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CN-116676420-B - Detection primer and detection method for cyanobacteria myotail phage

CN116676420BCN 116676420 BCN116676420 BCN 116676420BCN-116676420-B

Abstract

The invention discloses a detection primer and a detection method of cyanobacteria myotail phage. The invention relates to a specific primer designed for a ribonucleotide reductase Alpha subunit gene nrdA sequence of a T4-like group of marine cyanobacteria myophagium, which is named nrdA-F/nrdA-R, wherein the nrdA-F is 5'-GATGACACCCTCGATAGYAT-3', and the nrdA-R is 5'-TGWGTRCARCATCKGACAGT-3'. The primer disclosed by the invention can cover cyanobacteria myotail phage with high abundance and wide distribution in the ocean, is suitable for rapid and accurate quantification of cyanobacteria myotail phage in the ocean environment, and has a wide application prospect.

Inventors

  • HUANG SIJUN
  • ZHANG JIANDONG
  • LONG LIJUAN

Assignees

  • 南方海洋科学与工程广东省实验室(广州)
  • 中国科学院南海海洋研究所

Dates

Publication Date
20260505
Application Date
20220725

Claims (9)

  1. 1. A detection primer of cyanobacteria myotail phage, which is characterized in that the detection primer is as follows: nrdA-F:5’-GATGACACCCTCGATAGYAT-3’; nrdA-R:5’-TGWGTRCARCATCKGACAGT-3’。
  2. 2. A cyanobacterial myotail phage detection kit comprising the detection primer of claim 1.
  3. 3. A detection method of cyanobacteria myotail phage is characterized by comprising the following steps of extracting genome DNA of a sample to be detected as a template, carrying out qPCR by using the detection primer nrdA-F/nrdA-R as set forth in claim 1, and judging whether the sample to be detected contains cyanobacteria myotail phage or not according to an amplification curve after the reaction is finished.
  4. 4. The method according to claim 3, wherein the criterion for determining whether the sample to be tested contains cyanobacterial myotail phage according to the amplification curve is that if the amplification curve has a typical amplification curve, the sample to be tested contains cyanobacterial myotail phage, and if the amplification curve does not have a typical amplification curve, the sample to be tested does not contain cyanobacterial myotail phage.
  5. 5. The method according to claim 3, wherein the qPCR system comprises 10. Mu. Mol/L of the detection primers nrdA-F and nrdA-R each 1.0. Mu.L of the detection primers nrdA-F and nrdA-R per 25. Mu.L of the detection primers nrdA-F and nrdA-R, Green Pro Taq HS Premix 12.5.5 mu L, DNA template 1.0 mu L and sterile water 9.5 mu L, and qPCR reaction procedure was a two-step method of 95℃pre-denaturation for 30s, 95℃denaturation for 5s,60℃annealing and extension for 30s, collecting fluorescent signals, and repeating 45 cycles.
  6. 6. The quantification method of the marine cyanobacteria myotail phage abundance is characterized by comprising the following steps of: (1) Respectively connecting PCR amplification products obtained by taking seawater environment DNA as a template and taking the detection primer nrdA-F/nrdA-R as an amplification primer in claim 1 to plasmids to construct a plurality of recombinant plasmids, mixing the recombinant plasmids in equal quantity to obtain mixed recombinant plasmids, then carrying out gradient dilution on the mixed recombinant plasmids to serve as templates with different initial mixed standard concentration, and establishing a qPCR system by using the detection primer nrdA-F/nrdA-R in claim 1 to carry out qPCR detection; (2) After the reaction is finished, obtaining a circulation number Ct corresponding to each initial mixed recombinant plasmid concentration template, and establishing a standard curve by taking the logarithmic value of the mixed recombinant plasmid concentration as an abscissa and the circulation number Ct as an ordinate; (3) Extracting genome DNA of a sample to be detected as a template, establishing a qPCR system which is the same as that of the step (1) by using the detection primer nrdA-F/nrdA-R of claim 1, carrying out qPCR, obtaining cycle number Ct after the reaction is finished, substituting the cycle number Ct into a standard curve, and calculating to obtain the abundance of cyanobacteria myotail phage in the sample to be detected.
  7. 7. The method according to claim 6, wherein the qPCR system of step (1) comprises 10. Mu. Mol/L of the detection primers nrdA-F and nrdA-R each 1.0. Mu.L of the detection primers of claim 1 per 25. Mu.L of the detection primers, Green Pro Taq HS Premix 12.5.5 μ L, DNA template 1.0 μL, sterile water 9.5 μL.
  8. 8. The method according to claim 6 or 7, wherein the qPCR in steps (1) and (3) is performed in a two-step process of pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and extension, collecting fluorescent signals, and repeating 45 cycles.
  9. 9. Use of the detection primer of claim 1 for detecting the abundance of a marine cyanobacteria myotail phage.

Description

Detection primer and detection method for cyanobacteria myotail phage Technical Field The invention relates to the technical field of microorganisms, in particular to a detection primer and a detection method of cyanobacteria myotail phage. Background The virus is the most abundant biological particle in seawater, and has important ecological significance. Cyanobacterial myotail phages (cyanomyovirus) are an important group of viruses in the ocean that infect cyanobacteria as hosts. Since cyanobacteria is the primary producer in the ocean, its contribution to primary productivity can reach 50% in oligotrophic sea areas, and cyanobacteria phage infects and lyses hosts with important carbon cycle contributions, which is of great ecological importance. Cyanobacterial myotail phages include T4-like, TIM5-like, etc. groups, but the T4-like group is the main group currently discovered. Flow cytometry and fluorescence microscopy are the main methods for detecting the abundance of virus particles in a water sample, but the two methods detect the total abundance of viruses and cannot detect specific virus groups. The real-time fluorescent quantitative PCR (real time polymerase chain reaction, qPCR) method is the main method for detecting specific virus groups. Currently, the quantification method of cyanobacteria myotail phage is mainly based on real-time fluorescent quantitative PCR detection of g20 gene. However, the primers of the g20 gene currently used cause non-specific amplification, overestimating the amount of cyanobacteria myotail phage, resulting in inaccurate quantification. Disclosure of Invention In order to solve the design problem of the cyanobacteria myotail phage primer, the invention provides a detection primer of cyanobacteria myotail phage, which can better accurately quantify the cyanobacteria myotail phage. Aiming at ribonucleotide reductase (ribonucleotide reductase) Alpha subunit gene (nrdA) of cyanobacteria myophagium, the invention designs a specific primer based on the nrdA gene to realize qPCR detection of the group of phages. The marine cyanobacteria myotail phage has high diversity, so the coverage is wide, the specificity is strong, which is an important consideration factor for designing the primer, therefore, the primer with certain degeneracy is designed, and the method has a special important meaning for accurately quantifying the abundance of the cyanobacteria myotail phage. The design of the detection primer has degeneracy, the diversity of the covered marine cyanobacteria myotail phage is higher, the primer is applied to real-time fluorescent quantitative PCR detection, and the aim of promoting the application of a molecular biology technology in marine cyanobacteria myotail phage detection is to solve the problem of accurate quantification of the abundance of the cyanobacteria myotail phage. The invention is realized by the following technical scheme: The invention designs a pair of detection primers nrdA-F and nrdA-R capable of specifically amplifying marine cyanobacteria myotail phage, which can be used for quantifying the total abundance of cyanobacteria myotail phage T4-like group in a marine environment. The detection primer is a specific primer designed for the ribonucleotide reductase Alpha subunit gene (nrdA) sequence of the marine cyanobacteria myotail phage T4-like branch, and is named nrdA-F/nrdA-R, wherein the nrdA-F (forward primer sequence) is 5'-GATGACACCCTCGATAGYAT-3' (shown as SEQ ID NO. 1), and the nrdA-R (reverse primer sequence) is 5'-TGWGTRCARCATCKGACAGT-3' (shown as SEQ ID NO. 2). The second object of the invention is to provide a detection kit of cyanobacteria myotail phage, which comprises the detection primer nrdA-F/nrdA-R. The third object of the invention is to provide a detection method of cyanobacteria myotail phage, which comprises the following steps of extracting DNA of a sample to be detected as a template, carrying out qPCR by using the detection primer nrdA-F/nrdA-R, and judging whether the sample to be detected contains cyanobacteria myotail phage according to an amplification curve after the reaction is finished. Preferably, the standard for judging whether the sample to be tested contains the cyanobacteria myotail phage according to the amplification curve is that if the amplification curve has a typical amplification curve, the sample to be tested contains the cyanobacteria myotail phage, and if the amplification curve does not have a typical amplification curve, the sample to be tested does not contain the cyanobacteria myotail phage. Preferably, the qPCR system comprises 10 mu mol/L detection primers nrdA-F and nrdA-R of 1.0 mu L per 25 mu L,Green Pro Taq HS Premix 12.5.5 mu L, DNA template 1.0 mu L and sterile water 9.5 mu L, and qPCR reaction procedure was a two-step method of 95℃pre-denaturation for 30s, 95℃denaturation for 5s,60℃annealing and extension for 30s, collecting fluorescent signals, and repeating 45 cycles. A fourth object