RPA primer probe, kit, detection method and application for detecting Metschnikowia bicuspidatum

Abstract

A RPA primer probe, a kit and a detection method for detecting two-pointed meiqi yeast belong to the field of biotechnology. The kit is an RPA-LFD kit, which includes a composition of RPA primers and RPA probes. The primers are based on the target gene sequence of two-pointed meiqi yeast of Chinese mitten crab, and the 5' end of the downstream primer is labeled with a modification group-biotin; the upstream and downstream primers with a synthesized amplification fragment size of 292bp. The 5' end of the RPA probe is labeled with carboxyfluorescein FAM, the 3' end is added with an extension blocking group C3Spacer, and a tetrahydrofuran THF is added in the middle of the 5' end and the 3' end. The present invention can realize the RPA detection of two-pointed meiqi yeast, and can quickly, simply and specifically detect two-pointed meiqi yeast.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an RPA primer probe, a kit and a detection method for detecting Metschnikowia bicuspidatum. Background Art

[0002] Bicuspidata belongs to the genus Metschnikowia. It was first isolated by Metchnikoff (1884) from diseased fleas Daphnia magna. The ability of Bicuspidata to reproduce at low temperatures also causes a serious outbreak of "milk disease" during the wintering period. Bicuspidata has three subspecies: M.bicuspidata var.Bicuspidata, M.bicuspidatavar.Californica and M.bicuspidata var.Chathamia. M. bicuspidatum is a pathogenic yeast that can easily infect several economically important aquatic organisms, including shrimps: Macrobrachium rosenbergii, Palaemonetes sinensis, Exopalaemon carinicauda; crabs: Eriocheir sinensis, Portunus trituberculatus; fish: Chinook salmon Oncorhyncus tshawytscha; and food organisms: Daphnia and Artemia. M. bicuspidatum can cause high mortality in crustaceans. In recent years, severe M. bicuspidatum infection has occurred in Eriocheir sinensis in farms in northern China, resulting in a mortality rate of more than 30% in adult crabs in wintering ponds, causing great economic losses to the aquaculture industry.

[0003] Since the infection of Saccharomyces cerevisiae is latent in the early stage, obvious symptoms are usually found in the middle and late stages, and the accuracy of traditional bacterial isolation and biochemical identification is poor. Molecular detection methods have the advantages of high sensitivity, short time and simple operation. At present, methods for detecting Saccharomyces cerevisiae based on molecular technology have been reported, such as polymerase chain reaction (PCR), real-time fluorescence quantitative PCR reaction and loop-mediated isothermal amplification technology. However, the operation process of PCR and real-time fluorescence quantitative PCR reaction is complicated, time-consuming and dependent on expensive equipment (such as PCR instrument, etc.), which is not suitable for rapid on-site detection; loop-mediated isothermal amplification technology needs to be completed at 60-65℃, requiring a water bath or a constant temperature box, and the reaction at this temperature is prone to false positives due to aerosol contamination, so it is not suitable for the detection of Saccharomyces cerevisiae. Summary of the invention

[0004] In view of the above-mentioned technical problems, the present invention provides an RPA primer probe, a kit and a detection method for detecting Saccharomyces bicuspidatus, which has a short reaction time and can complete the detection under a constant temperature of 25-45°C, and has the advantages of good flexibility and strong practicality.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The present invention discloses an RPA primer for detecting the bicuspid yeast. According to the target gene sequence of the bicuspid yeast of Chinese mitten crab, a modification group-biotin is determined to be marked on the 5' end of the downstream primer; the primers are used to synthesize the upstream and downstream primers with an amplification fragment size of 292 bp; the sequence of the RPA primer is as follows:

[0007] The upstream primer was F: 5′-ATGGAAGAAATGGCCGTTGAAACCTCGCTGGAA-3′, and the downstream primer was R: 5′-GGACGTTAGAATTCGGGTCGATTTGGGTGAAAG-3′.

[0008] The present invention discloses an RPA probe for detecting bicuspid yeast. The 5' end of the RPA probe is labeled with carboxyfluorescein FAM, an extension blocking group C3 Spacer is added to the 3' end, and a tetrahydrofuran THF is added in the middle of the 5' end and the 3' end. The sequence of the probe is:

[0009] [FAM]-ATTTCTCCGAGTTCATTCAGAGCTCTACCGC[THF]CAGCACAGTAAACCCA

[0010] -[C3-spacer].

[0011] The present invention discloses a kit for detecting Metschnikova bicuspidatum. The kit is an RPA-LFD kit, comprising a combination of the RPA primer and the RPA probe.

[0012] Furthermore, the RPA-LFD kit also includes a positive control; that is, a template that the positive sample can refer to, such as Figure 5B The positive control and Figure 7 The positive control is represented by P, and the negative control is generally represented by N.

[0013] Furthermore, the positive control is the genomic DNA of Saccharomyces cerevisiae.

[0014] The application of the RPA primers, RPA probes or kit of the present invention in the detection of Saccharomyces cerevisiae in Eriocheir sinensis does not include the diagnosis and treatment of diseases.

[0015] The method for detecting the bicuspid yeast using the kit of the present invention comprises the following steps:

[0016] S1, extracting DNA of the sample to be tested;

[0017] S2, using the DNA extracted in step S1 as an amplification template, and using the RPA primer and probe combination of the kit to perform an RPA amplification reaction to obtain a product;

[0018] S3. Analyze the product obtained in step S2; the method does not include diagnosis and treatment of the disease.

[0019] Furthermore, in step S2, the amplification reaction is carried out at a temperature of 25-45°C and for a time of 15-30 minutes.

[0020] Furthermore, in step S3, the product is analyzed by agarose gel electrophoresis.

[0021] Further, in step S3, the product is analyzed using a dual nucleic acid detection test strip: if bands appear on both the quality control line and the detection line of the test strip, it is positive; if bands appear on the quality control line of the test strip and there are no bands on the detection line, it is negative.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention adopts recombinase polymerase amplification (RPA), which is an isothermal DNA amplification technology. Compared with other DNA amplification methods, it has many advantages, especially that it can be used in non-laboratory environments. The recombinase polymerase reaction has strong specificity and can reduce the reaction temperature. RPA can achieve the amplification of specific oligonucleotide sequences by reacting at a constant temperature of 25-45°C for 15-30 minutes, which is much lower than the 95°C of PCR; and the amplified product can be visualized by a double-layer nucleic acid detection test strip. This technology has extremely low requirements for hardware equipment, does not require large or expensive hardware equipment at all, has a short reaction time, does not require complex sample processing, has the advantages of good flexibility and strong practicality, and is particularly suitable for on-site pathogen detection.

[0024] 2. The present invention obtains the best combination of primers and probes by designing and screening the specific conservative regions of the two-pointed yeast, which has good detection specificity, high sensitivity, and stable detection effect; and cross-detection with six other bacteria, fungi, and parasites, without interfering signal generation, showing good specificity. Based on the RPA primers and probes established by the present invention, detection reagents and detection kits are designed, and RPA-LFD and RPA-AGE detection technologies are constructed. The detection sensitivity of two-pointed yeast is 4.8 copies / μL, which has high sensitivity and specificity, and only requires 37°C reaction for 10min and 30min, which can quickly and easily detect two-pointed yeast.

[0025] 3. The method of the present invention is simple and easy to carry: the enzymes and other necessary items required for amplification are freeze-dried and stored and can be placed at room temperature. During amplification, only hydrolysis buffer, primers, probes and templates need to be added, and the RPA-LFD kit only needs room temperature to complete the experiment. It has low professional requirements for operators and is suitable for on-site detection in fisheries. It can also be used for scientific research on the two-pointed yeast.

[0026] 4. The present invention has strong specificity: the probe is added to the kit of the present invention, which increases the specificity of the detection, and the conformity with the existing PCR method is 100%.

[0027] 5. The detection result of the present invention is simple and reliable: the color development of the test strip is directly observed by naked eyes, and if both the detection line and the quality control line are colored, it can be qualitatively judged that the yeast is positive. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Construct a result graph of the length of a standard plasmid conventional PCR amplification product for RPA, where:

[0029] Lane M: DL2000 Marker;

[0030] Lane 1: standard plasmid DNA template.

[0031] Lane N: negative control;

[0032] Figure 2A This is the RPA-AGE type temperature optimization diagram, where:

[0033] Lane M: DL1000 Marker;

[0034] The reaction temperatures in lanes 1-5 were 25°C, 30°C, 37°C, 39°C and 42°C, respectively.

[0035] Lane N: negative control;

[0036] Figure 2B This is the RPA-AGE time optimization diagram, where:

[0037] Lane M: DL1000 Marker;

[0038] The reaction times for lanes 1-4 were 10 min, 20 min, 30 min, and 40 min, respectively;

[0039] Lane N: negative control;

[0040] Figure 3A Optimize the reaction temperature profile for RPA-LFD where:

[0041] N: negative control;

[0042] 1-4 reaction temperatures were 30°C, 35°C, 37°C and 39°C respectively

[0043] Figure 3B Optimize reaction time graph for RPA-LFD, where:

[0044] N: negative control;

[0045] The reaction times of 1-3 are 10min, 15min, and 20min, respectively;

[0046] Figure 4A This is the sensitivity electrophoresis diagram of different dilution gradients of the recombinant plasmid of RPA-AGE, where:

[0047] Lane M: DL1000 Marker;

[0048] Lanes 1-6: 4.8 × 10 5 -4.8×10 0 copies / μL dilution gradients of the recombinant plasmid.

[0049] Lane N: negative control;

[0050] Figure 4B This is the sensitivity diagram of different dilution gradients of the RPA-LFD recombinant plasmid, where:

[0051] 1-6: 4.8×10 5 -4.8×10 0 copies / μL dilution gradients of the recombinant plasmid.

[0052] N: negative control;

[0053] Figure 5A This is the electrophoresis diagram of the specific fragment amplified by RPA-AGE, where:

[0054] Lane M: DL1000 Marker;

[0055] Lanes 1-7 are: DNA of Metschnikova bicuspidatum, DNA of Escherichia coli, DNA of Staphylococcus aureus, DNA of Aeromonas hydrophila, DNA of Candida albicans, DNA of Saccharomyces cerevisiae, and DNA of Microsporidia rotifer;

[0056] Lane N: negative control;

[0057] Figure 5B is the RPA-LFD specific detection diagram, where:

[0058] Positive: positive control;

[0059] N: negative control;

[0060] 1-7 are: Escherichia coli DNA, Staphylococcus aureus DNA, Aeromonas hydrophila DNA, Candida albicans DNA, Saccharomyces cerevisiae DNA, Microsporidia rotifer DNA, and Metschnikowia bicuspidatum DNA;

[0061] Fig. 6A Detection of clinical samples by RPA-AGE

[0062] Lane M: DL1000 Marker;

[0063] They are: positive control; DNA of 1-15 samples; negative control;

[0064] Figure 6B Detection of clinical samples by RPA-AGE

[0065] Lane M: DL1000 Marker;

[0066] They are: positive control; DNA of 16-30 samples; negative control;

[0067] Figure 7 For the detection of clinical samples by RPA-LFD, the RPA-LFD test strip method is used to perform all the detection and analysis of existing DNA samples to see the probability of positive;

[0068] P: positive control;

[0069] N: negative control;

[0070] Sequentially: 1-30 sample DNA

[0071] Fig. 8A PCR detection of clinical samples

[0072] Lane M: DL1000 Marker;

[0073] They are: positive control; DNA of 1-15 samples; negative control;

[0074] Figure 8B PCR detection of clinical samples

[0075] Lane M: DL1000 Marker;

[0076] They are: positive control; DNA of 16-30 samples; negative control. DETAILED DESCRIPTION

[0077] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0078] Example:

[0079] Design and screening of RPA primers and probes for the yeast Mycobacterium bicuspidatum of the present invention:

[0080] 1. Extraction of Genomic DNA

[0081] The strains of Saccharomyces cerevisiae, Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Candida albicans, Saccharomyces cerevisiae, and Microsporidia rotifera used in the present invention are all preserved in this laboratory. A genomic DNA extraction kit (purchased from EMI Biotechnology Co., Ltd.) was used to extract bacterial genomic DNA according to the instructions, and the DNA concentration was determined by an ultramicrospectrophotometer, and the extracted DNA was stored in a -20°C refrigerator for standby use.

[0082] 2. Design and synthesis of primers and probes

[0083] According to the histone acetyltransferase type B subunit 2 gene, according to the DNA constant temperature rapid amplification kit (basic type and colloidal gold type) instructions, 5 pairs of RPA primers were designed using Premier5.0 software. Primers of the present invention were determined by experimental screening. The primers and probes used in this test were synthesized by Shanghai Biotechnology Co., Ltd.

[0084] Table 1 Primer sequences and product lengths of different detection methods

[0085]

[0086] 3. Preparation of Plasmid Standards

[0087] 3.1 Conventional PCR amplification: Using the positive sample of Saccharomyces cerevisiae as a template, primers F3 / R3 were used for amplification. The primer RPA-plasmid conventional PCR reaction system and reaction procedure are shown in Table 2 below.

[0088] Table 2 Primer RPA-plasmid conventional PCR reaction procedure

[0089]

[0090] Table 3 Primer RPA-plasmid conventional PCR reaction system

[0091]

[0092]

[0093] 3.2 Purification and recovery of DNA fragments: Move the PCR amplification products described in Table 3 into the wells of 1.5% agarose gel, adjust the electrophoresis instrument to 120V, and run for 30 minutes. After the electrophoresis is completed, under ultraviolet light, use a clean scalpel to quickly cut off the strong positive target band gel at 141bp, and put the gel containing the target band into a 1.5ml centrifuge tube. Subsequently, use FastPure Gel DNA Extraction Mini Kit to recover DNA. At this time, care should be taken to cut off the gel without the target DNA part as much as possible to improve the DNA recovery rate.

[0094] 3.3 Vector Recombination: The recovered DNA fragments were connected to 1 μL pMD-19-T vector, and then the existing purchased kit (PMD TM 19-T Vector Cloning Kit) instructions, and then add 1 μL of recovered DNA sample and 3 μL ddH2O to form a total volume of 5 μL (as shown in Table 4). Add an equal amount of 5 μL Solution I to the prepared mixed reaction solution, and the total ligation system is 10 μL, and place it at 4°C overnight.

[0095] Table 4 Connection reaction system table is as follows:

[0096]

[0097] 3.4 Screening of strains - transformation of ligation products:

[0098] 3.4.1 Add 10 μL of the ligation solution after ligating the pMD-19-T vector to 50 μL of DH5α competent cells and place in a 1.5 ml centrifuge tube and place on ice for 30 minutes;

[0099] 3.4.2 Heat in a 42℃ water bath for 45 seconds, then transfer to ice and place for 1 minute;

[0100] 3.4.3 Add 890 μL LB medium to each tube, mix well, place in a shaker, and culture at 37°C for 60 min to culture the bacterial solution;

[0101] 3.4.4 Pipette 100 μL of bacterial solution and apply it to the solid medium containing 40 μL of X-Gal and 5 μL of IPTG dye solution added to AMP-LB, seal the plate with sealing film, place the plate upright at room temperature for 1 hour, then invert it and place it in a 37°C constant temperature incubator overnight (12-16 hours);

[0102] 3.4.5 Extraction of recombinant plasmid: Pick a single white positive colony from the plate, mix it with 100 μL ddH20, and take 1 μL as a DNA sample for sequencing identification. The purpose is to confirm whether the inserted fragment of the recombinant plasmid is consistent with the designed primer amplification fragment (141 bp). If they are consistent, it means that the construction is successful, ensuring that the extracted plasmid is not a false positive, so as to extract the recombinant plasmid with the correct sequence.

[0103] The remaining bacterial liquid was placed in a centrifuge tube containing 5 ml of AMP-LB liquid and placed in a shaker at 37°C.

[0104] Cultivate at 180 rpm for about 4-5 hours until the liquid becomes turbid and hyphae are visible. Use FastPurePlasmid Mini Kit to extract the recombinant plasmid from the amplified hyphae and store at -20℃ for later use.

[0105] 4. Calculation of the copy number of Metschnikowia bicuspidata plasmids

[0106] The concentration of the constructed recombinant plasmid was measured using an ultramicrospectrophotometer and was 172 ng / μL.

[0107] According to the following formula: copies / μL = [initial concentration (ng / μL) × 10 -9 ]×(6.02×10 23 ) / [(base number of vector fragment + base number of target gene) × 660], calculate the plasmid copy number as the plasmid standard, and the recombinant plasmid copy number is 4.8 × 10 10 copies / μL.

[0108] The amplification test was performed on the genomic DNA of the yeast Saccharomyces cerevisiae extracted in Example 1 according to the primer and probe combination selected above. The amplification conditions of RPA-AGE and RPA-LFD were established:

[0109] (1) Establishing RPA-LFD amplification conditions

[0110] DNA amplification was performed using a DNA constant temperature amplification kit (colloidal gold). The total reaction system was 50 μL, including: Abuffer 29.4 μL, upstream primer 2 μL (10 μ

[0111] mol), downstream primer 2μL (10μmol), probe 0.6μL (10μmol),

[0112] 9.5 μL of ddH2O, 4 μL of DNA template (200 ng), and 2.5 μL of B buffer. ddH2O was used instead of DNA template as a blank control. Other main mixtures were prepared in the form of dry enzyme granules in freeze-dried reaction tubes. The whole process was kept on ice. After the reaction was completed, aspirate

[0113] 10 μL of the reaction product was added to a centrifuge tube containing 190 μL of ddH2O. After thorough mixing, 50 μL of the diluted reaction product was pipetted and dropped into the sample well of the LFD test strip.

[0114] Observe the quality control line and test line and interpret the results within min.

[0115] (2) Establishment of RPA-AGE amplification conditions

[0116] DNA amplification was performed using a DNA constant temperature rapid amplification kit (electrophoresis type). The total reaction system was 50 μL, including: Abuffer 29.4 μL, upstream primer 2 μL

[0117] (10μmol), downstream primer 2μL (10μmol), ddH2O10.1μL, DNA template 4μL (200ng) and B buffer 2.5μL, ddH2O was used instead of DNA template as blank control. Other main mixtures were prepared in the form of dry enzyme particles in freeze-dried reaction tubes. After the reaction is completed, the amplified product needs to be added to the reaction system with an equal volume of 50mL DNA extraction solution

[0118] (phenol: chloroform: isoamyl alcohol = 25:24:1), centrifuge at 7780g (1200rpm) for 5 min, take 5 μl of supernatant, add 2 μL 6× Loading Buffer to mix, and perform electrophoresis on the mixed solution using 1.5% agarose gel at 120V for 30 min.

[0119] Optimization of RPA-AGE and RPA-LFD amplification conditions:

[0120] (1) Reaction temperature

[0121] In order to optimize the reaction temperature, the RPA-AGE reaction was incubated for 30 min at different temperatures (25°C, 30°C, 37°C, 39°C and 42°C). At 25°C, the target band ( Figure 2A), while the gel bands were more obvious at 37℃, 39℃, and 42℃. Therefore, 37℃ was selected as the optimal reaction temperature in this experiment, which is convenient for on-site temperature testing. In order to optimize the reaction time, the reaction was performed at 37℃ for 10, 20, 30, and 40 minutes respectively. The amplification intensity of the gel target band was the highest at 30 minutes ( Figure 2B ). Therefore, the optimal reaction condition for subsequent experiments in this experiment was 37°C for 30 min.

[0122] (2) Response time

[0123] In order to optimize the RPA-LFD reaction time, the reaction was carried out at different temperatures (30°C, 35°C, 37°C and 39°C) for 10 min. At 30°C, the detection line had a visible red line ( Figure 3A ), while the color of the detection line was more obvious at 35, 37, and 39°C. In order to optimize the reaction time, the reaction was performed at 37°C for 10, 15, and 20 minutes respectively. At 10 minutes, the color of the detection line was the darkest and the amplification intensity was the highest ( Figure 3B ). Therefore, the optimal reaction condition for the subsequent experiments in this experiment was 37°C for 10 min.

[0124] 6. RPA-AGE and RPA-LFD sensitivity detection

[0125] In order to test the sensitivity of RPA-AGE and RPA-LFD in this experiment, 4.8×10 5 -4.8×10 0 The recombinant plasmid with 100 copies / μL was used as the template for reaction amplification. The minimum detection limit of RPA-AGE and RPA-LFD was 4.8×10 0 copies / μL( Figure 4A , Figure 4B ).

[0126] 7.RPA-AGE and RPA-LFD specificity detection

[0127] The specificity of RPA was tested with other bacteria, fungi and parasites. Only the hepatopancreas DNA of Chinese mitten crab infected with Metschnikowia bicuspidata showed a bright target band ( Figure 5A ), indicating that the RPA primers are highly specific to this species. In addition, in the RPA-LFD amplification, only the Metschnikowia bicuspidata positive samples showed a red positive band in the LFD test line, while other pathogens did not produce positive bands ( Figure 5B ), indicating a high degree of specificity.

[0128] 8. Detection of Metschnikova bicuspidatum by RPA-AGE and RPA-LFD on clinical samples

[0129] RPA-AGE, RPA-LFD and PCR were used to detect and analyze 30 Chinese mitten crab samples from three farms. RPA-AGE and RPA-LFD (21 / 30, 70.0%) ( Figure 7 ) was higher than that of common PCR (16 / 30, 53.3%) ( Fig. 8A and Figure 8B ).

[0130] The components not described in detail in this application are all existing conventional technologies and will not be described in detail here.

[0131] It can be understood that the above specific description of the present invention is only used to illustrate the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents to achieve the same technical effects; as long as the use requirements are met, they are within the protection scope of the present invention.

Claims

1. An RPA primer and an RPA probe for detecting Metschnikowia bicuspidatum, characterized in that: The sequences of the RPA primers are as follows: The upstream primer was F: 5'-ATGGAAGAAATGGCCGTTGAAACCTCGCTGGAA-3', and the downstream primer was R: 5'-GGACGTTAGAATTCGGGTCGATTTGGGTGAAAG-3', The 5' end of the downstream primer is labeled with a modification group-biotin; The sequence of the RPA probe is: [FAM]-ATTTCTCCGAGTTCATTCAGAGCTCTACCGC[THF]CAGCACAGTAAACCCA -[C3-spacer].

2. A kit for detecting Mycosaccharomyces cerevisiae, characterized in that: The kit is an RPA-LFD kit, comprising the combination of the RPA primers and the RPA probes according to claim 1.

3. The kit according to claim 2, characterized in that: The RPA-LFD kit also includes a positive control.

4. The kit according to claim 3, characterized in that: The positive control is the genomic DNA of Saccharomyces cerevisiae.

5. Use of the RPA primers and RPA probes according to claim 1 or the kit according to claim 2 in the detection of Saccharomyces cerevisiae in Eriocheir sinensis, wherein the use does not include the diagnosis and treatment of diseases.

6. A method for detecting Mycosaccharomyces bicuspidatus using the kit according to any one of claims 2 to 4, characterized in that: The following steps are involved: S1, extracting DNA of the sample to be tested; S2, using the DNA extracted in step S1 as an amplification template, and using the RPA primer and probe combination to perform an RPA amplification reaction to obtain a product; S3, analyzing the product obtained in step S2; The method does not include diagnosis or treatment of disease.

7. The method for detecting Metschnikova bicuspidatum according to claim 6, characterized in that: In step S2, the amplification reaction is carried out at a temperature of 25-45°C and for a time of 15-30 minutes.

8. The method for detecting the bicuspid yeast according to claim 6, characterized in that: In step S3, the product is analyzed using a dual nucleic acid detection test strip: if bands appear on both the control line and the detection line of the test strip, it is positive; If there is a band on the control line of the test strip and no band on the test line, it is negative.

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