CN106119416A - The test kit of a kind of accurate quantification detection Astrovirus and detection method - Google Patents
The test kit of a kind of accurate quantification detection Astrovirus and detection method Download PDFInfo
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Abstract
The invention discloses test kit and the detection method of a kind of accurate quantification detection Astrovirus.Be specifically related to one group for the primer and the probe that detect Astrovirus, it has a nucleotide sequence shown in sequence table SEQ ID No.1 to SEQ ID No.3, and containing these primers and the test kit of probe and detection method thereof.The present invention provide a kind of utilize droplet type digital pcr technology accurate quantification detection Astrovirus detection method, the method without since certified reference material or other standards product, there is higher accuracy, sensitivity and repeatability, it is easy to standardization.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of test kit detecting Astrovirus and oligonucleotide,
More precisely, it is a kind of reverse transcription droplet type numeral polymerase chain reaction (droplet detecting Astrovirus
Digital reverse transcriptional polymerase chain reaction, RT-ddPCR)) fast quantification
Detection kit.
Background technology
Appleton is equal within 1975, finding Astrovirus (Human in the feces of gastroenteritis patient first
Astrovirus, HAstV), various countries the most all can occur that astrovirus infection breaks out situation to some extent.Astrovirus without
The single strand plus RNA virus of capsid, diameter 28～35nm, there are 5～6 little angles, make the star-shaped structure of whole virion.Sick
The long 6.8kb of virus gene, including 5 ' noncoding regions (5 ' non-coding region, NCR), three open reading frames
(open reading frams, ORFs) ORF1a, ORF1b and ORF2,3 ' noncoding regions of 80 nucleotide and one about 30
The poly-A tail of individual nucleotide).Astrovirus can be divided into 8 serotypes HAstV-1～HAstV-8.At present, starlike disease
Poison has been considered as one of important pathogen causing infant, old people and immunocompromised person's acute viral enteritis.
Although food-borne virus is strict cytozoon, irreproducible in food, but the food of trace in food
Borne virus will cause disease.And there may be in the food samples that food-borne virus pollutes, its virus titer is generally in
Than relatively low level.It is thus desirable to one is for food-borne virus precise and quantitative detection method, it is achieved food-borne virus is had
Effect monitoring.At present, the quantitative detecting method of Astrovirus mainly includes with polymerase chain reaction (Polymerase Chain
Reaction, PCR) basis real-time fluorescence quantitative PCR (Real-time Fluorescent Quantitative PCR, RT-
And reverse transcription-ring mediated constant temperature nucleic acid amplification (Realtime reverse transcription lood-qPCR)
Mediated isothermal amplification, RT-LAMP) method.Both approaches needs through carefully aligned standard
Curve and stable Astrovirus standard substance, and detect every time and all need Criterion curve, therefore potential in interpretation of result
The existence of subjective factors, causes the multiformity of result between the difference of result between repeated detection and different experiments room, affects starlike
The standardization of Viral Quantification detection and standardization.
Digital pcr (Digital PCR, the dPCR) technology that development in recent years is got up is a kind of brand-new nucleic acid quantification detection
Method.At present, digital pcr includes: micro-reative cell/orifice plate digital pcr (Chamber Digital PCR, cdPCR), microfluid
Digital pcr (Microfluidic Digital PCR, mdPCR) (large-scale integrated micro-fluidic chip) and droplet type digital pcr
(Droplet Digital PCR, ddPCR) three kinds of dPCR systems.At present, it is showed no that to utilize RT-ddPCR to carry out starlike both at home and abroad
The relevant report of Viral Quantification detection.
Summary of the invention
It is an object of the invention to provide strong, highly sensitive the drawing for detecting the RT-ddPCR of Astrovirus of a group-specific
Thing and probe.
It is a further object to provide a kind of simple to operate, result Astrovirus RT-ddPCR accurately quantitatively to examine
Test agent box.
For achieving the above object, the present invention is by the following technical solutions:
The present invention, by analyzing the composition of genome of Astrovirus, chooses its conservative fragments ORF2, designs specificity and draw
Thing and probe, the sequence of described primer and probe for detecting Astrovirus is shown in Table 1.Wherein, probe 5 ' flag F AM, 3 ' marks
Note BHQ.
Table 1RT-ddPCR primer probe sequence
The present invention also provides for the test kit of a kind of accurate quantification detection Astrovirus, and this test kit includes above-mentioned for detecting
The primer of Astrovirus and probe.Further, this test kit has also included RT-ddPCR reaction material requested and reagent, example
Such as Astrovirus positive control RNA, one-step RT-ddPCR supermix, acetolase, microdroplet generates oil, microdroplet generates
Card, 96 orifice plates and aluminium foil thermal sealer etc., above-mentioned material and reagent are preferably individually packaging.
Present invention also offers the detection method that a kind of Astrovirus is the most quantitative, i.e. RT-ddPCR method, it include with
Lower content:
1. extract testing sample RNA, it is possible to use extracting method well known in the prior art or commercial kit are carried out
Extract；
2. preparation RT-ddPCR reaction system, wherein, described reaction system includes SEQ ID NO:1 to SEQ in sequence table
Primer shown in ID NO:3 and probe.In one embodiment of the invention, RT-ddPCR reaction system such as table 2 institute
Show；
Table 2 Astrovirus ddPCR reaction system
3. RT-ddPCR reaction system step 2. prepared and microdroplet generate oil and add in microdroplet generation card, are placed in microdroplet
Generate and instrument generates microdroplet；
4. carrying out amplified reaction, reaction condition is shown in Table 3；
Table 3 Astrovirus RT-ddPCR reaction condition
Note: warming and cooling rate should≤2.5 DEG C/s
5. result judges.
96 orifice plates after amplification are placed in microdroplet and read in instrument (QX200, Bio-Rad, Pleasanton, CA), utilize
QuantaSoft software carries out result reading and analysis.Positive microdroplet containing amplified production is micro-with the feminine gender without amplified production
Drip the difference that can present fluorescence signal intensity, set threshold line with the peak of negative microdroplet bunch fluorescence amplitude for boundary.Press
Calculate according to Poisson distribution principle and obtain Astrovirus purpose content of fragment in RT-ddPCR reaction system, and then by RNA template
Sample-adding amount (2 μ L) and the RNA template body product of extraction, calculate as follows and obtain the most quantitative of sample stellate virus.
The invention have the advantage that 1) absolute quantitation of nucleic acid molecules avoided caused by PCR amplification efficiency difference
Deviation；2) without relying on certified reference material or other standards product；3) there is higher degree of accuracy, susceptiveness and repeatability, easily
In standardization；4) detection of nucleic acids of low copy number it is more suitable for；5) it is end point determination due to RT-ddPCR, the tolerance level to inhibitor
Higher, therefore can reduce the detection error caused by matrix type.
Below in conjunction with specification drawings and specific embodiments, the invention will be further described, all open according to the present invention
The equivalent of any this area that content is done, belongs to protection scope of the present invention.
Accompanying drawing explanation
Astrovirus purpose fragment RT-ddPCR testing result under the conditions of Fig. 1 different annealing temperature.
Fig. 2 RT-ddPCR test kit detection Astrovirus specificity analyses result.
Fig. 3 RT-ddPCR test kit detection Astrovirus sensitive analysis result.
Fig. 4 RT-ddPCR test kit detection Astrovirus repeatability analysis result.
Detailed description of the invention
Embodiment 1RT-ddPCR primer, probe design
For realizing the specific detection to Astrovirus and absolute quantification analysis, we choose Astrovirus open reading frame
The target sequence of 2 (Open Reading Frame, ORF2) coding caspid protein, carries out sequence by NCBI online tool
Analyze and comparison, utilize Prime Express software V4.0 (ABI, Foster City, CA, USA) to design more than 10 to primer
And probe combinations, finally obtaining high specificity through screening, be applicable to each 1 set of RT-ddPCR primer/probe combinations, sequence is shown in Table
1.Its middle probe 5 ' end flag F AM, 3 ' end labelling BHQ.Primer/probe is led to Trade Co., Ltd.'s synthesis by Beijing six directions.
The foundation of embodiment 2 detection method
(1) RNA extracts: utilize commercial kit to extract；Or utilize tradition Trizol method to extract RNA, specifically
Operate as follows: 1. adding 1mL Trizol in 100 μ L sample, after concussion 30s, room temperature places 5min；2. 250 μ L chlorine are added
Imitative, acutely shake 30s, room temperature places 5min；4 DEG C, 12000g, centrifugal 5min；3. supernatant is proceeded in new centrifuge tube, add
The isopropanol of 500 μ L acutely shakes mixing 30s, and room temperature places 5min；4. 4 DEG C, 5000g, centrifugal 5min；The most carefully remove
Clearly, precipitation 12000g after 1mL 70% ethanol purge, 4 DEG C, (siphoning away supernatant as far as possible, centrifuge tube is placed in ultra-clean centrifugal 5min
On platform, precipitation is dried up)；6. add 50 μ L and (in order to make viral RNA preferably dissolve, put 60 DEG C of heating without RNase water dissolution RNA
10min).The RNA extracted saves backup in-80 DEG C.
(2) the RT-ddPCR reaction system of 20 μ L is prepared according to table 2
(3) microdroplet generates
DdPCR reaction system and the 65 μ L microdroplets of 20 μ L are generated oil and are added separately in 8 hole microdroplets generation cards, is placed in micro-
Drip and generation instrument (QX200, Bio-Rad, Pleasanton, CA) generates microdroplet.
(4) amplified reaction
Slowly being transferred in 96 orifice plates by the water in oil microdroplet (40 μ L) generated, sealer is placed on PCR instrument (Veriti
Thermal cycler, Applied BioSystems, Foster City, CA) on carry out amplified reaction, amplification condition such as table 3
Shown in.
(5) result judges
96 orifice plates after amplification are placed in microdroplet and read in instrument (QX200, Bio-Rad, Pleasanton, CA), utilize
QuantaSoft software carries out result reading and analysis.Positive microdroplet containing amplified production is micro-with the feminine gender without amplified production
Drip and can present the difference of fluorescence signal intensity, can the peak of negative microdroplet bunch fluorescence amplitude be that boundary is to set threshold line.
Calculate according to Poisson distribution principle and obtain Astrovirus purpose content of fragment in RT-ddPCR reaction system, and then by RNA mould
Plate sample-adding amount (2 μ L) and the RNA template body product extracted, calculate as follows and obtain the most fixed of sample stellate virus
Amount.
RT-ddPCR testing conditions is also optimized by the present invention, such as annealing temperature, takes the Astrovirus RNA of extraction
As template, under the annealing temperature of 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C and 70 DEG C, carry out amplified reaction respectively, compare different condition
The testing result of lower RT-ddPCR.As seen from Figure 1, under the conditions of 50 DEG C, the amplification efficiency of Astrovirus genes of interest fragment is on the low side,
Positive microdroplet and negative microdroplet boundary are inconspicuous；Under the conditions of 55 DEG C and 60 DEG C, genes of interest fragment amplification efficiency is higher, may occur in which
Obvious positive microdroplet bunch, and under the conditions of 60 DEG C, the fluorescence signal of amplification is slightly above 55 DEG C, therefore, using 60 DEG C as optimal
The most fiery temperature.
Embodiment 3 test kit forms
1. the composition (being stored in-20 DEG C) of test kit
(1) primer and the probe SEQ ID No.1-3 for detecting Astrovirus of embodiment 1 design, by Beijing six directions
The synthesis of logical Trade Co., Ltd.；
(2) one-step RT-ddPCR supermix, 25mM magnesium acetate: purchased from BioRad company of the U.S., article No. 186-
3021；
(3) microdroplet generates oil: purchased from BioRad company of the U.S., article No. 186-3030；
(4) microdroplet generates card: purchased from BioRad company of the U.S., article No. 186-4007；
(5) aluminium foil thermal sealer: purchased from BioRad company of the U.S., article No. 181-4000；
(6) Twin Tec Semi-Skirted 96 orifice plate: purchased from Eppendorf company of Germany, article No. 0030128605；
(7) negative control: DEPC water；Purchased from the raw work in Shanghai, article No.: D1005；
(8) positive control: Astrovirus RNA standard substance；
(9) DEPC water: purchased from the raw work in Shanghai, article No.: D1005.
2. prepared by Astrovirus RNA standard substance
With Astrovirus RNA as template, utilize primer SEQ ID No.1-2, amplify the specificity segment of about 63bp, claim
For Ast (comprise RT-ddPCR expand target gene)；(pcDNA II carrier is purchased from construction recombination plasmid pcDNA II-Ast
Invitrogen company), carry out sequencing in Shanghai Sheng Gong company, and by sequencing result and GenBank stellate virus
Gene order carries out homology analysis, and its homology reaches more than 95%.Extract and plasmid DNA purification, with Promega company
Ribo MAXTMLarge Scale RNAProduction system-T7 test kit (article No.: P1300) carries out in vitro transcription
(carrying out by test kit operating instruction), in vitro transcription product DNase digests, and removes DNA therein.Utilize RNeasy
MiniElute Cleanup kit (purchased from QIAGEN company, article No. 74204) is further purified RNA.The RNA of purification is in Shanghai
Sheng Gong company carries out sequencing, and the gene order of sequencing result with GenBank stellate virus is carried out homology analysis,
Its homology reaches more than 95%.By cRNA subpackage, it is stored in-80 DEG C.The Astrovirus RNA standard substance of preparation is carried out uniformly
Property, stability test, all meet related request；Use many laboratory valued methods that RNA is carried out definite value research, finally prepare
Astrovirus RNA standard substance concentration be: 5.52 × 106copies/μl.-80 DEG C of preservations, the positive as test kit is right
According to.
Embodiment 4 detection method specificity and sensitivity evaluation
1. detection specificity analyses
(1) material: rotavirus, Astrovirus, hepatitis A virus (HAV), norovirus I type and norovirus II type RNA by
State's disease prevention and control center's virosis is provided.
(2) method: use test kit of the present invention, includes rotavirus, Astrovirus, first to common diarrhea virus
Hepatovirus, norovirus I type and norovirus II type RNA carry out RT-ddPCR augmentation detection, and observing this test kit has special nothing but
The generation of opposite sex reaction.
(3) result: rotavirus, Astrovirus, hepatitis A virus (HAV), norovirus I type and norovirus II type RNA are through PCR
After instrument (Veriti Thermal cycler, Applied BioSystems, Foster City, CA) amplification, microdroplet is used to read
Take instrument (QX200, Bio-Rad, Pleasanton, CA) detection, utilize QuantaSoft software to carry out result reading and analysis.Knot
Fruit shows, specific amplification occurs in only Astrovirus, and remaining virus is all negative through RT-ddPCR detection, it was demonstrated that the method has
There is good specificity.Result is shown in Fig. 2.
2. detection sensitivity analysis
(1) method: Astrovirus detection RNA standard substance prepared above is carried out 10 times of gradient dilutions and copies to 5.52
Shellfish/μ L, respectively takes 2 μ L, utilizes the test kit described in embodiment 3 to carry out RT-ddPCR detection, by analyzing reagent of the present invention
The minimum that box can be detected by, reaction system is shown in Table 2.
(2) result: find to utilize test kit of the present invention, can detect that the Astrovirus RNA standard of 5.52 copies/μ L
Material.Result is shown in Fig. 3.Visible test kit detection Astrovirus can reach 5.52 copies/μ L, substantially increases Astrovirus phase
Closing the sensitivity of detection method, be independent of standard substance, can realize absolute quantitation, visual result is reliable.
Embodiment 5 detection method accuracy and reproducibility
1. detection accuracy evaluation
Accuracy (truness) refers to the concordance journey between the meansigma methods of one group of test result and acceptable reference value
Degree.According to the standard of European Union Yu Codex, the criterion of accuracy is in whole detection dynamic range, measured value Ying Can
In the range of examining value ± 25%.
(1) method: utilize Astrovirus RNA standard substance (5.52 × 10 prepared above6Copies/ μ l) it is diluted to
5.52×103copies/μl、5.52×102copies/μl、5.52×101Copies/ μ l and 5.52copies/ μ l, is expressed as
Sample U1-U4 (table 4)
Each sample carries out RT-ddPCR detection respectively, and each sample sets 7 repetitions, arranges 1 blank (to go simultaneously
DEPC water replaces template ribonucleic acid).
(2) result: the RT-ddPCR measured value of Astrovirus RNA all with predicted value closely, the equal ＜ of its standard deviation
15% (table 4), the criterion less than 25%, illustrate that the method has extraordinary accuracy.
The accuracy of table 4 Astrovirus RT-ddPCR detection
2. detection repeatability is analyzed
(1) method: utilize Astrovirus RNA standard substance (5.52 × 10 prepared above6Copies/ μ l) it is diluted to
5.52×103Copies/ μ l, carries out RT-ddPCR detection, if 9 repetitions, arranges 1 blank (to remove DEPC water simultaneously
Replace template ribonucleic acid).
(2) result: from fig. 4, it can be seen that mensuration coefficient of variation CV of the Astrovirus RNA positive control of certain concentration is less than
25%, it was demonstrated that the method has good repeatability when Astrovirus detection by quantitative.
Obviously, the above embodiment of the present invention is only for clearly demonstrating example of the present invention, and is not right
The restriction of embodiments of the present invention.For those of ordinary skill in the field, the most also may be used
To make other changes in different forms.Here cannot all of embodiment be given exhaustive.Every belong to this
What bright technical scheme was extended out obviously changes or changes the row still in protection scope of the present invention.
Claims (6)
1. one group of primer and probe being used for detecting Astrovirus, it is characterised in that by sequence table SEQ ID No.1 to sequence table
Nucleotide sequence composition shown in SEQ ID No.3；The wherein nucleotide sequence shown in SEQ ID No.1 to SEQ ID No.2
For expanding the primer of Astrovirus RNA, nucleotides sequence shown in SEQ ID No.3 is classified as the probe for expanding Astrovirus RNA.
Primer for detecting Astrovirus the most according to claim 1 and probe, it is characterised in that: labelling held by probe 5 '
FAM, 3 ' end labelling BHQ.
3. the test kit of an accurate quantification detection Astrovirus, it is characterised in that described test kit includes claim 1 or 2
Described one group is for detecting primer and the probe of Astrovirus.
Test kit the most according to claim 3, it is characterised in that described test kit also includes Astrovirus positive control
RNA, one-step RT-ddPCR supermix, magnesium acetate, microdroplet generate oil, microdroplet generates card, 96 orifice plates and aluminium foil and seals
Film.
5. the detection method of an accurate quantification detection Astrovirus, it is characterised in that the method includes:
(1) testing sample RNA is extracted；
(2) preparation RT-ddPCR reaction system, wherein, described reaction system includes SEQ ID NO:1 to SEQ ID in sequence table
Primer and probe for expanding Astrovirus RNA shown in NO:3；
(3) RT-ddPCR reaction system step (2) prepared and microdroplet generate oil and add in microdroplet generation card, are placed in microdroplet raw
Cheng Yizhong generates microdroplet；
(4) amplified reaction is carried out；
(5) result judges.
Detection method the most according to claim 5, it is characterised in that in step (5), calculates according to Poisson distribution principle and obtains
Obtain the copy number of Astrovirus RNA, and then calculated the content obtaining testing sample stellate virus by equation below:
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| CN108085412A (en) * | 2017-11-06 | 2018-05-29 | 北京出入境检验检疫局检验检疫技术中心 | A kind of human astrovirus 1's type detection of nucleic acids standard substance and its preparation method and application |
| CN108103241A (en) * | 2017-11-27 | 2018-06-01 | 西南大学 | Citrus yellow vein virus droplet digital pcr immue quantitative detection reagent box and method |
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