Detection primer and detection method of cyanobacterial myovirus

Abstract

This invention discloses a primer and method for detecting cyanobacterial myotail phage. The specific primers designed for this invention target the nrdA sequence of the ribonucleotide reductase Alpha subunit gene of the T4-like group of marine cyanobacterial myotail phage, named nrdA-F / nrdA-R. nrdA-F is 5'-GATGACACCCTCGATAGYAT-3'; and nrdA-R is 5'-TGWGTRCARCATCKGACAGT-3'. The primers of this invention can cover the abundant and widely distributed cyanobacterial myotail phage in the ocean, making them suitable for rapid and accurate quantification of cyanobacterial myotail phage in marine environments, and have broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a primer and method for detecting cyanobacterial myotail phage. Background Technology

[0002] Viruses are the most abundant biological particles in seawater and have significant ecological importance. Cyanomyoviruses are an important group of viruses in the ocean, infecting cyanobacteria. Since cyanobacteria are the main primary producers in the ocean, contributing up to 50% to primary productivity in oligotrophic waters, cyanomyovirus infection and lysis of the host contribute significantly to carbon cycling and have important ecological significance. Cyanomyoviruses include T4-like and TIM5-like groups, but the T4-like group is currently the dominant group. Flow cytometry and fluorescence microscopy are the main methods for detecting viral particle abundance in water samples; however, these methods detect total viral abundance and cannot detect specific viral groups. Real-time quantitative PCR (qPCR) is the main method for detecting specific viral groups. Currently, the quantitative method for cyanomyoviruses is mainly based on real-time quantitative PCR detection of the g20 gene. However, the primers currently used for the g20 gene cause nonspecific amplification, overestimating the amount of cyanobacterial myotail phages and resulting in inaccurate quantification. Summary of the Invention

[0003] To address the aforementioned design challenges of primers for cyanobacterial myophylline phage and to improve the accurate quantification of cyanobacterial myophylline phage, this invention provides a detection primer for cyanobacterial myophylline phage.

[0004] This invention targets the ribonucleotide reductase Alpha subunit gene (nrdA) of cyanobacterial myocaud phage. Specific primers designed based on the nrdA gene enable qPCR detection of this group of phages. Marine cyanobacterial myocaud phages exhibit high diversity; therefore, broad coverage and high specificity are crucial considerations in primer design. Thus, primers with a certain degree of degeneracy are essential for accurately quantifying the abundance of cyanobacterial myocaud phages. The detection primers in this invention are degenerate and cover a high diversity of marine cyanobacterial myocaud phages. Applying these primers to real-time quantitative PCR detection aims to promote the application of molecular biology techniques in the detection of marine cyanobacterial myocaud phages and solve the problem of accurate quantification of cyanobacterial myocaud phage abundance.

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

[0006] This invention designs a pair of detection primers, nrdA-F and nrdA-R, capable of specifically amplifying marine cyanobacterial myophylline phage, which can be used to quantify the total abundance of the T4-like group of cyanobacterial myophylline phage in marine environments. The detection primers in this invention are specific primers designed against the ribonucleotide reductase Alpha subunit gene (nrdA) sequence of the T4-like branch of marine cyanobacterial myophylline phage, named nrdA-F / nrdA-R, where nrdA-F (forward primer sequence) is 5'-GATGACACCCTCGATAGYAT-3' (as shown in SEQ ID NO.1), and nrdA-R (reverse primer sequence) is 5'-TGWGTRCARCATCKGACAGT-3' (as shown in SEQ ID NO.2).

[0007] A second objective of this invention is to provide a detection kit for cyanobacterial myotail phage, comprising the aforementioned detection primers nrdA-F / nrdA-R.

[0008] The third objective of this invention is to provide a method for detecting cyanobacterial myotail phage, comprising the following steps: extracting DNA from the sample to be tested as a template, performing qPCR using the aforementioned detection primers nrdA-F / nrdA-R, and determining whether the sample to be tested contains cyanobacterial myotail phage based on the amplification curve after the reaction.

[0009] Preferably, the standard for determining whether the sample to be tested contains cyanobacterial myophylline based on the amplification curve is as follows: if the amplification curve has a typical amplification curve, it indicates that the sample to be tested contains cyanobacterial myophylline; if the amplification curve does not have a typical amplification curve, it indicates that the sample to be tested does not contain cyanobacterial myophylline.

[0010] Preferably, the qPCR system comprises the following components per 25 μL: 1.0 μL each of 10 μmol / L detection primers nrdA-F and nrdA-R. 12.5 μL Green Pro Taq HS Premix, 1.0 μL DNA template, and 9.5 μL sterile water were used. The qPCR reaction program was a two-step method: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, and fluorescence signal collection was performed. This was repeated for 45 cycles.

[0011] The fourth objective of this invention is to provide a method for quantifying the abundance of cyanobacterial myotail phage, comprising the following steps:

[0012] (1) Using genomic DNA of several different species of cyanobacterial myotail phage or marine environmental DNA as templates, and the above-mentioned detection primers nrdA-F / nrdA-R as amplification primers, the PCR amplification products obtained are respectively ligated to plasmids to construct several recombinant plasmids. The randomly selected recombinant plasmids are mixed in equal amounts to obtain mixed recombinant plasmids. Then, the mixed recombinant plasmids are serially diluted to be used as templates of different starting concentrations. The above-mentioned detection primers nrdA-F / nrdA-R are used to establish a qPCR system for qPCR.

[0013] (2) After the reaction is completed, the cycle number Ct corresponding to the initial mixed recombinant plasmid concentration template is obtained. 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) Extract genomic DNA from the sample to be tested as a template, and use the detection primers nrdA-F / nrdA-R to establish the same qPCR system as in step (1) for qPCR. After the reaction is completed, obtain the cycle number Ct, substitute it into the standard curve, and calculate the abundance of cyanobacterial myotail phage in the sample to be tested.

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

[0016] Preferably, the qPCR described in steps (1) and (3) has the following qPCR reaction program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; 60℃ annealing and extension for 30s; collection of fluorescence signal; and repeating for 45 cycles.

[0017] A fifth objective of this invention is to provide the application of the above-described detection primers in detecting the abundance of cyanobacterial myotail phage in a marine environment.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] In this invention, we designed a pair of primers for the quantitative detection of marine cyanobacterial myotail phages. These primers can cover the T4-like group of cyanobacterial myotail phages, which is abundant and widely distributed in the ocean, and are suitable for rapid and accurate quantification of cyanobacterial myotail phages in the marine environment. Attached Figure Description

[0020] Figure 1 This is for screening primer annealing and extension temperatures.

[0021] Figure 2 Myotail phage of cyanobacteria in the euphosphere of the South China Sea nrdA Gene abundance.

[0022] Figure 3 The amplification curve (A), melting peak (B), and standard curve (C) are obtained based on the mixed standard.

[0023] Figure 4 The abundance of cyanobacterial myotail phage in the euphotic zone shows a trend of first increasing and then decreasing with increasing sampling depth. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0025] Example 1 Primer Design

[0026] These primers were designed targeting the ribonucleotide reductase Alpha subunit gene (nrdA) sequence of the T4-like group of marine cyanobacterial myocaud phage. The inventors discovered a highly conserved region in the ribonucleotide reductase Alpha subunit gene sequence of the T4-like group of marine cyanobacterial myocaud phage and designed the forward primer nrdA-F: ​​5'-GATGACACCCTCGATAGYAT-3' (as shown in SEQ ID NO.1); and the reverse primer nrdA-R: 5'-TGWGTRCARCATCKGACAGT-3' (as shown in SEQ ID NO.2), Y (C / T), R (A / G), W (A / T), K (G / T). The primers nrdA-F / nrdA-R of this invention can specifically amplify the T4-like group of cyanobacterial myocaud phage, i.e., most of the myocaud phage groups in the marine environment. The forward primer nrdA-F contains one degenerate base, and the backward primer contains four degenerate bases. The introduction of degenerate bases ensures that the primer amplification can cover the diversity of the T4-like group of cyanobacterial myotail phage, which ensures that the primer can accurately quantify the abundance of marine cyanobacterial myotail phage.

[0027] Example 2: Selection of Annealing and Extending Temperatures

[0028] DNA from marine environmental samples containing T4-like cyanobacterial myotail phages, which were already present in the laboratory, was selected. Using the primers nrdA-F / nrdA-R designed in this study, temperature gradient (57.6℃-63.6℃) qPCR (Bio-RadCFX96) was performed. The qPCR system consisted of 25 μL samples, as detailed below:

[0029]

[0030] The qPCR reaction procedure is as follows:

[0031]

[0032] The results showed that the Ct value of the sample was the lowest when the annealing and extension temperature was 60℃. Figure 1 This means that the qPCR reaction is more sensitive at this temperature. Therefore, 60℃ was selected as the optimal annealing and extension temperature.

[0033] Example 3: Detection of diversity in primer amplification products

[0034] A cloning library was constructed using the designed primers nrdA-F / nrdA-R from environmental DNA collected from sea column samples in the South China Sea. The obtained nrdA gene sequence was aligned using Clustal X2 and then phylogenetically analyzed using MEGA7 with neighbor-joining. The results are as follows: Figure 2 As shown, all obtained nrdA gene sequences (sequence names starting with "nrdAF5R6") clustered with the known T4-like group of cyanobacterial myotail phages and exhibited broad sequence diversity. These results demonstrate that the PCR products of the designed primers nrdA-F / nrdA-R can effectively cover the target group and are specific, making them suitable for the quantitative detection of the T4-like group of marine cyanobacterial myotail phages.

[0035] Example 4: Establishment of the Standard Curve

[0036] DNA from marine environmental samples containing T4-like cyanobacterial myotail phages, already available in the laboratory, was selected. qPCR was performed using the designed primers nrdA-F / nrdA-R and the qPCR system and reaction procedure from Example 2. After gel recovery of the qPCR products, they were ligated into the pMD18-T vector (TaKaRa) and transformed into DH5α competent cells. Then, 15 clones were randomly selected, and positive results were verified by PCR using universal primers M13F (5'-TGTAAAACGACGGCCAGT-3') and M13R (5'-CAGGAAACAGCTATGACC-3') followed by sequencing. Ten positive clones were selected, cultured overnight in LB medium, and plasmid DNA was extracted (EZNAPlasmid Mini Kit I, OMEGA Bio-Tek). The plasmid DNA was verified by PCR using M13F and M13R primers and then gel recovered to obtain the recombinant plasmid. After the nucleic acid concentration of the recombinant plasmid was detected by the Qubit 3 instrument, 10 recombinant plasmids were mixed in equal volumes to obtain the mixed recombinant plasmid and its concentration. The mixed recombinant plasmid was then serially diluted 10-fold to obtain 7 standard gradients with concentrations of 9.87 × 10⁻⁶. 7 copies / μL, 9.87×10 6 copies / μL, 9.87×10 5 copies / μL, 9.87×10 4 copies / μL, 9.87×10 3 copies / μL, 9.87×10 2 copies / μL, 9.87×10 1 Copies / μL were collected, and then qPCR was performed using the designed primers nrdA-F / nrdA-R and the qPCR system and qPCR reaction program of Example 2. All seven standard gradient mixed recombinant plasmids exhibited typical amplification curves. Standard curves were obtained using CFX Manager Software (BIO-RAD version 3.1), with the logarithm of the mixed recombinant plasmid concentration as the x-axis and the cycle number (Ct) as the y-axis. In this invention, the amplification efficiency of the designed primers nrdA-F / nrdA-R was 91.5%, and no non-specific amplification or primer dimer formation occurred. Figure 3 The above results demonstrate that the standard curve was successfully established and can be used for the detection of marine environmental samples.

[0037] Example 5: Application of primers in the detection of marine environmental samples

[0038] Based on the primers nrdA-F / nrdA-R and the standard curve from Example 4, seawater samples from the South China Sea were selected to detect the abundance of the nrdA gene in cyanobacterial myotail phage. The sample collection process involved first filtering the solution through a 0.2 μm pore size carbonate membrane to remove bacteria and other cellular organisms. Then, the viral particles in the filtrate were concentrated using ultrafiltration. Total DNA was extracted from the concentrated viral particles, and qPCR was performed using the designed primers nrdA-F / nrdA-R and the qPCR system and reaction procedure from Example 2. The results are as follows: Figure 4 As shown, in the euphrates, the abundance of cyanobacterial myotail phage first increases and then decreases with increasing sampling depth. The abundance range of the nrdA gene of cyanobacterial myotail phage in the euphrates is 10. 2 -10 5 copies / mL.

[0039] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, 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 considered within the scope of protection of the present invention.

Claims

1. A primer for detecting cyanobacterial myotail phage, characterized in that, The detection primers are shown below: nrdA-F: ​​5'-GATGACACCCTCGATAGYAT-3'; nrdA-R:5'-TGWGTRCARCATCKGACAGT-3'.

2. A detection kit for cyanobacterial myotail phage, characterized in that, Includes the detection primers as described in claim 1.

3. A method for detecting cyanobacterial myotail phage, characterized in that, Includes the following steps: Genomic DNA was extracted from the sample to be tested and used as a template. qPCR was performed using the detection primers nrdA-F / nrdA-R described in claim 1. After the reaction, the presence of cyanobacterial myotail phage in the sample to be tested was determined based on the amplification curve.

4. The detection method according to claim 3, characterized in that, The criteria for determining whether a sample contains cyanobacterial myophylline based on the amplification curve are as follows: if the amplification curve has a typical amplification curve, it indicates that the sample contains cyanobacterial myophylline; if the amplification curve does not have a typical amplification curve, it indicates that the sample does not contain cyanobacterial myophylline.

5. The detection method according to claim 3, characterized in that, The qPCR system comprises the following components per 25 μL: 1.0 μL each of the 10 μmol / L detection primers nrdA-F and nrdA-R as described in claim 1; 12.5 μL Green Pro Taq HS Premix, 1.0 μL DNA template, and 9.5 μL sterile water were used. The qPCR reaction program was a two-step method: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, and fluorescence signal collection was performed. This was repeated for 45 cycles.

6. A method for quantitatively determining the abundance of marine cyanobacterial myotail phage, characterized in that, Includes the following steps: (1) Using marine environmental DNA as a template and the detection primers nrdA-F / nrdA-R as amplification primers as described in claim 1, the PCR amplification products obtained are respectively ligated to plasmids to construct several recombinant plasmids. The recombinant plasmids are mixed in equal amounts to obtain mixed recombinant plasmids. Then, the mixed recombinant plasmids are serially diluted to be used as templates with different initial mixed standard concentrations. A qPCR system is established using the detection primers nrdA-F / nrdA-R as described in claim 1 for qPCR detection. (2) After the reaction is completed, the cycle number Ct corresponding to the initial mixed recombinant plasmid concentration template is obtained. 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. (3) Extract genomic DNA from the sample to be tested as a template, and use the detection primers nrdA-F / nrdA-R described in claim 1 to establish the same qPCR system as in step (1) for qPCR. After the reaction is completed, obtain the cycle number Ct, substitute it into the standard curve, and calculate the abundance of cyanobacterial myotail phage in the sample to be tested.

7. The quantitative method according to claim 6, characterized in that, The qPCR system described in step (1) consists of the following components per 25 μL: 1.0 μL each of the 10 μmol / L detection primers nrdA-F and nrdA-R as described in claim 1; Green Pro Taq HS Premix 12.5μL, DNA template 1.0μL, sterile water 9.5μL.

8. The quantitative method according to claim 6 or 7, characterized in that, The qPCR described in steps (1) and (3) is a two-step reaction procedure: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s; 60℃ annealing and extension for 30s; collection of fluorescence signal; and repeating for 45 cycles.

9. The application of the detection primers according to claim 1 in detecting the abundance of marine cyanobacterial myotail phage.

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