CN116356058A - LAMP primer composition for detecting perennial isobornus and detection method thereof - Google Patents
LAMP primer composition for detecting perennial isobornus and detection method thereof Download PDFInfo
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Abstract
The invention discloses an LAMP primer composition for detecting perennial isobornus and a detection method thereof. The primer composition for detecting the perennial isobornus consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a loop primer LF and LB. The LAMP reaction procedure was: the isothermal condition of 63 ℃ is amplified for 60min, perennial heterobasidiomycetes in a sample to be detected can be detected rapidly, conveniently, efficiently, highly specifically and highly sensitively, complex instruments are not needed, the field detection of the perennial heterobasidiomycetes can be well met, the method is suitable for inspection and quarantine of entry and exit plants and plants, investigation, rapid diagnosis and monitoring of diseases and the like, and has important significance for preventing the perennial heterobasidiomycetes from being transmitted into China, and meanwhile, the establishment of the system also provides technical guidance and theoretical basis for detection of other pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an LAMP primer composition for rapidly detecting perennial isobornea and a macroscopic detection method thereof.
Background
Perennial heterobasidiomycetes (Heterobasidion annosum) belong to the basidiomycetes phylum, the agaricus (agaricus), the order Russulales, the family echinococcaceae, the genus heterobasidiomycetes (heteobasidiomycetes), mainly infest pine (Pinus) trees, cause conifer trunk rot, and cause great economic losses. Currently, few detection techniques are available for perennial heterobasidiomycetes. The traditional detection method mainly uses a plate separation method, and the identification is carried out according to the morphology of perennial heterobasidiomycetes. The method is time-consuming and labor-consuming, needs rich and professional bacteria identification experience, and is difficult to distinguish the perennial isobornus basidiomycetes from similar species according to morphological characteristics, so that the requirements of the woodland production cannot be met. With the development of molecular biology, particularly the popularization of PCR technology, more and more molecular biology techniques are being applied to the detection of perennial isobornus bacteria. However, these detection techniques often need to be performed in laboratories with specialized instruments, reagents and strict environmental conditions, the instruments and reagents are expensive, the operation requirements are high, the process is complex, the detection time is still long, the requirement of rapid detection cannot be met, and the method is not suitable for use and popularization in the basic environment.
Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification technology developed in recent years, and is a new nucleic acid amplification technology capable of replacing common PCR because of the advantages of simple operation, rapidness, high specificity, low cost and the like. The method 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 precipitation which is a byproduct when a large amount of target DNA is synthesized within 60min at 60-65 ℃. The color development dye such as SYBR Green, hydroxynaphthol blue (HNB) and the like is added into the reaction system, and the color change can be judged by naked eyes. Because the LAMP amplification process relies on identifying 6 independent areas of a target sequence, the reaction specificity is strong, the nucleic acid amplification process is carried out under the constant temperature condition, the common water bath or equipment with stable heat source can meet the reaction requirement, and the detection cost is greatly reduced. At present, the technology is widely applied to the rapid detection of fungi, bacteria, viruses and oomycetes, but is not reported at home and abroad in the detection of perennial isobornea.
The invention designs a specific LAMP primer composition of perennial isobornea by taking glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a target sequence for detection, and establishes a LAMP rapid detection method of the perennial isobornea on the basis.
Disclosure of Invention
The invention aims at solving the problems that the biological detection method of the perennial heterobasidiomycetes in the prior art is long in period, time-consuming, labor-consuming, tedious and poor in specificity, and the problems that a thermal cycle instrument is needed in the PCR detection technology and the perennial heterobasidiomycetes cannot be detected quickly, and provides an LAMP detection primer composition for detecting the perennial heterobasidiomycetes quickly and a molecular detection method thereof.
The aim of the invention can be achieved by the following technical scheme:
the LAMP primer composition for detecting the perennial isobornus comprises a forward inner primer FIP shown as SEQ ID N0.1, a reverse inner primer BIP shown as SEQ ID N0.2, a forward outer primer F3 shown as SEQ ID NO.3, a reverse outer primer B3 shown as SEQ ID N0.4, a forward loop primer LF shown as SEQ ID N0.5 and a reverse loop primer LB shown as SEQ ID N0.6.
The primer composition is applied to detection of perennial isobornus, and the detection is for non-disease diagnosis.
The primer composition is applied to preparation of LAMP detection kit of perennial isobornea.
A LAMP kit for detecting perennial isobornus comprises the primer composition.
As a preferred embodiment of the present invention, the LAMP kit for detecting a perennial isobornus further comprises 10×Thermopol Buffer, mgS0 4 dNTPs, betaine, bst DNApolymerase, hydroxynaphthol blue.
The kit is applied to detection of perennial isobornus strains, and the detection is for non-disease diagnosis.
A LAMP detection method for perennial heterobasidiomycetes is characterized in that DNA of a microorganism to be detected is extracted, a DNA solution is taken as a reaction template, LAMP reaction is carried out by using the primer composition, then the color change of an amplified product is observed, if the detection result is positive in sky blue, the perennial heterobasidiomycetes exists, and if the detection result is negative in purple, the perennial heterobasidiomycetes does not exist.
As a preferred aspect of the present invention, the LAMP reaction system: 2.5. Mu.L 10X ThermoPol Buffer,8 mmol.L -1 MgS0 4 ，1.2mmol·L -1 dNTPs, inner primers FIP and BIP each 1.6. Mu. Mol.L -1 Outer primers F3 and B3 each 0.4. Mu. Mol.L -1 Loop primers LF and LB were each 0.8. Mu. Mol.L -1 ，0.8μmol·L -1 Betaine, 8U.mu.L -1 Bst DNA polymerase，180mmol·L -1 Hydroxynaphthol blue, 2. Mu.L template DNA, and 26. Mu.L total system.
As a preferred embodiment of the present invention, the LAMP reaction is performed by the following procedure: the reaction was amplified at 63℃for 60min.
The method for detecting the perennial isobornus uses the extracted DNA as a template, and uses the LAMP primer composition to carry out LAMP reaction; hydroxylnaphthol blue (HNB) belongs to one of the metal ion indicators. HNB is Mg 2+ The color of the titrant changes with the pH change of the solution, so that the Mg in the LAMP reaction system can be monitored 2+ The change in concentration and the pH of the solution act as a color indicator. HNB is added into the reaction liquid before the reaction, the reaction system is purple, and Mg is added in the reaction process 2+ Combined with by-products of LAMP reaction to produce a large amount of precipitate, mg in solution 2+ The concentration was reduced and the pH was changed, thereby changing the color of HNB from purple to sky blue. Therefore, the presence or absence of the perennial isobornus is determined by the color change of the reaction system after the completion of the reaction: sky blue indicates positive detection, presence of perennial heterobasidiomycetes; purple indicates that the detection result is negative, and perennial heterobasidiomycetes are not present.
Compared with the prior art, the invention has the advantages and positive effects that:
(1) The practicability is good. Gel electrophoresis of products by common PCR reactions is easy to cause product diffusion, which is a major source of laboratory pollution; ethidium Bromide (EB) has huge toxicity and can accumulate and cause cancer; long-term observation of ultraviolet lamps also causes a degree of injury to the experimenters. The LAMP reaction is only carried out in a constant-temperature water bath kettle, and after the reaction is finished, whether the target strain exists or not is judged by observing the change of the color of the solution, so that the application value of the LAMP reaction in the field is improved.
(2) And (5) amplifying at constant temperature. Unlike PCR method, which needs thermal cycle, the method gets rid of the dependence on thermal cycle instrument, and the LAMP reaction can occur only by stable heat source, which greatly expands the application range of LAMP, and the LAMP can react under constant heat source because betaine is added into LAMP reaction liquid, so that double-chain DNA is in dynamic balance of melting, and amplification is realized under the action of Bst DNA polymerase.
(3) The accuracy is high: because the traditional method for detecting the perennial isobornus is only carried out according to morphological characteristics, the growth of the perennial isobornus is unstable due to the influence of the environment and is influenced by similar species, and the method is difficult to accurately identify. According to the genome sequence of the perennial heterobasidiomycete, blast software is utilized to compare the genome sequence of the perennial heterobasidiomycete with the genome sequences of other heterobasidiomycetes, the GAPDH gene sequence of the perennial heterobasidiomycete is selected to design a specific LAMP primer, and the gene is not used as a target in common PCR for designing an identification primer of the perennial heterobasidiomycete. The LAMP reaction specifically recognizes 6 independent regions on the target sequence by 4 primers (FIP, BIP, F, B3), with a relatively high specificity (Table 1). In addition, the forward loop primer LF and the reverse loop primer LB can improve the reaction rate, and the forward loop primer LF and the reverse loop primer LB can be used for rapidly detecting perennial isobornea together with other four primers under the condition of ensuring the reaction accuracy. The LAMP reaction specifically recognizes 6 independent regions on the target sequence by 4 primers, and has higher specificity and sensitivity than 2 independent regions of the target sequence recognized by PCR primers.
(4) The sensitivity is high: the LAMP detection method of the perennial isobornus has very high sensitivity and can reach 100pg DNA, which indicates that the detection method is sufficient for accurately and rapidly detecting the perennial isobornus under the condition of low DNA concentration.
For a better understanding and implementation, the present invention is described in detail below with reference to the drawings.
Drawings
FIG. 1 shows the nucleic acid sequence and primer positions of the GAPDH gene of a perennial heterobasidiomycete.
FIG. 2 and FIG. 3 are specific assays for detecting perennial isobornus LAMP primers:
the primers designed by the invention are used for carrying out LAMP amplification detection on DNA samples of different perennial heterobasidiomycete strains and other bacterial strains, and after the DNA samples are amplified in a water bath at the constant temperature of 63 ℃ for 60 minutes, the color change of the reaction solution is observed, and the results show that: different perennial heterobasidiomycete strains all showed sky blue positive reaction (figure 2), and solutions in reaction tubes of other reference bacterial strains all showed purple negative reaction (figure 3).
FIG. 3 sensitivity of LAMP detection of perennial isobornus:
LAMP amplifying genome DNA with different concentrations; the 26. Mu.L reaction system contained the amplification results of 10ng, 1ng, 100pg, 10pg, lpg, 100fg, and L0fgDNA, respectively (FIG. 4).
Detailed Description
For the purpose of making the objects and technical solutions of the present invention more clear, the present invention is further described with specific examples, not only these examples.
Example 1: detection of perennial isobornus by LAMP
1. LAMP primer composition for detecting perennial isobornus: forward inner primer FIP shown as SEQ ID N0.1, reverse inner primer BIP shown as SEQ ID N0.2, forward outer primer F3 shown as SEQ ID NO.3, reverse outer primer B3 shown as SEQ ID N0.4, forward loop primer LF shown as SEQ ID N0.5, and reverse loop primer LB shown as SEQ ID N0.6.
2. The detection method comprises the following steps: extracting DNA of a microorganism to be detected, taking a DNA solution as a reaction template, adding a detection solution to perform LAMP reaction, and detecting the perennial isobornus by using an LAMP reaction system as follows: 2.5. Mu.L 10X ThermoPol Buffer,8 mmol.L -1 MgS0 4 ，1.2mmol·L -1 dNTPs, inner primers FIP and BIP each 1.6. Mu. Mol.L -1 Outer primers F3 and B3 each 0.4. Mu. Mol.L -1 Loop primers LF and LB were each 0.8. Mu. Mol.L -1 ，0.8μmol·L -1 Betaine, 8U.mu.L -1 Bst DNA polymerase，180mmol·L -1 Hydroxy naphthol blue, 2. Mu.L template DNA, 26. Mu.L total system; the LAMP reaction is carried out by the following procedures: and (3) carrying out reaction amplification for 60min at 63 ℃, and observing the color change of the amplified product, wherein if the amplified product is sky blue, the detection result is positive, the perennial isobornus is present, and if the amplified product is purple, the detection result is negative, and the perennial isobornus is not present.
3. To verify the specificity of the LAMP method, 3 different perennial heterobasidiomycete strains and other 20 fungal strains were used as test materials (table 2), and LAMP detection results showed that: only the tube solutions with perennial isobornus as template exhibited sky blue positive reactions (FIG. 2), and the other reference fungal strains and negative controls all had purple negative reactions (FIG. 3).
TABLE 1
TABLE 2
a BJFC: specimen pavilion of Beijing university microbiological institute of forestry
b JSAFC: specimen library of Jiangsu agriculture and forestry occupational technology college
Example 2: sensitivity of LAMP detection of perennial isobornus
In order to determine the sensitivity of the LAMP detection method, the extracted DNA of the perennial isobornus is measured in concentration by a spectrophotometer and diluted in a 10-fold gradient so that the mass concentration thereof is 10 ng/. Mu.L in order -1 、1ng·μL -1 、100pg·μL -1 、10pg·μL -1 、1pg·μL -1 、100fg·μL -1 And 10 fg. Mu.L -1 LAMP was performed using 2. Mu.L of each of the templates. The reaction procedure was 63℃for 60min. HNB color development result shows that: when the DNA concentration of the perennial isobornus is up to 100 pg.mu.L -1 The solution in the reaction tube becomes sky blue (fig. 4).
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (9)
1. A LAMP primer composition for detecting a perennial isobornus, characterized in that the primer composition consists of: forward inner primer FIP shown as SEQ ID No.1, reverse inner primer BIP shown as SEQ ID No.2, forward outer primer F3 shown as SEQ ID No.3, reverse outer primer B3 shown as SEQ ID No.4, forward loop primer LF shown as SEQ ID No.5, and reverse loop primer LB shown as SEQ ID No. 6.
2. The use of the LAMP primer composition of claim 1 for detecting perennial isobornus, said detection being for non-disease diagnosis purposes.
3. The use of the LAMP primer composition of claim 1 in the preparation of a kit for detecting a perennial isobornus.
4. A LAMP kit for detecting a perennial isobornus, comprising the LAMP primer composition according to claim 1.
5. The LAMP kit for detecting a perennial isobornus according to claim 4, further comprising thermo pol Buffer, mgS0 4 dNTPs, betaine, bst DNA polymerase, hydroxynaphthol blue.
6. The use of the LAMP kit of claim 4 or 5 for detecting a perennial isobornus, said detection being for non-disease diagnosis purposes.
7. A LAMP detection method of perennial heterobasidiomycetes is characterized in that DNA of a microorganism to be detected is extracted, a DNA solution is taken as a reaction template, the primer composition of claim 1 is used for carrying out LAMP reaction, then color change of amplified products is observed, if the detection result is positive in sky blue, the perennial heterobasidiomycetes exists, and if the detection result is negative in purple, the perennial heterobasidiomycetes does not exist.
8. The LAMP detection method according to claim 7, wherein the LAMP reaction system: 2.5. Mu.L 10X ThermoPol Buffer,8 mmol.L -1 MgS0 4 ，1.2mmol·L -1 dNTPs, inner primers FIP and BIP each 1.6. Mu. Mol.L -1 Outer primers F3 and B3 each 0.4. Mu. Mol.L -1 Loop primers LF and LB were each 0.8. Mu. Mol.L -1 ，0.8μmol·L -1 Betaine, 8U.mu.L -1 Bst DNA polymerase，180mmol·L -1 Hydroxynaphthol blue, 2. Mu.L template DNA, and 26. Mu.L total system.
9. The LAMP detection method according to claim 7 or 8, characterized in that the procedure of the LAMP reaction is: the reaction was amplified at 63℃for 60min.
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