CN108060257A - It is a kind of that strong male rotten mould Primer composition and its detection method are detected based on loop-mediated isothermal amplification technique - Google Patents
It is a kind of that strong male rotten mould Primer composition and its detection method are detected based on loop-mediated isothermal amplification technique Download PDFInfo
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Abstract
Strong male rotten mould Primer composition and its detection method are detected based on loop-mediated isothermal amplification technique the invention discloses a kind of, extract the genomic DNA of microorganism to be checked, Genomic DNA solution is taken as reaction template, the detection solution added in LAMP kit carries out LAMP reactions, and the program of the LAMP reactions is：64 DEG C of reactions expand 60min, then observe the color change of amplified production, if color becomes sky blue from purple, represent mould in the presence of strong male corruption in object to be checked, if variation is not still purple to color, represent rotten mould there is no strong hero in object to be checked.Compared with strong male rotten mould detection technique is identified with traditional foundation morphological feature, the present invention possesses higher accuracy, sensitivity and actual effect, and it is easy to operate, practicability is good, and new technology platform is provided for strong male rotten mould detection, the quick detection mould available for strong male corruption.
Description
Technical field
The invention belongs to biological technical fields, and in particular to a kind of LAMP detection primer composition mould for strong male corruption,
Kit and its visible detection method.
Background technology
Strong male rotten mould (Pythium arrhenomane) is the important pathogenic bacteria of corn stalk rot disease, the most thermophilic of its growth
It spends for 23-25 DEG C, mainly causes the root-rot and stem rot of corn, there is larger harm to maize production.It is in view of strong male rotten mould
Harmfulness needs in production a kind of to fall ill that early stage is quick, the method for the precise Identification pathogen in corn.Maize Stem at present
The traditional identification by morphological characters method of the identification generally use of maize ear rot pathogen, that is, after sample of falling ill is adopted back from field, immediately into
Row pathogen is separately cultured, then carries out Morphological Identification.This method takes time and effort, it is necessary to abundant and specialty pathogen identification
Experience, and be difficult to distinguish allied species according to morphology, can not meet the needs of in the production of field.With molecular biology
Development, have been set up a series of molecular detecting methods based on PCR (PCR), including regular-PCR detect
Method, real-time PCR detection methods, multiplex PCR detection method etc..These detection methods are needed using expensive instrument and equipment such as
PCR instrument, fluorescence quantitative PCR instrument, and program is cumbersome, the grass-roots work being unsuitable on some resources limited laboratory and production
Person.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is near
A kind of new nucleic acid amplification technologies of year exploitation, the technology is by the BstDNA polymerases with strand-displacement activity in isothermy
It is expanded under (60~65 DEG C), 6 regions on target DNA is identified using 4 primer specificities, amplification efficiency is interior when 1 is small
109 copy numbers can be reached.In addition, the developing dyes such as SYBR Green, hydroxynaphthol blue (HNB) are added in the reaction system,
Reaction result can directly be judged by color change.LAMP detection method have easy to operate, specific height, high sensitivity,
The advantages that at low cost, becomes the new nucleic acid amplification technologies that can substitute regular-PCR.The technology is widely used to very at present
Bacterium, the quick detection of bacterium, virus and oomycetes.
The present invention devises the LAMP primer of strong male rotten mould specificity using β-tubulin genes as the target sequence of detection
Composition establishes strong male rotten mould LAMP rapid detection methods on this basis.
The content of the invention
For cycle length needed for strong male rotten mould biological detection method in the prior art, detection method poor specificity, sensitive
Spend the problem of low, the object of the present invention is to provide it is a kind of for strong male rotten mould LAMP detection primer composition, kit and its
Visible detection method
In order to realize foregoing invention purpose, the present invention is achieved through the following technical solutions：
It is a kind of to be included for detecting male rotten mould LAMP primer composition object, the Primer composition by force：Such as SEQ ID NO.1 institutes
The positive inner primer FIP shown, the reversed inner primer BIP as shown in SEQ ID NO.2, it is just outside as shown in SEQ ID NO.3
Primers F 3, the reversed outer primer B3 as shown in SEQ ID NO.4, the reversed ring primer LB as shown in SEQ ID NO.5.
Application of the above-mentioned LAMP primer composition object in the strong male corruption of detection is mould.
Application of the above-mentioned LAMP primer composition object in preparing to detect strong male rotten mould kit.
It is a kind of to be used to detect strong male rotten mould LAMP kit, include above-mentioned LAMP primer composition object in the kit.
As a kind of optimal technical scheme, using the genomic DNA of microorganism to be checked as template, using above-mentioned LAMP reagents
Male rotten mould LAMP reaction systems are by force for box detection：2.5 μ 10 × ThermoPol of L Buffer, 6mmolL-1MgSO4,
1.4mmol·L-1Each 1.6 μm of olL of dNTPs, inner primer FIP and BIP-1, each 0.2 μm of olL of outer primer F3 and B3-1, ring draws
0.8 μm of olL of object LB-1, 0.8molL-1Glycine betaine, 0.1%Triton X-100,20mmolL-1Tris-HCl(pH
8.8), 10mmolL-1KCl, 10mmolL-1(NH4)2SO4, 8U μ L-1Bst DNA polymerase, 192 μm of olL-1
Hydroxynaphthol blue (HNB), 2 μ L template DNAs, aqua sterilisa are mended to 25 μ L.
Application of the above-mentioned LAMP kit in the strong male corruption of detection is mould.
A kind of strong male rotten mould LAMP detection method, extracts the genomic DNA of microorganism to be checked, takes Genomic DNA solution
As reaction template, the detection solution added in above-mentioned LAMP kit carries out LAMP reactions, and the program of the LAMP reactions is：
64 DEG C of reaction amplification 60min, then observe the color change of amplified production, if color becomes sky blue from purple, represent to treat
It is mould to there is strong male corruption in inspection object, if it is still purple that color, which does not have to change, represents that there is no strong hero corruption is mould in object to be checked.
The strong male rotten mould method of detection of the present invention, using the DNA of extraction as template, utilizes the LAMP primer composition object
Carry out LAMP reactions；Hydroxylnaphthol blue (HNB) belong to one kind of Metal ion indicator.HNB is Mg2+Drop
Determine agent, color changes with solution PH and changed, therefore can be by monitoring Mg in LAMP reaction systems2+The variation of concentration and molten
Liquid pH and play the role of color indicator.HNB is added in reaction solution before reaction, reaction system is in purple, reaction process
Middle Mg2+Generation is combined with the by-product of LAMP reactions largely to precipitate, Mg in solution2+Concentration reduces, and pH changes, so that
The color of HNB becomes sky blue from purple.Therefore, it is strong male rotten to judge after reaction by the color change of reaction system
It is the presence or absence of mould：Sky blue represents test positive, exists strong male rotten mould；Purple represents testing result as feminine gender, and there is no strong heros
It is rotten mould.
Compared with prior art, the present invention its advantage and good effect are shown：
(1) it is easy to operate：The strong male rotten mould LAMP method of detection provided by the invention overcomes strong male rotten in the prior art
The problem of cycle needed for mould biological detection method is long, time-consuming and laborious, cumbersome, poor specificity and PCR detection techniques need heat
Instrument is cycled, can not quickly detect the problem of strong male rotten mould.Detection method only needs 60min under 64 DEG C of isothermys
Strong male rotten mould with regard to can accurately, fast and efficiently detect, the requirement to experimental place is simple, and other complex instruments, energy is not required
Preferably meet to strong male rotten mould Site Detection, be suitble to promote the use of in base and inlet and outlet inspection and quarantine department.
(2) accuracy is high：It is simply identified due to traditional strong male rotten mould detection method according to morphological feature, and it is strong male
It is unstable that rotten mould growth is affected by temperature form, while is influenced be subject to close kind, it is difficult to which precise Identification comes out.It is and of the invention
According to strong male rotten mould genome sequence, using Blast softwares by strong male rotten mould genome sequence and the mould gene of other corruption
Group sequence is compared, and is had chosen the distinctive terminal sequence of Pythium ultimum bacterium and is designed the LAMP primer of specificity.LAMP reacts
By 6 isolated areas on 4 primer (FIP, BIP, F3, B3) specific recognition target sequences, specificity is higher.This
Outside, reversed ring primer LB can improve reaction rate and other four primers together, in the case where ensuring to react accuracy,
It enables the invention to be rapidly performed by strong male rotten mould detection.
(3) high sensitivity：The strong male rotten mould LAMP detection method that the present invention establishes, sensitivity is very high, can reach
10pg DNA, show the detection method be enough in the case that DNA concentration it is relatively low quickly and accurately detect it is strong male rotten mould.
Description of the drawings
Fig. 1 is the specificity verification of strong male rotten mould LAMP primer：
LAMP detections are carried out to various pathogenic bacteria using strong male rotten mould LAMP specific primers, are expanded in 64 DEG C of constant temperature water baths
The color change of observing response solution, the results show after increasing 60min：Only with solution in the strong male rotten mould reaction tube for template
Color becomes sky blue (Fig. 1) by purple, and solution colour does not all change in other rotten mould reaction tubes, is still purple
Color.
Fig. 2 is the sensitivity verification of strong male rotten mould LAMP detection method：
The sensitivity of color change detection LAMP method based on reaction solution sets strong male rotten mould template concentrations scope
For 10ng μ L-1~1fg μ L-1.The results show：When strong male rotten mould template concentrations are respectively 10ng μ L-1、1ng·μL-1、
100pg·μL-1、10pg·μL-1When, solution colour becomes sky blue in corresponding reaction tube, is positive；And when strong
Male rotten mould template concentrations are respectively 1pg μ L-1、100fg·μL-1、10fg·μL-1、1fg·μL-1When, corresponding reaction
Solution colour is still purple in pipe, negative.
Specific embodiment
In order to make the purpose of the present invention, technical solution more clearly, the present invention is described further with specific example,
But it not only limits the use of in these examples.
Embodiment 1：It is detected using LAMP method strong male rotten mould
For detecting strong male rotten mould LAMP primer composition object：Positive inner primer FIP is as shown in SEQ ID NO.1, reversely
Inner primer BIP is as shown in SEQ ID NO.2, and positive outer primer F3 is as shown in SEQ ID NO.3, reversed outer primer B3 such as SEQ ID
Shown in NO.4, reversed ring primer LB is as shown in SEQ ID NO.5.
Each reagent concentration is in the strong male rotten mould LAMP kit of detection：2.5 μ L 10 × ThermoPol Buffer,
6mmol·L-1MgSO4, 1.4mmolL-1Each 1.6 μm of olL of dNTPs, inner primer FIP and BIP-1, outer primer F3 and B3 be each
0.2μmol·L-1, 0.8 μm of olL of ring primer LB-1, 0.8molL-1Glycine betaine, 0.1%Triton X-100,20mmol
L-1Tris-HCl (pH 8.8), 10mmolL-1KCl, 10mmolL-1(NH4)2SO4, 8U μ L-1Bst DNA
Polymerase, 192 μm of olL-1Hydroxynaphthol blue (HNB), 2 μ L template DNAs, aqua sterilisa are mended to 25 μ L.
LAMP detection method：The DNA of microorganism to be checked is extracted, 2 μ L DNA solutions is taken to add in 23 μ L as reaction template
Detection solution in LAMP kit carries out LAMP, and LAMP response procedures are：64 DEG C of reaction amplification 60min, then observation amplification
The color change of product if color becomes sky blue from purple, represents mould in the presence of strong male corruption in object to be checked, if color does not have
Variation is still purple, represents that there is no strong male rotten mould in object to be checked.
It is mould for material to be tested with strong male rotten mould and other 9 kinds of corruption in order to verify the specificity of LAMP method, LAMP detection knots
Fruit shows that strong male corruption is mould and sapphire positive reaction can be observed, and other rotten mould colour developing results are the negative reaction (figure of purple
1)。
Embodiment 2：The sensitivity test of strong male rotten mould LAMP reactions
It is in order to determine the sensitivity of LAMP detection method, the strong male rotten mould DNA spectrophotometric determinations of extraction is dense
10 doubling dilutions are carried out after degree, it is 10ng μ L to set DNA concentration scope-1~1fg μ L-1.Each concentration after dilution is taken respectively
2 μ L of DNA dilutions are as template, and the detection solution added in 23 μ L kits carries out LAMP reactions, and response procedures are：64 DEG C anti-
60min should be expanded.HNB chromogenic reactions show：When strong male rotten mould DNA concentration reaches 10pg μ L-1When, the solution in reaction tube
With regard to sky blue (Fig. 2) can be become.
Sequence table
<110>Agricultural University Of Nanjing
<120>It is a kind of that strong male rotten mould Primer composition and its detection method are detected based on loop-mediated isothermal amplification technique
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
acacaaggtg gttgaggtct cctatctgct tccgtacgct ca 42
<210> 2
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gctgccatgt ccggcatcac cggccaactt acgaagatcc 40
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
acaacgaagc cctctacga 19
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
agacgcggga aagggatc 18
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gcttgcgttt cccgggtca 19
Claims (7)
1. a kind of be used to detect strong male rotten mould LAMP primer composition object, which is characterized in that the Primer composition includes：Such as SEQ
Positive inner primer FIP shown in ID NO.1, the reversed inner primer BIP as shown in SEQ ID NO.2, as shown in SEQ ID NO.3
Positive outer primer F3, the reversed outer primer B3 as shown in SEQ ID NO.4, the reversed ring primer as shown in SEQ ID NO.5
LB。
2. application of the LAMP primer composition object described in claim 1 in the strong male corruption of detection is mould.
3. application of the LAMP primer composition object described in claim 1 in preparing to detect strong male rotten mould kit.
4. a kind of be used to detect strong male rotten mould LAMP kit, which is characterized in that comprising described in claim 1 in the kit
LAMP primer composition object.
5. according to claim 4 be used to detect strong male rotten mould LAMP kit, which is characterized in that with microorganism to be checked
Genomic DNA for template, use the LAMP kit described in claim 4 detect strong male rotten mould LAMP reaction systems for：
2.5 μ 10 × ThermoPol of L Buffer, 6mmolL-1MgSO4, 1.4mmolL-1DNTPs, inner primer FIP and BIP are each
1.6μmol·L-1, each 0.2 μm of olL of outer primer F3 and B3-1, 0.8 μm of olL of ring primer LB-1, 0.8molL-1Glycine betaine,
0.1%Triton X-100,20mmolL-1Tris-HCl, 10mmolL-1KCl, 10mmolL-1(NH4)2SO4, 8U μ
L-1Bst DNA polymerase, 192 μm of olL-1Hydroxynaphthol blue, 2 μ L template DNAs, aqua sterilisa are mended to 25 μ L.
6. application of the LAMP kit in the strong male corruption of detection is mould described in claim 4 or 5.
7. a kind of strong male rotten mould LAMP detection method, which is characterized in that extract the genomic DNA of microorganism to be checked, take gene
For group DNA solution as reaction template, the detection solution added in the LAMP kit of claim 4 or 5 carries out LAMP reactions,
The program of LAMP reaction is：64 DEG C reaction amplification 60min, then observe amplified production color change, if color by
Purple becomes sky blue, represents mould in the presence of strong male corruption in object to be checked, if variation is not still purple to color, represents in object to be checked
There is no strong male rotten mould.
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Cited By (7)
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| CN108531646A (en) * | 2018-07-10 | 2018-09-14 | 重庆文理学院 | The LAMP detection primer and detection method of curing ginger stalk rot P. myriotylum |
| CN111534626A (en) * | 2020-04-24 | 2020-08-14 | 中国农业科学院麻类研究所 | LAMP (loop-mediated isothermal amplification) detection primer composition for pythium bellatus, detection kit and visual detection method of LAMP detection primer composition |
| CN111621590A (en) * | 2020-06-29 | 2020-09-04 | 南京农业大学 | LAMP primer composition for detecting pythium terrestris, kit and detection method thereof |
| CN111676311A (en) * | 2020-06-29 | 2020-09-18 | 南京农业大学 | LAMP primer composition for detecting pythium aphanidermatum, kit and detection method thereof |
| CN114107539A (en) * | 2021-10-26 | 2022-03-01 | 江苏农林职业技术学院 | LAMP primer composition for detecting Pythium delavayi and application thereof |
| CN114317796A (en) * | 2021-10-26 | 2022-04-12 | 江苏农林职业技术学院 | LAMP primer composition for detecting pythium admittedly and detection method thereof |
| US11634783B2 (en) * | 2018-02-05 | 2023-04-25 | Syngenta Participations Ag | Methods for detecting fungi in turf grass with a lamp assay having novel primer sets |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11634783B2 (en) * | 2018-02-05 | 2023-04-25 | Syngenta Participations Ag | Methods for detecting fungi in turf grass with a lamp assay having novel primer sets |
| CN108531646A (en) * | 2018-07-10 | 2018-09-14 | 重庆文理学院 | The LAMP detection primer and detection method of curing ginger stalk rot P. myriotylum |
| CN111534626A (en) * | 2020-04-24 | 2020-08-14 | 中国农业科学院麻类研究所 | LAMP (loop-mediated isothermal amplification) detection primer composition for pythium bellatus, detection kit and visual detection method of LAMP detection primer composition |
| CN111621590A (en) * | 2020-06-29 | 2020-09-04 | 南京农业大学 | LAMP primer composition for detecting pythium terrestris, kit and detection method thereof |
| CN111676311A (en) * | 2020-06-29 | 2020-09-18 | 南京农业大学 | LAMP primer composition for detecting pythium aphanidermatum, kit and detection method thereof |
| CN114107539A (en) * | 2021-10-26 | 2022-03-01 | 江苏农林职业技术学院 | LAMP primer composition for detecting Pythium delavayi and application thereof |
| CN114317796A (en) * | 2021-10-26 | 2022-04-12 | 江苏农林职业技术学院 | LAMP primer composition for detecting pythium admittedly and detection method thereof |
| CN114107539B (en) * | 2021-10-26 | 2022-07-08 | 江苏农林职业技术学院 | LAMP primer composition for detecting pythium delicicola and application thereof |
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