CN106222301B - A kind of kit and detection method of accurate quantification detection II type of norovirus - Google Patents
A kind of kit and detection method of accurate quantification detection II type of norovirus Download PDFInfo
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- CN106222301B CN106222301B CN201610632899.8A CN201610632899A CN106222301B CN 106222301 B CN106222301 B CN 106222301B CN 201610632899 A CN201610632899 A CN 201610632899A CN 106222301 B CN106222301 B CN 106222301B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses the kits and detection method of a kind of accurate quantification detection II type of norovirus.More particularly to one group for detecting the primer and probe of II type of norovirus, with nucleotide sequence shown in sequence table SEQ ID No.1 to SEQ ID No.3, and kit and its detection method containing these primer and probes.The present invention provides a kind of detection method using droplet type digital pcr technology accurate quantification detection II type of norovirus, and this method has higher sensitivity, accuracy and repeatability, is easy to standardize.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a kind of kit and widow for detecting II type of norovirus
Nucleotide is more precisely a kind of reverse transcription-droplet type digital polymerase chain reaction for detecting II type of norovirus
(droplet digital reverse transcriptional polymerase chain reaction,RT-ddPCR))
Rapid quantitative detection reagent box.
Background technique
Norovirus (Norovirus) belongs to Caliciviridae, for no coating single strand plus RNA virus, full-length genome
For 7400-7700bp, including 3 open reading frame (Open Reading Frame, ORF), wherein ORF1 coding nucleotide three
RNA polymerase (RNA-dependent RNA polymerase, RdRp) since phosphatase, protease and RNA etc. is non-structural
Albumen, ORF2 encode major structural protein VP1, and ORF3 encodes secondary structure albumen.Based on RdRP and VP1 gene sequence information
Difference, NoV can be divided into 5 kinds of genotype G, I-G V, and wherein G I and G II are most commonly seen, and can infect people.
Since 1990s mid-term, II type of norovirus has become norovirus Major Epidemic type.Yin Qiyi morphs
And recombination, therefore per every about 2-3, it just will appear a novel variant strain, and cause worldwide generally popular.Promise
Such as the route of transmission of virus type II are as follows: the infection sources → excrement → water body (food) → human body is propagated, and the water and food of pollution are especially
Shellfish is the main carriers propagated；Moreover, norovirus has stronger tolerance to environment, in acid (pH 2.7) condition
Under, in heating (60 DEG C) condition and tap water under free chlorine (3.75~6.25mg/L) concentration conditions, still have infectivity, prevent hand
Section is extremely limited.Therefore, II type of norovirus in environment and food is detected and analyzed, is conducive to water body environment and shellfish
The development of marine product norovirus pollution risk assessment, and then effective early warning is carried out to breaking out for food-borne viral disease, protection
Human health ensures being normally carried out for food trade.
Although II type of norovirus is stringent cytozoon, irreproducible in food, it is infectious strong,
Even less than 102Copy number/mL virion leads to infect.There may be the food samples of II type of norovirus pollution
In, virus titer is generally in relatively low level.Therefore a kind of accurate quantification for II type of norovirus is needed to detect
Method realizes the effective monitoring to II type of norovirus.Currently, the quantitative detecting method of II type of norovirus mainly includes with poly-
Real-time fluorescence quantitative PCR (the Real-time on polymerase chain reaction basis (Polymerase Chain Reaction, PCR)
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.This two
Kind of method is needed by carefully aligned standard curve and stable II type standard substance of norovirus, and detects all need every time
Standard curve is established, therefore, the presence of potential subjective factor in interpretation of result causes between repeated detection the difference of result and not
With the diversity of laboratory monitoring result, the standardization and standardization of II type quantitative detection of norovirus are influenced.
Digital pcr (Digital PCR, dPCR) technology developed in recent years is a kind of completely new nucleic acid quantification detection
Method.Currently, digital pcr includes: micro- reaction chamber/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.Promise is carried out such as using RT-ddPCR currently, being showed no both at home and abroad
The relevant report of virus type II quantitative detection.
Summary of the invention
It is strong, high sensitivity for detecting the RT- of II type of norovirus that the object of the present invention is to provide a group-specifics
DdPCR primer and probe.
It is a further object to provide it is a kind of it is easy to operate, the accurate II type RT-ddPCR of norovirus of result is fast
Fast immue quantitative detection reagent box.
To achieve the above object, the invention adopts the following technical scheme:
The present invention chooses its parting gene segment RdRp-VP1 by the composition of genome of analysis II type of norovirus, if
Specific primer and probe are counted out, the sequence of the described group of primer and probe for detecting II type of norovirus is shown in Table 1.Wherein,
Probe 5 ' flag F AM, 3 ' label BHQ.
1 RT-ddPCR primer probe sequence of table
The present invention also provides a kind of kits of accurate quantification detection II type of norovirus, which includes above-mentioned be used for
Detect the primer and probe of II type of norovirus.Further, which further includes completing RT-ddPCR reaction material requested
And reagent, such as II type positive control RNA of norovirus, one-step RT-ddPCR supermix, acetolase, droplet generation
Oil, droplet generate card, 96 orifice plates and aluminium foil heat-sealing film etc., above-mentioned material and the preferably independent packaging of reagent.
The present invention also provides a kind of II type of norovirus precisely quantitative detection method, i.e. RT-ddPCR method, packets
Include the following contents:
1. extracting sample to be tested RNA, traditional Trizol method extracting method can be used or commercial kit extracts；
2. preparing RT-ddPCR reaction system, wherein the reaction system includes SEQ ID NO.1 to SEQ in sequence table
Primer and probe shown in ID NO.3.In one embodiment of the invention, RT-ddPCR reaction system such as table 2 institute
Show；
2 norovirus of table, II type ddPCR reaction system
3. RT-ddPCR reaction system and droplet that step 2. is prepared generate oil and be added in droplet generation card, it is placed in droplet
It generates in instrument and generates droplet；
4. carrying out amplification reaction, reaction condition is shown in Table 3；
3 norovirus of table, II type RT-ddPCR reaction condition
Note: warming and cooling rate answers≤2.5 DEG C/s
5. result judgement.
96 orifice plates after amplification are placed in droplet to read in instrument (QX200, Bio-Rad, Pleasanton, CA), are utilized
QuantaSoft software carries out result reading and analysis.Positive droplet containing amplified production and the feminine gender without amplified production are micro-
Drop can show the difference of fluorescence signal intensity, carry out given threshold line as boundary using the highest point of negative droplet cluster fluorescence amplitude.It presses
It is calculated according to Poisson distribution principle and obtains II type target fragment content of norovirus 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 the essence for II type of norovirus in sample that obtains as follows
Certainly measure.
The invention has the advantages that 1) caused by avoiding the absolute quantitation of nucleic acid molecules by PCR amplification efficiency variance
Deviation；2) without relying on certified reference material or other standards product；3) there is higher accuracy, sensitivity and repeatability, easily
In standardization；4) it is more suitable for the detection of nucleic acids of low copy number；5) since RT-ddPCR is end point determination, to the tolerance level of inhibitor
It is higher, therefore the detection error caused by can reducing by matrix type.
The invention will be further described with specific embodiment with reference to the accompanying drawings of the specification, all to disclose according to the present invention
The equivalent replacement of any this field that content is done, all belongs to the scope of protection of the present invention.
Detailed description of the invention
II type target fragment RT-ddPCR testing result of norovirus under the conditions of Fig. 1 different annealing temperature.
Fig. 2 RT-ddPCR kit detects II type specificity of norovirus and analyzes result.
Fig. 3 RT-ddPCR kit detects II type sensitivity analysis result of norovirus.
Fig. 4 RT-ddPCR kit detects II type repeatability of norovirus and analyzes result.
Specific embodiment
1 RT-ddPCR primer of embodiment, probe design
To realize that we choose RdRp-VP1 segment to the specific detection and absolute quantification analysis of II type of norovirus,
The segment is norovirus Genotyping segment, is located at norovirus full-length genome 4000bp-6500bp, online by NCBI
Tool carries out sequence analysis and comparison, is designed using Prime Express software V4.0 (ABI, Foster City, CA, USA)
More than 10 primer and probe is combined out, final obtains that high specificity, to be suitable for RT-ddPCR primer/probe combinations each by screening
1 set, sequence is shown in Table 1.Its middle probe 5 ' holds flag F AM, 3 ' end label BHQ.Primer/probe is limited by Beijing six directions promoting menstruation trade
Company's synthesis.
The foundation of 2 detection method of embodiment
(1) RNA is extracted: being extracted using commercial kit；Or RNA is extracted using tradition Trizol method, specifically
It operates as follows: 1mL Trizol being 1. added in 100 μ L samples, after shaking 30s, be placed at room temperature for 5min；2. 250 μ L chlorine are added
It is imitative, 30s is acutely shaken, 5min is placed at room temperature for；4 DEG C, 12000g, it is centrifuged 5min；3. supernatant is transferred in new centrifuge tube, it is added
The isopropanol of 500 μ L, which acutely shakes, mixes 30s, is placed at room temperature for 5min；4. 4 DEG C, 5000g, being centrifuged 5min；5. carefully removing
Clearly, 12000g after precipitating is cleaned with 70% ethyl alcohol of 1mL, 4 DEG C, centrifugation 5min (siphons away supernatant, centrifuge tube is placed in ultra-clean as far as possible
Precipitating is dried up on platform)；(in order to dissolve viral RNA preferably, 60 DEG C of heating are set without RNase water dissolution RNA 6. 50 μ L are added
10min).The RNA of extraction is saved backup in -80 DEG C.
(2) the RT-ddPCR reaction system of 20 μ L is prepared according to table 2
(3) droplet generates
The ddPCR reaction system of 20 μ L and 65 μ L droplets are generated oil to be added separately in 8 hole droplets generation card, are placed in micro-
Drop, which generates, generates droplet in instrument (QX200, Bio-Rad, Pleasanton, CA).
(4) amplified reaction
The droplet (40 μ L) of the Water-In-Oil of generation is slowly transferred in 96 orifice plates, sealer is placed on PCR instrument (Veriti
Thermal cycler, Applied BioSystems, Foster City, CA) on carry out amplification reaction, amplification condition such as table 3
It is shown.
(5) result judgement
96 orifice plates after amplification are placed in droplet to read in instrument (QX200, Bio-Rad, Pleasanton, CA), are utilized
QuantaSoft software carries out result reading and analysis.Positive droplet containing amplified production and the feminine gender without amplified production are micro-
Drop can show the difference of fluorescence signal intensity, can the highest point of negative droplet cluster fluorescence amplitude be that boundary carrys out given threshold line.
It is calculated according to Poisson distribution principle and obtains II type target fragment content of norovirus in RT-ddPCR reaction system, and then passed through
RNA template sample-adding amount (2 μ L) and the RNA template body product extracted, calculate obtain II type of norovirus in sample as follows
It is accurate quantitative.
The present invention is also optimized RT-ddPCR testing conditions, such as annealing temperature, takes the norovirus II of extraction
Type RNA is carried out amplification reaction under 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C and 70 DEG C of annealing temperature, as template than less respectively
The testing result of RT-ddPCR under the conditions of.As seen from Figure 1, under the conditions of 50 DEG C II type target gene fragment of norovirus amplification
Efficiency is relatively low, and positive droplet and negative droplet boundary are unobvious；Under the conditions of 55 DEG C and 60 DEG C target gene fragment amplification efficiency compared with
Height may occur in which obvious positive droplet cluster, and the fluorescence signal expanded under the conditions of 55 DEG C is slightly above 60 DEG C, therefore, by 55
DEG C be used as optimum annealing temperature.
3 kit forms of embodiment
1. the composition (being stored in -20 DEG C) of kit
(1) the primer and probe SEQ ID No.1-3 for being used to detect II type of norovirus that embodiment 1 designs, by Beijing
The six directions leads to Trade Co., Ltd.'s synthesis；
(2) U.S. BioRad company, article No. 186- one-step RT-ddPCR supermix, 25mM magnesium acetate: are purchased from
3021；
(3) droplet generates oil: being purchased from U.S. BioRad company, article No. 186-3030；
(4) droplet generates card: being purchased from U.S. BioRad company, article No. 186-4007；
(5) U.S. BioRad company, article No. 181-4000 aluminium foil heat-sealing film: are purchased from；
(6) Eppendorf company, Germany, article No. 0,030,128,605 96 orifice plate of Twin Tec Semi-Skirted: are purchased from；
(7) negative control: DEPC water；Purchased from the raw work in Shanghai, article No.: D1005；
(8) positive control: II type RNA standard substance of norovirus；
(9) DEPC water: purchased from the raw work in Shanghai, article No.: D1005.
2. prepared by II type RNA standard substance of norovirus
Using II type RNA of norovirus as template, using primer SEQ ID No.1-2, the specific piece of about 65bp is amplified
Disconnected, referred to as G II (expands target gene comprising RT-ddPCR)；(II carrier of pcDNA is purchased from II-G II of construction recombination plasmid pcDNA
Invitrogen company), sequencing is carried out in Shanghai Sangon Biotech Company, and by norovirus II in sequencing result and GenBank
The gene order of type carries out homology analysis, and homology is up to 100%.Simultaneously plasmid DNA purification is extracted, with Promega company
Ribo MAXTMLarge Scale RNA Production system-T7 kit (article No.: P1300) is transcribed in vitro
(carrying out by kit operational manual) is transcribed in vitro product and is digested with DNase, remove DNA therein.Utilize RNeasy
CRNA is further purified in MiniElute Cleanup kit (being purchased from QIAGEN company, article No. 74204).The cRNA of purifying is in upper
Hai Shenggong company carry out sequencing, and by sequencing result in GenBank II type of norovirus gene order progress it is homologous
Property analysis, homology is up to 100%.CRNA is dispensed, is stored in -80 DEG C.To the II type RNA standard substance of norovirus of preparation
Uniformity, stability test are carried out, related request is met；Using the laboratory Duo Jia valued methods to the standard substance of preparation into
Row definite value research, the II type RNA standard substance concentration of norovirus finally prepared are as follows: 5.93 × 104copies/μl.- 80 DEG C of guarantors
It deposits, the positive control as kit
4 detection method of embodiment specificity and sensitivity evaluation
1. detection specificity analysis
(1) material: rotavirus, astrovirus, II type of norovirus and II type RNA of norovirus are pre- by Chinese disease
Anti- control centre's virosis is provided.
(2) method: kit of the present invention is used, includes II type of norovirus, starlike disease to common diarrhea virus
Poison, II type of norovirus and II type RNA of norovirus carry out RT-ddPCR augmentation detection, and observing the kit, whether there is or not non-specificity
The generation of reaction.
(3) result: rotavirus, astrovirus, II type of norovirus and II type RNA of norovirus are through PCR instrument (Veriti
Thermal cycler, Applied BioSystems, Foster City, CA) amplification after, using droplet read instrument (QX200,
Bio-Rad, Pleasanton, CA) detection, result reading and analysis are carried out using QuantaSoft software.The result shows that only
There is positive droplet in II type of norovirus, remaining virus is negative droplet, it was demonstrated that this method has good specificity.As a result
See Fig. 2.
2. detection sensitivity is analyzed
(1) material: II type RNA standard substance of norovirus prepared above carries out 10 times of gradient dilutions to 5.93 copies/μ
L。
(2) method: each dilution gradient takes 2 μ L, carries out RT-ddPCR detection using kit described in embodiment 3, passes through
The minimum that kit of the present invention can be detected is analyzed, reaction system is shown in Table 2.
(3) result: discovery utilizes kit of the present invention, and the II type RNA of norovirus of 5.93 copies/μ L can be detected
Standard.As a result see Fig. 3.It can be seen that this kit detection II type of norovirus can reach 5.93 copies/μ L, promise such as disease is substantially increased
The sensitivity of malicious II type related detecting method, does not depend on standard items, it can be achieved that absolute quantitation, visual result are reliable.
5 detection method accuracy of embodiment and reproducibility
1. detection accuracy is analyzed
Accuracy (truness) refers to the consistency journey between the average value and acceptable reference value of one group of test result
Degree.According to the standard of European Union and Codex, the criterion of accuracy is the measured value Ying Can in entirely detection dynamic range
It examines in ± 25% range of value.
(1) material: II type of norovirus prepared above detects RNA positive control (5.93 × 104Copies/ μ l) respectively
It is diluted to 5.93 × 103copies/μl、5.93×102copies/μl、5.93×101Copies/ μ l and 5.93copies/ μ l,
It is expressed as sample U1-U4 (table 4).
(2) method: each sample carries out RT-ddPCR detection respectively, and each sample sets 7 repetitions, while 1 blank is arranged
Control (go DEPC water to replace template ribonucleic acid).
(3) result: the RT-ddPCR measured value of II type RNA of norovirus is close with predicted value, the equal < of standard deviation
15% (table 4), the criterion less than 25% illustrate that this method has extraordinary accuracy.
The accuracy of 4 norovirus of table, II type RT-ddPCR detection
2. detection repeatability analysis
(1) method: II type RNA positive control (5.93 × 10 of norovirus prepared above is utilized4Copies/ μ l) dilution
To 5.93 × 104Copies/ μ l carries out RT-ddPCR detection, if 9 repetitions, while 1 blank control is set (to remove DEPC
Water replaces template ribonucleic acid).
(2) result: from fig. 4, it can be seen that the measurement coefficient of variation CV of the II type RNA positive control of norovirus to certain concentration
Less than 25%, it was demonstrated that this method is used to have good repeatability when II type quantitative detection of norovirus.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention.For those of ordinary skill in the art, may be used also on the basis of the above description
To make other variations or changes in different ways.Here all embodiments can not be exhaustive.It is all to belong to this hair
The obvious changes or variations that bright technical solution is extended out are still in the scope of protection of the present invention.
Claims (2)
1. a kind of kit of accurate quantification detection II type of norovirus, which is characterized in that the kit includes: for detecting
Primer and probe, the II type positive control RNA of norovirus, one-step RT-ddPCR of II type RNA of norovirus
Supermix, magnesium acetate, droplet generate oil, droplet generates card, 96 orifice plates and aluminium foil heat-sealing film；
Wherein, the primer for detecting II type RNA of norovirus is nucleotide shown in SEQ ID No.1 to SEQ ID No.2
Sequence, the probe for detecting II type RNA of norovirus is nucleotide sequence shown in SEQ ID No.3, and the end of probe 5 ' is marked
Remember FAM, 3 ' end label BHQ.
2. a kind of detection method of non-diagnostic purpose accurate quantification detection II type of norovirus, which is characterized in that this method comprises:
(1) sample to be tested RNA is extracted；
(2) RT-ddPCR reaction system is prepared, wherein the reaction system includes shown in SEQ ID No.1 to SEQ ID No.2
For detecting the primer of II type RNA of norovirus, for detecting the spy of II type RNA of norovirus shown in SEQ ID NO.3
Needle, and probe 5 ' holds flag F AM, 3 ' end label BHQ；
(3) the RT-ddPCR reaction system and droplet prepared step (2) generate oil and are added in droplet generation card, and it is raw to be placed in droplet
Cheng Yizhong generates droplet；
(4) it carries out amplification reaction；
(5) result judgement calculates the copy number for obtaining II type RNA of norovirus according to Poisson distribution principle, and then by as follows
Formula calculates the content for obtaining II type of norovirus in sample to be tested:
II type content of norovirus in sample=RNA template
Volume number,
Wherein, RNA template sample-adding amount is 2 μ L.
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| CN107083327A (en) * | 2017-06-29 | 2017-08-22 | 浙江大学 | Determine kit, method and the application of the two of the gene copy numbers of DEFA1 3 |
| CN108034759A (en) * | 2017-11-06 | 2018-05-15 | 北京出入境检验检疫局检验检疫技术中心 | A kind of GII type norovirus detection of nucleic acids standard substance and its preparation method and application |
| CN113265487A (en) * | 2021-04-07 | 2021-08-17 | 拱北海关技术中心 | Aquatic product norovirus GI type and GII type combined quantitative detection kit |
| CN113215327A (en) * | 2021-06-25 | 2021-08-06 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Primer probe group, kit and method for detecting G II type norovirus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131798A (en) * | 2013-02-25 | 2013-06-05 | 湖北朗德医疗科技有限公司 | Norovirus real-time fluorescent RT-PCR detection kit and application thereof |
| CN105555972A (en) * | 2013-07-25 | 2016-05-04 | 伯乐生命医学产品有限公司 | Genetic assays |
-
2016
- 2016-08-04 CN CN201610632899.8A patent/CN106222301B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131798A (en) * | 2013-02-25 | 2013-06-05 | 湖北朗德医疗科技有限公司 | Norovirus real-time fluorescent RT-PCR detection kit and application thereof |
| CN105555972A (en) * | 2013-07-25 | 2016-05-04 | 伯乐生命医学产品有限公司 | Genetic assays |
Non-Patent Citations (2)
| Title |
|---|
| Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria,Cylindrospermopsis and Microcystis;Shu Harn Te et al.;《Applied and Environmental Microbiology》;20150529;第81卷(第15期);第5203-2511页 * |
| Microfluidic Quantitative PCR for Simultaneous Quantification of Multiple Viruses in Environmental Water Samples;Satoshi Ishii et al.;《Applied and Environmental Microbiology》;20140926;第80卷(第24期);第7505-7511页 * |
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