A reagent for detecting blue algae of the genus Zephyranthes and its application

Abstract

The present invention discloses a reagent for detecting cyanobacteria of the genus Zethionia and its application; the reagent comprises a primer set; the primer set comprises a forward primer having a sequence as shown in SEQ ID NO:8 and a reverse primer having a sequence as shown in SEQ ID NO:5. The present invention uses the rbcLX gene of Zethionia as a target sequence, creatively designs and screens to obtain a primer probe set for detecting cyanobacteria of the genus Zethionia, has high sensitivity and good specificity, only amplifies cyanobacteria of the genus Zethionia, and does not cross-react with algae of other genera such as Pseudo-Anabaena, Anabaena, Cryptomonas, Oscillatoria, Cylindrosporium, Splenidium, Nostoc, Pseudo-Cylindrosporium, Trilochromis, Microcystis, Sphingomyelia, Longisporium, Aphanizomenon, and Nodularia, providing a new means for rapid and quantitative detection of cyanobacteria of the genus Zethionia, and can effectively meet the relevant needs of water source water quality monitoring.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a reagent for detecting cyanobacteria of the genus Zephyranthes and an application thereof. Background Art

[0002] Limnothrix spp. is a common small cyanobacteria belonging to Cyanobacteria, Cyanophyceae, Synechococcales, Pseudanabaenaceae. As the degree of eutrophication of lakes increases, it often forms a dominant population and causes cyanobacterial blooms. A study analyzed the algae species composition of four typical reservoirs around cities in the Pearl River Delta region. The study showed that Limnothrix spp. is one of the dominant species. At present, the monitoring method of Limnothrix mainly adopts microscopic examination, which requires a lot of manual and meticulous work. This method still has the defects of cumbersome operation and individual data cannot reflect the overall situation. Accurate detection and monitoring of Limnothrix spp. is the key to understanding its ecological dynamics and managing potential harmful effects. Therefore, there is an urgent need for a reagent and method for efficiently detecting Limnothrix spp. cyanobacteria, which can quickly and accurately detect Limnothrix spp.

[0003] The present invention aims at typical harmful algae in Guangdong Province and establishes a molecular detection and identification method for blue-green algae of the genus Zephyranthes. The method can accurately detect Zephyranthes and be applied in routine monitoring, providing a new means for the rapid and quantitative detection of Zephyranthes, so as to establish an early assessment and early warning mechanism and take effective prevention and mitigation measures in time, thereby better carrying out the comprehensive prevention and control of harmful algal blooms and ensuring the ecological safety of water quality. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a reagent for detecting cyanobacteria of the genus Zephyranthes and its application.

[0005] The present invention also provides a kit having the above reagent.

[0006] The invention also provides the application of the above reagent and kit.

[0007] The invention also provides a method for detecting the algae.

[0008] According to one aspect of the present invention, a reagent for detecting cyanobacteria of the genus Zephyrus is provided, the reagent comprising a primer set; the primer set comprises a forward primer having a sequence as shown in SEQ ID NO:8 and a reverse primer having a sequence as shown in SEQ ID NO:5.

[0009] In some embodiments of the present invention, the reagent further comprises a probe sequence having a sequence as shown in SEQ ID NO:6.

[0010] In some embodiments of the present invention, the 5' end of the probe sequence is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group.

[0011] In some embodiments of the present invention, the fluorescent group includes one of FAM, CY5 and JOE.

[0012] In some embodiments of the present invention, the quenching group includes one of TAMRA, Dabcyl, BHQ1 and MGB.

[0013] According to a second aspect of the present invention, a kit is provided, wherein the kit contains the above reagent.

[0014] In some embodiments of the present invention, the kit further comprises a fluorescent PCR reaction mixture.

[0015] In some embodiments of the present invention, the kit further comprises sterile ultrapure water.

[0016] According to a third aspect of the present invention, an application of the above reagent and kit is provided, wherein the application is an application in detecting cyanobacteria of the genus Zephyron.

[0017] According to a fourth aspect of the present invention, a method for detecting cyanobacteria of the genus Zephyrus is provided, the method comprising the following steps: using the above reagent or kit to detect a sample to be detected.

[0018] In some embodiments of the present invention, the detection is performed using fluorescent quantitative PCR.

[0019] In some embodiments of the present invention, the final concentration of the primers in the reaction system of the fluorescent quantitative PCR is about 0.1 to 0.6 μM.

[0020] In some embodiments of the present invention, the final concentration of the primer in the reaction system of the fluorescent quantitative PCR is about 0.4 μM.

[0021] In some embodiments of the present invention, the amplification reaction system of the fluorescent quantitative PCR is:

[0022] Fluorescence PCR reaction mixture 2×1×;

[0023] Forward primer 0.1-0.6 μM;

[0024] Reverse primer 0.1-0.6 μM;

[0025] Probe 0.1-0.5 μM;

[0026] 4-6 μL of sample to be tested;

[0027] Add ddH2O to make up to 25 μL.

[0028] In some embodiments of the present invention, the amplification program of the fluorescent quantitative PCR is: pre-denaturation at 94-96°C for 4-6 minutes; then entering the cycling stage: denaturation at 94-96°C for 13-18 seconds, annealing and extension at 58-62°C for 20-40 seconds, 38-45 cycles, and collecting fluorescence data.

[0029] In some embodiments of the present invention, the amplification program of the fluorescent quantitative PCR is: pre-denaturation at 95°C for 5 minutes; then entering the cycling stage: denaturation at 95°C for 13-18 seconds, annealing and extension at 60°C for 20-40 seconds, 40 cycles, and collecting fluorescence data.

[0030] In some embodiments of the present invention, after the detection, the step of statistically analyzing the Ct value of the sample and interpreting the result is also included; the result interpretation is:

[0031] Positive: If the Ct of the sample is ≤35 and a typical S-shaped fluorescence curve appears, the sample is positive;

[0032] Negative: If the sample shows no fluorescence increase, the sample is negative.

[0033] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention uses the rbcLX gene of the Zeus genus as the target sequence, creatively designs and screens to obtain a primer probe set for detecting cyanobacteria of the genus Zeus, which has high sensitivity and good specificity, and only amplifies cyanobacteria of the genus Zeus, and does not cross-react with algae of other genera such as Pseudo-Anabaena, Anabaena, Cryptomonas, Oscillatoria, Columnar Spores, Chain Algae, Nostoc, Pseudo-Columnar Spores, Trilochromis, Microcystis, Sheathing Microcystis, Longisporium, Aphanizomenon, and Nodularia, providing a new means for rapid and quantitative monitoring of Zeus, and can effectively meet relevant needs such as water source water quality monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0035] Figure 1 This is a diagram of the amplification results of the F2R2P2 primer probe set in Example 2 of the present invention;

[0036] Figure 2 This is a diagram of the amplification results of the F3R3P3 primer probe set in Example 2 of the present invention;

[0037] Figure 3This is a diagram of the amplification results of the F4R3P3 primer probe set in Example 2 of the present invention;

[0038] Figure 4 This is a diagram of the amplification results of the F5R3P3 primer probe set in Example 2 of the present invention;

[0039] Figure 5 This is a diagram of the amplification results of the F6R3P3 primer probe set in Example 2 of the present invention;

[0040] Figure 6 This is a diagram of the amplification results of the F7R7P7 primer probe set in Example 2 of the present invention;

[0041] Figure 7 This is a diagram of the amplification results of the F8R8P8 primer probe set in Example 2 of the present invention;

[0042] Figure 8 This is a sensitivity test result diagram in Example 4 of the present invention;

[0043] Fig. 9 It is the standard curve diagram in Example 4 of the present invention;

[0044] Fig.10 This is a graph showing the results of conventional PCR electrophoresis detection in Example 6 of the present invention;

[0045] Fig.11 This is a diagram of the sequence alignment results in Example 6 of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0047] Main Materials:

[0048] 27 algae species including Zeselonia, Pseudo-Anabaena, Anabaena, Cryptomonas, Oscillatoria, Cylindrosporium, Splenidium, Nostoc, Pseudo-cylindrosporium, Trilothium, Microcystis, Sphingomyelia, Longisporium, Aphanizomenon, and Nodularia were purchased from the freshwater algae seed bank of the Chinese Academy of Sciences (see Table 2 for specific algae species);

[0049] The recombinant plasmid of Zeshima was synthesized by Liuhe BGI Biotechnology Co., Ltd.;

[0050] The extraction kit “NuClean Plant Genomic DNA Kit” was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.;

[0051] Pro Taq HS premixed probe qPCR kit was purchased from Aikerui Biotechnology Co., Ltd.

[0052] Main instruments:

[0053] QuantStudio 7 real-time fluorescence PCR instrument was purchased from Applied Biosystems, USA; 5424R high-speed refrigerated centrifuge was purchased from Eppendorf AG, Germany.

[0054] Example 1 Design and determination of primer probes for detection of cyanobacteria of the genus Zephyranthes

[0055] Design of primer probe sets:

[0056] According to the relevant published literature, representative sequences of different genera of the Zephyraceae were selected for comparison, and the conserved gene 16S fragment (NCBI No.: EF088335.1) was screened and selected, and then the primers were designed using the biological software Oligo 7.60, and 7 pairs of primer probe sets with the best performance such as amplification efficiency were screened for amplification of Zephyraceae cyanobacteria. The specific sequences are shown in Table 1. Primers and probes were synthesized by Liuhe BGI Biotechnology Co., Ltd.

[0057] Table 1

[0058]

[0059]

[0060]

[0061] Example 2 Optimization of primer probe set

[0062] DNA of 27 algae species (as shown in Table 2) including the genera of Zeselonia (Zeselonia cyanobacteria), Pseudo-Anabaena, Anabaena, Cryptomonas, Oscillatoria, Cylindrospermum, Splenidium, Nostoc, Pseudo-Cylindrospermum, Trilomicron, Microcystis, Scalycophyte, Longisporium, Aphanizomenon, and Nodularia were extracted, and specific experiments were performed using different sets of primers and probes designed in step (1) (F2R2P2, F3R3P3, F4R3P3, F5R3P3, F6R3P3, F7R7P7, and F8R8P8 primer-probe sets, respectively) to screen out the optimal detection primers and probes (three parallel tests were performed for each set). 6 copies / μL positive plasmid was used as positive control, and DEPC water was used as blank control.

[0063] Table 2

[0064]

[0065]

[0066] The reaction system was: 12.5 μL of real-time fluorescence PCR reaction reagent (2×), 1 μL of forward primer (concentration of 10 μmol / L), 1 μL of reverse primer (concentration of 10 μmol / L), 0.5 μL of probe (10 μmol / L) and 5 μL of DNA template (recombinant plasmid of Zeshima algae), and sterile ultrapure water was added to 25 μL.

[0067] The reaction cycle parameters of fluorescence PCR were: 95°C for 5 min; then entering the cycling stage: 95°C for 15 sec, 60°C for 30 sec (collecting fluorescence), and 40 cycles.

[0068] Test results such as Figure 1-7 As shown in the figure, it can be seen that primers F2R2P2, F3R3P3, F4R3P3, F6R3P3, F7R7P7 and F8R8P8 all showed non-specific amplification, while primer F5R3P3 only showed specific amplification curves in the Zeshima algae sample and the positive control, and no specific amplification curves were shown in the other 26 strains (see Figure 4 ), the specificity effect was good and met the requirements, and the subsequent performance test was carried out with the primer set F5R3P3.

[0069] Example 3 A kit for detecting cyanobacteria of the genus Zephyranthes

[0070] In this example, a kit for detecting cyanobacteria of the genus Zephyranthes was prepared, and the components included the detection primer probe set for cyanobacteria of the genus Zephyranthes prepared in Example 1 (the F5R3P3 primer probe set whose sequences are shown in SEQ ID NO: 8, SEQ ID NO: 5 and SEQ ID NO: 6, respectively), real-time fluorescent PCR reaction reagents (2×), and sterilized ultrapure water.

[0071] Judging criteria:

[0072] If the positive control Ct value is ≤35, and the negative control and blank control Ct values ​​are >35, the experiment is valid;

[0073] The Ct value of the test sample was ≤38, which was judged as positive;

[0074] If there is no typical "S" type amplification or the Ct value is greater than 35, it is judged as negative.

[0075] Example 4 Sensitivity test and establishment of standard curve

[0076] The recombinant plasmid of the cyanobacteria of the genus Zeus was diluted in a 10-fold gradient and 10 8 ~10 1The positive plasmid was detected using the established method. The kit for detecting cyanobacteria of the genus Zephyranthes prepared in Example 3 was used to perform the detection according to the reaction system and reaction procedure in Example 2.

[0077] Test results such as Figure 8 As shown in the figure, the minimum detection limit of the F5R3P3 primer probe set is 10 2 copies / uL. This is used to draw a standard curve (such as Fig. 9 As shown), the regression equation is y = -3.1973x + 44.669, R 2 =0.997, and the PCR amplification efficiency E was calculated to be 105%.

[0078] Example 5 Stability Test

[0079] The positive plasmid was diluted in 10-fold gradients, and 10 8 ~10 1 The kit for detecting cyanobacteria of the genus Zephyron prepared in Example 3 was used, and stability tests were performed according to the reaction system and reaction procedure in Example 2 to perform intra-batch and inter-batch stability tests.

[0080] Table 3

[0081]

[0082] The results are shown in Table 3. It can be seen from the table that the coefficient of variation (CV) of the intra-group and inter-group tests is less than 3%, indicating that the stability of this method is good.

[0083] Example 6 Actual detection of water bodies

[0084] From November 2022 to April 2023, water samples from key reservoirs in Zhuhai City will be collected every month and tested for cyanobacteria of the genus Zephyranthes using morphological and molecular biological methods.

[0085] Sampling method: Four key reservoirs in Zhuhai were selected: Nanping Reservoir, Qianwu Reservoir, Yangwei Reservoir and Dajingshan Reservoir. Every month, 1L of reservoir water was extracted from each reservoir using disposable sterile bottles as test samples, which were then refrigerated and sent to the laboratory.

[0086] Nucleic acid extraction from water samples: Place a 0.45um pore size polycarbonate membrane flat on the filter, slowly pour 500mL of water sample into the upper filter cup, and filter using a diaphragm vacuum pump. After filtering, use sterile tweezers to transfer the filter membrane to a 2mL centrifuge tube and cut the filter membrane into pieces with scissors. Complete nucleic acid extraction according to the instructions of the NuClean Plant Genomic DNA Kit.

[0087] Water sample detection: Morphology was determined by microscopic examination, and molecular biology was determined using the kit for detecting cyanobacteria of the genus Zephyranthes prepared in Example 3. The detection was performed according to the reaction system and reaction procedure in Example 2.

[0088] Water sample sequencing: Randomly select 8 positive water sample nucleic acids of the genus Zephyranthes, and use the upstream and downstream primers of fluorescent PCR for ordinary PCR. After electrophoresis to obtain the target band, purify and sequence it, and compare the measured sequence with the NCBI database to verify the consistency of the sequence.

[0089] Table 4 Fluorescence PCR test results of water samples (Ct value)

[0090]

[0091] Table 5 Water sample microscopic examination results (pieces / L)

[0092]

[0093] The results are shown in Tables 4 and 5. It can be seen from the table that the cyanobacteria of the genus Zethionia were detected in the water samples of the four reservoirs using the fluorescent PCR detection method every month. Using the microscopic method, the cyanobacteria of the genus Zethionia were detected in Dajingshan Reservoir in 2 months, and no detection was made in other months. The cyanobacteria of the genus Zethionia were detected in Qianwu Reservoir and Yangwei Reservoir in 1 month, and no detection was made in other months. No detection was made in Nanping Reservoir in any month.

[0094] The 8 selected water sample nucleic acids were amplified by PCR using primers, and the amplified products were subjected to 1% agarose gel electrophoresis. The results showed that the 8 water sample nucleic acids were successfully amplified into a 169 bp fragment, which was consistent with the size of the target band (such as Fig.10 The PCR product was sequenced, and the sequencing result was 100% consistent with the 16S gene of Zedoaria sp., and the detection result of the fluorescent PCR detection method was consistent with the sequencing result (as shown in Fig.11 shown).

[0095] Comparing the above results, it can be seen that the fluorescence PCR detection method has a higher detection rate than the traditional microscopy method, and has obvious advantages, which can greatly improve the detection rate and efficiency.

[0096] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A reagent for detecting cyanobacteria of the genus Zephyranthes, characterized in that: The reagents include a primer set; the primer set includes a forward primer whose sequence is shown in SEQ ID NO:8 and a reverse primer whose sequence is shown in SEQ ID NO:5; The reagent also includes a probe sequence as shown in SEQ ID NO:

6.

2. The reagent according to claim 1, characterized in that The 5' end of the probe sequence is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group.

3. The reagent according to claim 2, characterized in that The fluorescent group includes one of FAM, CY5 and JOE; the quenching group includes one of TAMRA, Dabcyl, BHQ1 and MGB.

4. A kit, characterized in that: The kit contains the reagent according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes a fluorescent PCR reaction mixture.

6. Use of at least one of the reagent according to any one of claims 1 to 3 and the kit according to any one of claims 4 to 5 in detecting cyanobacteria of the genus Zephyron.

7. A method for detecting cyanobacteria of the genus Zephyranthes, characterized in that: The method comprises the following steps: using the reagent described in any one of claims 1 to 3 or the kit described in any one of claims 4 to 5 to detect the sample to be tested.

8. The detection method according to claim 7, characterized in that: The detection is performed using fluorescent quantitative PCR.

9. The detection method according to claim 8, characterized in that: The amplification reaction system of the fluorescent quantitative PCR is: Add ddH2O to make up to 25 μL.

10. The detection method according to claim 8, characterized in that: The amplification program of the fluorescent quantitative PCR is: pre-denaturation at 94-96°C for 4-6 minutes; then entering the cycling stage: denaturation at 94-96°C for 13-18 seconds, annealing and extension at 58-62°C for 20-40 seconds, 38-45 cycles, and collecting fluorescence data.

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