CN107099613A - A kind of primer combination of probe and detection method for being used to detect real-time fluorescent RCR ulcer bacteria - Google Patents
A kind of primer combination of probe and detection method for being used to detect real-time fluorescent RCR ulcer bacteria Download PDFInfo
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- CN107099613A CN107099613A CN201710511735.4A CN201710511735A CN107099613A CN 107099613 A CN107099613 A CN 107099613A CN 201710511735 A CN201710511735 A CN 201710511735A CN 107099613 A CN107099613 A CN 107099613A
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses a kind of primer combination of probe and detection method for being used to detect real-time fluorescent RCR ulcer bacteria.The invention provides a kind of primer pair, it is made up of primers F and primer R；The primers F is as shown in the sequence 1 of sequence table；The primer R is as shown in the sequence 2 of sequence table.The present invention also protects primer combination of probe, is made up of the primer pair and probe；The probe is as follows：Section A (dT fluorophors) tetrahydrofuran residue (dT quenchers) section B；Modified at the 3' ends of the probe；The section A is as shown in the sequence 3 of sequence table；The section B is as shown in the sequence 4 of sequence table.The method that the present invention has also set up detection real-time fluorescent RCR ulcer bacteria, whole detection process is in 1 hour, and the characteristics of its is quick, specific and sensitive can meet the quick initial survey of inward rapeseed sample and the Rapid identification of germ isolate.
Description
Technical field
The present invention relates to agricultural and technical field of plant quarantine, and in particular to one kind is used to detect real-time fluorescent RCR ulcer bacteria
Primer combination of probe and detection method.
Background technology
Real-time fluorescent RCR canker is one of fungal disease of most serious on rape, and its pathogenic bacteria is the rape of Leptosphaeria
Stem foot ulcer bacteria (Leptosphaeria maculans), the germ pathogenicity is strong, and real-time fluorescent RCR portion can be caused to fester,
The rape underproduction can be caused to reach 30%-50%, even more high when serious.Real-time fluorescent RCR ulcer bacteria is in Australia, Canada, U.S.
The rape main production country such as state, Europe is found, and has no report in China, belongs to the inward plant quarantine harmful organism of China.Closely
Nian Lai is more in China's entry and exit inspection and quarantine bureau enters the territory rapeseed from states such as import Australia, Ukraine and Canada
Secondary intercepting and capturing stem foot ulcer bacteria L.maculans.China rape producing region weather is similar to Australia, the U.S., European three big states,
And the current rape Main Cultivars of China are highly susceptible.In addition, with trade globalization, the incoming risk aggravation of germ.
To prevent the incoming of real-time fluorescent RCR ulcer, efficient quick detection turns into the incoming Chinese technology of prevention real-time fluorescent RCR canker and closed
Key.
Being presently used for the detection method of oil base stem foot ulcer bacteria mainly has filter paper cultivation, PCR method and ring mediation etc.
Temperature amplification (Loop-Mediated Isothermal Amplification, LAMP) detection method.Standard PCR has to pass through change
Property, annealing, extension three steps, the used time was at 90 minutes or so.LAMP amplification methods are directed to 6 different zones of target-gene sequence
4 species specificity LAMP primers are designed, are incubated using a kind of Bst archaeal dna polymerases of strand-displacement activity in isothermy (63 DEG C or so)
30-60min is educated, amplification is completed.Recombinase-mediated isothermal duplication (RPA) technology is considered as that can replace PCR detection of nucleic acids
Technology.RPA technologies depend on three kinds of enzymes：The recombinase of single-chain nucleic acid (Oligonucleolide primers), single stranded DNA can be combined to combine
Albumen (SSB) and strand displacement archaeal dna polymerase.The mixture of these three enzymes is also active at normal temperatures, and optimum temperature is at 37 DEG C -42
Between DEG C, without denaturation, it can carry out at normal temperatures.This can undoubtedly greatly speed up nucleic acid amplification speed.The sensitivity of RPA detections
It is very high, it can be obtained greatly from single template molecule by nucleic acid (especially DNA) template amplification of trace to the level that can be detected
About 1012 amplified productions.And RPA does not need the sample treatment of complexity also, it is adaptable to the detection on the spot of nucleic acid can not be extracted.This
Outside, due to not needing temperature control device, RPA can really realize portable Rapid nucleic acid detection.
The content of the invention
It is an object of the invention to provide a kind of primer combination of probe and detection side for being used to detect real-time fluorescent RCR ulcer bacteria
Method.
The invention provides a kind of primer pair, it is made up of primers F and primer R；
The primers F is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the substitution and/or missing and/or addition of one or several nucleotides and with sequence 1
The DNA molecular of identical function；
The primer R is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is had by the substitution and/or missing and/or addition of one or several nucleotides and with sequence 2
The DNA molecular of identical function.
The present invention also protects primer combination of probe, is made up of the primer pair and probe；
The probe is as follows：
Section A- (dT- fluorophors)-tetrahydrofuran residue-(dT- quenchers)-section B；
Modified at the 3' ends of the probe；
The section A is as shown in the sequence 3 of sequence table；
The section B is as shown in the sequence 4 of sequence table.
The fluorophor is FAM；The quenching group is BHQ1.
Described " being modified at the 3' ends of the probe " is to use C3-spacer or phosphate or biotin-TEG or amine
Modified.
The purposes of the primer pair or the primer combination of probe is at least one of following (b1)-(b4)：
(b1) identify or aid in identify whether strain to be tested is real-time fluorescent RCR ulcer bacteria；
(b2) whether real-time fluorescent RCR ulcer bacteria is contained in detection sample to be tested；
(b3) prepare for identify or aid in identify strain to be tested whether be real-time fluorescent RCR ulcer bacteria kit；
(b4) prepare for detect in sample to be tested whether the kit containing real-time fluorescent RCR ulcer bacteria.
The present invention also protects the application of the primer pair or the primer combination of probe, be in following (b1)-(b4) extremely
Few one kind：
(b1) identify or aid in identify whether strain to be tested is real-time fluorescent RCR ulcer bacteria；
(b2) whether real-time fluorescent RCR ulcer bacteria is contained in detection sample to be tested；
(b3) prepare for identify or aid in identify strain to be tested whether be real-time fluorescent RCR ulcer bacteria kit；
(b4) prepare for detect in sample to be tested whether the kit containing real-time fluorescent RCR ulcer bacteria.
The present invention also kit first of the protection containing the primer pair, or, the kit containing the primer combination of probe
Second；The purposes of the kit is following (c1) or (c2)：
(c1) identify or aid in identify whether strain to be tested is real-time fluorescent RCR ulcer bacteria；
(c2) whether real-time fluorescent RCR ulcer bacteria is contained in detection sample to be tested.
The present invention also protects the preparation method of the kit first or the kit second, including each bar primer is individually wrapped
The step of dress.
The present invention also protection identify strain to be tested whether be real-time fluorescent RCR ulcer bacteria method, comprise the following steps：With
The STb gene of strain to be tested is template, and recombinase-mediated isothermal duplication is carried out using the primer combination of probe, if at 6-20 points
Have that fluorescence signal rises, strain to be tested is or candidate is real-time fluorescent RCR ulcer bacteria between clock, if equal between 6-20 minute
There is no that fluorescence signal rises, strain to be tested is or candidate is non-real-time fluorescent RCR ulcer bacteria.
In the present invention also protection detection sample to be tested whether the method containing real-time fluorescent RCR ulcer bacteria, including following step
Suddenly：Using the STb gene of sample to be tested as template, recombinase-mediated isothermal duplication is carried out using the primer combination of probe, if
There are fluorescence signal rise, sample to be tested to contain or doubtful containing real-time fluorescent RCR ulcer bacteria between 6-20 minutes, if in 6-20
No fluorescence signal rises between minute, sample to be tested does not contain or doubtful do not contain real-time fluorescent RCR ulcer bacteria.
Strain to be tested described in any of the above concretely real-time fluorescent RCR ulcer bacteria, rape black shank bacterium, branch top spore bacterium or
Verticillium albo atrum reinke ＆ berthold.
The reaction system of recombinase-mediated isothermal duplication described in any of the above is concretely：The μ L of rehydration buffer solution 29.5,
Primers F and primer R each 2.1 μ L, probe P 0.6 μ L, DNA profiling (0.05-20ng), deionized water are mended to 47.5 μ L, after mixing
Add in the 0.2mL TwistAmp Basic reaction tubes containing lyophilized enzyme powder, fully dissolving, add magnesium acetate solution
(280mmol/L) 2.5 μ L, add a magnetic bead.
In reaction system, primers F, primer R, probe P are added in reaction system in working solution form, primers F, primer R
It is 10 μm of ol/L with concentration of the probe P in working solution.
In reaction system, the rehydration buffer solution, lyophilized enzyme powder and TwistAmp Basic reaction tube bacterium come fromKit.
It is describedKit is TwistDx companies, and article No. is TAEX002KIT product.
The operating method of recombinase-mediated isothermal duplication described in any of the above is concretely：39 DEG C are reacted 30 minutes.
Recombinase-mediated isothermal duplication described in any of the above specifically can using TwistDX T8 isothermal duplications instrument (TwistDX,
Britain) carry out.
The magnetic bead magnetic bead that concretely TwistDX T8 isothermal duplications instrument is carried.
The invention provides a kind of primer combination of probe and detection method for being used to detect real-time fluorescent RCR ulcer bacteria, experiment
As a result show, the real-time fluorescent RCR ulcer bacteria from states such as Canada is carried out using the primed probe and detection method of the present invention
Detection, can obtain positive amplification in 10 minutes, and other bacterial strains such as rape black shank bacterium do not have obvious fluorescence signal increase.
Whole detection process is in 1 hour, and the characteristics of its is quick, specific and sensitive can meet the quick first of inward rapeseed sample
Inspection and the Rapid identification of germ isolate.
Brief description of the drawings
Fig. 1 is specificity experiments result.
Fig. 2 is sensitivity experiment result.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetition experiments, as a result makes even
Average.
Kit：TwistDx companies, article No.：TAEX002KIT.
Strains tested information in embodiment is as shown in table 1.
The strains tested information of table 1
| Sequence number | Bacterial strain | Numbering | Acquiring way |
| 1 | Real-time fluorescent RCR ulcer bacteria | CBS275.63 | Fungal organism center of diversity (CBS) |
| 2 | Real-time fluorescent RCR ulcer bacteria | ATCC200782 | ATCC |
| 3 | Rape black shank bacterium | 9912-4 | Intercepted and captured in Italian import cabbage seed |
| 4 | Rape black shank bacterium | 9907-2 | Intercepted and captured in Italian import cabbage seed |
| 5 | Rape black shank bacterium | SS-13 | Chinese Jian Ke institutes Culture Collection |
| 6 | Rape black shank bacterium | CBS303.51 | Fungal organism center of diversity (CBS) |
| 7 | Rape black shank bacterium | SS-13-2 | Chinese Jian Ke institutes Culture Collection |
| 8 | Branch top spore bacterium | 9-7-4 | Chinese Jian Ke institutes Culture Collection |
| 9 | Rape black shank bacterium | CBS11951 | Fungal organism center of diversity (CBS) |
| 10 | Branch top spore bacteria strain 6a | ZS-5 | Chinese Jian Ke institutes Culture Collection |
| 11 | Verticillium albo atrum reinke ＆ berthold separates vaa2 | M1 | Chinese Jian Ke institutes Culture Collection |
Embodiment 1, the RPA primed probes for identifying real-time fluorescent RCR ulcer bacteria
According to quarantine real-time fluorescent RCR ulcer bacteria Leptosphaeria maculans ITS region sequences, set using primer
The Software for Design feasible RPA primers of multigroup theory (including three sense primers and three anti-sense primers) is counted, as shown in table 2.
The RPA candidate drugs of table 2
| F1 | 5'-AAGCACTGCCGCCTCGATCAGTGGCGGC-3' |
| F2 | 5'-ACTGCCGCCTCGATCAGTGGCGGCAGTCTAC-3' |
| F3 | 5'-CACTGCCGCCTCGATCAGTGGCGGCAGTC-3' |
| R1 | 5'-TTGCAAGTGGTTTTAGGGGATCCAATTGGTG-3' |
| R2 | 5'-AATTGCAAGTGGTTTTAGGGGATCCAATTGGTGGG-3' |
| R3 | 5'-CAATTGCAAGTGGTTTTAGGGGATCCAATTGGTGGGC-3' |
By the middle and upper reaches primer of table 1 and primer composition various combination (F1/R1, F1/R2, F1/R3, F2/R1, F3/R1, F3/
R3 compliance test result, sensitivity analysis and specificity analysis) are carried out, wherein F1/R1, F1/R2, F1/R3 detection is unsatisfactory for specificity
It is required that, not only detect that oil base stem foot ulcer bacteria produces positive findings, detect that other bacterium (such as rape black shank bacterium) can also produce
Positive findings.In remaining primer, F2/R1, F3/R1, F3/R3 are satisfied by specific requirements, but F2/R1 Detection results and sensitivity
It is optimal.
By comprehensive analysis, a pair of optimal RPA primers for identifying oil base stem foot ulcer bacteria, following institute are filtered out
Show：
F (sequence 1 of sequence table)：5'-ACTGCCGCCTCGATCAGTGGCGGCAGTCTAC-3'；
R (sequence 2 of sequence table)：5'-TTGCAAGTGGTTTTAGGGGATCCAATTGGTG-3'.
The exo probes for identifying oil base stem foot ulcer bacteria are further devised, it is as follows：
P:ACTGCCGCCTCGATCAGTGGCGGCAGTCTAC(FAM-dT)(THF)(BHQ1-dT)
GATTCTGCCCATGT-C3spacer
THF represents tetrahydrofuran residue, sometimes referred to as " dSpacer ".
In practical application, the C3-spacer of the 3' ends of exo probes also can be replaced phosphate, biotin-TEG or amine
Deng modification group.
The foundation of embodiment 2, RPA detection methods
1st, the STb gene of strain to be tested is extracted.
2nd, the STb gene obtained using step 1 is template, and primers F, primer R and the probe P prepared using embodiment 1 carries out RPA
Reaction；Concrete operations are as follows：
Reaction system：Take rehydration buffer solution (Kit is carried) 29.5 μ L, primers F and primer
R each 2.1 μ L, probe P 0.6 μ L, DNA profiling (0.05-20ng), deionized water is mended to 47.5 μ L, is added to after mixing containing lyophilized
Enzyme powder (Kit is carried) 0.2mL TwistAmp Basic reaction tubes (
Kit is carried) in, fully dissolving adds magnesium acetate solution (280mmol/L) 2.5 μ L.
In reaction system, primers F, primer R, probe P are added in reaction system in working solution form, primers F, primer R
It is 10 μm of ol/L with concentration of the probe P in working solution.
A magnetic bead (TwistDX T8 isothermal duplication instrument is carried) is added into reaction system, TwistDX T8 etc. are positioned over
In warm amplification instrument (TwistDX, Britain), 39 DEG C are reacted 30 minutes, are recorded the fluorescence signal of amplified production and are made the following judgment：
If between 6-20 minutes, there is fluorescence signal rise, then the strain to be tested is or candidate is that real-time fluorescent RCR is burst
Ulcer germ；If unstressed configuration signal rises between 6-20 minutes, the strain to be tested is or candidate is non-real-time fluorescent RCR ulcer
Germ.
Embodiment 3, specificity
Two kinds of detection methods of the foundation of embodiment 2 are respectively adopted to the strains tested shown in table 1 and oil base stem foot has been infected
The rapeseed pod shell of ulcer is detected.
As a result it is as shown in Figure 1.In Fig. 1,1 is the real-time fluorescent RCR ulcer bacteria bacterial strain of serial number 1 in table 1, and 2 be sequence in table 1
Number real-time fluorescent RCR ulcer bacteria bacterial strain for being 2,3 be to have infected the rapeseed pod shell of oil base stem foot ulcer to adopt indicated by the solid line, 4-
12 are followed successively by the bacterial strain of serial number 3-11 in table 1.
As a result show, only real-time fluorescent RCR ulcer bacteria bacterial strain and the rapeseed pod shell for having infected oil base stem foot ulcer
DNA sample had fluorescence signal rise between 6-20 minutes when detecting, and at 6-20 minutes during the detection of the DNA sample of remaining bacterial strain
Between without fluorescence signal rise.
The above results show that method of the invention has higher specificity.
Embodiment 4, sensitivity
1st, the STb gene of the real-time fluorescent RCR ulcer bacteria bacterial strain of serial number 1 in table 1 is extracted, DNA concentration is obtained for 2.16ng/ μ
L DNA solution；
2nd, the DNA solution obtained with 10 times of gradient dilution steps 1 of water, obtains each dilution；
3rd, take each dilution that step 2 is arrived as template, two kinds of detection methods of the foundation of embodiment 2 are respectively adopted；
Because the dilution factor of the dilution of use is different, following different reaction system is formed：
In reaction system 1, bacterial strain STb gene initial content is：2.16ng；
In reaction system 2, bacterial strain STb gene initial content is：0.216ng；
In reaction system 3, bacterial strain STb gene initial content is：0.0216ng；
In reaction system 4, bacterial strain STb gene initial content is：0.00216ng.
As a result it is as shown in Figure 2.In Fig. 2,1-4 is corresponding in turn to reaction system 1-4.As a result show, the PRA inspections that the present invention is set up
Survey method can successfully be detected 0.0216ng genomic DNA templates.
<110>China Inst. of Quarantine Inspection Sciences
<120>A kind of primer combination of probe and detection method for being used to detect real-time fluorescent RCR ulcer bacteria
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ttgcaagtgg ttttagggga tccaattggt g 31
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Claims (9)
1. primer pair, is made up of primers F and primer R；
The primers F is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by the substitution of one or several nucleotides of the process of sequence 1 and/or missing and/or addition and with sequence 1 identical
The DNA molecular of function；
The primer R is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) have by the substitution of one or several nucleotides of the process of sequence 2 and/or missing and/or addition and with sequence 2 identical
The DNA molecular of function.
2. primer combination of probe, is made up of the primer pair and probe described in claim 1；
The probe is as follows：
Section A- (dT- fluorophors)-tetrahydrofuran residue-(dT- quenchers)-section B；
Modified at the 3' ends of the probe；
The section A is as shown in the sequence 3 of sequence table；
The section B is as shown in the sequence 4 of sequence table.
3. primer combination of probe as claimed in claim 2, it is characterised in that：The fluorophor is FAM；The quenching group
For BHQ1.
4. primer combination of probe as claimed in claim 2 or claim 3, it is characterised in that：It is described " to be repaiied at the 3' ends of the probe
Decorations " are to be modified using C3-spacer or phosphate or biotin-TEG or amine.
5. the application of any described primer combination of probe of primer pair or claim 2-4 described in claim 1, is as follows
(b1) at least one of-(b4)：
(b1) identify or aid in identify whether strain to be tested is real-time fluorescent RCR ulcer bacteria；
(b2) whether real-time fluorescent RCR ulcer bacteria is contained in detection sample to be tested；
(b3) prepare for identify or aid in identify strain to be tested whether be real-time fluorescent RCR ulcer bacteria kit；
(b4) prepare for detect in sample to be tested whether the kit containing real-time fluorescent RCR ulcer bacteria.
6. the kit first containing primer pair described in claim 1, or, contain any primed probe groups of claim 2-4
The kit second of conjunction；The purposes of the kit is following (c1) or (c2)：
(c1) identify or aid in identify whether strain to be tested is real-time fluorescent RCR ulcer bacteria；
(c2) whether real-time fluorescent RCR ulcer bacteria is contained in detection sample to be tested.
7. the preparation method of kit first or kit second described in claim 6, including the step of each bar primer is individually packed.
8. identify strain to be tested whether be real-time fluorescent RCR ulcer bacteria method, comprise the following steps：With the STb gene of strain to be tested
For template, recombinase-mediated isothermal duplication is carried out using any described primer combination of probe of claim 2-4, if in 6-20
Have that fluorescence signal rises, strain to be tested is or candidate is real-time fluorescent RCR ulcer bacteria between minute, if between 6-20 minutes
No fluorescence signal rises, strain to be tested is or candidate is non-real-time fluorescent RCR ulcer bacteria.
9. detect sample to be tested in whether the method containing real-time fluorescent RCR ulcer bacteria, comprise the following steps：With sample to be tested
STb gene is template, and recombinase-mediated isothermal duplication is carried out using any described primer combination of probe of claim 2-4, if
There are fluorescence signal rise, sample to be tested to contain or doubtful containing real-time fluorescent RCR ulcer bacteria between 6-20 minutes, if in 6-
No fluorescence signal rises between 20 minutes, sample to be tested does not contain or doubtful do not contain real-time fluorescent RCR ulcer bacteria.
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