CN106701927B - Method and kit for rapidly detecting chrysophyceae through loop-mediated isothermal amplification - Google Patents
Method and kit for rapidly detecting chrysophyceae through loop-mediated isothermal amplification Download PDFInfo
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Abstract
The invention firstly provides a chrysophyceae CoI gene as a molecular marker for applying to the rapid detection of chrysophyceae by using a loop-mediated isothermal amplification technology, and continuously provides a group of loop-mediated isothermal amplification primers which can specifically amplify the molecular marker by using loop-mediated isothermal amplification. Finally, a method for rapidly detecting golden algae by using a loop-mediated isothermal amplification technology is provided, wherein a sample plasmid to be detected and genome DNA are used as templates, and the specific primer group is used for carrying out loop-mediated isothermal amplification reaction. The primer has the advantages of strong specificity, high sensitivity and good repeatability. The lowest concentration which can be detected by the method can respectively reach the existence of 100 copies/mu L chrysophyceae mitochondrion CoI gene and the existence of 1000cells/mL chrysophyceae cell, the sensitivity is improved by 1000 times compared with the sensitivity of the traditional PCR technology, the sensitivity problem is well solved, and the method has important significance for early monitoring of microalgae culture pollutants.
Description
Technical Field
The invention relates to the technical field of microbial molecular detection, in particular to a method and a kit for rapidly detecting golden algae by adopting a loop-mediated isothermal amplification technology.
Background
Chrysophyceae, a flagellate-Poterioochromonas sp, is a most severely contaminated protozoa present in large-scale culture of Chlorella vulgaris. The flagellate has the characteristics of autotrophy and mixed nutrition, is very easy to infect and fast phagocytize chlorella, and once infected, the chlorella can be completely defeated in a short time in large-scale culture, thereby bringing great harm to the large-scale culture of the chlorella. Therefore, the method is very important for the early rapid monitoring and the determination of a molecular identification method of the golden algae in the chlorella culture.
The study on golden algae is currently carried outThe research on the separation and culture and the phagocytic habit of the algal bloom is limited, but the research on the early monitoring is very little, and only the traditional microscopic observation, DNA first generation sequencing and Polymerase Chain Reaction (PCR) stage are remained. The traditional morphological observation sensitivity is low and is as low as 104cells/mL are suitable for the existence of a large number of protozoa, and because protozoa are various in types and large in quantity, besides known morphological knowledge, a large number of species are difficult to distinguish, while DNA first-generation sequencing and Polymerase Chain Reaction (PCR) have the defects of low detection efficiency, complex operation, time consumption, high false positive probability, frequent influence on the judgment of actual conditions, missing the time of optimal pollution control, and are not suitable for basic level and field detection. Therefore, it is important to establish a simple and sensitive early-stage monitoring method for chrysophyceae.
LAMP (Loop-mediated isothermal amplification) is a novel nucleic acid amplification technology developed by Notomi in 2000, which utilizes 4 different specific primers to recognize 6 specific regions of a target gene, performs high-efficiency amplification of a target sequence under a constant isothermal condition by means of Bst DNA polymerase with a strand displacement effect, and can synthesize 109-1010An order of magnitude of target DNA. The technology avoids the special requirement of the conventional PCR on the cycle temperature, and improves the application value of the PCR detection method due to the characteristics of high efficiency, simplicity, low cost, short detection period and the like. To date, LAMP technology has not been effectively used for early monitoring of chrysophyceae, an important contaminant in large-scale microalgae culture.
Disclosure of Invention
The invention aims to provide a method for simply, conveniently, quickly and accurately detecting golden algae in an environment with limited instrument and equipment so as to realize early monitoring of golden algae which is an important pollutant in large-scale microalgae culture.
In order to achieve the above object, a first technical problem solved by the present invention is: the chrysophyceae CoI gene is applied to the rapid detection of the chrysophyceae by using the loop-mediated isothermal amplification technology. Generally, when selecting and detecting target genes, 18S rRNA which is ubiquitous in eukaryotes is preferably selected for eukaryotes, but the genes have high conservation and are not suitable for species-specific analysis. After repeated sequence comparison, the invention discovers that the design of isothermal amplification primers by using the CoI gene as a molecular marker can effectively identify the chrysophyceae even under the condition that other polluted protozoa exist in the process of culturing the microalgae. The DNA sequence of the CoI gene, cytochrome oxidase I (CoI cytochrome c oxidase I) gene can accurately distinguish species differences, and has the specificity of each species, just like the ID card number of the species. In the process of algae culture, pollution of different kinds of microorganisms can be dealt with, and in order to clarify information of the pollutants, the invention takes the chrysophyceae CoI gene as a molecular marker for target monitoring. Meanwhile, because the protozoan gene sequence has high similarity, if direct DNA sequencing and Polymerase Chain Reaction (PCR) are directly adopted, the detection efficiency is low, and the detection result has the condition of false positive. Therefore, the invention continues to adopt the loop-mediated isothermal amplification technology, and uses the chrysophyceae CoI gene as a molecular marker to carry out specificity screening (as shown in a sequence SEQ ID No. 7) and primer design, so that the second technical problem is solved, and a group of loop-mediated isothermal amplification primers are further provided, and can specifically carry out loop-mediated isothermal amplification on the molecular marker.
The primer set is preferably: comprises a pair of outer primers, a pair of inner primers and a pair of circular primers; the outer primers are F3 and B3, and the nucleotide sequences of the outer primers are shown as SEQ ID No. 1-2; the inner primers are FIP and BIP, and the nucleotide sequences of the inner primers are shown as SEQID No. 3-4; the circular primers are LF and LB, and the nucleotide sequences are shown in SEQ ID No. 5-6.
Furthermore, the invention provides a method for rapidly detecting golden algae by using a loop-mediated isothermal amplification technology, which takes plasmids and genome DNA of samples to be detected as templates and utilizes the specific primer group to carry out loop-mediated isothermal amplification reaction.
The method further comprises the steps of: the specific outer primers F3 and B3 are adopted to clone the gene containing the target amplification fragment into a plasmid pGEM-T, and the constructed recombinant plasmid pGEMT-CoI is used as the basis for detecting the sensitivity of the gold alga copy number.
The method further comprises the steps of: and (3) carrying out gel electrophoresis on the amplification product, and if a specific stepped strip appears, judging that the sample contains the chrysophyceae, namely, the chrysophyceae is positive.
Preferably, the reaction system of the loop-mediated isothermal amplification reaction comprises 1 muL of Bst DNA polymerase of 8U/muL, 2.5 muL of Bst DNA polymerase buffer solution of 10 ×, 1.5 muL of 25 mmol/muL MgSO4, 2.5 muL of 10mM dNTPs, 4 muL of 5mol/L Betain, 0.5 muL of 10mM F3/B3 primer, 1.5 muL of 20mM FIP/BIP primer, 1 muL of 10mM LF/LB primer, 1 muL of template, and 1 muL of H2O：6.5μL。
Preferably, the reaction conditions of the loop-mediated isothermal amplification reaction are as follows: the reaction is carried out for 60min at 65 ℃ and for 7min at 80 ℃.
Preferably, the gel electrophoresis is performed by using 2% agarose gel by mass and volume and performing constant-pressure 120V electrophoresis for 30 min.
The method can also be designed into a kit for quickly identifying the chrysophyceae at the early stage. The method has the characteristics of low requirement on samples, high efficiency, accurate detection, time and labor saving, cost saving, obvious effect and the like. The kit comprises the specific primer.
Specifically, the kit can comprise a detection reagent and a genomic DNA extraction reagent. The detection reagent comprises a specific primer group and a positive standard substance. The genomic DNA extraction reagent preferably comprises one of High Puretemplate Preparation Kit by Roche and DNeasy Plant Mini Kit by Qiagen.
The invention provides a group of loop-mediated isothermal amplification primers for rapidly detecting golden algae and a detection method. The primer group has strong specificity, high sensitivity and good repeatability. The lowest concentration which can be detected by the method can respectively reach the existence of 100 copies/mu L chrysophyceae mitochondrion CoI gene and the existence of 1000cells/mL chrysophyceae cell, the sensitivity is improved by 1000 times compared with the sensitivity of the traditional PCR technology, the sensitivity problem is well solved, and the method has important significance for early monitoring of microalgae culture pollutants. In addition, the method is simple and convenient to operate, does not need special instruments, and is convenient for carrying out a large number of sample detections in an environment with limited conditions.
The sensitivity, stability and specificity of the method and the effectiveness of the environmental sample are comprehensively evaluated as follows:
(1) sensitivity: diluting the plasmid quantitative standard pGEMT-CoI by using sterilized double distilled water 10 respectively9、108、107、106、105、104、103、102、101cpies/mu L, and the optimized system is adopted for detection, so as to verify the sensitivity of the established method. The detection range of the method can reach 8 orders of magnitude, and the sensitivity can reach 100 copies.
(2) Specificity: the invention selects 22 samples to carry out specific primer verification: 5 rotifer samples, 12 ciliate samples, 2 amoeba samples, 1 flagellate sample, one chlorella sample. And (3) detecting by adopting a loop-mediated isothermal amplification method, wherein the detection result shows that the rest samples except the golden algae are negative results.
Drawings
FIG. 1 is a preliminary screen of feasibility experiments for three pairs of primers selected by design, where M: 100bp DNAladder; 1: a first pair of primers; 2: a second pair of primers; 3: a third pair of primers; a: using chrysophyceae genome DNA as a template; b: using tetrahymena genome DNA as a template; c: water control;
FIG. 2 shows the results of a pair of specific primers for CoI gene in genomic DNA of Chrysophyta through loop-mediated isothermal amplification;
wherein M: 100bp DNA Ladder; 1: brachionus plicatilis in Jiangxi; 2: brachionus plicatilis; 3: brachionus rufimbriae in Yunnan province; 4: blood corpuscle algae rotifer; 5: water wheel insect of Beijing; 6: tetrahymena; 7: hainan Abelmoschus manihot; 8: beijing Sterkiella; 9: yunnan frontispora; 10: caterpillar of Beijing grass; 11: cutting insects; 12 tip caterpillar; 13: vorticella, Beijing beetle; 14, Hainan fanshaped plande; 15, the double eyebrows are grown; 16, the membrane bag worms; 17: CBN Vannella; 18: heterolobean, wuhan; 19: hainan flagellate suigesumonas clinomogenisis; 20: chlorella; 21, water; 22: golden algae;
FIG. 3 shows the sensitivity results of conventional PCR amplification of chrysophyceae plasmid: from left to right 1-9 are respectively 109Samples were diluted in copical gradients of copies/μ L of plasmid standards at 10 concentrations9～101copies/mu L, M is a DNA molecular weight marker, and N is a negative control;
FIG. 4 shows the sensitivity results of the loop-mediated isothermal amplification of chrysophyceae plasmids: from left to right 1-9 are respectively 109Samples were diluted in copical gradients of copies/μ L of plasmid standards at 10 concentrations9～101copies/mu L, M is a DNA molecular weight marker, and N is a negative control;
FIG. 5 shows the sensitivity results of conventional PCR amplification of genomic DNA from Chrysophyta: from left to right 1-8 are respectively 107The cell concentration of cells/mL is ten times of the gradient dilution sample, the concentration is 10 respectively7～100cells/mL, M is a DNA molecular weight marker, and N is a negative control;
FIG. 6 shows the result of the sensitivity of the genomic DNA of Chrysophyta through loop-mediated isothermal amplification: from left to right 1-8 are respectively 107The cell concentration of cells/mL is ten times of the gradient dilution sample, the concentration is 10 respectively7～100cells/mL, M is a DNA molecular weight marker, and N is a negative control;
FIG. 7 shows the cell setting of Chlorella vulgaris at 106The ratio of the chlorella to the chrysophyte cells from left to right is 10 respectively in the fluorescence signal diagram detected by the addition experiment of each chlorella2:1、103:1、104:1、105:1、106:1；
FIG. 8 shows the cell setting of Chlorella vulgaris at 107The ratio of the chlorella to the chrysophyte cells from left to right is 10 respectively in the fluorescence signal diagram detected by the addition experiment of each chlorella2:1、103:1、104:1、105:1、106:1、107:1；
FIG. 9 is a graph showing fluorescence signals detected in an addition experiment in which 108 cells of Chlorella were set, and the cell ratios of Chlorella from left to right to Chlorella and Chlorella are 10 respectively2:1、103:1、104:1、105:1、106:1、107:1、108:1。
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: design and Synthesis of primers
(1) Designing and screening primers: several pairs of primers were designed and synthesized based on the gene sequence of chrysophyceae cytochrome oxidase I (CoI cytochrome c oxidase I), and primers among them, which were useful in subsequent experiments, were screened based on the following conditions.
a. The sequence GC content is defined as a common sequence with the content of 40-65%, the sequence GC content of more than 65% is defined as a GC-rich sequence, and the sequence AT content of less than 40% is defined as an AT-rich sequence;
b. primer lengths were set based on GC content, with different primer lengths as follows:
c. the melting temperatures (Tm values) of the different primers are set according to the GC content of the sequence.
d. The primer spacing, F2 and B2 primer spacing, is 120-180nt, F3 and F2 primer spacing is 0-20nt, and F1c and B1c primer spacing is 0-100 nt.
e. The stability of the primer end is measured by the difference value (delta G) of the free energy before and after the junction of 6 bases at the primer end and a target sequence. The free energy difference was calculated using the SantalLucia method. The 3 'short free energy difference of the F2, B2, F3, B3, LF and LB primers should be less than or equal to-4 kcal/mol, and the 5' free energy difference of the F1c and B1c primers should be less than or equal to-3 kcal/mol.
(2) The primer composition is as follows: the method mainly utilizes 4 different specific primers to identify 6 specific regions of a target gene, and can carry out amplification reaction under isothermal condition. The amplification of the gene and the detection of the product can be finished in one step, the amplification efficiency is high, and the amplification can be carried out for 10 min to 60min9-1010Doubling; the specificity is high, and the detection of the target gene sequence can be judged only by the existence or nonexistence of an amplification product. The presence or absence of amplification reaction is judged by using a stepwise band of electrophoresis.
Amplification principle: DNA is in a state of dynamic equilibrium at about 65 ℃ and when any one of the primers is extended by base pairing to the complementary part of the double-stranded DNA, the other strand is dissociated and becomes a single strand. Under the action of strand displacement DNA polymerase, the 3' -end of the F2 segment of the FIP primer is used as the origin and is paired with the complementary sequence of the template DNA, thereby initiating strand displacement DNA synthesis. The F3 primer is complementary to the F3c sequence at the tip of F2c, and extends forward by synthesizing its own DNA while displacing the DNA strand synthesized by the FIP primer before the DNA strand is synthesized by the action of a strand displacement type DNA polymerase from the 3' end. Finally, the DNA strand synthesized from the F3 primer forms a double strand with the template DNA. The DNA strand synthesized first by the FIP primer is replaced by the F3 primer to generate a single strand, and the single strand has complementary F1c and F1 segments at the 5' end, and self-base pairing occurs to form a ring structure. Meanwhile, the BIP primer is hybridized and combined with the single strand, a complementary strand is synthesized by taking the 3' end of the BIP primer as a starting point, and a ring structure is opened in the process. Then, similarly to F3, the B3 primer is inserted from the outside of the BIP primer to form base pairing, and a new complementary strand is synthesized from the 3' end as the origin by the action of polymerase. Through the above two processes, a double-stranded DNA is formed. And complementary sequences exist at two ends of the replaced single-stranded DNA, self base pairing occurs naturally to form a ring structure, and then the whole strand presents a dumbbell-shaped structure. The structure is the initial structure of the gene amplification cycle of the loop-mediated isothermal amplification method. All the processes so far are intended to form the starting point structure of the gene amplification cycle of the loop-mediated isothermal amplification method. Loop-mediated isothermal amplification gene amplification cycle: first, in the dumbbell structure, DNA synthesis and extension were performed using the F1 segment at the 3' end as the starting point and itself as a template. At the same time, FIP primer F2 hybridized to the single-stranded loop F2c, initiating a new round of strand displacement reaction. Through the process, the complementary sequences on the same strand form structures with different sizes repeatedly until the template replication and amplification are completed.
According to the design and screening, three pairs of specific outer primers, three pairs of specific inner primers and three pairs of specific circular primers are finally selected. The outer primers are F3 and B3, and the nucleotide sequences are shown as SEQ ID No. 1-2; the inner primers are FIP (consisting of sequences of F1c and F2) and BIP (consisting of sequences of B1c and B2), and the nucleotide sequences of the inner primers are shown in SEQ ID No. 3-4; the circular primers are LF and LB, and the nucleotide sequences are shown in SEQ ID No. 5-6:
F3：5’-GTTCTTGTAACAGGACATGC-3’；
B3：5’-AAACAGTCCATCCGGTAC-3’；
FIP：5’-GGCTCCTATCATTAATGGAGCAAAATAATGATTTTCTTCATGGTTATGCC-3’；
BIP：5’-GGCGTTCCCAAGGCTAAATAATATTAGCTTCTACTAATGCTGATGA-3’；
LF：5’-GTTTCCAAATCCTCCAATT-3’；
LB：5’-GTTATTACCACCTTCTTTAT-3’。
example 2: construction and preparation of plasmid quantitative standard
The method comprises the following steps:
1. construction of plasmid Standard
Cloning by a PCR method to obtain a CoI gene fragment containing a target gene sequence, recombining the CoI gene fragment into a vector pGEM-T, and determining a DNA sequence. The constructed recombinant plasmid was named pGEMT-CoI as a quantitative standard.
2. Preparation of quantitative standards
The plasmid pGEMT-CoI was extracted and purified with an Omega plasmid extraction kit, and the copy number was converted from the concentration and mass of the plasmid determined by Nanodrop8000 and the Avogastron constant.
The calculation formula is as follows:
the average molecular weight of one base pair is 660 g/mol;
the total length of the plasmid is the total length of the vector plus the length of the insert;
n represents the Avogastron constant (6.02 × 10)23copies/mol)。
Example 3: extraction of genomic DNA
And counting the cells of the culture solution of the pure cultured golden algae and chlorella to obtain the cell density of the golden algae and the chlorella. 2mL of each cell culture solution was subjected to High Pure Template fractionation Kit (Roche) to extract Pure gold algae DNA, and DNeasy Plant Mini Kit (QIAGEN) DNA extraction Kit was used to extract DNA mixed therewith, and the concentration and quality of DNA were measured using Nanodrop 8000. And selecting DNA with the best concentration and quality for storage and standby.
Example 4: establishment of loop-mediated isothermal amplification reaction system
1. Through a large amount of experimental exploration, an optimal loop-mediated isothermal amplification reaction system and reaction conditions are established.
The specific components and contents of the optimum reaction system are as follows (25. mu.L)
The reaction conditions of the loop-mediated isothermal amplification reaction are as follows: the reaction is carried out for 60min at 65 ℃ and for 7min at 80 ℃.
The gel electrophoresis is performed for 30min by using 2% agarose gel by mass and volume and performing constant-pressure 120V electrophoresis.
Example 5: preliminary screening of feasibility of loop-mediated isothermal amplification reaction primers
In order to test the feasibility of the designed primers, three groups of synthesized primers are subjected to sample feasibility analysis, and the invention selects a positive sample of chrysophyceae genomic DNA, a negative tetrahymena genomic DNA and a water control. The loop-mediated isothermal amplification method is adopted for detection, the detection result shows that except the result obtained by the first group of primers meets the requirement, the other samples are negative results, and the result is shown in figure 1.
Example 6: verification of specificity of primer in loop-mediated isothermal amplification reaction
In order to test the specificity of the loop-mediated isothermal amplification reaction primers, 20 samples are selected for detection: 5 rotifer samples, 12 ciliate samples, 2 amoeba samples, 1 flagellate sample, one chlorella, one water control. The loop-mediated isothermal amplification method is adopted for detection, the detection result shows that other samples except the golden algae are negative results, and the result is shown in figure 2.
Example 7: sensitivity of Loop-mediated isothermal amplification reactions
1. Template plasmid and genomic DNA
The quantitative standard pGEMT-CoI was diluted with sterilized double distilled water in 10-fold gradient, calculated according to the formula in example 2. The plasmid concentration after dilution was 10 respectively9、108、107、106、105、104、103、102、101And (4) reactions are carried out by adopting the optimized system and conditions.
According to the counting result, 10 times of double distilled water is used for diluting the chrysophyte cell sample in a gradient way, the genome DNA is extracted, and the number of the diluted cells is 10 respectively7、106、105、104、103、102、101、100cells/mL, using optimized system and conditions for reaction. 2. Conventional PCR
And (3) carrying out PCR amplification by using the gradient diluents as templates, and carrying out gel electrophoresis detection after the reaction is finished.
3. Loop-mediated isothermal amplification reaction
The above-mentioned gradient dilutions were used as templates to perform loop-mediated isothermal amplification reaction using the reaction system described in example 4, and after the reaction was completed, gel electrophoresis detection was performed.
The results show thatSensitivity detection bottom line for plasmid standard after PCR amplification is 105copies/. mu.L, as in FIG. 3; the sensitivity of the detection on the DNA of the golden algae genome is 105cells/mL, as in FIG. 5.
The primer provided by the invention is used for carrying out loop-mediated isothermal amplification, and the sensitivity detection bottom line for plasmid standard substances is 102copies/μ L, visible sensitivity is 1000 times higher than that of traditional PCR, as shown in FIG. 4; the sensitivity of the detection on the DNA of the golden algae genome is 103cells/mL, 100-fold higher sensitivity than conventional PCR, as shown in FIG. 6.
Example 8: chlorella and Chlorella addition test
The method comprises the following steps:
1. cell setting of Chlorella is 106An
Respectively take 100、101、102、103、104The golden algae cells are 10 in sequence according to the ratio of the chlorella to the golden algae cells6:1、105:1、104:1、1031 and 1021, extracting nucleic acid according to the methods described in examples 2 and 3. And carrying out reaction by adopting an optimized system and conditions. The results show that when the ratio of chlorella to chrysophyceae cells is equal to 1031, the existence of the golden algae can be effectively detected, and the detection result is shown in figure 7.
2. Cell setting of Chlorella is 107An
Respectively take 100、101、102、103、104、105The golden algae cells are 10 in sequence according to the ratio of the chlorella to the golden algae cells7:1、106:1、105:1、104:1、1031 and 1021, extracting nucleic acid according to the method described in example 3. And carrying out reaction by adopting an optimized system and conditions. The results show that when the ratio of chlorella to chrysophyceae cells is equal to 1041, the existence of the golden algae can be effectively detected, and the detection result is shown in figure 8.
3. Cell setting of Chlorella is 108An
Respectively take 100、101、102、103、104、105、106The golden algae cells are 10 in sequence according to the ratio of the chlorella to the golden algae cells8:1、107:1、106:1、105:1、104:1、1031 and 1021, extracting nucleic acid according to the methods described in examples 2 and 3. And carrying out reaction by adopting an optimized system and conditions. The result shows that when the ratio of the chlorella to the chrysophyceae cells is less than or equal to 1061, the existence of the golden algae can be effectively detected, and the detection result is shown in figure 9.
As can be seen from the experiments of the above examples, the detection method of the invention can rapidly and accurately detect the existence of the chrysophyte, and the primer has strong specificity, high sensitivity and good repeatability. The lowest concentration which can be detected by the method can respectively reach the existence of 100 copies/mu L chrysophyceae mitochondrion CoI gene and the existence of 1000cells/mL chrysophyceae cell, the sensitivity is improved by 1000 times compared with the sensitivity of the traditional PCR technology, the sensitivity problem is well solved, and the method has important significance for early monitoring of microalgae culture pollutants.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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gagacccaat actatatcaa catttattct gattttttgg tcaccctgaa gttt 714
Claims (12)
1. The loop-mediated isothermal amplification primer group can specifically amplify the molecular marker shown as SEQ ID No.7 by loop-mediated isothermal amplification; comprises a pair of outer primers, a pair of inner primers and a pair of circular primers; the outer primers are F3 and B3, and the nucleotide sequences of the outer primers are shown as SEQ ID No. 1-2; the inner primers are FIP and BIP, and the nucleotide sequences of the inner primers are shown in SEQ ID No. 3-4; the circular primers are LF and LB, and the nucleotide sequences are shown in SEQ ID No. 5-6.
2. The primer set of claim 1, wherein the primer set is used in a method for rapidly detecting chrysophyceae by using a loop-mediated isothermal amplification technology.
3. A method for rapidly detecting golden algae by a loop-mediated isothermal amplification technology is characterized in that a sample plasmid to be detected and genome DNA are taken as templates, and the primer group of claim 1 is utilized to carry out loop-mediated isothermal amplification reaction.
4. The method for rapidly detecting golden algae by the loop-mediated isothermal amplification technology according to claim 3, further comprising the steps of: and (3) carrying out gel electrophoresis on the amplification product, and if a specific stepped strip appears, judging that the sample contains the chrysophyceae, namely, the chrysophyceae is positive.
5. The method for rapidly detecting golden algae by the loop-mediated isothermal amplification technology according to claim 3, wherein the reaction system of the loop-mediated isothermal amplification reaction comprises 1 μ L of Bst DNA polymerase with concentration of 8U/μ L, 2.5 μ L of Bst DNA polymerase buffer solution with concentration of 10 ×, and 25mmol/μ L of MgSO 254: 1.5 mu L; 10mM dNTPs: 2.5 mu L; 5mol/L of Betain: 4 mu L of the solution; 10mM F3/B3 primer: 0.5 mu L; 20mM FIP/BIP primer: 1.5 mu L; 10mM LF/LB primer: 1 mu L of the solution; template: 1 mu L of the solution; h2O：6.5μL。
6. The method for rapidly detecting golden algae by the loop-mediated isothermal amplification technology according to claim 3 or 5, wherein the reaction conditions of the loop-mediated isothermal amplification reaction are as follows: the reaction is carried out for 60min at 65 ℃ and for 7min at 80 ℃.
7. The method for rapidly detecting golden algae by the loop-mediated isothermal amplification technology according to claim 4, wherein the gel electrophoresis is performed by using 2% agarose gel by mass-volume ratio and performing constant-pressure 120V electrophoresis for 30 min.
8. The application of the primer group in the claim 1 in the preparation of the kit for rapidly detecting chrysophyceae by the loop-mediated isothermal amplification technology.
9. A kit for rapidly detecting chrysophyceae by using a loop-mediated isothermal amplification technology, which comprises the primer set of claim 1.
10. The kit of claim 9, wherein: comprises a detection reagent and a genome DNA extraction reagent.
11. The kit of claim 10, wherein: the detection reagent comprises: the primer set and the positive standard substance according to claim 1.
12. The kit of claim 10, wherein: the genomic DNA extraction reagent includes one of High Pure Template Preparation Kit of Roche and DNeasy Plant Mini Kit of Qiagen.
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| CN105177135A (en) * | 2015-09-09 | 2015-12-23 | 宁波大学 | Detection method of karlodinium micrum |
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| CN101838697B (en) * | 2010-05-12 | 2012-06-20 | 中国水产科学研究院东海水产研究所 | Method for fast detecting Kerina breve by using loop-mediated isothermal amplification technology |
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| CN105177135A (en) * | 2015-09-09 | 2015-12-23 | 宁波大学 | Detection method of karlodinium micrum |
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