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RPA (recombinase polymerase amplification) detection primer, probe and detection method for bacterial fruit blotch of cucurbit

A technology for the detection of fruit spot bacteria and detection methods, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of low sensitivity, expensive reagents and instruments, and time-consuming, etc., to achieve high sensitivity, Good specificity and results visible to the naked eye

Active Publication Date: 2022-04-05
SHANGHAI ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Common detection methods for this bacterial disease include symptom identification, PCR detection, nested PCR detection, enzyme-linked immunosorbent assay, quantitative PCR (Qpcr) detection and other methods. These methods are either low in sensitivity, time-consuming, or expensive reagents and instruments

Method used

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  • RPA (recombinase polymerase amplification) detection primer, probe and detection method for bacterial fruit blotch of cucurbit
  • RPA (recombinase polymerase amplification) detection primer, probe and detection method for bacterial fruit blotch of cucurbit
  • RPA (recombinase polymerase amplification) detection primer, probe and detection method for bacterial fruit blotch of cucurbit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 designs and screens specific primer pair

[0033] (1) Design primers:

[0034] For the 651bp ORF specific sequence CP029373.1 (896040-896693) of cucurbit bacterial fruit spot bacterium, the specific nucleotide sequence is shown in SEQ ID NO.1, and 12 pairs of different RPA primers were designed, wherein the primers Set the TM value between 65-75°C and the amplification length between 150-294bp, and configure the alternative RPA primer pairs into solutions with a concentration of 10μM.

[0035] The specific sequences of the designed 13 pairs of different RPA primer pairs are shown in Table 1, from 5' to 3'.

[0036] Table 1

[0037]

[0038]

[0039] (2) The screening process is as follows:

[0040]Isolate and purify the bacterial fruit spot disease of melons, extract DNA according to the conventional method, and use the extracted DNA as a template for PRA detection. The RPA reaction system is: complex solution 29.5 μL, sterile deionized water 13.0 ...

Embodiment 2

[0043] The specificity test of embodiment 2 melon bacterial fruit spot pathogen RPA detection primer

[0044] The DNA templates were from reference bacteria: Pseudomonas syringae (bacterial leaf spot of melon), Erwinia carotovora (soft rot of Chinese cabbage), E.chrysanthemi (bottom rot of rice), Xanthomonasoryzae (bacterial leaf spot of rice), Microbacterium arborescens (Microbacterium dendriticum), Pectobacterium carotovorum subsp. brasiliense (cucumber stem rot fungus), Escherichia coli (Escherichia coli), Agrobacterium sp. (Agrobacterium).

[0045] Purify the melon bacterial fruit spot bacteria and each reference bacteria, configure the primer pair Ac-F1 and Ac-R1 respectively into a solution with a concentration of 10.0 μmol / L, extract the DNA according to the conventional method for use, and use the extracted DNA as a template for RPA Detection, wherein, the RPA reaction system is: complex solution 29.5 μL, sterile deionized water 13.0 μL, 10 μmol / L forward / reverse prime...

Embodiment 3

[0049] Embodiment 3 The sensitivity test of the detection primer of melon bacterial fruit spot pathogen RPA and the sensitivity test of test paper detection

[0050] 1) Purify the bacterial fruit spot pathogen of melons, and prepare 2.5 × 10 with sterile water after cultivation. 9 -2.5×10 1 cfu / mL bacterial suspension;

[0051] 2) According to the primer specificity results of examples 1 and 2, design and detect the probe in the amplified fragment of the primer pair Ac-F1 and Ac-R1, and modify it, name it Ac-nfo, and use the primer pair Ac-F1 and Ac-R1, probe Ac-nfo were respectively configured into a solution with a concentration of 10 μM;

[0052] 3) Use the bacterial suspension prepared in step 1) as a template for PRA detection. The RPA reaction system is: 29.5 μL of complex solution, 12.4 μL of sterile deionized water, 0.6 μL of 10 μmol / L Ac-nfo, 0.6 μL of 10 μmol / L Ac-F1 / Ac-R1 each 2.0 μL, 280 mmol / L magnesium acetate solution 2.5 μL, DNA template 1.0 μL, total volume...

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Abstract

The invention relates to an RPA detection primer, a probe and a detection method of cucurbit bacterial fruit blotch, the RPA detection primer comprises an RPA primer pair Ac-F1 and Ac-R1 of cucurbit bacterial fruit blotch and a detection probe Ac-nfo, the amplification product size of the RPA primer pair Ac-F1 and Ac-R1 is 342bp, the 5'terminal of the primer Ac-R1 is labeled with biotin, the 5 'terminal of the primer Ac-R1 is labeled with biotin, and the 5' terminal of the detection probe Ac-nfo is labeled with biotin. The 5'tail end of the detection probe Ac-nfo is marked with FAM, the 3 'tail end of the detection probe Ac-nfo is marked with C3Spacer, and the position, 32 bp away from the 5' tail end, of the probe is modified with dSpacer; the RPA primer group is high in detection sensitivity, and the detection limit reaches 2.5 * 10 < 4 > cfu / mL; the invention provides a detection method for detecting the melon bacterial fruit blotch based on RPA combined with lateral flow test paper, establishes a rapid isothermal visual detection method for the melon bacterial fruit blotch, can complete sample detection within 15-20 min, realizes rapid diagnosis, identification and detection of the melon bacterial fruit blotch in a constant-temperature environment, and has a wide application prospect. The kit has the advantages of high sensitivity, good specificity and the like, and provides technical support for quarantine, detection, prevention and control of the disease.

Description

technical field [0001] The invention belongs to the technical field of RPA detection, and in particular relates to a RPA detection primer, probe and detection method of melon bacterial fruit spot pathogen. Background technique [0002] Bacterial fruit blotch (BFB) of melons is caused by Acidovorax citrulli (A.citrulli for short), which seriously damages watermelon, melon and other Cucurbitaceous crops, and is an important bacterial quarantine disease. . The disease is a disease that can be transmitted by seeds. The leaves, fruits and seeds in the growth period can be infected, which greatly affects the yield and quality of fruits, and can cause devastating damage in severe cases. In the 1990s, it was first reported in my country, and then there were occasional reports in Xinjiang, Hainan, Shanghai, Zhejiang and other places. In the "National List of Agricultural Plant Quarantine Pests Occurring in Various Regions (2016)", my country has already had 12 occurred in provinces ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12N15/11C12Q1/6844
CPCY02A50/30
Inventor 曾蓉戴富明徐丽慧宋志伟高萍高士刚
Owner SHANGHAI ACAD OF AGRI SCI
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