CN117230235A - False-positive-free amplification rapid detection method and kit for plant disease alternaria alternata - Google Patents

Abstract

The invention discloses a false-positive-free amplification rapid detection method and a kit aiming at Alternaria alternata, belonging to the technical field of plant fungal disease detection. The invention utilizes a hydrogel system to limit the nucleic acid loop-mediated isothermal amplification reaction to the micro-nano scale, improves the activity of isothermal amplification enzyme, shortens the detection time, designs an inner primer sequence, prevents the primer from forming a G-C complementary structure by more than or equal to 2 continuous bases at the 3' end of the inner primer FIP or BIP from being G or C, effectively inhibits the generation of non-specific products, thoroughly eliminates false positive results and ensures the accuracy of detecting Alternaria alternata. The method is simple in integral operation, high in detection speed and high in portability, and is suitable for rapid counting of the Alternaria alternata on the base layer site.

Description

False-positive-free amplification rapid detection method and kit for plant disease alternaria alternata

Technical Field

The invention relates to the technical field of plant fungal disease detection, in particular to a false positive amplification-free rapid detection method and a kit for plant disease alternaria alternata.

Background

Alternaria alternata (Alternaria alternate) is a global problem that causes economic losses of agricultural products, fruits and vegetables because of black spot disease caused by plant infection. The traditional detection method for A. Alternata mainly comprises the steps of purifying monospore of a colony according to the purely cultured micro morphology, and analyzing and identifying the monospore of the colony by comparing the morphology of conidium and spore-forming structure. However, the determination of morphological characteristics is easily affected by the environment, and a set of standard identification methods under various growth environments cannot be found out.

Modern molecular rapid detection methods amplify target nucleic acid genomes based on specific primers, allowing fungal detection from morphology-based phenotypes into molecular-system-based genotypes. The third generation molecular detection technology refers to digital detection of amplification by placing single nucleic acid molecules in a reaction chamber with independent limiting fields, and the technology does not depend on a standard curve, and can calculate the absolute copy number of target nucleic acid by only reading the number of positive systems at the end point. However, the most commonly used liquid drop type and chip type digital nucleic acid detection products in the market have complex operation in practical application, require professional personnel, limit the application of the liquid drop type and chip type digital nucleic acid detection products, find a medium or technology with high biocompatibility and portability, and have very important significance for improving the rapid detection efficiency of plant disease fungi.

The nano-domain-limited characteristic of the hydrogel material has the characteristics of simplicity and high efficiency in the field of molecular research, and has the advantages of good biocompatibility, adjustable biodegradability, porous structure and the like. For example, patent document CN 112410405A discloses that a hydrogel system is added into an LAMP reaction system, and a nano pore canal in the gel can play a role in isolating organic matters, heavy metals and other inhibitors, so that quick detection of bacteria in a complex sample is realized. The identification of eukaryotes with high molecular weight genome is obviously different from prokaryotes, and the target high molecular weight gene to be detected brings serious false positive, so that a great research space is still provided for how to apply the advantages of the hydrogel to the fields of fungus accurate analysis and the like.

The nonspecific false positive amplification is a common phenomenon in the rapid detection process of nucleic acid, has adverse effects on the accuracy and sensitivity of fungus identification, and 4-6 LAMP primers increase the possibility of forming primer dimers in a reaction system. Meanwhile, the digital LAMP technology improves the enzyme activity under the limited space, shortens the detection time, and increases the self-assembly or non-specific hybridization of the primer under the high enzyme activity. Thus, eliminating non-specific false positive products remains a critical challenge for rapid detection techniques of nucleic acids.

The existing mode for inhibiting the nonspecific signals generally adopts nano materials such as graphene oxide or molybdenum disulfide to physically adsorb redundant nucleic acid chains (Anal. Chem.2019,91,15694-15702,ACS Appl.Mater.Interfaces 2018,10,4409-4418), but the method does not eliminate a molecular layer from the self-amplification process, can influence the amplification of an LAMP positive system, and reduces the detection sensitivity and speed. The addition of nucleic acid probes can be used to increase the specificity of the assay (Anal. Chem.2019,91, 12852-12858), but does not eliminate non-specific amplification and competes with specific amplification.

Therefore, how to realize rapid monitoring of plant disease alternaria alternata and effectively eliminate nonspecific amplification is a problem that needs to be solved by researchers in the field.

Disclosure of Invention

The invention aims to provide a detection method for rapidly and accurately identifying plant disease Alternaria alternata, overcomes the defect of nonspecific amplification in digital molecular detection, and realizes accurate digital counting of Alternaria alternata on post-harvest agricultural products.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides a false-positive-free amplification rapid detection method for alternaria alternata, which aims at plant diseases, and comprises the following steps of:

(1) Extracting genome DNA in a sample to be detected, and adding the genome DNA serving as a template into a loop-mediated isothermal amplification reaction system containing hydrogel monomers to form a hydrogel system; the reaction system comprises a primer for specifically amplifying the Alternaria alternata DNA fragment, wherein the primer comprises an inner primer FIP and a BIP, and more than or equal to 2 continuous bases at the 3' -end of the inner primer FIP or the BIP are not G or C;

(2) Placing the hydrogel system under a constant temperature condition for isothermal amplification reaction;

(3) After the reaction is finished, fluorescent signals in the hydrogel system are analyzed by utilizing a fluorescent imaging technology, fluorescent points are calculated, and the absolute molecular concentration of the amplified Alternaria alternata DNA in the sample to be detected is calculated.

The sample to be tested can be pure culture of Alternaria alternata, or agricultural products such as fruits and vegetables. The picked agricultural products are easy to be infected by Alternaria alternata, and the fungus diseases are rapidly identified in early stage by adopting the detection method provided by the invention, so that economic loss caused by the occurrence of the diseases is reduced.

In step (1), the Alternaria alternata nucleic acid genome is first extracted. Alternaria alternata is a eukaryotic organism whose cell wall is more difficult to lyse than prokaryotes such as bacteria and viruses, and has a nucleic acid genome of greater molecular weight. Therefore, it is necessary to perform cell disruption against Alternaria alternata, sufficiently release nucleic acids in fungal cells, purify and extract the nucleic acids, and then perform isothermal amplification count of hydrogels.

Preferably, liquid nitrogen cryo-milling samples to break cells is selected, and FH Plant DNA Kit is selected for extraction and purification of the Alternaria alternata nucleic acid genome.

After the nucleic acid extraction is finished, fully mixing the nucleic acid sample to be detected with each reagent of the LAMP reaction system, adding a hydrogel monomer into the system, standing and crosslinking at room temperature to form hydrogel, so that the extracted nucleic acid molecules are randomly distributed in a nano porous channel of the hydrogel, and limiting the template molecules to be detected in the inner region.

The invention utilizes the limited space inside the nano porous hydrogel to carry out isothermal amplification reaction of the fungal nucleic acid sample. Due to the presence of the cross-linked network, the hydrogels allow for the relative separation of nucleic acids without complex manipulation. Thanks to the high density nanopore structure of the hydrogel, the template molecules can be amplified inside the nanopore, and the cross-linked hydrogel walls strongly block the diffusion of the nucleic acid product.

In order to eliminate non-specific products caused by nucleic acid amplification, the invention observes the difference of non-specific amplification caused by different primers by improving the sequence of the inner primer, and definitely determines the specific base sequence causing false positive signals. The results of the study showed that when two or more consecutive bases at the 3' end of a single inner primer FIP or BIP (from the 3' end to the 5' end) are G or C (e.g., GG, CC, GC, CG), the end readily forms a G-C complementary structure with CC, GG, CG, GC in the primer sequence, resulting in primer self-assembly and nonspecific hybridization, thus giving rise to false positive results. Thus, in designing the inner primers, the G, C base number and G/C base ratio contained at the 3' ends of the inner primers FIP and BIP are reduced, so that the formation of two G-C complementary structures between a single inner primer itself or two inner primers is avoided, for example, two G or C continuous bases are not present at the 3' end, namely, two continuous bases at the 3' end of FIP or BIP are not GG, CC, GC, CG, or when the 3' end base of the inner primer FIP (or BIP) is G (or C), then the 3' end base of the other primer BIP (or FIP) is avoided being designed as C (or G). The primers designed by the strategy are used for hydrogel LAMP amplification, so that nonspecific products caused by nucleic acid amplification can be eliminated on a molecular level, and false positive results are avoided.

Preferably, primers are designed for the cohesive galacturonase (aagp) gene fragment of Alternaria alternata, specifically comprising:

F3：5'-AAGATCACTGTCAAGGGCG-3'(SEQ ID NO.1)；

B3：5'-ATGGTAAGACCATCGCAGC-3'(SEQ ID NO.2)；

FIP：5'-TGGGCTTGGTCTTTCCACCATTCCGAGGGATCTGTTCTCA AC-3'(SEQ ID NO.3)；

BIP:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGACGGGA-3' (SEQ ID NO. 4); or 5'-TTCTCCGCTCACAAACTGACCGAACGACTTG GAC-3' (SEQ ID NO. 5);

LF：5'-ACCAACGAGCACCATCACC-3'(SEQ ID NO.6)；

LB：5'-ACTCCACCATCACCGGCAT-3'(SEQ ID NO.7)。

according to the invention, through designing the 3' -end sequence of the inner primer, the primer is prevented from forming a G-C complementary structure, the nonspecific amplification of nucleic acid can be effectively inhibited, and the false positive result is eliminated.

Furthermore, in order to obtain the quick, stable and high-detection-rate alternaria alternata nucleic acid LAMP, the invention optimizes various factors influencing the hydrogel LAMP system.

Preferably, the loop-mediated isothermal amplification reaction system comprises: 1 xLAMP buffer, 6-8 mM MgSO ₄ 1.0 to 1.4mM dNTP,640 to 1280U/mL Bst2.0DNA polymerase, 0.2 to 1.6. Mu.M FIB and BIP,0.2 to 0.3. Mu.M F3 and B3,0.4 to 0.8. Mu.M LF and LB, and fluorescent dye.

More preferably, the total magnesium ion concentration is 7mM; dNTP concentration was 1.4mM; bst2.0 isothermal amplification enzyme concentration was 960U/mL, inner primer FIB and BIP concentration was 1.6. Mu.M, outer primers F3 and B3 concentration was 0.2. Mu.M, and loop primers LF and LB concentration was 0.6. Mu.M.

The fluorescent dye is any one of Eva Green, STBR Green, syto 9 and SYTOX Orange.

Preferably, the invention employs polyethylene glycol based hydrogels, and the hydrogel monomers include mercapto-polyethylene glycol-mercapto and tetra-arm ethylene glycol acrylate. Mixing the two monomers in water solution, and forming gel at room temperature for 3-5 min.

Preferably, the molecular weight of the mercapto-polyethylene glycol-mercapto group is 3400MW and the molecular weight of the four-arm ethylene glycol acrylate is 10000MW.

Preferably, the molar mass ratio of the mercapto-polyethylene glycol-mercapto group to the four-arm ethylene glycol acrylate is 2:1.

more preferably, 1.1mg of mercapto-polyethylene glycol-mercapto group, 1.6mg of 4-arm-polyethylene glycol-acrylate is added to 25. Mu.L of the hydrogel system.

In the step (2), the hydrogel system is placed in a sealed space for preventing evaporation of water to perform isothermal amplification reaction. The sealing space is utilized to prevent the water in the hydrogel from evaporating, so that the accuracy of the detection result is ensured. Specifically, the mixed system solution is dripped into a transparent sealing small chamber, and is stood for forming glue, and is sealed by a film.

The isothermal amplification reaction is carried out under the condition of heating at 60-70 ℃ for 5-60 min. To enhance the signal to noise ratio of the fluorescent amplified spot signal, the sample is then heated at 75-80℃for 3-7 min.

Preferably, the conditions of the isothermal amplification reaction include: the first step, 63-65 ℃ for 9-15 min; and secondly, heating at 80 ℃ for 5min.

In the step (3), fluorescent spots generated after LAMP in-situ amplification in the hydrogel system are imaged by utilizing a fluorescence imaging technology. In the invention, the digital hydrogel LAMP visualizes the detection result by means of fluorescence signals under the environment of providing good nano-confinement. The template DNA is amplified in the internal nanopore, and the products accumulate over time, forming a single nucleic acid bolus. When the wavelength of the fluorescent light source is in the excitation wavelength range of the dye, the single nucleic acid groups in the hydrogel hole can generate corresponding fluorescent bright spots.

Preferably, the hydrogel system after the LAMP amplification is irradiated by a portable fluorescent light source to excite the amplified group of the Alternaria alternata nucleic acid genome so as to emit fluorescence, the portable intelligent mobile phone shoots a picture, and the number of fluorescent spots is analyzed to obtain the absolute molecular concentration of Alternaria alternata.

The portable fluorescent light source can be a handheld fluorescent flashlight or a small gel fluorescent gel cutting instrument. And recording an analysis result by using the smart phone. The method is suitable for realizing rapid field detection in areas with lack of resources and crude conditions.

The invention also provides a kit for detecting the alternaria alternata, which comprises a loop-mediated isothermal amplification reaction reagent, a hydrogel monomer and a primer for specifically amplifying the alternaria alternata DNA fragment.

Specifically, the loop-mediated isothermal amplification reaction reagent comprises isothermal amplification reaction buffer solution, dNTPs, magnesium sulfate, isothermal amplification DNA polymerase and fluorescent dye.

The hydrogel monomer comprises sulfhydryl-polyethylene glycol-sulfhydryl and four-arm ethylene glycol acrylate.

The primers for specifically amplifying the Alternaria alternata DNA fragment comprise the inner primer, the outer primer and the loop primer.

The invention has the beneficial effects that:

(1) The invention provides a false-positive-free amplification rapid detection method for plant disease Alternaria alternata, which utilizes a hydrogel system to fix template Alternaria alternata nucleic acid molecules at positions in an inner pore canal to form a single physical segmentation chamber, and the amplification is carried out under isothermal conditions, so that the concentration of amplified products is gradually increased along with time, and the amplification can be rapidly observed by naked eyes to form a single Alternaria alternata fluorescent amplification point, thereby realizing absolute quantitative counting of Alternaria alternata.

(2) The invention adopts a digital isothermal amplification mode to detect the nucleic acid molecular sample, so that the nucleic acid amplification reaction is limited in micro-nano scale, the activity of isothermal amplification enzyme is improved, the detection time is shortened, meanwhile, the inner primer sequence is modified to effectively inhibit the generation of nonspecific products, the false positive result is thoroughly eliminated, and the accuracy of Alternaria alternata detection is ensured.

(3) The detection method provided by the invention has specificity for detecting the Alternaria alternata template, and can not amplify other common agricultural product fungus strains. In a nucleic acid sample mixed with a plurality of fungi, the target nucleic acid of Alternaria alternata can be effectively distinguished without being interfered by other kinds of nucleic acid molecules. In addition, the statistical analysis result of the multi-batch test count is stable, and the reproducibility is good.

(4) The quick and accurate counting method of the alternaria alternata not only can be applied to the accurate molecular counting of pure culture of the alternaria alternata, but also can be applied to the accurate counting of the nucleic acid of the alternaria alternata on the peel or pulp of agricultural products. The method has the advantages of simple integral operation, high detection speed, small fluorescent imaging and mobile phone shooting record, higher portability, suitability for quick counting of the Alternaria alternata on the base layer site, and higher commercial potential.

Drawings

FIG. 1 is a flow chart of rapid digital counting of hydrogels of Alternaria alternata for post-harvest agricultural products.

FIG. 2 is a graph of digital nonspecific amplification products in hydrogels, wherein (a) and (b) are the original positive and negative system endpoint amplification plots and the real-time fluorescence plots, respectively; (c) A fluorescence profile of a negative system without FIP or BIP inner primer; (d) A fluorescence profile for a negative system containing FIP and BIP, or only FIP or BIP; (e) The positive fluorescence profile for BIP-free, 0.2. Mu.M BIP hydrogel and aqueous solution.

FIG. 3 is a comparison of non-specific amplification product elimination using modified BIP primer pairs, wherein (a) and (b) are a negative system fluorescence curve comparison plot and a hydrogel endpoint count comparison plot, respectively; (c) Hydrogel endpoint count comparison plots for positive amplification systems; (d) To improve the comparison of fluorescence curves of the primers BIP-G2 and BIP-G4 in hydrogel and aqueous systems; (e) For a comparison of the electrophoresis patterns after amplification of the original BIP primer and the modified BIP primer, M: DNAmarker, molecular weight of the lowest noodle band is 150bp,1: original BIP primer positive amplification, 2: original BIP primer negative amplification, 3: improvement of BIP-G4 primer positive amplification, 4: BIP-G4 primer negative amplification was improved.

FIG. 4 shows the optimization of the reaction time (a), amplification temperature (b), magnesium ion concentration (c), isothermal amplification enzyme concentration (d) and loop primer concentration (e) of the Alternaria alternata counting system.

FIG. 5 shows hydrogel endpoint counts (a) and standard plots (b) for sensitivity evaluation of Alternaria alternata counting system.

FIG. 6 is a specific detection of Alternaria alternata counting system against common fungi of agricultural products.

FIG. 7 is a reproducibility evaluation of the Alternaria alternata counting system in different batches of test samples.

FIG. 8 shows the hydrogel count of fungus contaminated Alternaria alternata during storage of jujube fruit, with the left panel showing whole jujube fruit and the right panel showing fresh cut jujube.

Detailed Description

The invention will be better understood by further describing the details of the invention in the following description, in order to make the purpose, overall flow and application of the invention more clear, but not to limit the scope of the invention. Those skilled in the art will recognize that many alternative modifications may be made to this architecture scheme flow, which would still be within the spirit and scope of this disclosure, and which is intended to be included within the scope of this disclosure.

The following sections illustrate the preparation and use of standard reagents to determine the accuracy of the results of the Alternaria alternata counting system based on a comparison of hydrogel system counts of the extracted Alternaria alternata nucleic acid genome to theoretical concentrations.

Thiol-polyethylene glycol-thiol (MW 3400), 4-arm-polyethylene glycol-acrylate (MW 10000) used in the examples were purchased from Laysan Bio Inc. A sealed incubation chamber (frame-seal) was purchased from Bio-Rad company. Primers were synthesized by the division of biological engineering (Shanghai). 10X Isorhermal Amplification Buffer, bst2.0 WarmStart ^TM DNA polymerase, mgSO ₄ dNTPs were purchased from New England Biolab. The rapid non-toxic Plant DNA extraction kit FH Plant DNAKit was purchased from beijing manter biotechnology limited. SYBR Green I is purchased from Shanghai flash molecular Biotechnology Inc. Liquid nitrogen was purchased from Hangzhou Jinjingjingjingjingjingjinggao, inc. Potato Dextrose Agar (PDA) medium was purchased from Hangzhou Kiriluz lake Biotechnology Co.

Alternaria alternata BNCC 185706, alternaria alternata WH1-21 BNCC 122814, alternaria alternata BNCC 338228, alternaria alternata BNCC 120440, alternaria alternata BNCC 113562, aspergillus fumigatus BNCC 185786, alternaria alternata BNCC 369805 and Fusarium oxysporum BNCC 120618 are purchased from Henan province industrial microorganism strain engineering technology research center.

The nucleotide sequence of the present invention is written from left to right in the 5 'to 3' direction.

Example 1

The embodiment provides a detailed implementation process of a rapid detection method for plant fungal disease Alternaria alternata, which comprises the following steps:

1. alternaria alternata culture and nucleic acid extraction: taking out Alternaria alternata strain stored in test tube from refrigerator at 4deg.C, transferring a small piece of mycelium from test tube onto PDA plate on ultra clean bench, sealing with sealing film, culturing in constant temperature incubator at 25deg.C for about one week, and culturing until mycelium grows on the surface of culture dish. The grown Alternaria alternata hyphae are gently scraped into a mortar by a toothpick or an inoculating needle, poured into liquid nitrogen, quickly ground into powder, filled into a centrifuge tube with 1.5mL, and the Alternaria alternata genome DNA is extracted according to the operation steps described in the specification of a quick nontoxic Plant DNA extraction kit FH Plant DNAKit and is stored at the temperature of minus 20 ℃ for standby.

2. Construction of a molecular counting system: mixing the extracted Alternaria alternata nucleic acid genome sample into an LAMP reaction system containing hydrogel monomers, adding the LAMP reaction system into a sealed small chamber, spreading, attaching a cover plate by using tweezers, sealing the whole reaction system, standing at room temperature, gelling and crosslinking, and enabling Alternaria alternata DNA to be randomly distributed in the nano-pores of the hydrogel matrix.

The primer sequences for the Alternaria alternata molecular counts were as follows:

F3：5'-AAGATCACTGTCAAGGGCG-3'；

B3：5'-ATGGTAAGACCATCGCAGC-3'；

LF：5'-ACCAACGAGCACCATCACC-3'；

LB：5'-ACTCCACCATCACCGGCAT-3'；

FIP：5'-TGGGCTTGGTCTTTCCACCATTCCGAGGGATCTGTTCTCAAC-3'；

BIP：5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGACGGGAGG-3'；

the hydrogel LAMP reaction system mixture (25. Mu.L) consisted of 1.1mg of thiol-polyethylene glycol-thiol, 1.6mg of 4-arm-polyethylene glycol-acrylate, 1.4mM of dNTPs, 1X Isothermal Amplification Buffer, 1.6. Mu.M of FIB and BIP, 0.2. Mu.M of F3 and B3, 0.6. Mu.M of LF and LB, total magnesium ion concentration of 7mM, 960U/mL of Bst2.0 WarmStart ^TM DNA polymerase, 1 XSYBR Green I and 2. Mu.L template DNA samples.

3. Isothermal amplification reaction: the sealed chamber containing the hydrogel was heated at a constant temperature of 65℃for 9min of LAMP amplification reaction, and then heated at 80℃for 5min to enhance the signal-to-noise ratio of the fluorescent amplification spot signal.

4. Amplification product observations and counts: and (3) waiting for the end of the heating process, exciting amplification products of the Alternaria alternata nucleic acid to generate fluorescence by using a portable gel fluorescence mini-imager, counting the number of amplification spots by using Image J software, and digitally quantifying the molecular concentration of Alternaria alternata DNA.

The schematic diagram of the operation flow is shown in fig. 1. The detection method provided by the invention can obtain the digital amplification result only through two experimental steps, specifically, the first step is that firstly, the nucleic acid sample extracted from the agricultural product after being picked is mixed with the hydrogel LAMP reaction system and then added into a sealed small chamber for sealing; and secondly, heating the whole hydrogel system for a certain time, imaging in a portable small gel fluorescence imager, and taking a photo by using a mobile phone. Because the amplification primer provided by the embodiment has very high molecular binding specificity to the Alternaria alternata, the counted fluorescent amplification points are the accurate number of Alternaria alternata. The operation flow is simple and easy to realize, large-scale instruments and equipment are not needed in the operation process, the requirements of non-professional technicians on-site detection are met, and the counting work of the alternaria alternata for the post-harvest agricultural products is realized.

Example 2

This example analyzes the cause of the formation of nonspecific digital isothermal amplification in the hydrogel LAMP reaction. The operation is as follows:

1. alternaria alternata culture and nucleic acid extraction were the same as in example 1.

2. The procedure for the preparation of the molecular counting system was the same as in example 1, in particular: hydrogel LAMP reaction System (25. Mu.L): 1.1mg of mercapto-polyethylene glycol-mercapto, 1.6mg of 4-arm-polyethylene glycol-acrylate, 1.4mM dNTPs, 1X Isothermal Amplification Buffer, 0.2. Mu.M F3 and B3, 0.6. Mu.M LF and LB,7mM total magnesium ion concentration, 960U/mL Bst2.0 WarmStart ^TM The DNA polymerase and 1 XSYBR Green I, FIB and BIP primer concentrations were determined according to the actual requirements, and the primer sequences were the same as in example 1. 2 mu L of template Alternaria alternata DNA sample is added in a positive system, no nucleic acid template molecule is added in a negative system, and whether amplification products appear or not is observed.

3. And respectively carrying out hydrogel counting amplification and real-time fluorescent amplification curve experiments. Hydrogel count amplification reaction conditions: the sealed chamber containing the hydrogel was heated at a constant temperature of 65℃for 15min of LAMP amplification reaction, and then heated at 80℃for 5min to enhance the signal-to-noise ratio of the fluorescent amplification spot signal. Real-time fluorescent amplification curve experimental reaction conditions: the amplification system is placed in a real-time fluorescent quantitative PCR instrument, and is heated for 60min at 65 ℃ by a set program.

4. The observation and counting of fluorescent amplification spots were the same as in example 1.

5. Analysis of results:

as shown in FIG. 2 (a), after 15min incubation, the negative group without the template produced obvious false positive non-specific amplification products, and the positive group at the same time had suspected false positive amplification points. The real-time fluorescence curve of FIG. 2 (b) shows that negative amplification produced a severe false positive signal.

FIP and BIP inner primers are essential components for initiating the initial amplification of LAMP, and as shown in FIG. 2 (c), the fluorescence intensity of the above-mentioned nonspecific false positive signal is greatly reduced in the absence of BIP primer.

As shown in FIG. 2 (d), according to the peak time of the LAMP reaction fluorescence curve, when only BIP primer is present in the negative system, significant fluorescence amplification can be generated, and the addition of FIP does not raise the false positive signal. Thus, false positive signals are caused by a single BIP primer.

As shown in FIG. 2 (e), the amplification of Alternaria alternata is not performed without the BIP primer, and thus the BIP primer is an essential structure for amplification in both hydrogel and aqueous solution.

Example 3

This example compares the effect of eliminating non-specific isothermal amplification products by improving the BIP primers. The operation is as follows:

1. alternaria alternata culture and nucleic acid extraction were the same as in example 1.

2. The procedure for the preparation of the molecular counting system was the same as in example 1. Hydrogel LAMP reaction System (25. Mu.L): 1.1mg of mercapto-polyethylene glycol-mercapto, 1.6mg of 4-arm-polyethylene glycol-acrylate, 1.4mM dNTPs, 1X Isothermal Amplification Buffer, 0.2. Mu.M F3 and B3,1.6mM of FIB and modified BIP, 0.6. Mu.M LF and LB,7mM total magnesium concentration, 960U/mL Bst2.0 WarmStart ^TM DNA polymerase and 1 XSYBR Green I. 2 mu L of template Alternaria alternata DNA sample is added in a positive system, and no template Alternaria alternata DNA sample is added in a negative systemAdding nucleic acid template molecules, and observing whether amplification products appear.

The improved BIP primer sequences for comparison of the effect of non-specific amplification product elimination are as follows:

original BIP:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGACGGGAGG-3';

improvement of BIP-G1:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGACGGGAG-3';

improvement of BIP-G2:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGACGGGA-3';

improvement of BIP-G3:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGACGG-3';

improvement of BIP-G4:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGA C-3';

improvement of BIP-G5:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGA-3';

improvement of BIP-G6:5'-TTCTCCGCTCACAAACTGACCGAACGACTTG-3';

3. and respectively carrying out hydrogel counting amplification and real-time fluorescent amplification curve experiments. Hydrogel count amplification reaction conditions: the sealed chamber containing the hydrogel is heated at a constant temperature of 65 ℃ to respectively carry out LAMP amplification reaction for 10min and LAMP amplification reaction for 60min, and then the temperature is heated at 80 ℃ for 5min to enhance the signal-to-noise ratio of fluorescent amplification point signals. Real-time fluorescent amplification curve experimental reaction conditions: the amplification system is placed in a real-time fluorescent quantitative PCR instrument, and is heated for 60min at 65 ℃ by a set program.

4. The observation and counting of fluorescent amplification spots were the same as in example 1.

5. Preparing an aqueous solution system for LAMP reaction, and carrying out a real-time fluorescent amplification curve experiment by adopting the improved BIP-G2 and the improved BIP-G4 as primers under the same conditions. The LAMP amplification products generated by the BIP primer and the modified BIP-G4 primer were subjected to agarose gel electrophoresis experiments, respectively, and were photographed by a fluorescence imager using 2% agarose for 20 min.

6. Analysis of results:

the present example further eliminates false positive amplification products of Alternaria alternata LAMP detection system from the molecular point of view of BIP base sequence improvement. As shown in FIG. 3 (a), the negative systems using the modified BIP-G1 to BIP-G6 primers all inhibit the false positive signal intensity to different degrees, wherein the inhibition effect of the modified BIP-G2 and BIP-G4 primers is most remarkable, and it is possible that the removed molecular structure portions are more likely to produce non-specific amplification products.

As shown in FIG. 3 (b), after 60min of the heating reaction of the negative system, the original BIP primer generated a large number of nonspecific fluorescent amplification spots, and the system of the improved BIP-G2 primer and BIP-G4 primer completely eliminated the false positive amplification spots of the hydrogel. Thus, the above results demonstrate that hydrogel false positive amplification signals can be eliminated by modifying the BIP-G2 and BIP-G4 primers, i.e., the GG or GGGAGG structure of the original BIP primer is a sequence that causes non-specific amplification of a single primer.

As shown in FIG. 3 (c), the number of positive hydrogel amplification spots of the BIP-G2 and BIP-G4 primers was improved compared with the original BIP, and after heating for 10min, non-specific amplification did not occur at this time, and the amplification efficiency of the improved BIP-G2 and BIP-G4 primers remained the same as that of the original BIP primers.

As shown in FIG. 3 (d), the hydrogel amplification efficiency of BIP-G2 and BIP-G4 primers is improved to be consistent compared with that of the hydrogel and the aqueous solution, however, the amplification in the aqueous solution is affected when the aqueous solution is amplified to generate different amplification efficiencies, namely, false positive signals are eliminated, and the amplification in the hydrogel is more stable than that in the aqueous solution and cannot be affected to the detection sensitivity of the detection positive system.

As shown in FIG. 3 (e), in the positive amplification system, the original BIP primer lane 1 and the modified BIP-G4 primer lane 3 each present a clear amplified band, whereas in the negative amplification system, the original BIP primer lane 2 presents a false positive electrophoresis band, while the modified BIP-G4 primer lane 4 does not have any band. The hydrogel LAMP reaction using the improved BIP-G4 primer can accurately and absolutely quantitatively detect the quantity of Alternaria alternata in a sample while shortening the detection time, does not generate any false positive signal even after long-time heating reaction, and thoroughly eliminates the non-specific digital isothermal amplification product from the aspect of molecular level.

Example 4

This example optimizes the reaction conditions of the Alternaria alternata counting system. The operation is as follows:

1. alternaria alternata culture and nucleic acid extraction were the same as in example 1.

2. The construction flow of the molecular counting system and the proportion of each component of the hydrogel LAMP reaction system are the same as in example 1. The BIP primer was modified BIP-G4 among the primers for molecular counting of Alternaria alternata, as in example 1.

The effect of different heating times (3 min, 6min, 9min, 12min and 15 min), amplification temperatures (61 ℃,63 ℃, 65 ℃, 67 ℃ and 69 ℃), total magnesium ion concentrations (5 mM, 6mM, 7mM and 8 mM), isothermal amplification enzyme concentrations (320U/mL, 640U/mL, 960U/mL and 1280U/mL), and loop primer concentrations (0. Mu.M, 0.2. Mu.M, 0.4. Mu.M, 0.6. Mu.M, 0.8. Mu.M and 1. Mu.M) of the amplification system in the hydrogel LAMP chip was tested, respectively, using different gradients of reaction conditions to optimize the whole Alternaria alternata counting system.

3. And (5) performing isothermal amplification reaction.

4. The observation and counting of fluorescent amplification spots were the same as in example 1.

5. Analysis of results:

as shown in FIG. 4 (a), fluorescent amplification spots were already present at the time of amplification for 6min, and the amplification spots increased slightly with the extension of the amplification time, and there was no significant change in the fluorescent spot count at the time of reaction to 9min, indicating that the amplification reaction was substantially completed. When the heating time is 6min, the detection rate of the Alternaria alternata reaches more than 70%, and accurate quantification can be realized within 9 min.

As shown in FIG. 4 (b), as the amplification temperature increased from 63℃to 69 ℃, the end point fluorescence count of the hydrogel LAMP showed a tendency to gradually rise and then fall, and the detection amount at 65℃reached the maximum value.

As shown in FIG. 4 (c), when the total magnesium ion concentration was low (5 mM), the hydrogel LAMP reaction was not performed, and as the total magnesium ion concentration increased, the reaction started to proceed, the amplification efficiency gradually increased, and at a concentration of 6mM and 7mM, the number of amplification spots was not significantly different.

As shown in FIG. 4 (d), the number of amplified spots at 320U/mL of enzyme concentration was significantly lower than 640U/mL and above, and the number of amplified spots at 960U/mL of enzyme concentration was the largest, and the increase in the concentration of amplified enzyme did not increase the number of amplified spots.

As shown in FIG. 4 (e), as the loop primer concentration increases from 0. Mu.M to 1. Mu.M, the number of LAMP amplification points of the hydrogel generally tends to increase and decrease, and a maximum value is obtained at 0.6. Mu.M.

In summary, the optimal reaction system for Alternaria alternata enumeration was 9min heating time, 65℃amplification temperature, 7mM total magnesium ion concentration, 960U/mL isothermal amplification enzyme concentration, and 0.6. Mu.M loop primer concentration.

Example 5

This example evaluates the sensitive detection of the Alternaria alternata counting system. The operation is as follows:

1. alternaria alternata culture and nucleic acid extraction were the same as in example 1.

2. The construction flow of the molecular counting system and the proportion of each component of the hydrogel LAMP reaction system are the same as in example 1. The BIP primer was modified BIP-G4 among the primers for molecular counting of Alternaria alternata, as in example 1.

The Alternaria alternata genome DNA is subjected to gradient dilution, and hydrogel LAMP counting of Alternaria alternata is carried out by adopting the same operation flow.

3. The heating process of the isothermal amplification reaction was the same as in example 1.

4. The observation and counting of fluorescent amplification spots were the same as in example 1. The hydrogel amplification results were counted for observation and analyzed by linear fit to the expected theoretical concentration.

5. Analysis of results:

as shown in FIG. 5 (a), as the concentration of Alternaria alternata genomic DNA in the sample increases, so does the fluorescent amplification spots that the hydrogel system can observe, and by counting the fluorescent amplification spots using Image J, the relationship between the number of hydrogel amplification spots and the number of expected amplification spots is shown in FIG. 5 (b). Analysis found that the number of amplification spots measured exhibited a good linear relationship with the number of expected amplification spots (R ² = 0.9964), the limit of detection is 0.5 copy/. Mu.l, demonstrating the reliability of this counting system in absolute quantification of alternaria alternata nucleic acid.

Example 6

This example tests the specificity of the Alternaria alternata counting system under the interference of common agricultural product fungal nucleic acids. The operation is as follows:

1. the same procedure as in example 1 was followed using as control strain the contaminated fungi of 6 commonly used harvested agricultural products selected from the group consisting of Botrytis cinerea, anthrax mucilaginosa, brown rot of America and Aspergillus fumigatus, erysiphe graminis and Fusarium oxysporum.

2. The construction flow of the molecular counting system and the proportion of each component of the hydrogel LAMP reaction system are the same as in example 1. The BIP primer was modified BIP-G4 among the primers for molecular counting of Alternaria alternata, as in example 1.

The specificity detection experiment is provided with the independent verification of nucleic acid of each strain and the verification of mixed nucleic acid samples.

3. The heating process of the isothermal amplification reaction was the same as in example 1.

4. The observation and counting of fluorescent amplification spots were the same as in example 1.

5. Analysis of results:

as shown in FIG. 6, only two samples of Alternaria alternata nucleic acid genome can obtain fluorescent amplification spots, and none of the other 6 agricultural product fungi and the template-free blank have hydrogel LAMP amplification signals. In the detection of the nucleic acid sample mixed with a plurality of strains, the number of the target alternaria can be accurately counted, the interference of other fungus nucleic acid can not be caused, and the hydrogel counting system has high specificity for the alternaria.

Example 7

This example evaluates the reproducibility of the test batches of the Alternaria alternata counting system. The operation is as follows:

1. alternaria alternata culture and nucleic acid extraction were the same as in example 1.

2. The construction flow of the molecular counting system and the proportion of each component of the hydrogel LAMP reaction system are the same as in example 1. The BIP primer was modified BIP-G4 among the primers for molecular counting of Alternaria alternata, as in example 1.

And the hydrogel counting systems prepared in different batches are adopted to respectively verify the Alternaria alternata with the same concentration, so that other components and concentration conditions are consistent.

3. The heating process of the isothermal amplification reaction was the same as in example 1.

4. The observation and counting of fluorescent amplification spots were the same as in example 1.

5. Analysis of results:

as shown in FIG. 7, the count of batches within a group is more stable, but a more stable number of Alternaria alternata nucleic acid molecule counts can be formed, both between and within groups. The hydrogel counting system of the Alternaria alternata has good reproducibility for detecting nucleic acid genome molecules, and can be adopted to accurately and stably count Alternaria alternata for a plurality of times.

Application example 1

The detection system is practically applied to count Alternaria alternata on jujube fruit samples. The operation is as follows:

1. and respectively selecting whole jujube fruits and fresh-cut jujubes, standing and storing at room temperature, respectively extracting nucleic acid genome by using a kit after fungi are infected, and counting and extracting Alternaria alternata in a nucleic acid sample.

2. The construction flow of the molecular counting system and the proportion of each component of the hydrogel LAMP reaction system are the same as in example 1. The BIP primer was modified BIP-G4 among the primers for molecular counting of Alternaria alternata, as in example 1.

And (3) respectively adding different nucleic acid genome samples extracted from the jujube fruit samples as templates into a hydrogel counting system to amplify and count the subsequent alternaria alternata.

3. The heating process of the isothermal amplification reaction was the same as in example 1.

4. The observation and counting of fluorescent amplification spots were the same as in example 1.

5. Analysis of results:

as shown in FIG. 8, the number of Alternaria alternata on whole jujube fruits and fresh-cut jujubes has a great tendency to increase with the extension of the storage time, and the pollution amount is not only accumulated with the storage days, which indicates that Alternaria alternata will propagate and grow in a larger amount after the Alternaria alternata infects post-harvest agricultural products.

The invention provides an effective counting method for monitoring the alternaria alternata in the post-harvest agricultural products.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims (10)

1. A false positive amplification-free rapid detection method for plant disease alternaria alternata, which is characterized by comprising the following steps:

(1) Extracting genome DNA in a sample to be detected, and adding the genome DNA serving as a template into a loop-mediated isothermal amplification reaction system containing hydrogel monomers to form a hydrogel system; the reaction system comprises a primer for specifically amplifying the Alternaria alternata DNA fragment, wherein the primer comprises an inner primer FIP and a BIP, and more than or equal to 2 continuous bases at the 3' -end of the inner primer FIP or the BIP are not G or C;

(2) Placing the hydrogel system under a constant temperature condition for isothermal amplification reaction;

(3) After the reaction is finished, fluorescent signals in the hydrogel system are analyzed by utilizing a fluorescent imaging technology, fluorescent points are counted, and the absolute molecular concentration of the amplified Alternaria alternata DNA in the sample to be detected is calculated.

2. The rapid detection method for false positive amplification against alternaria alternata plant disease according to claim 1, wherein the two linked bases at the 3' -end of the inner primer FIP or BIP are not GG, CC, GC, CG.

3. The rapid detection method for false positive amplification of alternaria alternata as claimed in claim 1, wherein in the step (1), the loop-mediated isothermal amplification reaction system comprises: 1 xLAMP buffer, 6-8 mM MgSO ₄ ，1.0～1.4mM dNTP, 640-1280U/mL Bst2.0DNA polymerase, 0.2-1.6mu.M FIB and BIP, 0.4-0.8mu.M LF and LB, 0.2-0.3mu.M F3 and B3, and fluorescent dye.

4. The rapid detection method for false positive amplification against alternaria alternata plant disease according to claim 1, wherein in step (1), the primer comprises:

F3：5'-AAGATCACTGTCAAGGGCG-3'；

B3：5'-ATGGTAAGACCATCGCAGC-3'；

FIP：5'-TGGGCTTGGTCTTTCCACCATTCCGAGGGATCTGTTCTCAAC-3'；

BIP:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGACGGGA-3'; or 5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGAC-3'.

LF：5'-ACCAACGAGCACCATCACC-3'；

LB：5'-ACTCCACCATCACCGGCAT-3'。

5. The rapid detection method for false positive amplification against alternaria plant disease of claim 1, wherein in step (1), the hydrogel monomer comprises thiol-polyethylene glycol-thiol and tetra-arm ethylene glycol acrylate.

6. The rapid detection method for false positive amplification of alternaria alternata as claimed in claim 5, wherein the molecular weight of thiol-polyethylene glycol-thiol is 3400MW, the molecular weight of four-arm ethylene glycol acrylate is 10000MW, and the molar mass ratio of the two is 2:1.

7. the rapid detection method for false positive amplification against alternaria alternata plant disease according to claim 1, wherein in the step (2), the hydrogel system is placed in a sealed space to prevent evaporation of water for isothermal amplification reaction.

8. The rapid detection method for false positive amplification against alternaria alternata plant disease according to claim 1, wherein the conditions of the isothermal amplification reaction comprise: the first step, 63-65 ℃ for 9-15 min; and secondly, heating at 80 ℃ for 5min.

9. The rapid detection method for false positive amplification of alternaria alternata as claimed in claim 1, wherein in the step (3), the hydrogel system is irradiated by a portable fluorescent light source, a picture is taken by a smart phone, and the number of fluorescent spots is analyzed.

10. A kit for detecting alternaria alternata, which is characterized by comprising a loop-mediated isothermal amplification reaction reagent, a hydrogel monomer and a primer for specifically amplifying a alternaria alternata DNA fragment, wherein the primer comprises:

F3：5'-AAGATCACTGTCAAGGGCG-3'；

B3：5'-ATGGTAAGACCATCGCAGC-3'；

FIP：5'-TGGGCTTGGTCTTTCCACCATTCCGAGGGATCTGTTCTCAAC-3'；

BIP:5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGACGGGA-3'; or 5'-TTCTCCGCTCACAAACTGACCGAACGACTTGGAC-3'.

LF：5'-ACCAACGAGCACCATCACC-3'；

LB：5'-ACTCCACCATCACCGGCAT-3'。