membrane is extracted using QIAamp Viral RNA Mini Kit kits, first
560 μ l AVL lysates are added in the EP pipes equipped with filter membrane fragment, during oscillator is put into after mixing, 30min is acutely vibrated,
Supernatant is taken into another EP pipe after 8000 × g centrifugations, remaining steps carry out the extraction of RNA according to its specification, and extraction is finished
Viral RNA save backup in -20 DEG C.
3.2. one-step method quantitative fluorescent PCR
NoV GI and NoV GII primers and probe synthesize (table 1) by Shanghai Sheng Gong biotech firms.Viral RNA template is adopted
One Step PrimescriptTMRT-PCR Kit (TaKaRa) carry out reverse transcription amplification (RT-PCR), react in fluorescent quantitation
Carry out in PCR instrument (ABI 7500Real Time, American AB I), and to result fluoroscopic examination.Amplification system (25 μ L)：2×
12.5 μ L of buffer, reverse transcriptase and each 0.5 μ L of Taq enzyme, upstream and downstream primer (20 μm of ol/L) each 0.6 μ L, probe (20 μ
Mol/L) 0.3 μ L, 5 μ L of RNA templates, ddH2O 5μL.Amplification condition：42 DEG C of reverse transcription reaction 30min；95 DEG C of denaturations 2min；
95 DEG C of denaturation 5s, 55 DEG C of annealing, extension 35s, 39 circulations.
3.3. make calibration curve
With embodiment 1.
3.4. the amount (PCRU/mL) of fluorescence quantitative PCR detection concentrating virus
With embodiment 1.
6. the calculating of norovirus wash-out and thickening efficiency
With embodiment 1.
7. conclusion
According to the result of quantitative fluorescent PCR, positive control be initially added in blueberry sample norovirus NoV GII and
The amount of NoV GI is 6.5 × 10 respectively10PCRU/15g and 7.8 × 108Promise such as disease after PCRU/15g, blueberry sample elution and concentration
The result of calculation of the eluting rate, enrichment factor and detection limit of poison concentrates promise in blueberry sample using the method as shown in table 2 and table 3
Eluting rate, enrichment factor and detection limit such as virus N oV GII is respectively 55.23%, 22.43% and 65 PCRU/15g；NoV GI
Eluting rate, enrichment factor and detection limit are respectively 43.65%, 18.35% and 78 PCRU/15g.
Embodiment 3：Norovirus concentration and the foundation of detection method in ham sample
In concentrated ham sample before virus, first to the disinfection (autoclave sterilization such as viral concentration container
Process).
1st, using the norovirus in BE elution ham samples
Every part of 15g packing of the instantaneity prepared food such as meat (ham).Add in every portion of ham 140 μ L norovirus NoV GI and
NoV GII positive excrement supernatants, point-like are coated surface of ham (5 μ l are per point), are placed in 30-60min in two stage biological safety cabinet.
After air-drying, ham is shredded, load in 50ml centrifuge tubes, add 30ml eluent ALK (KH2PO46.8g, NaCl 58.5g,
Trinton X-100 1mL, ddH2O 1L, pH=9.2), eluent pH value is adjusted between 9.2-9.5, under room temperature, whirlpool shakes
30min is swung, 10000rpm centrifugation 15min, supernatant are transferred in another 50ml centrifuge tube, such as find that grease-like material is floated on
Supernatant, can (supernatant in specific proportions：Mixed liquor=3:1, v/v) add chloroform-n-butanol mixed liquor (volume ratio 1:1), vibrate
Fetch water after centrifugation mutually standby.
2nd, using norovirus in negatively charged membrane concentrate eluant
Supernatant or water are adjusted with the hydrochloric acid of 1mol/L mutually makes pH=3.5, the supernatant after adjustment is moved in ultrafiltration cup, is adopted
Aperture is the Milliflex PLUS vavuum pump negative pressure leachings of 0.45 μm of negative electrical charge filter membrane, adds 500mL again toward ultrafiltration cup
PBS (0.1mol/L, pH=3.5) buffer solution is run through filter membrane, and (step can reach and remove RNA inhibitor in Partial Food
Purpose), filter membrane is taken out, after shredding, is put in the EP pipes of 1.5mL, is treated next step nucleic acid extraction.
3rd, the detection of norovirus copy number
3.1.RNA extract
Norovirus RNA on negative electrical charge filter membrane is extracted using QIAamp Viral RNA Mini Kit kits, first
560 μ L AVL lysates are added in the EP pipes equipped with filter membrane fragment, during oscillator is put into after mixing, 30min is acutely vibrated,
Supernatant is taken after 8000g centrifugations into another EP pipe, remaining steps carry out the extraction of RNA according to its specification, what extraction was finished
Viral RNA is saved backup in -20 DEG C.
3.2. one-step method quantitative fluorescent PCR
NoV GI and NoV GII primers and probe synthesize (table 1) by Shanghai Sheng Gong biotech firms.Viral RNA template is adopted
One Step PrimescriptTMRT-PCR Kit (TaKaRa) carry out reverse transcription amplification (RT-PCR), react in fluorescent quantitation
Carry out in PCR instrument (ABI 7500Real Time, American AB I), and to result fluoroscopic examination.Amplification system (25 μ L)：2×
12.5 μ L of buffer, reverse transcriptase and each 0.5 μ L of Taq enzyme, upstream and downstream primer (20 μm of ol/L) each 0.6 μ L, probe (20 μ
Mol/L) 0.3 μ L, 5 μ L of RNA templates, ddH2O 5μL.Amplification condition：42 DEG C of reverse transcription reaction 30min；95 DEG C of denaturations 2min；
95 DEG C of denaturation 5s, 55 DEG C of annealing, extension 35s, 39 circulations.
3.3. make calibration curve
With embodiment 1.
3.4. the copy number of fluorescence quantitative PCR detection concentrating virus
With embodiment 1.
4th, the calculating of norovirus wash-out and thickening efficiency
With embodiment 1.
5th, conclusion
According to the result of quantitative fluorescent PCR, positive control be initially added in ham sample norovirus NoV GII and
The amount of NoV GI is 6.5 × 10 respectively10PCRU/15g and 7.8 × 108Promise such as disease after PCRU/15g, ham sample elution and concentration
Poison eluting rate, enrichment factor and detection limit result of calculation as shown in table 2 and table 3, using promise in the method concentrated ham sample
Eluting rate, enrichment factor and detection limit such as virus N oV GII is respectively 34.61%, 13.64% and 650 PCRU/15g；NoV
GI eluting rates, enrichment factor and detection limit are respectively 39.77%, 11.88% and 780 PCRU/15g.
Embodiment 4：Norovirus concentration and the foundation of detection method in salad sample
Before virus in concentration salad sample, first to the disinfection (autoclave sterilization such as viral concentration container
Process).
1st, using the norovirus in BE elution salad samples
Salad prepares (tomato by a certain percentage by cucumber, tomato, carrot and mayonnaise：Cucumber：Carrot：Salad
Sauce=1：1：1：2, w/w).Every part of 15g packing.140 μ L norovirus NoV GI and NoV GII positive excrement is added in every portion of salad
Just supernatant, point-like are coated salad surface (5 μ l are per point), are placed in 30-60min in two stage biological safety cabinet.After air-drying, shred
Mix, load in 50ml centrifuge tubes, add 30ml eluent GE (Glycine 3.75g, NaCl 8.775g, ddH2O 1L, pH
=9.5), vortex oscillation 15min under room temperature, 10000rpm centrifugation 5min, supernatant are transferred in another 50ml centrifuge tube, by one
Certainty ratio (filtrate：Vertrel XF=2：1, volume ratio) add reagent Vertrel XF (HFC, Dupont Chemical, purchase
In Shenzhen Eubo New Material Technology Co., Ltd.), after vibration 1-5min, stand 15min water intakings mutually standby.
2nd, using norovirus in negatively charged membrane concentrate eluant
Water is adjusted with the hydrochloric acid of 1mol/L mutually makes pH=3.5, and the supernatant after adjustment is moved in ultrafiltration cup, adopt aperture for
The Milliflex PLUS vavuum pump negative pressure leachings of 0.45 μm of negative electrical charge filter membrane, again toward filter tunnel add 500mL PBS
(0.1mol/L, pH=3.5) buffer solution is run through filter membrane, and (step can reach the mesh for removing RNA inhibitor in Partial Food
), filter membrane is taken out, after shredding, is put in the EP pipes of 1.5mL, is treated next step nucleic acid extraction.
3. the detection of norovirus copy number
3.1.RNA extract
Norovirus RNA on negative electrical charge filter membrane is extracted using QIAamp Viral RNA Mini Kit kits, first
560 μ L AVL lysates are added in the EP pipes equipped with filter membrane fragment, during oscillator is put into after mixing, 10min is acutely vibrated,
Supernatant is taken into another EP pipe after 8000 × g centrifugation 30s, remaining steps carry out the extraction of RNA according to its specification, have extracted
Complete viral RNA is saved backup in -20 DEG C.
3.2. one-step method quantitative fluorescent PCR
NoV GI and NoV GII primers and probe synthesize (table 1) by Shanghai Sheng Gong biotech firms.Viral RNA template is adopted
One Step PrimescriptTMRT-PCR Kit (TaKaRa) carry out reverse transcription amplification (RT-PCR), react in fluorescent quantitation
Carry out in PCR instrument (ABI 7500Real Time, American AB I), and to result fluoroscopic examination.Amplification system (25 μ L)：2×
12.5 μ L of buffer, reverse transcriptase and each 0.5 μ L of Taq enzyme, upstream and downstream primer (20 μm of ol/L) each 0.6 μ L, probe (20 μ
Mol/L) 0.3 μ L, 5 μ L of RNA templates, ddH2O 5μL.Amplification condition：42 DEG C of reverse transcription reaction 30min；95 DEG C of denaturations 2min；
95 DEG C of denaturation 5s, 55 DEG C of annealing, extension 35s, 39 circulations.
3.3. make calibration curve
With embodiment 1.
3.4. the copy number of fluorescence quantitative PCR detection concentrating virus
With embodiment 1.
4th, the calculating of norovirus wash-out and thickening efficiency
With embodiment 1.
5th, conclusion
According to the result of quantitative fluorescent PCR, positive control is initially added norovirus NoVGII and NoV in romaine lettuce sample
The amount of GI is 6.5 × 10 respectively10With 7.8 × 108PCRU/15g, romaine lettuce sample elution and concentration after norovirus eluting rate,
The result of calculation of enrichment factor and detection limit concentrates norovirus NoV in romaine lettuce sample using the method as shown in table 2 and table 3
The eluting rate of GII, enrichment factor and detection limit are respectively 28.64%, 7.76% and 650PCRU/15g；NoV GI eluting rates, concentration
Rate and detection limit are respectively 18.94%, 4.56% and 780PCRU/15g.
1 norovirus of table (NoV GI and GII) specific primer and probe
R=A or G；Y=C or T.
2 four kinds of elution buffer formula of liquid of table
The eluting rate and the rate of recovery of norovirus (NoV GI and GII) in 3 four kinds of instant food of table
4 negatively charged membrane absorption method of table is to norovirus in four kinds of instant food (NoV GI and GII) detection limit
Above content is to further illustrating that the present invention is done with reference to specific preferred embodiment, it is impossible to assert this
It is bright to be embodied as being confined to these explanations.For general technical staff of the technical field of the invention, do not taking off
On the premise of present inventive concept, its steel structure can be flexible and changeable, can be with subseries product.Simply make and simply pushing away
Replacement living is drilled, the scope of patent protection that the present invention is determined by the claims submitted to should be all considered as belonging to.
Claims (10)
1. a kind of method that norovirus in detection food are concentrated based on negatively charged membrane, it is characterised in that methods described is：(1) disease
Poison wash-out：Testing sample is soaked with eluent, shaken at room temperature wash-out, centrifugation obtain supernatant；The testing sample is fresh
Veterinary antibiotics, salad or cooked meat, if testing sample is fruit, add pectase and cellulase in eluent；Institute
It is one of following to state eluent：Eluent BE, eluent ALK, eluent SPE or eluent GE；The eluent BE is consisted of：
Trishydroxymethylaminomethane 12.11g/L, glycine 3.75g/L, beef extract 10-30g/L, solvent are ddH2O, pH=9.5；
The eluent GE is consisted of：Glycine 3.75g/L, NaCl 8.775g/L, solvent is ddH2O, pH=9.5；The eluent
ALK is consisted of：KH2PO46.8g/L, NaCl 58.5g/L, Trinton X-1001mL/L, solvent is ddH2O, pH=9.2；Institute
State eluent SPE to consist of：NaHCO384g/L, soybean protein 10-20g/L, solvent are ddH2O, pH=9.3；(2) virus is dense
Contracting：Take supernatant and pH value is adjusted to 2～4, adopt aperture for 0.45 μm of negative electrical charge filter membrane suction filtration, obtain the filter paper after suction filtration；(3)
Virus concentration is detected：Filter paper after suction filtration is shredded, extracting RNA carries out real-time fluorescence quantitative PCR reaction, obtains testing sample Ct
Value, according to norovirus NoV GI calibration curves and norovirus NoV GII calibration curves, it is final obtain testing sample in promise such as
Viral level；The norovirus NoV GI calibration curves are with 1.27～1.27 × 105PCRU/mL norovirus NoV GI standards
Product concentration is ordinate, is made with the Ct values of real-time fluorescence quantitative PCR as abscissa, the norovirus NoV GII marks
Directrix curve is with 0.86～0.86 × 108PCRU/mL norovirus NoV GII standard concentrations are ordinate, fixed with real-time fluorescence
The Ct values of amount PCR are made for abscissa.
2. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 1, it is characterised in that step
(1), when the testing sample is fresh vegetables or fruit, the eluent is eluent BE；The eluent BE is consisted of：Three
Hydroxymethyl aminomethane 12.11g/L, glycine 3.75g/L, beef extract 10g/L, solvent are ddH2O, pH=9.5.
3. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 1, it is characterised in that step
(1), when the testing sample is salad, the eluent is eluent GE, and the eluent GE consists of：Glycine 3.75g,
NaCl 8.775g, ddH2O 1L, pH=9.5.
4. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 1, it is characterised in that described
Pectase addition is calculated as 1-18U/mL with effluent volume, and the cellulase addition is calculated as 1- with effluent volume
5.6U/mL。
5. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 4, it is characterised in that described
When testing sample is salad, the supernatant that shaken at room temperature wash-out is obtained is with volume ratio 2：1 addition reagent Vertrel XF, vibration extraction
Take, collecting water is mutually used for viral concentration.
6. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 1, it is characterised in that step
(1) testing sample is cooked meat, and the eluent is eluent ALK or eluent SPE；The eluent SPE compositions
For：NaHCO384g/L, soybean protein 10g/L, solvent are ddH2O, pH=9.3.
7. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 6, it is characterised in that described
If the supernatant after eluent vibration wash-out contains grease-like floating thing, by supernatant volume ratio 1:1 chloroform and positive fourth
Alcohol mixed liquor is extracted, and collecting water is mutually used for viral concentration.
8. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 1, it is characterised in that step
(2) supernatant adjusts pH value to 3-3.5.
9. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 1, it is characterised in that step
(2) the secondary suction filtration of PBS of 0.1mol/L, pH=3.5 after the suction filtration, is added, the filter paper after suction filtration is obtained.
10. the method for norovirus in detection food being concentrated based on negatively charged membrane as claimed in claim 1, it is characterised in that step
(3) AVL lysates are added after the filter paper is shredded, 5-8min is acutely vibrated on turbula shaker, RNA is extracted.
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