A detection primer and detection method for picocyanobacteria

Abstract

The invention discloses a detection primer and a detection method for picocyanobacteria. The invention discloses a specific primer designed for the 16S-23S rRNA ITS sequence of the marine picocyanobacteria "Syn / Pro" group, named Picocya-Ala-F / Picocya-boxA-R. The primer of the invention can cover the cyanobacteria groups with high abundance and wide distribution in the ocean, and is suitable for the rapid and accurate quantification of picocyanobacteria in different media (seawater, surface sediments, columnar sediments, etc.) of the marine environment. The invention is the first to use molecular biological methods (qPCR) to quantify the total abundance of various groups of marine picocyanobacteria, so it has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a detection primer and a detection method for picocyanobacteria. Background Art

[0002] Marine micro-cyanobacteria, mainly including Prochlorococcus and Synechococcus, are the main primary producers in the ocean. In oligotrophic seas, their contribution to primary productivity can reach 50%. At present, there are three main quantitative methods for micro-cyanobacteria, namely microscopic counting, flow cytometry and molecular biology counting. Among them, the microscopic counting method is aimed at all cyanobacterial groups, it is difficult to distinguish different groups, and the observation is time-consuming and the result error is large; the flow cytometry method is fast and accurate, but it also has its own defects. First, for samples with low abundance of micro-cyanobacteria, flow cytometry is more difficult and has large errors, especially for samples at the bottom of the ocean euphotic layer; secondly, flow cytometry cannot be applied to seawater and sediment environmental conditions with different particle size conditions (such as 0.2-3μm, >3μm), and can only be used for large groups such as Prochlorococcus and Synechococcus, but cannot distinguish more detailed cyanobacterial groups. Microscopic counting can quantify cyanobacteria in sediments and seawater with different particle size conditions, but its throughput is relatively low, time-consuming and sample pretreatment is relatively complicated.

[0003] Molecular biological counting methods (such as real time polymerase chain reaction, qPCR) can overcome the shortcomings of the above two methods. Using this method, the distribution patterns of different ecotypes of cyanobacteria (Prochlorococcus, Synechococcus) in the ocean have been described in detail. At the same time, some specific primers (CYAN108F-CYAN377R, CYA359F-CYA781R, etc.) have been designed to quantify the abundance of cyanobacteria in freshwater. However, so far, few pairs of specific primers have been used to quantify the total abundance of various groups of marine micro-cyanobacteria. Marine micro-cyanobacteria have diverse groups and some species have low abundance. Therefore, wide coverage and strong specificity are important considerations for the design of cyanobacterial primers, which are of particular significance for our accurate quantification of the abundance of cyanobacteria. Summary of the invention

[0004] In order to solve the design problem of the above-mentioned cyanobacteria primers and better solve the problem of accurate quantification of cyanobacteria, the present invention provides a detection primer for picocyanobacteria. The diversity of marine picocyanobacteria covered by the detection primers in the present invention is high. By applying the primers to real-time fluorescence quantitative PCR detection, it is intended to promote the application of molecular biology in marine cyanobacteria detection and solve the problem of accurate quantification of cyanobacteria abundance.

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

[0006] The present invention finds a pair of detection primers Picocya-Ala-F and Picocya-boxA-R that can specifically amplify the 16S-23S ribosomal RNA transcribed spacer (ITS) of marine picocyanobacteria, and can be used for the quantification of the total abundance of various groups of picocyanobacteria in marine environments. The detection primers in the present invention are specific primers designed for the 16S-23S rRNA ITS sequence of the "Syn / Pro" branch of marine picocyanobacteria, named Picocya-Ala-F / Picocya-boxA-R, wherein Picocya-Ala-F (forward primer sequence) is 5'-GCTTTGCAAGCAGGATGTCAG-3' (as shown in SEQ ID NO.1), and Picocya-boxA-R (reverse primer sequence) is 5'-CTATGCAGTTGTCAAGGTTC-3' (as shown in SEQ ID NO.2).

[0007] The second object of the present invention is to provide a detection kit for picocyanobacteria, comprising the above-mentioned detection primers Picocya-Ala-F / Picocya-boxA-R.

[0008] The third object of the present invention is to provide a method for detecting picocyanobacteria, comprising the following steps: extracting genomic DNA of a sample to be tested as a template, performing qPCR using the above-mentioned detection primers Picocya-Ala-F / Picocya-boxA-R, and after the reaction is completed, judging whether the sample to be tested contains picocyanobacteria according to the amplification curve.

[0009] Preferably, the standard for judging whether the sample to be tested contains picocyanobacteria according to the amplification curve is: if the amplification curve has a typical amplification curve, it means that the sample to be tested contains picocyanobacteria; if the amplification curve has no typical amplification curve, it means that the sample to be tested does not contain picocyanobacteria.

[0010] Preferably, the qPCR system is as follows: each 25 μL includes the following components: 10 μmol / L of the detection primers Picocya-Ala-F and Picocya-boxA-R described in claim 1, 1.0 μL each, 2× Green Pro Taq HS Premix 12.5 μL, DNA template 1.0 μL, sterile water 9.5 μL; qPCR reaction program: 95°C pre-denaturation for 30 s; 95°C denaturation for 5 s, 61.3°C annealing for 30 s, 72°C extension for 45 s, collect fluorescence signals, and repeat 45 cycles.

[0011] A fourth object of the present invention is to provide a method for quantifying the abundance of picocyanobacteria, comprising the following steps:

[0012] (1) using genomic DNA of several different species of cyanobacteria as templates and the above-mentioned detection primers Picocya-Ala-F / Picocya-boxA-R as amplification primers to obtain PCR amplification products respectively connected to plasmids to construct several recombinant plasmids, mixing equal amounts of the recombinant plasmids to obtain mixed recombinant plasmids, then gradiently diluting the mixed recombinant plasmids to use as templates of different starting mixed recombinant plasmid concentrations, and using the above-mentioned detection primers Picocya-Ala-F / Picocya-boxA-R to establish a qPCR system for qPCR;

[0013] (2) After the reaction is completed, the cycle number Ct corresponding to each initial mixed recombinant plasmid concentration template is obtained, and a standard curve is established with the logarithm of the mixed recombinant plasmid concentration as the abscissa and the cycle number Ct as the ordinate;

[0014] (3) Extracting genomic DNA of the sample to be tested as a template, using the detection primers Picocya-Ala-F / Picocya-boxA-R described in claim 1 to establish the same qPCR system as step (1) for qPCR, and obtaining the cycle number Ct after the reaction is completed, substituting it into the standard curve, and calculating the abundance of picocyanobacteria in the sample to be tested.

[0015] Preferably, the qPCR system described in step (1) comprises the following components per 25 μL: 10 μmol / L of the detection primers Picocya-Ala-F and Picocya-boxA-R described in claim 1, 1.0 μL each, 2× Green Pro Taq HS Premix 12.5μL, DNA template 1.0μL, sterile water 9.5μL.

[0016] Preferably, the qPCR reaction procedure of steps (1) and (3) is as follows: pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, annealing at 61.3°C for 30s, extension at 72°C for 45s, collecting fluorescence signals, and repeating 45 cycles.

[0017] The fifth object of the present invention is to provide the use of the above-mentioned detection primers in detecting the abundance of micro-cyanobacteria in different media (surface sediments, columnar sediments, seawater of different particle sizes) in the marine environment.

[0018] The sixth object of the present invention is to provide the use of the above detection primers in detecting the diversity of picocyanobacteria.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] In the present invention, we designed a pair of primers for the quantitative detection of marine picocyanobacteria, which can cover the cyanobacteria groups with high abundance and wide distribution in the ocean, and are suitable for the rapid and accurate quantification of picocyanobacteria in different media (seawater, surface sediments, columnar sediments, etc.) in the marine environment. The present invention is the first to use molecular biological methods (qPCR) to quantify the total abundance of various groups of marine picocyanobacteria, so it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Primer design based on the ITS sequence of the marine cyanobacteria "Syn / Pro" group.

[0022] Figure 2 Phylogenetic diversity map of picocyanobacteria produced based on designed primers.

[0023] Figure 3 Amplification curve (A), melting peak (B) and standard curve (C) obtained based on mixed standards.

[0024] Figure 4 The abundance of cyanobacterial ITS genes in bottom seawater (A) and surface sediments (B) in the coastal waters of the Pearl River Estuary. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0026] Example 1 Primer design

[0027] This primer is designed for the ITS sequence of the "Syn / Pro" group. First of all, the "Syn / Pro" group mainly includes Synechococcus, Prochlorococcus and Cyanobium, which are highly abundant and widely distributed in the ocean, covering almost all marine micro-cyanobacteria. We found that there is a highly conserved region in the ITS sequence of the "Syn / Pro" group ( Figure 1The reverse primer position) was designed based on this, and the reverse primer Picocya-boxA-R: 5'-CTATGCAGTTGTCAAGGTTC-3' (as shown in SEQ ID NO.2); the forward primer Picocya-Ala-F: 5'-GCTTTGCAAGCAGGATGTCAG-3' (as shown in SEQ ID NO.1) can well match the various groups in "Syn / Pro" ( Figure 1 Therefore, the primers Picocya-Ala-F / Picocya-boxA-R in the present invention can specifically amplify the "Syn / Pro" group, i.e., most of the cyanobacteria groups in the marine environment, which ensures that the primers can accurately quantify the abundance of marine picocyanobacteria.

[0028] Example 2 Primer verification

[0029] (1) Selection of annealing temperature

[0030] Environmental sample DNA available in the laboratory was selected, and temperature gradient (53°C-63°C) qPCR (Bio-Rad CFX96) was performed using the primers Picocya-Ala-F / Picocya-boxA-R designed this time.

[0031] Among them, the qPCR system is 25 μL, as follows:

[0032]

[0033] The qPCR reaction procedure is as follows:

[0034]

[0035]

[0036] The results showed that when the annealing temperature was 61.3℃, the Ct value of the sample was the smallest, that is, the qPCR reaction was more sensitive at this temperature. Therefore, 61.3℃ was selected as the optimal annealing temperature.

[0037] (2) Verification of PCR product diversity

[0038] The designed primers Picocya-Ala-F / Picocya-boxA-R were used to construct clone libraries from genomic DNA of water column samples collected from Sanya Bay and the Indian Ocean. All ITS sequences were aligned by Clustal X2 and phylogenetic analysis was performed by MEGA 7 using the maximum likelihood method. Figure 2As shown in the figure, all ITS sequences belong to the "Syn / Pro group" and show a certain pattern. The cyanobacterial community in the offshore waters of Sanya Bay is dominated by Clade II Synechococcus; in the water column samples of the Indian Ocean, the HLII ecotype Prochlorococcus is mainly distributed in the upper sea area (5-25m), and the LL ecotype Prochlorococcus is mainly distributed in the middle to bottom layers of the euphotic layer (50-150m). The LL ecotype Prochlorococcus has a high diversity, covering not only the four ecotypes of LLI-LLIV, but also a variety of sequences that are not clustered into the above four ecotypes ( Figure 2 The above results show that the PCR product of the designed primers Picocya-Ala-F / Picocya-boxA-R can well cover the "Syn / Pro group" and is suitable for the quantitative detection of marine micro-cyanobacteria.

[0039] Example 3 Establishment of standard curve

[0040] 9 different pure strains of cyanobacteria (Prochlorococcus strains MED4, MIT9312, MIT9313, NATL1A and Synechococcus strains CB0101, CC9311, WH7803, WH7805, YX02-1) cultured in the laboratory were selected, and after extracting the genomic DNA of cyanobacteria, qPCR was performed using the primers Picocya-Ala-F / Picocya-boxA-R designed this time and the qPCR system and qPCR reaction program of Example 2. After the qPCR product gel was recovered, it was connected with the pMD18-T vector (TaKaRa) and transformed into DH5a competent cells. Then, 5 clones were selected for each strain, and positive test and sequencing verification were performed after PCR using universal primers M13F (5'-TGTAAAACGACGGCCAGT-3', as shown in SEQ ID NO.3) and M13R (5'-CAGGAAACAGCTATGACC-3', as shown in SEQ ID NO.4). One positive clone of each of the nine cyanobacterial strains was selected and cultured overnight in LB medium. Plasmid DNA was extracted (EZNAPlasmid Mini Kit I, OMEGA Bio-Tek). The plasmid DNA was verified by PCR with M13F and M13R primers and then recovered by gel to obtain the recombinant plasmid. After the nucleic acid concentration of the recombinant plasmid was detected by the Qubit 3 instrument, the nine recombinant plasmids were mixed in equal amounts to obtain a mixed recombinant plasmid and its concentration. After the mixed recombinant plasmid was diluted 10 times in a gradient, 7 standard gradients were obtained, and their concentrations were 3.51×10 7 copies / μL、3.51×10 6 copies / μL、3.51×10 5 copies / μL、3.51×10 4copies / μL、3.51×10 3 copies / μL、3.51×10 2 copies / μL、3.51×10 1 copies / μL, and then qPCR was performed using the designed primers Picocya-Ala-F / Picocya-boxA-R and the qPCR system and qPCR reaction program of Example 2. The mixed recombinant plasmids of the 7 standard gradients all had typical amplification curves, and the Ct value was less than 35. The standard curve was obtained by using CFX Manager Software (BIO-RADversion 3.1) with the logarithmic value of the mixed recombinant plasmid concentration as the horizontal axis and the cycle number (Ct) as the vertical axis. The standard curve equation is: y=-3.504x+38.409. In this invention, the amplification efficiency of the designed primers Picocya-Ala-F / Picocya-boxA-R was 92.9%, and there was no nonspecific amplification or primer dimers, and the sample was not contaminated ( Figure 3 ). The above results show that the establishment of this standard curve is very successful and can be used for the detection of marine environmental samples.

[0041] Example 4 Application of primers in marine environment sample detection

[0042] Based on the above primers Picocya-Ala-F / Picocya-boxA-R and the standard curve of Example 3, we selected a section from the nearshore to the open sea in the Pearl River Estuary and detected the abundance of cyanobacterial genes in the bottom seawater and surface sediments. After extracting the genomic DNA from the bottom seawater and surface sediments, qPCR was performed using the designed primers Picocya-Ala-F / Picocya-boxA-R and the qPCR system and qPCR reaction procedure of Example 2. The results are shown in Figure 2. Figure 4 As shown, in the bottom seawater, the abundance of cyanobacteria shows a "U" trend from nearshore to offshore, with the highest abundance nearshore, lower abundance offshore, and the lowest abundance in the middle sea area between nearshore and offshore; in the surface sediments, the abundance of cyanobacterial ITS genes shows a gradually decreasing trend with increasing distance from the shore.

[0043] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Sequence Listing <110> Institute of South China Sea Oceanology, Chinese Academy of Sciences <120> A detection primer and detection method for picocyanobacteria <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <400> 1 gctttgcaag caggatgtca g 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ctatgcagtt gtcaaggttc 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 tgtaaaacga cggccagt 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 caggaaacag ctatgacc 18

Claims

1. A detection primer for picocyanobacteria, characterized in that: The detection primers are composed of 2 primers as shown below: Picocya-Ala-F: 5'-GCTTTGCAAGCAGGATGTCAG-3'; Picocya-boxA-R: 5'-CTATGCAGTTGTCAAGGTTC-3'.

2. A detection kit for picocyanobacteria, characterized in that: Comprising the detection primer described in claim 1.

3. A method for detecting picocyanobacteria, characterized in that: The following steps are involved: Extract the genomic DNA of the sample to be tested as a template, use the detection primers Picocya-Ala-F / Picocya-boxA-R described in claim 1 to perform qPCR, and after the reaction is completed, determine whether the sample to be tested contains picocyanobacteria according to the amplification curve.

4. The detection method according to claim 3, characterized in that: The standard for judging whether the sample to be tested contains picocyanobacteria according to the amplification curve is: if the amplification curve has a typical amplification curve, it means that the sample to be tested contains picocyanobacteria; if the amplification curve has no typical amplification curve, it means that the sample to be tested does not contain picocyanobacteria.

5. The detection method according to claim 3, characterized in that: The qPCR system is as follows: each 25 μL includes the following components: 10 μmol / L of the detection primers Picocya-Ala-F and Picocya-boxA-R described in claim 1, 1.0 μL each, 2×SYBR ® Green Pro Taq HS Premix 12.5 μL, DNA template 1.0 μL, sterile water 9.5 μL; qPCR reaction program was: 95 ℃ pre-denaturation for 30 s; 95 ℃ denaturation for 5 s, 61.3 ℃ annealing for 30 s, 72 ℃ extension for 45 s, collecting fluorescence signals, and repeating 45 cycles.

6. A method for quantifying the abundance of picocyanobacteria, characterized in that: The following steps are involved: (1) The PCR amplification products obtained by using genomic DNA of several different species of cyanobacteria as templates and the detection primers Picocya-Ala-F / Picocya-boxA-R described in claim 1 as amplification primers are respectively connected to plasmids to construct several recombinant plasmids, and the recombinant plasmids are mixed in equal amounts to obtain mixed recombinant plasmids, and then the mixed recombinant plasmids are gradiently diluted to be used as templates of different starting mixed standard subconcentrations, and the detection primers Picocya-Ala-F / Picocya-boxA-R described in claim 1 are used to establish a qPCR system for qPCR; (2) After the reaction is completed, the cycle number Ct corresponding to each initial mixed recombinant plasmid concentration template is obtained, and a standard curve is established with the logarithm of the mixed recombinant plasmid concentration as the horizontal axis and the cycle number Ct as the vertical axis; (3) Extracting genomic DNA from the sample to be tested as a template, using the detection primers Picocya-Ala-F / Picocya-boxA-R described in claim 1 to establish the same qPCR system as step (1) for qPCR, and obtaining the cycle number Ct after the reaction is completed. Substituting it into the standard curve, the abundance of picocyanobacteria in the sample to be tested is calculated.

7. The quantitative method according to claim 6, characterized in that The qPCR system described in step (1) is as follows: each 25 μL includes the following components: 10 μmol / L of the detection primers Picocya-Ala-F and Picocya-boxA-R described in claim 1, 1.0 μL each, 2×SYBR ® Green Pro Taq HS Premix 12.5 μL, DNA template 1.0 μL, sterile water 9.5 μL.

8. The quantitative method according to claim 6 or 7, characterized in that: The qPCR reaction procedure of steps (1) and (3) is as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 61.3°C for 30 s, extension at 72°C for 45 s, collecting fluorescence signals, and repeating 45 cycles.

9. Use of the detection primer according to claim 1 in detecting the abundance of picocyanobacteria.

10. Use of the detection primers according to claim 1 in detecting the diversity of picocyanobacteria.

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