Lamp primer, kit and detection method for rapid detection of apple scab

A detection method and kit technology are applied to LAMP primers, kits and detection fields for rapid detection of apple scab bacteria, which can solve the problems affecting the accuracy of detection results, no detection, false positives, etc., and achieve long detection time, Low accuracy, high specificity effects

Active Publication Date: 2022-08-05
QINGDAO AGRI UNIV
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AI Technical Summary

Problems solved by technology

[0005] Although the loop-mediated isothermal amplification technology has the above advantages, due to the high requirements for primer specificity, conditions and systems, this detection method is prone to false positives due to contamination problems and non-specific binding of loop primers. problem, which affects the accuracy of the detection results; and no LAMP primers have been found for the specific detection of apple scab

Method used

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  • Lamp primer, kit and detection method for rapid detection of apple scab
  • Lamp primer, kit and detection method for rapid detection of apple scab
  • Lamp primer, kit and detection method for rapid detection of apple scab

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Primer Design and Synthesis

[0058] According to the specificity of the β-tubulin gene sequence of Venturiainaequalis (the sequence is shown in SEQ ID No. 7 in the sequence listing), the LAMP primer online design software PrimerExplorer (http: / / primerexplorer.jp / lampv5e / index) was used. .html) designed a set of LAMP primers specific for apple scab bacteria and carried out sequence synthesis. The LAMP primers included outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. The primer sequences were : F3: 5'-GCTTCCGTCTACCTTCCTGT-3'; (SEQ ID No. 1)

[0059] B3: 5'-CCGGTCTGGAGATGAACCTA-3'; (SEQ ID No. 2)

[0060] FIP: 5'-AGTGGACGTACAATTTCGCGCA-ACTCCCGTACCCAATTCTCT-3'; (SEQ ID No. 3)

[0061] BIP: 5'-CGTCCCCTGAATCCTCTGCC-CCACGAAGTACACGTTAGCT-3'; (SEQ ID No. 4)

[0062] LF: 5'-TGTTGACGGTGGTGAGGT-3'; (SEQ ID No. 5)

[0063] LB: 5'-CCGCGTCCAGGCTTTTCA-3'. (SEQ ID No. 6)

[0064] The above-synthesized primers were diluted with sterile doub...

Embodiment 2

[0065] Example 2 LAMP detection of apple scab

[0066] 1. Extraction of genomic DNA of apple scab to be tested

[0067] Take an appropriate amount of the sample to be tested in 400 μL of DNA lysis buffer (0.15M Tris-HCl, 0.04M EDTA-Na 2 , 0.2M NaCl, 3mM SDS, PH=8.0), add a steel ball, shake at 65Hz for 1min, centrifuge at 12000rpm for 2min, take 200μL of supernatant, add 400μL of absolute ethanol, stand at -20°C for 30min, and centrifuge at 12000rpm After 2 min, the supernatant was discarded, air-dried, and 30 μL of sterile double-distilled water was added to dissolve the DNA to obtain a genomic DNA extract.

[0068] 2. Establishment of LAMP reaction detection system:

[0069] Take the genomic DNA of the sample to be tested extracted above as a template, and use primers F3, B3, FIP, BIP, LF and LB to carry out LAMP amplification. The LAMP detection reaction system is 26 μL as an example. The specific reaction system includes:

[0070] 10 μM F3: 0.5 μL

[0071] 10 μM B3: 0....

Embodiment 3

[0088] Example 3 Specificity test of LAMP primers

[0089] A LAMP reaction detection system was established according to the method of Example 2, and successive tests were performed for Venturiainaequalis, Marssonina coronaria, Valsamali, Botryosphaeria dothidea, and anthracnose ( Colletotrichumgloeosporioides), Alternaria mali and Sclerotiumrolfsii were tested by LAMP, numbered A1-A7 respectively. Simultaneously, synchronous experiments were performed with sterile double distilled water as the negative control template. .

[0090] Test result: as figure 1 As shown, only the reaction product in the reaction tube of sample A1 (apple scab) is sky blue, and the electrophoresis result is a trapezoidal band, and the test shows a positive result; the test results of the other 6 samples and the negative control are all negative, The product in the reaction tube is purple. The results show that: the three pairs of primers provided by the present invention have high specificity for ...

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Abstract

The invention discloses a LAMP primer, a kit and a detection method for rapid detection of apple scab. The LAMP primer consists of a pair of outer primers F3 and B3, a pair of inner primers FIP and BIP, and a pair of loop primers LF and LB. The outer primer F3 is shown in SEQ ID No.1, the outer primer B3 is shown in SEQ ID No.2, the inner primer FIP is shown in SEQ ID No.3, and the inner primer BIP is shown in SEQ ID No.4, The loop primer LF is shown in SEQ ID No.5, and the loop primer LB is shown in SEQ ID No.6. The rapid detection of apple scab can be realized by using the above three pairs of specific LAMP primers to perform LAMP amplification on the sample to be tested. The early diagnosis of apple scab and the detection and identification of pathogenic bacteria are of great significance to the timely and effective prevention and control of apple scab.

Description

technical field [0001] The invention relates to the technical field of detection methods for apple scab, in particular to a LAMP primer, a kit and a detection method for rapid detection of apple scab. Background technique [0002] Apple scab, also known as apple scab, caused by Venturiainaequalis, is one of the important diseases in apple producing areas around the world. The disease mainly harms apple leaves and fruits, and can also infect petioles, flowers, sepals, pedicels, young shoots and bud scales, etc. In severe cases, it will cause defoliation, fruit drop, cracking and deformity of the affected fruits, and directly affect the yield and quality of apples. Apple scab has the characteristics of fast epidemic speed, great harm and difficult to control. In recent years, due to the extensive orchard management model, the occurrence of apple scab in the main apple producing areas of my country is on the rise. Apple scab overwintered on bud scales or diseased leaves with ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2531/119
Inventor 任维超李保华
Owner QINGDAO AGRI UNIV
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