CN114107526A - RPA primer pair and crRNA for detecting citrullus lanatus acidophilus, kit and using method thereof - Google Patents

RPA primer pair and crRNA for detecting citrullus lanatus acidophilus, kit and using method thereof Download PDF

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CN114107526A
CN114107526A CN202111440174.6A CN202111440174A CN114107526A CN 114107526 A CN114107526 A CN 114107526A CN 202111440174 A CN202111440174 A CN 202111440174A CN 114107526 A CN114107526 A CN 114107526A
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焦健
郑先波
冯建灿
黄松
白团辉
宋春辉
王苗苗
宋尚伟
庞宏光
杨孟莉
李明
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Henan Agricultural University
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Abstract

The invention belongs to the field of rapid detection of pathogenic microorganisms, and particularly relates to a method for detecting acidophilic bacteria (a) of watermelonAcidovorax citrulli) The RPA primer pairs and crRNA of (a), and kits and methods of use thereof. The RPA primer pair sequence is shown as SEQ ID No.1 and SEQ ID No.2, and the crRNA target sequence is shown as SEQ ID No. 3. The invention adopts a Crispr/LbCas12a nucleic acid detection platform, designs specific detection sites aiming at the genome of acidophilic watermelon bacteria (Acidovorax citrulli), and combines a fluorescence visualization technology to develop a simple diagnostic method suitable for detecting the pathogenic bacteriaThe method is carried out. And simultaneously, the sensitivity is further improved by combining an RPA isothermal amplification technology.

Description

RPA primer pair and crRNA for detecting citrullus lanatus acidophilus, kit and using method thereof
Technical Field
The invention belongs to the field of rapid detection of pathogenic bacteria, and particularly relates to an RPA primer pair and crRNA for detecting acidophilic watermelon bacteria (Acidovorax citrulli), a kit and a using method thereof.
Background
Bacterial pathogens often cause significant reductions in the yield and quality of vegetable and fruit production areas worldwide, posing a serious threat to the horticultural industry. Wherein, Acidophilic watermelon bacterium (Acidovorax citrulli) Bacterial fruit blotch can be caused in cucurbit crops, is a main yield-limiting pathogenic bacterium of melon crops in the world, and can cause destructive damage to fruit industries such as watermelon, melon and the like in the market. Because of its destructive influence on horticultural crops, Acidophilic watermelon is classified as a quarantine pest in agricultural plant disease and pest quarantine lists at home and abroad (EPPO global database; https:// gd. epo. int/datasheets /). At present, the acidophilic bacteria of the watermelon become endemic diseases in most planting areas of the world, the most main transmission and diffusion mode of the acidophilic bacteria is through seeds, and the elimination of batches of seeds polluted by pathogenic bacteria by adopting a simple, quick and sensitive on-site detection technology is an important quarantine measure.
Successful quarantine measures require an accurate, timely, local, and simple pathogenA bacteria detection method to destroy infectious material in situ. According to the identification standards of pathogenic bacteria recommended by the guidelines of the European Plant Protection Organization (EPPO),A. citrullithe identification requires a series of steps such as separation, purification, culture and physiological and biochemical analysis, and further requires molecular techniques such as PCR or RT-PCR for confirmation. However, while these methods are time consuming, they also require the establishment of laboratories, the purchase of expensive instruments and the training of professional operators, which makes them not portable and suitable for immediate testing in the basic sector.
Nucleic acid detection systems based on CRISPR-Cas proteins (Cas 12, Cas13, and Cas 14) also have great application prospects in molecular diagnostics in recent years, because they show trans-cleavage on non-specific targets after binding targets to trigger cis-cleavage. Among them, the Cas12a system performs non-specific cleavage of ssDNA by recognizing specific dsDNA sequences, and is particularly suitable for detection of target sites from viruses, pathogenic bacteria, or transgenic organisms.
Disclosure of Invention
The invention provides an RPA primer pair and crRNA for detecting Acidovorax citrulli (Acidovorax citrulli), a kit and a using method thereof, and aims at Acidovorax citrulli (Acidovorax citrulli) by adopting a Crispr/LbCas12a nucleic acid detection platformAcidovorax citrulli) The genome of (1) designs specific detection sites, and combines a fluorescence visualization technology to develop a simple diagnostic method suitable for detecting the pathogenic bacteria. And simultaneously, the sensitivity is further improved by combining an RPA isothermal amplification technology.
The technical scheme of the invention is realized as follows:
an RPA primer pair and a crRNA for detecting acidophilic watermelon are disclosed, wherein the sequences of the RPA primer pair are shown as SEQ ID No.1 and SEQ ID No.2, and the sequence of the crRNA is shown as SEQ ID No. 3.
A kit containing the above RPA primer pair and crRNA target, the kit comprising 2 μ L RPA amplification product system and 50 μ L crRNA/LbCas12a mixed system.
The RPA amplification product system comprises RPA enzyme powder, 29.5 mu L of rehydration buffer solution, 11 mu L of nuclease-free water, 280 nM magnesium acetate and 2 mu L of genome DNA, and RPA pre-amplification is carried out by using a commercial twist Amp Basic kit (twist Dx).
The crRNA/LbCas12a mixed system comprises 100 nM LbCas12a, 120 nM crRNA, 400 nM ssDNA FQ-reporter, 20U RNase inhibitor, 2. mu.L of RPA amplification product, 5. mu.L of 10 XNEBuffer 3.1 and ddH2O amounted to 50. mu.L.
The concentration of the positive primer and the negative primer in the RPA primer pair in the kit is 10 mu M, and the content is 2.5 mu L.
The sequence of the FQ-reporter in the kit is 5 '-FAM-TTATT-Quencher-3'.
The use method of the kit comprises the following steps:
(1) mixing the freeze-dried RPA enzyme powder with 10 mu M of RPA primer pair, a rehydration buffer solution, nuclease-free water and microbial genome DNA of a seed to be detected to obtain a reaction system;
(2) adding 280 nM magnesium acetate into the reaction system in the step (1), centrifuging to start reaction, and incubating to obtain an initial product;
(3) a crRNA/LbCas12a mixed system is prepared, 2 mu L of the primary product obtained in the step (2) is added, and a trans-cleavage reaction is carried out on a Bio-Tek FLx800 microplate fluorescent reader and the result is observed.
In the step (1), the volume of the 10 mu M RPA primer pair is 2.5 mu L, the volume of the rehydration buffer solution is 29.5 mu L, the volume of the nuclease-free water is 11 mu L, and the volume of the microbial genome DNA of the seed to be detected is 2 mu L.
The volume of the 280 nM magnesium acetate in step (2) is 2.5. mu.L, and the incubation condition is 37 ℃ for 30 min.
The preparation method of the crRNA/LbCas12a mixed system in the step (3) comprises the following steps: the formation of the crRNA/LbCas12a complex was promoted by pre-mixing 100 nM LbCas12a and 120 nM crRNA and pre-incubating for 10 min at 25 deg.C, then adding 400 nM ssDNA FQ-reporter, 20U RNase inhibitor, 2. mu.L of RPA amplification product, 5. mu.L of 10 XNEBuffer 3.1 to make up the reaction system and adding ddH2O to 50. mu.L.
The temperature of the trans-cutting reaction in the step (3) is 37 ℃; the observation was made at λ ex every 30 s: 485 nm; and lambda em: fluorescence was measured once at 535 nm or visually observed under 254nm ultraviolet light.
The invention has the following beneficial effects:
the invention is based on a nucleic acid detection platform of Crispr/LbCas12a and aims at acidophilic watermelon bacterium (C: (C)Acidovorax citrulli) The genome of (1) designs specific detection sites, and combines a fluorescence visualization technology to develop a simple diagnostic method suitable for detecting the pathogenic bacteria. And simultaneously, the sensitivity is further improved by combining an RPA isothermal amplification technology. Compared with the conventional PCR detection, the minimum detection bacterial liquid concentration of LbCas12a fluorescence detection is 40 CFU/reaction (FIG. 2B), which is 100 times that of PCR (FIG. 2C).
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows acidophilic bacteria of watermelonA. citrulliLbCas12a fluorescence detection scheme (a) and detection effect (B);
FIG. 2 is a diagram of analysis of specificity and sensitivity of LbCas12a fluorescence detection, wherein A is specificity analysis of LbCas12a fluorescence detection A. citrulli, B is sensitivity of LbCas12a fluorescence detection A. citrulli, and C is sensitivity of PCR detection A. citrulli.
FIG. 3 shows the LbCas12a fluorescence detection-based detection of watermelon seeds of 40 batchesA. citrulliVisual detection of (2).
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Examples
Strains and culture conditions
Acidophilic bacterium of watermelonA. citrulli The GDMCC 803619 strain was grown on King's medium B. Grown aerobically at 28 ℃ for 72 h and genomic DNA was extracted using TIANAmp Bacteria DNA Kits (Tiangen, Beijing, China) according to the instructions.
To prepare bacterial suspensions of different concentrations, single colonies of the strain were inoculated into LB and cultured at 28 ℃ for 24 h at 120 r/min. Bacterial cells were harvested by centrifugation and resuspended in sterile 0.01M PBS buffer, adjusting the concentration to OD600= 0.8. The cell concentration was determined by serial 10-fold dilution in 1mL of sterile 0.01M PBS buffer and plating 100. mu.L of the suspension on agar medium, which was repeated 3 times. The original cell concentration was adjusted to about 1X 10 after calculation based on the growth on the medium after serial dilution8 CFU mL-1。
2、A. citrulliDesign of specific RPA primers and crRNA
Extraction from the software package of metagenomic analysis software Metadehlan (v 3.0)A. citrulliThe program previously analyzed all genome sequences in a known database, about 13500 bacteria and 3500 viruses, and screened for species-specific gene markers. The extracted gene markers were further aligned with the NCBI database by BLAST analysis to confirm that they hadA. citrulliThe intra-species conservation and inter-species specificity, the coding gene of the specific protein C8E07_2517 (NCBI accession number REG 69369) was finally selected, and specific RPA primers and crRNA target sites were designed.
Figure 678921DEST_PATH_IMAGE002
。
RPA isothermal amplification and LbCas12a fluorescence analysis
RPA pre-amplification was performed using a commercial twist amp Basic kit (twist dx) prior to LbCas12a fluorescence analysis. First, the lyophilized RPA enzyme powder was mixed with 2.5. mu.L of forward and reverse primers (10. mu.M), 29.5. mu.L of reconstitution buffer, 11. mu.L of nuclease-free water and 2. mu.L of genomic DNA. The reaction was then initiated by centrifugation by adding 2.5. mu.L of magnesium acetate (280 nM) to the lid of the tube and after incubation at 37 ℃ for 30 min, 2. mu.L of the product was used in the following LbCas12a detection reaction.
Using 100 nM LbCas12a, 120 nM crRNA, 400 nM ssDNA FQ-reporter (5 '-FAM-TTATT-Quencher-3', biosynthesized by worker), 20U RNase inhibitor, 2. mu.L of RPA product, 5. mu.L of NEBuffer 3.1 (10X) to compose the reaction system and add ddH2O to 50. mu.L, and the trans-cleavage activity assay was performed. Wherein the LbCas12a and crRNA are premixed and preincubated at 25 ℃ for 10 min to promote the formation of the crRNA/LbCas12a complex prior to adding other components to the reaction system. The reaction was carried out at 37 ℃ on a Bio-Tek FLx800 microplate fluorescence reader, and the fluorescence was measured every 30 s (λ ex: 485 nm; λ em: 535 nm). In addition, the fluorescence signal was visually observed with a hand-held ultraviolet lamp (254 nm) with the lowest concentration capable of producing visible positive green fluorescence being the limit of detection (LOD).
Acidiphilium citrullum shown in figure 1A. citrulliThe LbCas12a fluorescence detection procedure and final detection effect. The extracted microbial DNA is first pre-amplified with RPA and the number of target templates is amplified and detected. The RPA product was then added to the LbCas12a fluorescence detection system in the presence of UV ultraviolet light (254 nm)A. citrulliThere was a clear green fluorescence signal in the EP tube for the genome, whereas there was no visible green fluorescence in the blank tube without the genome. The results show that the RPA primer and the crRNA designed by the research can effectively detect the acidophilic bacteria of the watermelonA. citrulliIs present.
Wherein FIG. 1A is 2. mu.LA. citrulliThe genomic DNA was pre-amplified with RPA. After 20 min reaction at 37 ℃, 2. mu.L of RPA product was taken for LbCas12a fluorescence detection. FIG. 1B shows that the endpoint fluorescence was obtained 20 min after LbCas12a reaction. The centrifuge tube above the bar graph shows the visualized signal after uv irradiation. Data shown are mean ± standard deviation from 6 independent reactions. Blank control represents no addition to the reactionA. citrulliThe DNA of (1).
To confirm that the designed RPA primer and crRNA can only bind specificallyA. citrulliThe other 5 kinds of the above-mentioned herbs were selectedA. citrulliThe strains with closer relativity were analyzed for specificity. The results show (fig. 2A) that only a. citrulli can excite the green fluorescence signal. In addition, the microbial community of healthy, non-diseased watermelon and melon leaves also did not produce false positive results, whereas leaf microbial DNA taken from watermelon fruit blotch was able to excite green fluorescent signals. The results show that the developed A. citrulli detection method has high specificity and can be used for specially detecting pathogenic bacteria. And analyzing and comparing the detection sensitivity. Compared with the conventional PCR detection, the minimum detection bacterial liquid concentration of LbCas12a fluorescence detection is 40 CFU/reaction (FIG. 2B), which is 100 times that of PCR (FIG. 2C).
The embodiment has the following effects: detection of watermelon seed A, citrulli
For detecting pathogenic bacteria transmitted through seedsA. citrulliAnd randomly selecting 40 seed samples from the market. 50 watermelon seeds of each product were gently crushed, soaked in 40 mL sterile water (0.1% Tween 80 added) and vortexed gently for 10 min to facilitate bacterial elution. 1mL of the soaked eluate was centrifuged for 5 min in a small centrifuge (9660 Xg) to remove the supernatant, and the resulting precipitate was used for subsequent DNA extraction. To enable sample preparation in situ, bacterial genomic DNA was rapidly prepared using commercial lysis buffer MightyPrep reagent for DNA (TaKaRa, large, china). Briefly, the pellet was resuspended in 50. mu.L of extraction buffer and then heated at 95 ℃ for 10 min using a small thermostatted metal bath. After short centrifugation, 2. mu.L of supernatant was taken as substrate directly, and pathogenic bacteria were detected by RPA isothermal amplification and LbCas12a visualization. The DNA template extracted by the kit can also be used for qPCR detection for sensitivity comparison.
In the final method validation, a total of 40 batches of watermelon seed samples were collected from the market to test for new method pairsA. citrulliThe detection effect of the pathogen. The LbCas12a fluorescence detection results showed (fig. 3), most of the samples were negative and 6 samples were positive. The PCR results were identical to LbCas12a fluorescence detection, indicating thatThe diagnosis results of the two methods are consistent, but the LbCas12a fluorescence detection does not need a complex instrument, and the detection time is shorter, and only needs about 40 minutes.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> Henan university of agriculture
<120> RPA primer pair and crRNA for detecting citrullus lanatus acidophilus, and kit and using method thereof
<141> 2021-11-30
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Acidovorax citrulli
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gagatgtgct ttggagccct tgaggaattg tt 32
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<213> Acidovorax citrulli
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gaacgcatgg atgatgttga agttgtgttc tg 32
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<212> DNA/RNA
<213> Acidovorax citrulli
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uaauuucuac uaaguguaga ugccgaaagg uugcgcgacg u 41

Claims (10)

1. An RPA primer pair and crRNA for detecting acidophilic watermelon are characterized in that: the RPA primer pair has sequences shown as SEQ ID No.1 and SEQ ID No.2, and the crRNA has a sequence shown as SEQ ID No. 3.
2. A kit comprising the RPA primer pair of claim 1 and a crRNA target, wherein: the kit comprises 2 mu L of RPA amplification product system and 50 mu L of crRNA/LbCas12a mixed system.
3. The kit of claim 2, wherein: the RPA amplification product system comprises RPA enzyme powder, 29.5 mu L of rehydration buffer solution, 11 mu L of nuclease-free water, 280 nM magnesium acetate and 2 mu L of genome DNA.
4. The kit of claim 2, wherein: the crRNA/LbCas12a mixed system comprises 100 nM LbCas12a, 120 nM crRNA, 400 nM ssDNA FQ-reporter, 20U RNase inhibitor, 5 μ L10 XNEBuffer 3.1 and ddH2O amounted to 50. mu.L.
5. The kit of claim 2, wherein: the concentration of the positive primer and the negative primer in the RPA primer pair in the kit is 10 mu M, and the content is 2.5 mu L; the FQ-reporter sequence is 5 '-FAM-TTATT-Quencher-3'.
6. The method of using the kit of any one of claims 2 to 5, characterized in that the steps are as follows:
(1) mixing the freeze-dried RPA enzyme powder with 10 mu M of RPA primer pair, a rehydration buffer solution, nuclease-free water and microbial genome DNA of a seed to be detected to obtain a reaction system;
(2) adding 280 nM magnesium acetate into the reaction system in the step (1), centrifuging to start reaction, and incubating to obtain an initial product;
(3) a crRNA/LbCas12a mixed system is prepared, 2 mu L of the primary product obtained in the step (2) is added, and a trans-cleavage reaction is carried out on a Bio-Tek FLx800 microplate fluorescent reader and the result is observed.
7. Use according to claim 6, characterized in that: in the step (1), the volume of the 10 mu M RPA primer pair is 2.5 mu L, the volume of the rehydration buffer solution is 29.5 mu L, the volume of the nuclease-free water is 11 mu L, and the volume of the microbial genome DNA of the seed to be detected is 2 mu L.
8. Use according to claim 6, characterized in that: the volume of the 280 nM magnesium acetate in step (2) is 2.5. mu.L, and the incubation condition is 37 ℃ for 30 min.
9. The use method of claim 6, wherein the step (3) of preparing the crRNA/LbCas12a mixed system comprises the following steps: the formation of the crRNA/LbCas12a complex was promoted by pre-mixing 100 nM LbCas12a and 120 nM crRNA and pre-incubating for 10 min at 25 deg.C, then adding 400 nM ssDNA FQ-reporter, 20U RNase inhibitor, 2. mu.L of RPA amplification product, 5. mu.L of 10 XNEBuffer 3.1 to make up the reaction system and adding ddH2O to 50. mu.L.
10. Use according to claim 6, characterized in that: the temperature of the trans-cutting reaction in the step (3) is 37 ℃; the observation was made at λ ex every 30 s: 485 nm; and lambda em: fluorescence was measured once at 535 nm or visually observed under 254nm ultraviolet light.
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JIAN JIAO ET AL.: ""A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system"", 《JOURNAL OF HAZARDOUS MATERIALS》, vol. 426, pages 1 - 9 *

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