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CN-122012787-A - Primer probe combination for detecting fusarium on detection layer, RCA (RCA visualization) kit and detection method

CN122012787ACN 122012787 ACN122012787 ACN 122012787ACN-122012787-A

Abstract

The invention belongs to the field of biological detection, and particularly relates to a primer probe combination for detecting fusarium, an RCA (RCA visual kit) and a detection method. Comprising the following sequences of a lock probe PLP, an RCA amplification upstream primer, an RCA amplification downstream primer and a ZIP sequence. The sensitivity was 100-fold higher than that of conventional PCR.

Inventors

  • LIU FUGUO
  • XIA WEI
  • LI XIAOFANG
  • JIAO JINTAO
  • SUN HAO

Assignees

  • 潍坊科技学院

Dates

Publication Date
20260512
Application Date
20260310

Claims (8)

  1. 1. The primer probe combination for detecting fusarium on the basis of rolling circle amplification technology is characterized by comprising the following sequences: lock probe PLP: 5'P-CAAGATGTACCCCGCCAGATCTTGGTCGGATTAGACGACTGCTGGGAGCAGCAACTTCTTCGTCTTCAACGCTCCCGCTATCAGCATGTTGTCTTC-3',SEQ ID NO.1; The RCA amplification upstream primer is 5'-Bio-CCAGCAGTCGTCTAATCC-3', SEQ ID NO.2; RCA amplification downstream primer 5'-GAGCAGCAACTTCTTCGT-3', SEQ ID No.3.
  2. 2. The RCA visualization kit for detecting fusarium on a layer is characterized by comprising the primer probe combination of claim 1, and further comprising a buffer solution, a connecting sequence and an LFD lateral flow immunity test strip.
  3. 3. The RCA visualization kit for detecting fusarium in a layer according to claim 2, wherein the linker sequence is a ZIP sequence as follows: 5’-FAM-CTTCAACGCTCCCGCTAT-3’,SEQ ID NO.4。
  4. 4. the fusarium-out RCA visualization kit of claim 2, wherein the buffer comprises a ligation buffer and an amplification buffer.
  5. 5. A method of detecting fusarium in a layer of RCA visualization kit of claim 2, comprising: And (3) using the primer probe combination, performing RCA reaction on nucleic acid of a sample to be detected in a buffer environment, and detecting a reaction product by adopting an LFD lateral flow immunity test strip.
  6. 6. The method for detecting fusarium in a RCA visualization kit according to claim 5, wherein the RCA reaction comprises a ligation reaction and an amplification reaction.
  7. 7. The method for detecting fusarium on the detection layer of the RCA visualization kit according to claim 6, wherein the temperature of the connection reaction is 55-65 ℃, the cycle number is 6-15, the temperature of the amplification reaction is 62-68 ℃, and the reaction time is 30-90 min.
  8. 8. The method of claim 5, wherein the temperature of the LFD lateral flow immunoassay strip is room temperature for 5min to 15min.

Description

Primer probe combination for detecting fusarium on detection layer, RCA (RCA visualization) kit and detection method Technical Field The invention belongs to the field of biological detection, and particularly relates to a primer probe combination for detecting fusarium, an RCA (RCA visual kit) and a detection method. Background Fusarium layering (Fusariumproliferatum) diseases reduce the yield and quality of various crops. The most abundant methods for detecting Fusarium bacteria in layers are still traditional morphological characterization methods. The method has the defects of low efficiency, poor timeliness and strong subjectivity. The prior art polymerase chain reaction PCR is one of the techniques for detecting a variety of pathogenic microorganisms, but in PCR, primers are consumed in each cycle. Thus, starting from one template DNA molecule, a maximum of 2 n copies are theoretically produced over n cycles. At very low concentrations of starting template, the amount of target product is very small in the early cycle and may be masked by background noise in the reaction system, non-specific activity of the enzyme or primer dimer. Even if eventually detected, a greater number of cycles is required and the reaction may have entered an inefficient stage before this time. There is a theoretical upper limit on the signal amplification per template molecule. In addition, PCR primers, especially when the sequence is not completely specific or annealing conditions are not optimized, may bind to non-target sequence portions, resulting in primer dimers or non-specific products. PCR primers/probes are typically very short, 20nt, and mutation of any one of the critical bases in their binding region can severely affect annealing efficiency and extension, resulting in a dramatic decrease in amplification efficiency or even complete failure. Therefore, the PCR primer has high efficiency, high timeliness and objectivity when performing PCR detection, but has low sensitivity. Disclosure of Invention In order to solve the technical problem of low detection sensitivity in the prior art, the invention provides a primer probe combination for detecting fusarium on a layer, an RCA (radar cross section) visualization kit and a detection method. A primer probe combination for detecting fusarium in a disease layer comprises sequences SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The RCA visualization kit for detecting fusarium comprises a primer probe combination, a buffer solution, a connecting sequence and an LFD lateral flow immunity test strip. Further, the linker sequence is a ZIP sequence as follows: 5’-FAM-CTTCAACGCTCCCGCTAT-3’,SEQ ID NO.4。 further, the buffers include ligation buffers and amplification buffers. The primer probe combination or the RCA visualization kit is applied to detecting fusarium on a layer. Further, the method for detecting fusarium by the RCA visualization kit detection layer is as follows: And (3) using the primer probe combination, performing RCA reaction on nucleic acid of a sample to be detected in a buffer environment, and detecting a reaction product by adopting an LFD lateral flow immunity test strip. Further, the RCA reaction includes a ligation reaction and an amplification reaction. Further, the temperature of the connection reaction is 55-65 ℃, the cycle times are 6-15 cycles, the temperature of the amplification reaction is 62-68 ℃, and the reaction time is 30-90 min. Further, the temperature for detection by adopting the LFD lateral flow immunity test strip is room temperature, and the time is 5 min-15 min. The principle of the invention is as follows: RCA is an isothermal amplification technology simulating viral nucleic acid and plasmid replication processes, and the core principle of rolling circle amplification can be summarized as that a special DNA polymerase is utilized, a circular DNA template is taken as a core, and a very long single-stranded DNA consisting of hundreds to thousands of repeated units is continuously replicated and synthesized like 'traveling around a circle'. This extra long DNA strand can be conveniently detected, indirectly evidencing the presence of the original target molecule. The technique relies mainly on a lock-probe PLP. PLP consists of approximately 100 bases, flanked by complementary mating regions for the target sequence, a junction sequence in the middle, amplified primer binding sites, and a zipcode sequence for hybridization. Linear padlock probes can be efficiently ligated into a circular form by the action of a ligase only if the PLP and the corresponding detection target DNA are present in the ligation system simultaneously and are perfectly complementary to each other. After entering the amplification reaction stage, the circular PLP is amplified by the action of polymerase and primers. Compared with the prior art, the invention has the following beneficial effects: 1. the invention provides a primer probe combination for detecting fusarium b