CN107641660A - A kind of LAMP HNB primer sets, kit and method for being used to detect real-time fluorescent RCR ulcer bacteria - Google Patents
A kind of LAMP HNB primer sets, kit and method for being used to detect real-time fluorescent RCR ulcer bacteria Download PDFInfo
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Abstract
A kind of application the invention provides LAMP HNB primer sets and its in the product of detection real-time fluorescent RCR ulcer bacteria or preparation detection real-time fluorescent RCR ulcer bacteria, present invention also offers a kind of kit and its application for being used to detect real-time fluorescent RCR ulcer bacteria, a kind of method for detecting real-time fluorescent RCR ulcer bacteria still further provides based on this present invention.It the experiment proved that, the present invention LAMP HNB primers group-specifics are good, high sensitivity, detection time are short, and the detection method for the real-time fluorescent RCR ulcer bacteria established based on the LAMP HNB primer sets, can it is inexpensive, sensitive, accurate, easy and it is quick detection real-time fluorescent RCR ulcer bacteria whether there is or sample in whether contain real-time fluorescent RCR ulcer bacteria nucleic acid, greatly improve detection efficiency, more effective way is provided for the detection of real-time fluorescent RCR ulcer bacteria, and to passing in and out corresponding plants and examination and test of products quarantine has directive significance.
Description
Technical field
The invention belongs to biological technical field, and in particular to the LAMP-HNB primer sets of detection real-time fluorescent RCR ulcer bacteria,
Kit and method.
Background technology
Real-time fluorescent RCR canker is one of fungal disease of rape most serious, and its pathogenic bacteria is the rape stem of Leptosphaeria
Base ulcer bacteria (Leptosphaeria maculans), the germ pathogenicity is strong, real-time fluorescent RCR portion can be caused to fester, sternly
During weight the rape underproduction can be caused to reach 30%-50%, even more high.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 bacteria, establish a kind of accuracy is high, the strong and simple and rapid detection method of sensitiveness will be into
One of key factor for being passed to China for prevention real-time fluorescent RCR canker.
The method of detection real-time fluorescent RCR ulcer bacteria is mainly two methods of quantitative fluorescent PCR and regular-PCR at present, but general
Logical PCR detection complex operations and time-consuming, at the same need the expensive laboratory apparatus such as PCR instrument, gel electrophoresis and imaging system with
And molecular biology professional operator, applicability limitation is larger, and real-time fluorescence RT-PCR technology needs expensive detection
Equipment, testing cost is higher, is not suitable for basic unit and small-size laboratory is promoted the use of.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of inexpensive, sensitive, accurate, easy and quick detection rape
The LAMP-HNB methods of stem foot ulcer bacteria, present invention also offers when the specific good, high sensitivity for this method, detection
Between it is short, do not need special instrument and LAMP-HNB primer sets applied widely and the reagent containing the LAMP-HNB primer sets
Box.
In order to solve the above problems, the present invention is achieved through the following technical solutions：
First aspect present invention provides a kind of LAMP-HNB primer sets for being used to detect real-time fluorescent RCR ulcer bacteria, and this draws
Thing group includes outer primer pair and inner primer pair, and described outer primer is to for the nucleotide sequence and SEQ shown in SEQ ID NO.1
Nucleotide sequence shown in ID NO.2, described inner primer is to for the nucleotide sequence and SEQ ID shown in SEQ ID NO.3
Nucleotide sequence shown in NO.4.
Second aspect of the present invention provides described LAMP-HNB primer sets and is preparing detection real-time fluorescent RCR ulcer bacteria
Application in product.
Third aspect present invention provides a kind of kit for being used to detect real-time fluorescent RCR ulcer bacteria, including above-mentioned
LAMP-HNB primer sets.
In a preference, in addition to indicator.
In a preference, described indicator is hydroxynaphthol blue.
In a preference, described hydroxynaphthol blue is that the article No. from Sigma companies is in H991331 kits
Reagent.
In a preference, in addition to archaeal dna polymerase.
In a preference, described archaeal dna polymerase is Bst archaeal dna polymerases.
In a preference, described Bst archaeal dna polymerases are that the article No. from NEB companies is in M0538S kits
Reagent.
In a preference, in addition to isothermal amplification buffer solution, dNTPs, contain Mg2+Soluble compound.
In a preference, described contains Mg2+Soluble compound be MgSO4。
In a preference, the isothermal amplification buffer solution, MgSO4It is M0538S for the article No. from NEB companies
Reagent in kit.
In a preference, in addition to positive control.
In a preference, in addition to negative control.
In a preference, in addition to nucleic acid extracting reagent.
In a preference, described nucleic acid extracting reagent is that the article No. of TIANGEN Biotech (Beijing) Co., Ltd. is
Reagent in DP305 DNA extraction kit.
Fourth aspect present invention provides a kind of kit and burst in detection real-time fluorescent RCR ulcer bacteria or containing real-time fluorescent RCR
Application in ulcer germ nucleic acid samples.
Fifth aspect present invention provides a kind of the real-time fluorescent RCR ulcer bacteria for being used to detect in sample or real-time fluorescent RCR and burst
The method of ulcer germ nucleic acid, the step of including the use of above-mentioned LAMP-HNB primer sets or kit.
In a preference, described method also include obtain sample nucleic acid the step of.
In a preference, described nucleic acid is DNA or RNA.
In a preference, described DNA is to be by using the article No. of TIANGEN Biotech (Beijing) Co., Ltd.
Method in DP305 DNA extraction kit obtains.Optional, when described nucleic acid is RNA, in addition to RNA reverse transcriptions are
The step of cDNA.
In a preference, described method, which is additionally included in detection sample nucleic acid, adds the progress of LAMP-HNB reaction reagents
The step of LAMP-HNB amplified reactions.
In a preference, described LAMP-HNB reaction reagents include archaeal dna polymerase, isothermal amplification buffer solution,
DNTPs and contain Mg2+Soluble compound.
In a preference, described contains Mg2+Soluble compound be MgSO4.
In a preference, the isothermal amplification buffer solution, MgSO4It is M0538S for the article No. from NEB companies
Reagent in kit.
In a preference, described archaeal dna polymerase is Bst archaeal dna polymerases.
In a preference, described Bst archaeal dna polymerases are that the article No. from NEB companies is in M0538S kits
Reagent.
In a preference, described method also include LAMP-HNB amplified reactions before add indicator the step of.
In a preference, described indicator is hydroxynaphthol blue.
In a preference, described hydroxynaphthol blue is that the article No. from Sigma companies is in H991331 kits
Reagent.Optional, the upstream and downstream primer of the outer primer pair is set to F3 and B3, the upstream and downstream primer of described inner primer pair
FIP and BIP are set to, final concentration relation of four kinds of primers in LAMP-HNB amplified reactions is：([F3]=[B3]):
([FIP]=[BIP])=1:8.
In a preference, the described four kinds of final concentrations of primer in LAMP-HNB amplified reactions respectively [F3]=
[B3]=0.2 μm ol/L, [FIP]=[BIP]=1.6 μm ol/L.
In a preference, final concentration of 150 μm of ol/ in LAMP-HNB amplified reactions of described hydroxynaphthol blue
L。
In a preference, described method is also included pattern detection result and positive control and/or negative control knot
The step of fruit is compared, when testing result is identical with the result that positive control detects, the sample for illustrating to detect contains rape stem
Base ulcer bacteria or containing real-time fluorescent RCR ulcer bacteria nucleic acid, when testing result is identical with the result that negative control detects, explanation
The sample of detection is without real-time fluorescent RCR ulcer bacteria or does not contain real-time fluorescent RCR ulcer bacteria nucleic acid.
In a preference, described positive control testing result is sky blue, and negative control result is light blue.
In a preference, the reaction system of methods described is as follows：
In a preference, the response procedures of methods described are：Reacted 80 minutes at 62 DEG C.
The indicator being related in the present invention may be selected from SYBR Green I, EvaGreen, PicoGreen, Peko Green,
One kind in propidium iodide, calcein or hydroxynaphthol blue.
" detection sample " in the present invention can be plant tissue, fungi, may contain the other of real-time fluorescent RCR ulcer bacteria
The tissue and nucleic acid in source.
" detection real-time fluorescent RCR ulcer bacteria " refers to detecting in sample whether contain real-time fluorescent RCR canker in the present invention
Bacterium or detection sample in whether the nucleic acid containing real-time fluorescent RCR ulcer bacteria.
The invention has the advantages that：
(1) present invention designs specific LAMP-HNB primer sets according to real-time fluorescent RCR ulcer bacteria ITS sequence, and is based on
LAMP-HNB primer sets establish the LAMP-HNB methods of detection real-time fluorescent RCR ulcer bacteria, can be achieved to real-time fluorescent RCR canker
The qualitative detection of bacterium.
(2) present invention for the design of real-time fluorescent RCR ulcer bacteria LAMP-HNB primers group-specific is good, high sensitivity and
Detection time is short.
(3) detection method for the real-time fluorescent RCR ulcer bacteria that the present invention establishes, can be inexpensive, sensitive, accurate, easy and fast
Speed detection real-time fluorescent RCR ulcer bacteria whether there is or sample in whether contain real-time fluorescent RCR ulcer bacteria nucleic acid, greatly carry
High detection efficiency, more effective way is provided for the detection of real-time fluorescent RCR ulcer bacteria, and to pass in and out corresponding plants and
Examination and test of products quarantine has directive significance.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings：
Fig. 1：LAMP-HNB testing results in embodiment 3.Wherein ,+：Group 2, real-time fluorescent RCR ulcer bacteria；-：Group 1, water
(negative control).
Fig. 2：The specific test testing result of LAMP-HNB primer sets in embodiment 4.Wherein+：Group 1, real-time fluorescent RCR is burst
Ulcer germ；-：Group 2, negative control；1：Group 3, the thin sickle-like bacteria of oat；2：Group 4, Alternaria；3：Group 5, Phoma；4：Group
6, sclerotinite；5：Group 7, rape black shank bacterium.
Fig. 3：The sensitivity test testing result of LAMP-HNB primer sets in embodiment 5.Wherein-：Group 1, negative control；
100:Group 2, add template 100ng；10:Group 3, add template 10ng；1:Group 4, add template 1ng；0.1:Group 5, add template
100μg；0.01:Group 6, add the μ g of template 10；0.001:Group 7, add the μ g of template 1.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail, it is necessary to which explanation, these embodiments are only
It is illustrative, and is not considered as limiting the invention.Unless otherwise specified, the technological means employed in embodiment is
Conventional meanses well-known to those skilled in the art, used reagent, product and instrument are also available commercial.It is not detailed
The various processes and method carefully described are conventional methods as known in the art, such as Sambrook equimolecular Cloning: A Laboratory Manuals
(Sambrook J&Russell DW,Molecular cloning:A laboratory manual, 2001), or according to manufacture
The condition of manufacturers instruction suggestion.
Real-time fluorescent RCR ulcer bacteria, the thin sickle-like bacteria of oat, Alternaria, Phoma, the core used in following examples
Cup fungi and rape black shank bacterium provide by Zhanjiang Entry-Exit Inspection and Quarantine Bureau.
Embodiment 1
Present embodiments provide a kind of LAMP-HNB primer sets for detecting real-time fluorescent RCR ulcer bacteria and its application.
First, the design of LAMP-HNB primer sets
According to real-time fluorescent RCR ulcer bacteria ITS sequence (GenBank ID:KT225526.1), a kind of LAMP-HNB is devised
Primer sets, include a pair of outer primers：F3 and B3, a pair of inner primers：FIP and BIP, particular sequence are as follows：
F3:5’-GTTCGAGCGTCATTTGTACC-3’(SEQ ID NO.1)；
B3:5’-AGTTCAGCGGGTATCCCTA-3’(SEQ ID NO.2)；
FIP:5’-TGCCGGCTGCCAATTGTTTCCTCTGCTTGGTGTTGGGT-3’(SEQ ID NO.3)；
BIP:5’-GCAGCACATTTTGCGCCTCTCTGATCCGAGGTCAAGAGC-3’(SEQ ID NO.4)
Above-mentioned primer is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd..
It is demonstrated experimentally that above-mentioned primer pair is used in combination high specificity, high sensitivity and repeatability very well, and use
The several LAMP-HNB primer pairs that primer-design software randomly selects combine then more or less presence specificity not
The various defects such as by force, sensitivity is not high and repeatability is bad.
Above-mentioned LAMP-HNB primers can be applied to prepare the product of detection real-time fluorescent RCR ulcer bacteria, for detecting rape
The sample of stem foot ulcer bacteria or detection containing real-time fluorescent RCR ulcer bacteria nucleic acid.
Embodiment 2
A kind of kit and its application are present embodiments provided, the kit includes：
(1) the LAMP-HNB primer sets in embodiment 1；
(2) HNB that the article No. from Sigma companies is H991331；
(3) 10 × isothermal amplification buffer solution that article No. from NEB companies is M0538S, Bst archaeal dna polymerases,
MgSO4；
(4) article No. from TIANGEN Biotech (Beijing) Co., Ltd. is the examination in DP305 DNA extraction kit
Agent.
It is demonstrated experimentally that the reagent of above-mentioned (1), (2), (3) and (4) is used in combination, effect is more superior, is embodied in province
When, economy, repeatability be high, high sensitivity and the characteristics of high specificity.And use the LAMP-HNB primer sets of (1) of the present invention
Combination with other some reagents randomly selected then more or less presence specificity is not strong, sensitivity is not high and it is repeated not
Good various defects.
Mentioned reagent box can be applied to detect real-time fluorescent RCR ulcer bacteria or detection contains real-time fluorescent RCR canker sclerotium
The sample of acid.
Embodiment 3
Present embodiments provide and whether deposited in a kind of method for detecting real-time fluorescent RCR ulcer bacteria, such as detection testing sample
In real-time fluorescent RCR ulcer bacteria or the nucleic acid with the presence or absence of real-time fluorescent RCR ulcer bacteria, the LAMP- of embodiment 1 the method use
The kit of HNB primer sets and embodiment 2.
Above-mentioned testing sample can be plant origin sample or non-plant origin sample.Described nucleic acid can be DNA,
RNA, cDNA or cDNA fragment, or be the reagent containing DNA, cDNA, RNA or cDNA fragment, or be plasmid.
When described sample contains RNA, also need to carry out reverse transcription to the sample, be translated into as DNA, cDNA or
CDNA fragments are detected again.
The above method comprises the following steps：
1st, DNA is extracted
With reference to kit specification operation (plant genome DNA extracts kit, article No. DP305, Tiangeng biochemical technology
(Beijing) Co., Ltd) extraction real-time fluorescent RCR ulcer bacteria genomic DNA, it is 100ng/ μ L real-time fluorescent RCRs that concentration, which is prepared,
The genomic DNA of ulcer bacteria.
2nd, LAMP-HNB is expanded
Using the LAMP-HNB primer sets in embodiment 1, LAMP-HNB amplifications are carried out using following two groups as template：
1 (-) of group：Water (negative control)；
2 (+) of group：The genomic DNA for the real-time fluorescent RCR ulcer bacteria that step 1 is extracted.Carried out using the genomic DNA of group 2
LAMP-HNB has done when detecting and repeated twice.
LAMP-HNB reaction systems are as shown in table 1：
Wherein, 10 × isothermal amplification buffer solution, Bst archaeal dna polymerases and MgSO4Purchased from NEB companies, article No. is
M0538S；HNB is purchased from Sigma companies, article No. H991331.
The LAMP-HNB reaction systems of table 1
| Component | System |
| 10 × isothermal amplification buffer solution | 2.5μL |
| MgSO4 | Final concentration 4.0mmol/L |
| dNTP Mix | Final concentration 1.0mmol/L |
| F3 primers | 0.2 μm of ol/L of final concentration |
| B3 primers | 0.2 μm of ol/L of final concentration |
| FIB primers | 1.6 μm of ol/L of final concentration |
| BIP primers | 1.6 μm of ol/L of final concentration |
| HNB | 150 μm of ol/L of final concentration |
| Bst archaeal dna polymerases | Final concentration 0.16U/ μ L |
| Template (group 1 or group 2) | 1μL |
| ddH2O | Complement to 25 μ L |
LAMP-HNB reaction conditions：Reacted 80 minutes at 62 DEG C.
3rd, interpretation of result
After LAMP-HNB reactions terminate, product is developed the color with naked eyes and carries out result judgement, be light blue representing sample not during color
Ulcer bacteria containing real-time fluorescent RCR, contain real-time fluorescent RCR ulcer bacteria or containing real-time fluorescent RCR canker in expression sample during sky blue
Sclerotium acid.
As a result as shown in figure 1, the amplified production of 1 (negative control) of group is light blue color；Group 2 be (real-time fluorescent RCR ulcer bacteria
Genomic DNA) amplified production be in sky blue, this explanation the present invention LAMP-HNB methods can fast and effeciently detect rape
Stem foot ulcer bacteria.
Embodiment 4
The present embodiment has carried out specificity verification to the LAMP-HNB primer sets of embodiment 1, specifically comprises the following steps：
1st, DNA extraction
With reference to kit explanation operation (plant genome DNA extracts kit, article No. DP305, Tiangeng biochemical technology
(Beijing) Co., Ltd) real-time fluorescent RCR ulcer bacteria, the thin sickle-like bacteria of oat, Alternaria, Phoma, nuclear disk are extracted respectively
Bacterium, the genomic DNA of rape black shank bacterium.
2nd, LAMP-HNB is expanded
Using the genomes of 6 groups of fungies and the DNA of 1 group of negative control extracted in step 1 as template, using in embodiment 3
The method of the detection real-time fluorescent RCR ulcer bacteria of step 2 carries out LAMP-HNB amplifications, and used 7 groups of amplification templates are as follows：
1 (+) of group：Real-time fluorescent RCR ulcer bacteria DNA (positive control)；
2 (-) of group：Water (negative control)；
3 (1) of group：The genomic DNA of the thin sickle-like bacteria of oat；
4 (2) of group：The genomic DNA of Alternaria；
5 (3) of group：The genomic DNA of Phoma；
6 (4) of group：The genomic DNA of sclerotinite；
7 (5) of group：The genomic DNA of rape black shank bacterium；
3rd, interpretation of result
After LAMP-HNB reactions terminate, product is developed the color with naked eyes and carries out result judgement, be light blue representing sample not during color
Ulcer bacteria containing real-time fluorescent RCR, contain real-time fluorescent RCR ulcer bacteria in expression sample during sky blue.
Testing result is as shown in Fig. 2 testing result is：Sky blue is presented in group 1, and other groups of presentations are light blue, illustrates to utilize
The method carries out detection energy specific detection to 7 groups of samples and goes out real-time fluorescent RCR ulcer bacteria, and this absolutely proves that this method has height
Specificity.
Embodiment 5
The present embodiment has carried out sensitivity checking to the LAMP-HNB primer sets of embodiment 1, specifically comprises the following steps：
1st, DNA extraction
With reference to kit explanation operation (plant genome DNA extracts kit, article No. DP305, Tiangeng biochemical technology
(Beijing) Co., Ltd) genomic DNA of real-time fluorescent RCR ulcer bacteria is extracted, and obtained genomic DNA is subjected to 10 times of ladders
Degree dilution, concentration is prepared respectively as 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L and 0.001ng/
μ L genomic DNA.
2nd, LAMP-HNB-HNB is expanded
Using the method for the detection real-time fluorescent RCR ulcer bacteria in the step 2 of embodiment 3, with following 7 groups of genomic DNA
LAMP-HNB-HNB amplifications are carried out for template：
1 (-) of group：The μ L of water (negative control) 1；
2 (100) of group：The genomic DNA of the 100ng/ μ L prepared from step 1 real-time fluorescent RCR ulcer bacteria, 1 μ L；
3 (10) of group：The genomic DNA of the 10ng/ μ L prepared from step 1 real-time fluorescent RCR ulcer bacteria, 1 μ L；
4 (1) of group：The genomic DNA of the 1ng/ μ L prepared from step 1 real-time fluorescent RCR ulcer bacteria, 1 μ L；
5 (0.1) of group：The genomic DNA of the 0.1ng/ μ L prepared from step 1 real-time fluorescent RCR ulcer bacteria, 1 μ L；
6 (0.01) of group：The genomic DNA of the 0.01ng/ μ L prepared from step 1 real-time fluorescent RCR ulcer bacteria, 1 μ L；
7 (0.001) of group：The genomic DNA of the 0.001ng/ μ L prepared from step 1 real-time fluorescent RCR ulcer bacteria, 1 μ
L；
3rd, interpretation of result
After LAMP-HNB-HNB reactions terminate, product is developed the color with naked eyes and carries out result judgement, is light blue representing sample during color
Product are free of real-time fluorescent RCR ulcer bacteria, contain real-time fluorescent RCR ulcer bacteria in expression sample during sky blue.
Testing result is as shown in figure 3, testing result is：Group 1 and group 7 are light blue color, do not detect real-time fluorescent RCR canker
Bacterium；Group 2~6 is in sky blue, detects real-time fluorescent RCR ulcer bacteria, thus proves the LAMP-HNB primer pairs of the embodiment of the present invention 1
With high sensitivity, and the method for the detection real-time fluorescent RCR ulcer bacteria established based on LAMP-HNB primer sets and kit
LAMP-HNB has higher sensitivity, can detect that the micro real-time fluorescent RCR ulcer bacteria DNA only containing 0.01ng in sample.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Zhanjiang Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center
<120>A kind of LAMP-HNB primer sets, kit and method for being used to detect real-time fluorescent RCR ulcer bacteria
<130> 20171016
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gttcgagcgt catttgtacc 20
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
agttcagcgg gtatcccta 19
<210> 3
<211> 38
<212> DNA
<213>Artificial sequence
<400> 3
tgccggctgc caattgtttc ctctgcttgg tgttgggt 38
<210> 4
<211> 39
<212> DNA
<213>Artificial sequence
<400> 4
gcagcacatt ttgcgcctct ctgatccgag gtcaagagc 39
Claims (10)
1. a kind of LAMP-HNB primer sets for being used to detect real-time fluorescent RCR ulcer bacteria, it is characterised in that the LAMP-HNB draws
Thing group includes outer primer pair and inner primer pair, and described outer primer is to for the nucleotide sequence and SEQ shown in SEQ ID NO.1
Nucleotide sequence shown in ID NO.2, described inner primer is to for the nucleotide sequence and SEQ ID shown in SEQ ID NO.3
Nucleotide sequence shown in NO.4.
2. the LAMP-HNB primer sets described in claim 1 are burst in detection real-time fluorescent RCR ulcer bacteria or preparation detection real-time fluorescent RCR
Application in the product of ulcer germ.
3. a kind of kit for being used to detect real-time fluorescent RCR ulcer bacteria, it is characterised in that described kit will including right
Seek the LAMP-HNB primer sets described in 1.
4. kit according to claim 3, it is characterised in that described kit also includes indicator；
Optional, described indicator is hydroxynaphthol blue；
Optional, described hydroxynaphthol blue is reagent that the article No. from Sigma companies is in H991331 kits；
Optional, in addition to archaeal dna polymerase；
Optional, described archaeal dna polymerase is Bst archaeal dna polymerases；
Optional, described Bst archaeal dna polymerases are reagent that the article No. from NEB companies is in M0538S kits；
It is optional, in addition to isothermal amplification buffer solution, dNTPs, contain Mg2+Soluble compound；
Optional, described contains Mg2+Soluble compound be MgSO4；
Optional, the isothermal amplification buffer solution, MgSO4It is the examination in M0538S kits for the article No. from NEB companies
Agent；
Optional, in addition to positive control；
Optional, in addition to negative control；
Optional, in addition to nucleic acid extracting reagent；
Optional, described nucleic acid extracting reagent is the DNA extracts reagents that the article No. of Tiangeng biochemical technology Co., Ltd is DP305
Reagent in box.
5. the kit described in claim 3 or 4 in detection real-time fluorescent RCR ulcer bacteria or contains real-time fluorescent RCR canker sclerotium
Application in sour sample.
6. a kind of method for being used to detect real-time fluorescent RCR ulcer bacteria or real-time fluorescent RCR ulcer bacteria nucleic acid in sample, its feature
It is, described method is including the use of LAMP-HNB primer sets in claim 1 or the examination any one of claim 3-4
The step of agent box.
7. according to the method for claim 6, it is characterised in that the step of described method also includes obtaining sample nucleic acid；
Optional, described nucleic acid is DNA or RNA；
Optional, described DNA is that the DNA for being DP305 by using the article No. of TIANGEN Biotech (Beijing) Co., Ltd. is carried
The method in kit is taken to obtain；
It is optional, when described nucleic acid is RNA, in addition to the step of RNA reverse transcriptions are cDNA.
8. according to the method for claim 7, it is characterised in that described method is additionally included in detection sample nucleic acid and added
LAMP-HNB reaction reagents carry out the step of LAMP-HNB amplified reactions；
Optional, described LAMP-HNB reaction reagents include archaeal dna polymerase, isothermal amplification buffer solution, dNTPs and contained
Mg2+Soluble compound；
Optional, described contains Mg2+Soluble compound be MgSO4；
Optional, the isothermal amplification buffer solution, MgSO4It is the examination in M0538S kits for the article No. from NEB companies
Agent；
Optional, described archaeal dna polymerase is Bst archaeal dna polymerases；
Optional, described Bst archaeal dna polymerases are reagent that the article No. from NEB companies is in M0538S kits；
It is optional, the step of described method also includes adding indicator before LAMP-HNB amplified reactions；
Optional, described indicator is hydroxynaphthol blue；
Optional, described hydroxynaphthol blue is reagent that the article No. from Sigma companies is in H991331 kits；
Optional, the upstream and downstream primer of the outer primer pair is set to F3 and B3, the upstream and downstream primer of described inner primer pair
FIP and BIP are set to, final concentration relation of four kinds of primers in LAMP-HNB amplification reaction systems is：([F3]=[B3]):
([FIP]=[BIP])=1:8；
It is optional, the final concentration of described four kinds of primers in LAMP-HNB amplification reaction systems be respectively [F3]=[B3]=
0.2 μm of ol/L, [FIP]=[BIP]=1.6 μm ol/L；
It is optional, final concentration of 150 μm of ol/L in LAMP-HNB amplification reaction systems of described hydroxynaphthol blue.
9. according to the method for claim 8, it is characterised in that described method is also included pattern detection result and the positive
Control and/or negative control result the step of being compared, when testing result is identical with the result that positive control detects, illustrate to examine
The sample of survey contains real-time fluorescent RCR ulcer bacteria or containing real-time fluorescent RCR ulcer bacteria nucleic acid, when testing result and negative control are examined
The result of survey is identical, illustrates the sample of detection without real-time fluorescent RCR ulcer bacteria or does not contain real-time fluorescent RCR ulcer bacteria nucleic acid；
Optional, described positive control testing result is sky blue, and negative control result is light blue.
10. according to the method for claim 8, it is characterised in that the reaction system of methods described is as follows：
Optional, the response procedures of methods described are as follows：
Reacted 80 minutes at 62 DEG C.
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| CN201710971911.2A Pending CN107641660A (en) | 2017-10-18 | 2017-10-18 | A kind of LAMP HNB primer sets, kit and method for being used to detect real-time fluorescent RCR ulcer bacteria |
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| CN109371164A (en) * | 2018-12-21 | 2019-02-22 | 华中农业大学 | Application of the specific sequence of rape black shank bacterium subspecies in the detection of rape black shank bacterium |
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| CN109371164A (en) * | 2018-12-21 | 2019-02-22 | 华中农业大学 | Application of the specific sequence of rape black shank bacterium subspecies in the detection of rape black shank bacterium |
| CN109371164B (en) * | 2018-12-21 | 2020-04-10 | 华中农业大学 | Application of specific sequence of rape black shank bacterium subspecies in rape black shank bacterium detection |
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