A specific detection target of Phytophthora syringae psyri_s00018g00015.1 and its application

A Phytophthora syringae, specific technology, applied in the application, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve the problems of long cycle, poor specificity and low sensitivity of detection methods, and achieve fast and accurate detection. high sex effect

Active Publication Date: 2022-07-12
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the problems in the prior art that the biological detection method of Phytophthora syringae requires a long cycle, the detection method has poor specificity and low sensitivity, the purpose of the present invention is to provide a new detection target for Phytophthora syringae Psyri_s00018g00015.1 RPA-LFD detection primer composition established by its target

Method used

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  • A specific detection target of Phytophthora syringae psyri_s00018g00015.1 and its application
  • A specific detection target of Phytophthora syringae psyri_s00018g00015.1 and its application
  • A specific detection target of Phytophthora syringae psyri_s00018g00015.1 and its application

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Experimental program
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Effect test

Embodiment 1

[0040] More than 1,100 Phytophthora syringae-specific genes were obtained by whole-genome sequence alignment, and some genes were randomly selected from more than 1,000 Phytophthora syringae specific genes as candidate genes ( Psyri_s00018g00015.1, Psyri_ s00001g00003.1, Psyri_s00001g00023.1Psyri_s00016g00009.1, Psyri_ s00024g00013.1 ), designed and screened specific primers, and Table 1 lists the five target genes and upstream and downstream detection primers (Table 1).

[0041] Table 1 Sequences and primers of five specific genes of Phytophthora syringae

[0042]

[0043] Select different species from Phytophthora syringae (Phytophthora cedar; .

[0044] Sample detection: To a 0.2 mL TwistAmp reaction unit tube (TwistAmp Basickits, Twist) containing lyophilized enzyme powder, add Buffer 29.5 μL, 10 μM upstream primer 2.1 μL, 10 μM downstream primer 2.1 μL, probe 0.6 μL, Add 2.0 μL of DNA and 2.5 μL of MgAc to the inside of the PCR tube lid, and make up to 50 μL of ...

Embodiment 2

[0050] To further verify the specificity of the RPA lateral flow chromatography test strip detection method, Phytophthora syringae strains and other Phytophthora and pathogenic bacteria were used as test materials (Table 2). The results of the RPA lateral flow chromatography test strip detection method showed that the The test strips for Phytophthora show two brown bands, one in the Control Line and the other in the Test Line, then the result is positive, while the test strips for other Phytophthora and pathogenic bacteria only have quality control A brown band appears in the test area, and there is no band in the test area (TestLine), the result is negative, indicating that the sample does not contain Phytophthora syringae.

[0051] Select different species from Phytophthora syringae (Phytophthora cedar; Fusarium; anthracnose flathead; Verticillium dahliae; Rhizoctonia solani; M. oryzae, etc.) DNA was used as a template for RPA-LFD (lateral flow chromatography test strip dete...

Embodiment 3

[0057]The genomic DNA of the standard strain of Phytophthora syringae with the same concentration was used as the amplification template, and the upstream and downstream primers Psyri015.1F / Psyri015.1R were used for PCR amplification reaction to compare the sensitivity of the two methods; repeat the experiment 3 times to determine the PCR Sensitivity of Phytophthora syringae genomic DNA detection by the method; the results of three repeated experiments were consistent. The genomic DNA concentration is 1 ng μL -1 , PCR detection can detect specific bands, and the results are as follows Figure 5 As shown, it was proved that specific amplification occurred, and the test result was judged to be positive. After utilizing the RPA amplification reaction of the present invention, the detection result of the LFD lateral flow chromatography test strip is as follows: Image 6 The indicated target bands are resolved. It can be seen that based on the same new excavation target Psyri_...

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Abstract

The invention discloses a new detection target of Phytophthora syringae, Psyri_s00018g00015.1, a detection primer, a detection kit and a detection method. The protein sequence of the detection target Psyri_s00018g00015.1 is shown in SEQ ID NO: 1, The DNA sequence encoding this protein is shown in SEQ ID NO:2. At the same time, the present invention also discloses the specific primer and probe combination of the RPA detection technology for the specific detection target Psyri_s00018g00015.1, the forward primer sequence is as shown in SEQ ID NO: 3, and the reverse primer sequence is as shown in SEQ ID NO: 4, the probe sequence is shown in SEQ ID NO:5. The invention discovers a new detection target of Phytophthora syringae, provides a new detection method for the detection of Phytophthora syringae, and at the same time, the RPA-LFD detection primers and probes developed based on the target can realize the specific detection of Phytophthora syringae, high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of gene detection, in particular to a specific detection target of Phytophthora syringae Psyri_ s00018g00015.1 and its applications. Background technique [0002] Phytophthora ( Phytophthora ) There are more than 100 species officially reported, and many Phytophthora species have records that seriously endanger the safety of agricultural production. It can be said that each of them is the destroyer of crops, causing shortage of seedlings and monarchy at light, serious yield loss and even extinction. Produce. Therefore, it is of great significance to prevent foreign harmful quarantine Phytophthora from being introduced into my country. Phytophthora lilac ( P. syringae ) is a pathogen of the genus Phytophthora, which can cause symptoms such as root rot, sticky sticks and other symptoms on host plants, and cause brown rot of citrus fruits. At present, the pathogen is widely distributed in all citrus-ri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/31C12N15/11C07K14/37
CPCC12Q1/6895C12Q1/6844C07K14/37C12Q2531/119C12Q2565/625Y02A50/30
Inventor 戴婷婷周紫薇焦彬彬俞萱徐洁莹
Owner NANJING FORESTRY UNIV
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