CN106119417A - The test kit of a kind of accurate quantification detection norovirus I type and detection method - Google Patents
The test kit of a kind of accurate quantification detection norovirus I type 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 norovirus I type.Be specifically related to one group for the primer and the probe that detect norovirus I type, 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 also provides for a kind of detection method utilizing droplet type digital pcr technology accurate quantification detection norovirus I type, and the method, without relying on certified reference material or other standards product, has higher degree of 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 for detecting norovirus I type and widow
Nucleotide, more precisely, is a kind of reverse transcription droplet type numeral polymerase chain reaction detecting norovirus I type
(droplet digital reverse transcriptional polymerase chain reaction,RT-ddPCR))
Rapid quantitative detection reagent box.
Background technology
Norovirus (Norovirus) belongs to Caliciviridae, for without peplos single strand plus RNA virus, full-length genome
For 7.5-7.7kp, including 3 open reading frame (Open Reading Frame, ORF), wherein ORF1 coding nucleotide three phosphorus
The non-structural proteins such as the RNA polymerase (RNA-dependent RNA polymerase, RdRp) that acid enzyme, protease and RNA rely on
In vain, ORF2 encodes major structural protein VP1, ORF3 and encodes secondary structure albumen.Based on RdRP and VP1 gene sequence information not
With, norovirus can be divided into 5 kinds of genotype G I-G V.Wherein G I and G II is most commonly seen, for infecting the main viral gene of people
Type.
Norovirus is the main food-borne virus causing nonbacterial gastroenteritis to break out in worldwide, can infect each
Age group crowd, especially child, cause patient's Nausea and vomiting, stomachache, and even low grade fever, headache, myalgia, weak and appetite subtracts
The symptom such as move back.Its route of transmission is: the source of infection → feces → water body (food) → human body is propagated, the water of pollution and food especially shellfish
Class is the main carriers propagated.Norovirus has stronger toleration to environment, under the conditions of acid (pH 2.7), heating
In (60 DEG C) condition and tap water under free chlorine (3.75～6.25mg/L) concentration conditions, still having infectivity, preventive means is very
Limited.Therefore, being detected and analyzed norovirus in environment and food, beneficially water body environment and shellfish by nest RT-PCR promise are such as
Carrying out of virus pollution risk assessment, and then food-borne viral disease is broken out carry out effective early warning, protect human health, protect
Barrier food trade is normally carried out.
Although norovirus I type is strict cytozoon, irreproducible in food, but it is infectious strong,
Even less than 102The virion of copy number/mL leads to infect.And there may be the food sample that norovirus I type pollutes
In product, its virus titer is generally in than relatively low level.It is thus desirable to a kind of accurate quantification for norovirus I type detects
Method, it is achieved the effective monitoring to norovirus I type.At present, the quantitative detecting method of norovirus I type mainly includes with polymerization
Real-time fluorescence quantitative PCR (the Real-time on polymerase chain reaction (Polymerase Chain Reaction, PCR) basis
Fluorescent Quantitative PCR, RT-qPCR) and reverse transcription-ring mediated constant temperature nucleic acid amplification (Realtime
Reverse transcription lood-mediated isothermal amplification, RT-LAMP) method.These are two years old
The method of kind needs through carefully aligned standard curve and stable norovirus I type standard substance, and detects every time and all need
Criterion curve, therefore, the existence of potential subjective factors in interpretation of result, cause the difference and not of result between repeated detection
With the multiformity of laboratory monitoring result, affect standardization and the standardization of norovirus I type detection by quantitative.
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 both at home and abroad and utilizes RT-ddPCR to carry out promise such as
The relevant report of virus I-form detection by quantitative.
Summary of the invention
It is an object of the invention to provide the RT-for detecting norovirus I type that a group-specific is strong, highly sensitive
DdPCR primer and probe.
It is a further object to provide a kind of simple to operate, result norovirus I type RT-ddPCR accurately is fast
Speed immue quantitative detection reagent 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 norovirus I type, chooses its parting gene fragment RdRp-VP1, design
Going out specific primer and probe, the sequence of described primer and probe for detecting norovirus I type is shown in Table 1, wherein, and probe 5 '
Flag F AM, 3 ' labelling BHQ.
Table 1 RT-ddPCR primer and probe sequence
The present invention also provide for a kind of accurate quantification detection norovirus I type test kit, this test kit include above-mentioned for
The primer of detection norovirus I type and probe.Further, this test kit also included RT-ddPCR reaction material requested and
Reagent, such as norovirus I type positive control RNA, one-step RT-ddPCR supermix, magnesium acetate, microdroplet generate oil,
Microdroplet generates card, 96 orifice plates and aluminium foil thermal sealer etc., above-mentioned material and reagent and is preferably individually packaging.
Present invention also offers the detection method that a kind of norovirus I type is the most quantitative, i.e. RT-ddPCR method, it includes
Herein below:
1. extract testing sample RNA, it is possible to use tradition Trizol method extracting method or commercial kit are extracted；
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 norovirus I type 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 norovirus I type 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 norovirus I type 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, during calculating obtains sample as follows, norovirus I type is accurate
Quantitatively.
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
Norovirus I type purpose fragment RT-ddPCR testing result under the conditions of Fig. 1 different annealing temperature.
Fig. 2 RT-ddPCR test kit detection norovirus I type specificity analysis result.
Fig. 3 RT-ddPCR test kit detection norovirus I type sensitive analysis result.
Fig. 4 RT-ddPCR test kit detection norovirus I type repeatability analysis result.
Detailed description of the invention
Embodiment 1 RT-ddPCR primer, probe design
For realizing the specific detection to norovirus I type and absolute quantification analysis, we choose norovirus typing base
Because of fragment RdRp-VP1, carry out sequence analysis and comparison by NCBI online tool, utilize Prime Express software V4.0
(ABI, Foster City, CA, USA) designs more than 10 to primer and probe combinations, through screening finally obtain high specificity,
Being 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.Draw
Thing/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 norovirus I type purpose content of fragment in RT-ddPCR reaction system, and then pass through RNA
Template sample-adding amount (2 μ L) and the RNA template body product extracted, calculate as follows and obtain the essence of norovirus I type in sample
Certainly measure.
RT-ddPCR testing conditions is also optimized by the present invention, such as annealing temperature, takes norovirus I type of extraction
RNA, as template, carries out amplified reaction respectively under the annealing temperature of 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C and 70 DEG C, the most different
Under the conditions of the testing result of RT-ddPCR.As seen from Figure 1, the amplification effect of norovirus I type genes of interest fragment under the conditions of 50 DEG C
Rate is on the low side, and 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 55 DEG C, the fluorescence signal of amplification is slightly above 60 DEG C, therefore, by 55 DEG C of works
For optimum annealing 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 norovirus I type of embodiment 1 design, by Beijing six
He Tong Trade Co., Ltd. synthesizes；
(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: norovirus I type RNA standard substance；
(9) DEPC water: purchased from the raw work in Shanghai, article No.: D1005.
2. prepared by norovirus I type RNA standard substance
With norovirus I type RNA as template, utilize primer SEQ ID No.1-2, amplify the specificity sheet of about 68bp
Disconnected, referred to as G I (comprise RT-ddPCR and expand target gene)；(pcDNA II carrier is purchased from construction recombination plasmid pcDNA II-G I
Invitrogen company), carry out sequencing in Shanghai Sheng Gong company, and by sequencing result and norovirus I type in GenBank
Gene order carry out homology analysis, its homology reaches 100%.Extract and plasmid DNA purification, with Promega company
Ribo MAXTMLarge Scale RNA Production 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 cRNA.The cRNA of purification is in upper
Hai Shenggong company carries out sequencing, and with the gene order of norovirus I type in GenBank, sequencing result is carried out homology
Analyzing, its homology reaches 100%.By cRNA subpackage, it is stored in-80 DEG C.The norovirus I type RNA standard substance of preparation is entered
Row uniformity, stability test, all meet related request；Use many laboratory valued methods that the standard substance of preparation is carried out
Definite value research, the norovirus I type RNA standard substance concentration of final preparation is: 5.43 × 107copies/μl.-80 DEG C of preservations,
Positive control as test kit
Embodiment 4 detection method specificity and sensitivity evaluation
1. detection specificity analyses
(1) material: rotavirus, Astrovirus, norovirus I type and norovirus II type RNA are pre-by China's disease
Anti-control centre virosis is provided.
(2) method: use test kit of the present invention, includes rotavirus, Astrovirus, promise to common diarrhea virus
As virus I-form and norovirus II type RNA carry out RT-ddPCR augmentation detection, observe this test kit with or without nonspecific reaction
Occur.
(3) result: rotavirus, Astrovirus, norovirus I type and norovirus II type RNA are through PCR instrument (Veriti
Thermal cycler, Applied BioSystems, Foster City, CA) amplification after, use microdroplet read instrument (QX200,
Bio-Rad, Pleasanton, CA) detection, utilize QuantaSoft software to carry out result reading and analysis.Result shows, only
There is positive microdroplet in norovirus I type, and remaining virus is negative microdroplet, it was demonstrated that the method has good specificity.Result
See Fig. 2.
2. detection sensitivity analysis
(1) material: norovirus I type RNA standard substance prepared above carries out 10 times of gradient dilutions to 5.43 copies/μ
L。
(2) method: each dilution gradient takes 2 μ L, utilizes the test kit described in embodiment 3 to carry out RT-ddPCR detection, passes through
Analyzing the minimum that test kit of the present invention can be detected by, reaction system is shown in Table 2.
(3) result: find to utilize test kit of the present invention, can detect that 5.43 copies/μ L norovirus I type RNA mark
Quasi-material.Result is shown in Fig. 3.Visible test kit detection norovirus I type can reach 5.43 copies/μ L, substantially increases promise such as
The sensitivity of virus I-form related detecting method, is independent of standard substance, can realize absolute quantitation, and visual result is reliable.
Embodiment 5 detection method accuracy and reproducibility
1. detection accuracy analysis
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) material: norovirus I type detection RNA positive control (5.43 × 10 prepared above7Copies/ μ l) respectively
It is diluted to 5.43 × 103copies/μl、5.43×102copies/μl、5.43×101Copies/ μ l and 5.43copies/ μ l,
It is expressed as sample U1-U4 (table 4).
(2) method: each sample carries out RT-ddPCR detection respectively, each sample sets 7 repetitions, arranges 1 blank simultaneously
Comparison (to go DEPC water to replace template ribonucleic acid).
(3) result: the RT-ddPCR measured value of norovirus I type RNA is all close with predicted value, 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 norovirus I type RT-ddPCR detection
2. detection repeatability is analyzed
(1) method: utilize norovirus I type RNA positive control (5.43 × 10 prepared above7Copies/ μ l) dilution
To 5.43 × 103Copies/ μ l, carries out RT-ddPCR detection, if 8 repetitions, arranges 1 blank (to remove DEPC simultaneously
Water replaces template ribonucleic acid).
(2) result: from fig. 4, it can be seen that mensuration coefficient of variation CV of the norovirus I type RNA positive control to certain concentration
Less than 25%, it was demonstrated that the method has good repeatability when norovirus I type 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 norovirus I type, it is characterised in that by sequence table SEQ ID No.1 to sequence
Nucleotide sequence composition shown in list SEQ ID No.3；The wherein nucleotide shown in SEQ ID No.1 to SEQ ID No.2
Sequence is the primer for expanding norovirus I type RNA, and the nucleotides sequence shown in SEQ ID No.3 is classified as expanding promise such as
The probe of virus I-form RNA.
Primer for detecting norovirus I type the most according to claim 1 and probe, it is characterised in that: probe 5 ' is held
Flag F AM, 3 ' end labelling BHQ.
3. the test kit of accurate quantification detection norovirus I type, it is characterised in that described test kit includes claim 1
Or a group described in 2 is for detecting primer and the probe of norovirus I type.
Test kit the most according to claim 3, it is characterised in that described test kit also includes that the norovirus I type positive is right
According to RNA, one-step RT-ddPCR supermix, magnesium acetate, microdroplet generates oil, microdroplet generates card, 96 orifice plates and aluminium foil thermal
Sealer.
5. the detection method of accurate quantification detection norovirus I type, 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 norovirus I type 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 norovirus I type RNA, and then by the content of norovirus I type in equation below calculating acquisition testing sample:
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988425A (en) * | 2017-11-06 | 2018-05-04 | 北京出入境检验检疫局检验检疫技术中心 | A kind of I type norovirus detection of nucleic acids standard substances of G and its preparation method and application |
| CN108220401A (en) * | 2018-03-16 | 2018-06-29 | 张家港出入境检验检疫局检验检疫综合技术中心 | A kind of campylobacter jejuni recombinant plasmid standard items quantitative detecting method based on droplet type digital pcr |
| CN108676920A (en) * | 2018-07-07 | 2018-10-19 | 广东省实验动物监测所 | It is a kind of quickly to detect mouse norovirus primer, kit and its RT-RPA methods |
| CN113025758A (en) * | 2021-04-07 | 2021-06-25 | 拱北海关技术中心 | Primer, probe and kit for detecting GI type norovirus of aquatic product |
| CN114622038A (en) * | 2022-03-28 | 2022-06-14 | 广西壮族自治区兽医研究所 | Primer group and probe for detecting avian circovirus type 2 by micro-drop digital PCR (polymerase chain reaction) and application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988425A (en) * | 2017-11-06 | 2018-05-04 | 北京出入境检验检疫局检验检疫技术中心 | A kind of I type norovirus detection of nucleic acids standard substances of G and its preparation method and application |
| CN108220401A (en) * | 2018-03-16 | 2018-06-29 | 张家港出入境检验检疫局检验检疫综合技术中心 | A kind of campylobacter jejuni recombinant plasmid standard items quantitative detecting method based on droplet type digital pcr |
| CN108676920A (en) * | 2018-07-07 | 2018-10-19 | 广东省实验动物监测所 | It is a kind of quickly to detect mouse norovirus primer, kit and its RT-RPA methods |
| CN108676920B (en) * | 2018-07-07 | 2021-07-20 | 广东省实验动物监测所 | Primer and kit for rapidly detecting mouse norovirus and RT-RPA method thereof |
| CN113025758A (en) * | 2021-04-07 | 2021-06-25 | 拱北海关技术中心 | Primer, probe and kit for detecting GI type norovirus of aquatic product |
| CN114622038A (en) * | 2022-03-28 | 2022-06-14 | 广西壮族自治区兽医研究所 | Primer group and probe for detecting avian circovirus type 2 by micro-drop digital PCR (polymerase chain reaction) and application |
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| CN106119417B (en) | 2019-12-13 |
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