CN111676311B - LAMP primer composition for detecting pythium aphanidermatum, kit and detection method thereof - Google Patents
LAMP primer composition for detecting pythium aphanidermatum, kit and detection method thereof Download PDFInfo
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- CN111676311B CN111676311B CN202010604316.7A CN202010604316A CN111676311B CN 111676311 B CN111676311 B CN 111676311B CN 202010604316 A CN202010604316 A CN 202010604316A CN 111676311 B CN111676311 B CN 111676311B
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- 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
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- 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
Abstract
The invention discloses an LAMP primer composition, a kit and a detection method for detecting pythium aphanidermatum. An LAMP primer composition for detecting pythium aphanidermatum: the forward inner primer FIP is shown as SEQ ID NO.1, the reverse inner primer BIP is shown as SEQ ID NO.2, the forward outer primer F3 is shown as SEQ ID NO.3, the reverse outer primer B3 is shown as SEQ ID NO.4, the forward loop primer LF is shown as SEQ ID NO.5, and the reverse loop primer LB is shown as SEQ ID NO. 6. Compared with the traditional detection technology for identifying the pythium aphanidermatum according to morphological characteristics, the method has higher accuracy, sensitivity and practicability, is convenient to operate, provides a new technical platform for the detection of the pythium aphanidermatum, and can be used for the high-sensitivity rapid detection of the pythium aphanidermatum.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an LAMP primer composition for detecting pythium aphanidermatum, a kit and a detection method thereof.
Background
Pythium spinosum belongs to the class of Oomycetes (oxomycetes), the order peronosporales (peronosporales), the family of Pythiaceae (Pythiaceae), the genus Pythium (Pythium), and is a relatively common pathogenic Pythium. The pathogenic bacteria can infect seeds and plant roots to cause plant wilting, necrosis and other symptoms, and become one of the important pathogenic bacteria of many crop diseases. At present, no effective control measures are available for the disease, and strengthening quarantine and preventing the spread of pathogenic bacteria are the most effective measures for controlling the disease. Therefore, epidemic situation monitoring is enhanced, a rapid detection method is established, and basis is provided for disease risk and research decision, so that loss caused by pythium aphanidermatum is reduced.
At present, detection technologies for pythium aphanidermatum are few. The traditional detection method mainly adopts a plate separation method and a bait method and carries out identification according to the morphology of the pythium aphanidermatum. The growth of the pythium is affected by the temperature, and the shape is unstable, so that great difficulty is brought to detection. With the development of molecular biology, particularly the popularization of PCR technology, more and more molecular biology technologies are applied to the detection of Pythium aphanidermatum, but the detection time is still longer, and meanwhile, a precise temperature cycle device is relied on, the detection process is complex, and the requirement of rapid detection cannot be met.
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, and is a novel nucleic acid amplification technology capable of replacing PCR due to the advantages of simple operation, rapidness, high specificity, low cost and the like. It designs 4 specific primers aiming at 6 regions of a target gene, causes self-circulation strand displacement reaction under the action of Bst large-fragment polymerase, and generates white magnesium pyrophosphate precipitate as a byproduct while synthesizing a large amount of target DNA within 80min at the temperature of 60-65 ℃. Because the LAMP amplification process depends on 6 independent areas for identifying the target sequence, the reaction specificity is very strong, the nucleic acid amplification process is carried out under the constant temperature condition, a common water bath or equipment with a stable heat source can meet the reaction requirement, and the detection cost is greatly reduced. The LAMP has the characteristics of simple reaction, rapidness, high efficiency, economy and the like, so the LAMP has a very wide application prospect. Since the LAMP detection technology was established for 14 years, the technology has been widely applied to detection research of pathogenic bacteria such as viruses, bacteria, parasites, fungi and the like, but few reports are made on detection of plant pathogenic oomycetes, and detection of pythium aphanidermatum is not reported at home and abroad.
The invention analyzes the difference of the genomic sequences of the pythium aphanidermatum and other pythium moulds through sequence comparison, designs the LAMP primer composition of the pythium aphanidermatum specificity by taking the PysM90 gene sequence as a target, and establishes the LAMP rapid detection method of the pythium aphanidermatum on the basis.
Disclosure of Invention
Aiming at the problems of long required period, poor specificity and low sensitivity of a detection method in the prior art, the invention aims to provide the LAMP primer composition, the kit and the detection method for detecting the pythium aphanidermatum.
In order to achieve the purpose, the invention is realized by the following technical scheme:
an LAMP primer composition for detecting pythium aphanidermatum: the forward inner primer FIP is shown as SEQ ID NO.1, the reverse inner primer BIP is shown as SEQ ID NO.2, the forward outer primer F3 is shown as SEQ ID NO.3, the reverse outer primer B3 is shown as SEQ ID NO.4, the forward loop primer LF is shown as SEQ ID NO.5, and the reverse loop primer LB is shown as SEQ ID NO. 6.
The LAMP detection primer composition disclosed by the invention is applied to detection of pythium aphanidermatum.
The LAMP primer composition disclosed by the invention is applied to preparation of a reagent for detecting pythium aphanidermatum.
The LAMP kit for detecting the pythium aphanidermatum is characterized in that the concentration of each reagent in the kit is as follows: 0.8. mu.M forward inner primer FIP, 0.8. mu.M reverse inner primer BIP, 0.1. mu.M forward outer primer F3, 0.1. mu.M reverse outer primer B3, 0.1. mu.M forward loop primer LF, 0.1. mu.M reverse loop primer LB, 0.8M betaine, 1.4mM dNTPs, 20mM Tris-HCl, 10mM KCl, 10mM (NH)4)2SO4、6mM MgSO40.1% Triton X-100, Bst DNA polymerase 8U/. mu.l, and 1mL of a detection solution was prepared using ultrapure water.
The LAMP kit for detecting the pythium aphanidermatum is applied to the detection of the pythium aphanidermatum.
The LAMP detection method of the pythium aphanidermatum comprises the step of carrying out LAMP reaction by using the LAMP primer composition or the LAMP kit for detection.
One preferable LAMP detection method for Pythium erythraeum comprises the steps of extracting DNA of a microorganism to be detected, taking 4 mu L of DNA solution as a reaction template, and adding 21 mu L of detection solution in an LAMP kit to carry out LAMP, wherein the LAMP reaction program comprises the following steps: and (3) carrying out reaction amplification at 62 ℃ for 70min, then adding 0.25 mu L of SYBR Green I into an amplification product, observing color change, if the color is changed from orange to yellow Green, or fluorescence exists under an ultraviolet lamp, indicating that pythium aphanidermatum exists in the object to be detected, and if the color is not changed, still orange, or no fluorescence exists under the ultraviolet lamp, indicating that pythium aphanidermatum does not exist in the object to be detected.
The method for detecting the pythium aphanidermatum takes the extracted DNA as a template, and the LAMP primer composition is utilized to carry out LAMP reaction; after the reaction, 0.25. mu.L of SYBR Green I was added to the amplified product, and the color change of the reaction solution or the presence or absence of fluorescence under an ultraviolet lamp was observed. SYBR Green I is a highly sensitive DNA fluorescent dye and has extremely high affinity with double-stranded DNA. Adding SYBR Green I after the reaction is finished, and judging whether the pythium aphanidermatum exists or not through the color change and the fluorescence of a reaction system: the reaction solution is yellow green or has fluorescence under an ultraviolet lamp to indicate that the reaction solution is positive, and pythium aphanidermatum exists; the reaction solution is orange or has no fluorescence under an ultraviolet lamp, which indicates that the detection result is negative and pythium aphanidermatum does not exist.
Application of PysM90 gene as a detection target in detecting the acanthomonas.
Application of PysM90 gene as a detection target in preparation of a trichoderma detection reagent.
Advantageous effects
Compared with the prior art, the invention has the advantages and positive effects that:
(1) the operation is convenient: the LAMP method for detecting the pythium aphanidermatum provided by the invention overcomes the problems that the biological detection method of the pythium aphanidermatum in the prior art needs long period, wastes time and labor, is complex and has poor specificity, and the PCR detection technology needs a thermal cycler and cannot quickly detect the pythium aphanidermatum. The detection method can accurately, quickly and efficiently detect the pythium aphanidermatum in 70min under the isothermal condition of 62 ℃, has simple requirements on experimental places, does not need other complex instruments, can better meet the field detection of the pythium aphanidermatum, and is suitable for popularization and use in basic level and import and export inspection and quarantine departments.
(2) The accuracy is high: because the traditional pythium aphanidermatum detection method is only used for identification according to morphological characteristics, the growth of the pythium aphanidermatum is influenced by temperature and has unstable morphology, and is simultaneously influenced by similar species, so that the pythium aphanidermatum detection method is difficult to accurately identify. According to the genome sequence of the pythium aphanidermatum, the genome sequence of the pythium ultimum is compared with the genome sequences of other pythium by using Blast software, and the PysM90 gene sequence of the pythium aphanidermatum is selected to design a specific LAMP primer, and the gene is not used as a target in common PCR (polymerase chain reaction) to design an identification primer of the pythium aphanidermatum. The LAMP reaction specifically recognizes 6 independent regions on the target sequence (FIG. 1) by 4 primers (FIP, BIP, F3, B3), and the specificity is relatively high. In addition, the reverse loop primer LB can improve the reaction rate, and together with other four primers, the method can rapidly detect the pythium aphanidermatum under the condition of ensuring the reaction accuracy.
(3) The sensitivity is high: the LAMP detection method of the pythium aphanidermatum, which is established by the invention, has very high sensitivity and can reach 100pg DNA, and the LAMP detection method is proved to be enough to accurately and quickly detect the pythium aphanidermatum under the condition of lower DNA concentration.
Drawings
FIG. 1 shows the target region in the PysM90 gene sequence for the design of Pythium erythraeum LAMP primers:
specific LAMP primers are designed by taking PysM90 gene sequence of Pythium aphanidermatum as a target
FIG. 2 is a sensitivity verification of the Pythium erythrinum LAMP detection method:
the concentration range of the template of the pythium aphanidermatum is set to be 100ng-10fg based on the color change of the reaction solution and the sensitivity of the LAMP method for detecting the presence or absence of fluorescence under an ultraviolet LAMP. The results show that: when the template concentrations of the pythium aphanidermatum are respectively 100ng, 10ng, 1 ng and 100pg, the color of the solution in the corresponding reaction tube is changed into yellow green (figure 2), and the positive reaction is realized; when the template concentrations of Pythium erythrinum are 10pg, 1pg, 100fg and 10fg, respectively, the color of the solution in the corresponding reaction tube is still orange (FIG. 2), and the reaction tube shows a negative reaction, and further observation under an ultraviolet lamp verifies the above results
Detailed Description
In order to make the purpose and technical solution of the present invention more clear, the present invention is further described with specific examples, but is not limited to these examples.
Example 1: detection of Pythium spinosum by LAMP method
LAMP primer composition for detecting Pythium erythrinum: the forward inner primer FIP is shown as SEQ ID NO.1, the reverse inner primer BIP is shown as SEQ ID NO.2, the forward outer primer F3 is shown as SEQ ID NO.3, the reverse outer primer B3 is shown as SEQ ID NO.4, the forward loop primer LF is shown as SEQ ID NO.5, and the reverse loop primer LB is shown as SEQ ID NO. 6.
The concentration of each reagent in the LAMP kit for detecting the pythium aphanidermatum is as follows: 0.8. mu.M forward inner primer FIP, 0.8. mu.M reverse inner primer BIP, 0.1. mu.M forward outer primer F3, 0.1. mu.M reverse outer primer B3, 0.1. mu.M forward loop primer LF, 0.1. mu.M reverse loop primer LB, 0.8M betaine, 1.4mM dNTPs, 20mM Tris-HCl, 10mM KCl, 10mM (NH)4)2SO4、 6mM MgSO40.1% Triton X-100, Bst DNA polymerase 8U/. mu.l, and 1mL of a detection solution was prepared using ultrapure water.
The LAMP detection method comprises the following steps: extracting DNA of a microorganism to be detected, taking 4 mu L of DNA solution as a reaction template, adding 21 mu L of detection solution in an LAMP kit to carry out LAMP, wherein the LAMP reaction procedure is as follows: and (3) carrying out reaction amplification at 62 ℃ for 70min, then adding 0.25 mu L of SYBR Green I into an amplification product, observing color change, if the color changes from orange to yellow Green or has fluorescence under an ultraviolet lamp, indicating that pythium aphanidermatum exists in the object to be detected, and if the color does not change, still remains orange or has no fluorescence under the ultraviolet lamp, indicating that the pythium aphanidermatum does not exist in the object to be detected.
In order to verify the specificity of the LAMP method, 4 strains of Pythium erythraea and other non-target strains are used as test materials, and the LAMP detection result shows that only Pythium erythraea can observe a yellow-green positive reaction, and the other non-target strains show a negative reaction (Table 1).
TABLE 1 specificity verification of Pythium erythrinum LAMP primers
aNIMR national microbial resource platform
bNJAU-Nanjing university of agriculture
Example 2: sensitivity test of Pythium spinosum LAMP reaction
In order to determine the sensitivity of the LAMP detection method, the extracted DNA of the Pythium erythraeum is diluted by 10 times after the concentration is measured by a spectrophotometer, the concentration range of the DNA is set to be 100ng-10fg, 4 mu L of each diluted DNA dilution solution with each concentration is taken as a template, 21 mu L of kit solution is added for LAMP reaction, and the reaction program is as follows: amplification was carried out at 62 ℃ for 70 min. The results show that: when the DNA concentration of P.erythraea reached 100pg, the solution in the reaction tube became yellow-green, and further fluorescence was observed under an ultraviolet lamp to obtain a uniform result (FIG. 2).
Sequence listing
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Claims (7)
1. An LAMP primer composition for detecting Pythium erythrinum, characterized in that the primer composition comprises: a forward inner primer FIP shown as SEQ ID NO.1, a reverse inner primer BIP shown as SEQ ID NO.2, a forward outer primer F3 shown as SEQ ID NO.3, a reverse outer primer B3 shown as SEQ ID NO.4, a forward loop primer shown as SEQ ID NO.5, and a reverse loop primer LB shown as SEQ ID NO. 6.
2. The LAMP primer composition of claim 1, for use in detection of P.aculeatus.
3. The LAMP primer composition of claim 1, which is used for preparing a reagent for detecting Pythium erythraeum.
4. The LAMP kit for detecting the pythium aphanidermatum is characterized in that the concentration of each reagent in the kit is as follows: 0.8. mu.M forward inner primer FIP, 0.8. mu.M reverse inner primer BIP, 0.1. mu.M forward outer primer F3, 0.1. mu.M reverse outer primer B3, 0.1. mu.M forward loop primer LF, 0.1. mu.M reverse loop primer LB, 0.8M betaine, 1.4mM dNTPs, 20mM Tris-HCl, 10mM KCl, 10mM (NH)4)2SO4、6 mM MgSO40.1% Triton X-100, Bst DNA polymerase 8U/mul, and preparing 1mL detection solution by using ultrapure water; wherein the forward inner primer FIP is shown as SEQ ID NO.1, the reverse inner primer BIP is shown as SEQ ID NO.2, the forward outer primer F3 is shown as SEQ ID NO.3, the reverse outer primer B3 is shown as SEQ ID NO.4, the forward loop primer LF is shown as SEQ ID NO.5, and the reverse loop primer LB is shown as SEQ ID NO. 6.
5. The LAMP kit for detecting Pythium erythraeum of claim 4, for use in detecting Pythium erythraeum.
6. An LAMP detection method of Pythium erythraeum, characterized in that the LAMP kit of claim 4 is used for detection by LAMP reaction.
7. The LAMP detection method according to claim 6, characterized by comprising extracting DNA of a microorganism to be detected, taking 4. mu.L of DNA solution as a reaction template, adding 21. mu.L of the detection solution according to claim 4 to carry out LAMP, and the LAMP reaction procedure is as follows: and (3) carrying out reaction amplification at 62 ℃ for 70min, then adding 0.25 mu L of SYBR Green I into an amplification product, observing color change, if the color is changed from orange to yellow Green, or fluorescence exists under an ultraviolet lamp, indicating that pythium aphanidermatum exists in the object to be detected, and if the color is not changed, still orange, or no fluorescence exists under the ultraviolet lamp, indicating that pythium aphanidermatum does not exist in the object to be detected.
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| KR101719794B1 (en) * | 2014-07-23 | 2017-03-28 | 대한민국 | Primer for diagnosis of Pythium ultimum and uses thereof |
| CN104232782B (en) * | 2014-09-29 | 2016-02-10 | 云南省烟草农业科学研究院 | A kind of detect tobacco soil-borne fungus pathogen PCR primer and application and method |
| CN105316427A (en) * | 2015-12-10 | 2016-02-10 | 中国农业科学院植物保护研究所 | Fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum |
| CN106222263B (en) * | 2016-07-30 | 2019-12-06 | 南京农业大学 | LAMP primer composition for detecting pythium ultimum, kit and detection method thereof |
| CN108060257B (en) * | 2018-01-17 | 2020-01-10 | 南京农业大学 | Primer composition for detecting pythium androsaceus based on loop-mediated isothermal amplification technology and detection method thereof |
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| 基于环介导等温扩增技术快速检测瓜果腐霉菌;李庆玲等;《南京农业大学学报》;20180130(第01期);第79-87页 * |
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