CN109735664A - The detection method of norovirus in a kind of water - Google Patents
The detection method of norovirus in a kind of water Download PDFInfo
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- CN109735664A CN109735664A CN201910216955.3A CN201910216955A CN109735664A CN 109735664 A CN109735664 A CN 109735664A CN 201910216955 A CN201910216955 A CN 201910216955A CN 109735664 A CN109735664 A CN 109735664A
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Abstract
The invention discloses a kind of detection methods of norovirus in water, comprising the following steps: (1) enrichment of water sample processing and norovirus；(2) extraction of norovirus RNA, the viral nucleic acid solution purified；(3) RT-PCR reaction solution is configured；(4) fluorescence quantitative PCR instrument detects；The program of the fluorescence quantitative PCR instrument detection are as follows: 50 DEG C of 30min；95℃5min；95 DEG C of 15s, 60 DEG C of 30s acquire signal；65 DEG C of 30s are recycled 45 times, and fluorescence channel detects and selects the channel FAM, VIC.The detection method of norovirus, is detected using fluorescence quantitative PCR instrument in water of the invention, and sensitivity and accuracy are higher, and can quantitative analysis.
Description
Technical field
The present invention relates to the detection method of Inactivation of Enteric Viruses In Water, in particular to the detection method of norovirus in a kind of water.
Background technique
Norovirus belongs to mankind's Caliciviridae, norovirus category, and norovirus genome is single-stranded positive RNA, base
Because of group overall length about 7kb~7.5kb.Norovirus can be divided into I~G of G, V 5 genotype altogether, wherein mainly I type of G of infection people
With II type of G.Norovirus infectious diarrhea has prevalence in worldwide, and whole year can infect, and infects object master
If adult and school-ager, cold season presents high-incidence.The U.S. is every year in all Non-bacterial diarrheas are broken out, 60-
90% is caused by norovirus.Also there are similar results in the developed countries such as Holland, Britain, Japan, Australia.In China 5
Year old or less in diarrhea children, norovirus recall rate is 15% or so, and antibody level of serum investigation shows that promise is such as in Chinese population
The infection of virus is also very universal.
Currently, the domestic detection report for norovirus in water is less, although norovirus is more stable in water
It can not rise in value, cause to be difficult to detect, so, develop the method for norovirus in accurate, the stable detection water sample of one kind especially
It is important.
Summary of the invention
The present invention provides a kind of detection method of norovirus in water, this method can be carried out the norovirus in water
Accurately, quantitative detection.
In order to solve the above technical problems, the present invention provides technical solutions below: the detection of norovirus in a kind of water
Method, comprising the following steps:
(1) enrichment of water sample processing and norovirus；
(2) extraction of norovirus RNA, the viral nucleic acid solution purified；
(3) RT-PCR reaction solution is configured；
(4) fluorescence quantitative PCR instrument detects；
The program of the fluorescence quantitative PCR instrument detection are as follows: 50 DEG C of 30min；95℃5min；95 DEG C of 15s, 60 DEG C of 30s acquisitions
Signal；65 DEG C of 30s are recycled 45 times；Fluorescence channel detects and selects the channel FAM, VIC.
Further, the water sample handles the step of enrichment with norovirus are as follows: MgCl is added in water sampling2Solution makes
Its concentration reaches 0.03~0.05mol/L, and adjusting water sample makes pH to 3.0~5.0, and water sample is made to pass through mixed nitrate cellulose membrane,
With beef extract-glycine elution liquid concussion elution filter membrane；Filter membrane is removed, eluent pH to 6.0~8.0 is adjusted, into eluent
PEG8000 solution is added to stand overnight, centrifugation retains precipitating；The ultrapure water without RNA enzyme is added and dissolves precipitating, centrifuged supernatant
For viral crude extract.
Further, the extraction step of the norovirus RNA are as follows: viral crude extract is taken, lysate is added, it is sufficiently mixed
Even, then for several times, eluent, the viral nucleic acid solution purified after centrifugation is added in rinsing liquid rinsing.
Further, the MgCl2The concentration of solution is 2.5mol/L.
Further, the beef extract-glycine elution liquid configuration method are as follows: by 30.0g beef extract and the sweet ammonia of 3.75g
Sour stirring and dissolving adjusts pH to 9.5, is settled to 1000mL with ultrapure water in ultrapure water, dispenses, 121 DEG C of high pressure sterilizations
15min。
Further, the water sample was handled in the step of enrichment with norovirus, the body of eluent and PEG8000 solution
Product is than being 1:0.25.
Further, the configuration method of the PEG8000 solution are as follows: stir PEG8000 400.0g and sodium chloride 87.0g
It mixes and is dissolved in ultrapure water, be settled to 1000mL with ultrapure water, dispense, 121 DEG C of high pressure sterilization 15min.
Further, the volume that lysate is added in the viral crude extract is 600 μ L.
Compared with prior art, the detection method of norovirus has the beneficial effect that in water of the invention
The detection method of norovirus, is detected using fluorescence quantitative PCR instrument in water of the invention, sensitivity and accurate
Degree is higher, and can quantitative analysis.
Detailed description of the invention
Specific embodiment in reference to the accompanying drawing is described in further detail technical solution of the present invention, but not structure
At any limitation of the invention.
Fig. 1 is the RNA canonical plotting of Viral extraction process control in the embodiment of the present invention.
Specific embodiment
Embodiment 1
The detection method of norovirus in water, comprising the following steps:
(1) enrichment of water sample processing and norovirus
2.5mol/L MgCl is added in water sampling2Solution makes its concentration reach 0.03~0.05mol/L, with 1mol/L hydrochloric acid
Adjusting water sample makes pH to 3.0~5.0, and vacuum filtration is used to be run through aperture for 0.22 μm of mixed nitrate cellulose membrane, so
Afterwards with 10mL beef extract-glycine elution liquid concussion elution 20~30min of filter membrane；Filter membrane is removed, is washed with the adjusting of 1mol/L hydrochloric acid
De- liquid pH to 6.0~8.0, the PEG8000 solution of 0.25 times of volume is added into eluent, and the centrifuge tube that turns upside down is with sufficiently mixed
It is even.Centrifuge tube and sample liquid are stood overnight at 4 DEG C together.Then at 4 DEG C, 10000g is centrifuged 5min, discards supernatant, and it is heavy to retain
It forms sediment.Then 200 ultrapure waters of the μ L without RNA enzyme are added, 60 DEG C of heating 5min sufficiently dissolve precipitating.4 DEG C, 10000g is centrifuged 3min,
Supernatant is taken, virus is obtained and slightly mentions product.It separately takes a water sample that 10 μ L process control viruses are added, shakes up, operated with method, as process
Control 1.
(2) extraction of norovirus RNA:
Illustrate according to Viral nucleic acid extraction reagent box (being purchased from Jiangsu Shuoshi Biological Technology Co., Ltd.): 200 μ L being taken to contain
The liquid of virus is added 600 μ L lysates, mixes well, be placed at room temperature for 5 minutes, during which overturns pipe and helps to crack for several times.Add
Enter 600 μ L buffers into cracked sample solution, mixes well.650 μ L mixed liquors are shifted to being cased with the affine of adapter
In column, 13000rpm is centrifuged 30 seconds, discards filtered fluid, repeats this operation until having filtered remaining liquid.500 μ L rinsing liquids are added
Into affinity column, 13000rpm is centrifuged 30 seconds, discards filtered fluid, and it is primary to repeat this step.Affinity column is nested into again
In the centrifuge tube of RNase-free, 50 μ L eluents are added in the centre of the column bottom Xiang Qinhe adsorbed film, are placed at room temperature for 2 points
Clock, 13000rpm are centrifuged the viral nucleic acid solution of harvest purifying in 2 minutes.
(3) RT-PCR reaction solution is configured
Illustrate that norovirus is prepared in operation according to norovirus (GI/GII) kit for detecting nucleic acid (RT-PCR sonde method)
GI/GII reaction system.Illustrate that MS2 reaction system is prepared in operation according to MS2 process control kit (RT-PCR sonde method).Separately
20 μ L MS2 process control are taken, are cooled to room temperature on ice after 95 DEG C of heating 5min, it is dilute to carry out gradient according to the ratio of 1:9
It releases, controls standard curve for manufacturing process.Wherein, norovirus (GI/GII) kit for detecting nucleic acid is raw purchased from the large generation in Jiangsu
Object Science and Technology Ltd., MS2 process control kit are purchased from Beijing Longrun Biological Technology Co., Ltd..
It is controlled according to the method setting up procedure control of table 1 with additional amplification.It is calculated according to reaction system, upper press proof quality control number
Each reagent institute expense is as shown in Table 3 and Table 4.
Table 1
Reaction solution, every 20 μ L of pipe are dispensed into PCR pipe；Corresponding reaction template, reaction solution and template are added in each pipe
It mixes, carries out PCR reaction immediately.
(4) fluorescence quantitative PCR instrument detects
It is detected using LightCycler96 type fluorescence quantitative PCR instrument.Response procedures are as follows: 50 DEG C of 30min；95℃
5min；95 DEG C of 15s, 60 DEG C of 30s acquire signal；65 DEG C of 30s are recycled 45 times.Fluorescence channel detects and selects: the channel FAM, VIC.
Test example
1. the configuration of solution
(1) PEG8000 solution: by 400.0g PEG8000,87.0g sodium chloride, for stirring and dissolving in ultrapure water, use is ultrapure
Water is settled to 1000mL, packing, and 121 DEG C of high pressure sterilization 15min are spare.
(2)2.5mol/L MgCl2Solution: by 238.0g magnesium chloride (MgCl2) stirring and dissolving in ultrapure water, uses ultrapure water
It is settled to 1000mL, is dispensed, 121 DEG C of high pressure sterilization 15min are spare.
(3) beef extract-glycine elution liquid (containing 3% beef extract, 0.05mol/L glycine, pH 9.5)
By 30.0g beef extract and 3.75g glycine stirring and dissolving in ultrapure water, pH to 9.5 is adjusted, with ultrapure water constant volume
To 1000mL, packing, 121 DEG C of high pressure sterilization 15min are spare.
2. sample treatment and virus enrichment
Two bottles of water sampling, every bottle of 500mL is separately added into the 2.5mol/L MgCl of 10mL2Solution reaches its concentration
0.03~0.05mol/L.Then adjusting water sample with 1mol/L hydrochloric acid makes pH to 3.0~5.0, wherein one bottle of 10 μ L of addition is excessively program-controlled
System virus, shakes up, as process control 1.
It uses vacuum filtration to be run through aperture as 0.22 μm of mixed nitrate cellulose membrane, then uses 10mL beef extract-
Glycine elution liquid concussion elution 20~30min of filter membrane；Remove filter membrane, with 1mol/L hydrochloric acid adjust eluent pH to 6.0~
8.0, the PEG8000 solution of 0.25 times of volume is added into eluent, turns upside down centrifuge tube to mix well.By centrifuge tube and
Sample liquid is stood overnight at 4 DEG C together.Then at 4 DEG C, 10000g is centrifuged 5min, discards supernatant, and retains precipitating.Then it is added
200 μ L dissolve precipitating without the ultrapure water of RNA enzyme, and 60 DEG C of heating 5min sufficiently dissolve precipitating.4 DEG C, 10000g is centrifuged 3min, takes
Clear liquid (V1) obtains virus and slightly mentions product.
The extraction of 3.RNA
It takes 200 μ L (V2) containing virulent liquid according to Viral nucleic acid extraction reagent box explanation, is cracked.600 μ are added
L nucleic acid precipitating reagent is mixed well into cracked sample solution.650 μ L mixed liquors are shifted to the affinity column for being cased with adapter
Interior, 13000rpm is centrifuged 30 seconds, discards filtered fluid, repeats this operation until having filtered remaining liquid.It is slow that 500 μ L Tris are added
It rushes in salting liquid to affinity column, 13000rpm is centrifuged 30 seconds, discards filtered fluid, and it is primary to repeat this step.Affinity column is covered again
Enter into the centrifuge tube of RNase-free, the centre of the column bottom Xiang Qinhe adsorbed film is added 50 μ L (V3) and goes to RNA enzyme water, room
Temperature is placed 2 minutes, and the 2 minutes viral nucleic acid solution purified is centrifuged under the revolving speed of 13000rpm.
4. configuring RT-PCR reaction solution
4.1 by norovirus (GI/GII) kit for detecting nucleic acid (RT-PCR sonde method) and MS2 process control kit
(RT-PCR sonde method) is taken out from -20 DEG C of refrigerators, is placed on ice chest and thaws.Oscillation mixes the several seconds after defrosting, and 3000 leave calculation
Second.
4.2 take 20 μ L MS2 process control, are cooled to room temperature on ice after 95 DEG C of heating 5min.According to the ratio of 1:9
Gradient dilution is carried out, does 3 dilution gradients, altogether 4 concentration, for manufacturing process control standard curve (process control 2-5)
It is controlled according to the method setting up procedure control of table 2 with additional amplification.
Table 2
It is as shown in Table 3 and Table 4 according to reaction system, upper press proof quality control number calculating each reagent institute expense.
3 norovirus GI/GII reaction system of table
| Reaction solution title | Additional amount (μ L)/every reaction |
| Nov premixed liquid (no MS2) | 17.5 |
| Enzyme mixation | 2.5 |
| Total volume | 20 |
4 MS2 reaction system of table
| Reaction solution title | Additional amount (μ L)/every reaction |
| MS2 premixed liquid | 17.5 |
| Enzyme mixation | 2.5 |
| Total volume | 20 |
Reagent is sequentially added into clean centrifuge tube according to sequence described above, after vortex oscillation mixes, is put on ice chest for use,
Entire configuration process need to operate on ice chest.It takes out clean eight union of PCR to be fixed on PCR pipe box, by the reaction on ice chest
After the of short duration centrifugation of liquid, lid is gently opened, dispenses reaction solution into PCR pipe, every 20 μ L of pipe avoids bubble in pipe as far as possible.Each
Corresponding reaction template is added in pipe, adding template sequence is negative control → measuring samples → amplification control → process control → sun
Property control, avoid bubble to exist as far as possible in pipe, cover pipe lid.PCR pipe box is gently beaten, reaction solution and template are mixed, and with
The revolving speed of 3000rpm is centrifuged the several seconds, carries out PCR reaction immediately.
5. fluorescence quantitative PCR detection
It is detected using LightCycler96 type fluorescence quantitative PCR instrument.Response procedures are as follows: 50 DEG C of 30min；95℃
5min；95 DEG C of 15s, 60 DEG C of 30s acquire signal；65 DEG C of 30s are recycled 45 times.Fluorescence channel detects and selects: the channel FAM, VIC.
6. experimental data
The PCR detection data of each process is as shown in table 5.
5 PCR detection data of table
6.1 standard curve
Using the RNA concentration lg value of Viral extraction process control 2-5 as X-axis, standard curve is established using its Ct value as Y-axis, is marked
It is as shown in Figure 1 to infuse curve.Obtained standard curve is y=-3.657x+22.282, r2=0.9994, it is more than or equal to 0.98.Not
It is 1 that dilution, which controls viral RNA concentration, and gradient dilution process control RNA concentration is respectively 10-1、10-2、10-3。
6.2 extraction efficiency
The Ct value of Viral extraction process control 1 is substituted into standard curve, calculates the process after Viral extraction
Control viral RNA concentration.
Extraction efficiency calculation formula: extraction efficiency
Calculate gained: process control 1, E=12% meets process control requirements.
6.3. inhibition index
RNA is controlled by additional amplification, amplification inhibition index is calculated, is controlled as amplification.
Inhibition index=(amplification control 1) Ct value-(amplification control 3) Ct value.Such as inhibition index >=2.00,10 times need to be compared
Dilute the inhibition index of food samples, i.e. inhibition index=(amplification control 2) Ct value-(amplification control 3) Ct value.
Gained: inhibition index=25.04-25.22=-0.18 is calculated, meets amplification control and requires.
Wherein, full it need to be enough lower quality control requirement, detection side is effective.Blank control is negative, and negative control is negative, sun
Property control it is positive.
Process control needs to meet: extraction efficiency>=1%, such as extraction efficiency<1%, need to detect again, but as extraction efficiency<
1%, testing result is the positive, can also take the circumstances into consideration to be determined as the positive.
Amplification control needs to meet: inhibition index < 2.00；Such as inhibition index >=2.00,10 times of dilution food samples need to be compared
Inhibition index；Such as inhibition index < 2.00 of 10 times of dilution food samples amplifications, then amplification is effective, and need to be using 10 times of dilutions
The Ct value of food samples RNA is as a result；The inhibition index of 10 times of dilution food samples amplifications also >=2.00 when, expanding may nothing
Effect, needs to detect again；But such as inhibition index >=2.00, testing result is the positive, can also take the circumstances into consideration to be determined as the positive.
Test sample: when the Ct value of sample to be tested is more than or equal to 45, it is determined as norovirus feminine gender；The Ct value of sample to be tested
When less than or equal to 38, it is determined as the norovirus positive；The Ct value of sample to be tested is greater than 38, when less than 45, should detect again；Again
When testing result is more than or equal to 45, it is determined as norovirus feminine gender；When less than or equal to 38, it is determined as the norovirus positive.
The present invention is detected using fluorescence quantitative PCR instrument, and sensitivity and accuracy are higher, and Data Processing in Experiment is simple,
Detection efficiency is greatly saved.
The above is the preferred embodiment of the present invention, is not restricted to the present invention.It will be understood by those skilled in the art that
The several improvements and modifications made without departing from the principle of the present invention, also should be regarded as protection scope of the present invention.
Claims (8)
1. the detection method of norovirus in a kind of water, which comprises the following steps:
(1) enrichment of water sample processing and norovirus；
(2) extraction of norovirus RNA, the viral nucleic acid solution purified；
(3) RT-PCR reaction solution is configured；
(4) fluorescence quantitative PCR instrument detects；
The program of the fluorescence quantitative PCR instrument detection are as follows: 50 DEG C of 30min；95℃ 5min；95 DEG C of 15s, 60 DEG C of 30s acquisitions
Signal；65 DEG C of 30s are recycled 45 times；Fluorescence channel detects and selects the channel FAM, VIC.
2. the detection method of norovirus in a kind of water according to claim 1, which is characterized in that water sample processing with
The step of enrichment of norovirus are as follows: MgCl is added in water sampling2Solution makes its concentration reach 0.03~0.05mol/L, adjusts water
Sample makes pH to 3.0~5.0, makes water sample by mixed nitrate cellulose membrane, with beef extract-glycine elution liquid concussion elution filter
Film；Filter membrane is removed, eluent pH to 6.0~8.0 is adjusted, PEG8000 solution is added into eluent and stands overnight, centrifugation retains
Precipitating；The ultrapure water without RNA enzyme is added and dissolves precipitating, centrifuged supernatant is viral crude extract.
3. the detection method of norovirus in a kind of water according to claim 1, which is characterized in that the norovirus
The extraction step of RNA are as follows: take viral crude extract, lysate is added, mixes well, then for several times, addition is washed for rinsing liquid rinsing
De- liquid, the viral nucleic acid solution purified after centrifugation.
4. the detection method of norovirus in a kind of water according to claim 2, which is characterized in that the MgCl2Solution
Concentration is 2.5mol/L.
5. the detection method of norovirus in a kind of water according to claim 2, which is characterized in that the beef extract-is sweet
The configuration method of propylhomoserin eluent are as follows: by 30.0g beef extract and 3.75g glycine stirring and dissolving in ultrapure water, adjust pH to
9.5, it is settled to 1000mL with ultrapure water, is dispensed, 121 DEG C of high pressure sterilization 15min.
6. according to the detection method of norovirus in a kind of water as claimed in claim 2, which is characterized in that the water sample processing and promise
In such as the step of viral enrichment, the volume ratio of eluent and PEG8000 solution is 1:0.25.
7. according to the detection method of norovirus in a kind of water as claimed in claim 2, which is characterized in that the PEG8000 solution
Configuration method are as follows: by 400.0g PEG8000 and 87.0g sodium chloride stirring and dissolving in ultrapure water, be settled to ultrapure water
1000mL, packing, 121 DEG C of high pressure sterilization 15min.
8. the detection method of norovirus in a kind of water according to claim 3, which is characterized in that the addition lysate
Volume be 600 μ L.
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