Kit for rapidly diagnosing peach bacterial shot hole disease

A kit, peach and plum technology, applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of diagnostic errors, cumbersome operation, inapplicability, etc., and meet the requirements of low and high detection equipment. The effect of amplification efficiency and fast extraction speed

Pending Publication Date: 2021-08-10
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

The traditional morphological identification method requires practitioners to have solid professional theoretical knowledge of plant pathology and rich experience in disease identification. The identification cycle is long and the operation is cumbersome, and it is easy to be affected by different varieties, different tissues, different growth stages, disease symptoms or subjective symptoms. Cognition and other influences cause diagnostic errors, which are not applicable in field testing and production practice
Most of the existing molecular identification methods, such as conventional PCR, real-time fluorescence quantitative PCR, Bio-PCR, LAMP, etc., require cyclers or water baths, which cannot truly realize real-time detection in the field. Application space is limited
At present, the detection methods related to PCR technology ba

Method used

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  • Kit for rapidly diagnosing peach bacterial shot hole disease
  • Kit for rapidly diagnosing peach bacterial shot hole disease
  • Kit for rapidly diagnosing peach bacterial shot hole disease

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0049] Example 1 Screening of XAP RPA Detection Primers

[0050] When collecting peach bacterial perforations from all major in my country, it is often separated from Pantoea Sp. Bacteria, which is much larger than XAP, and is shown in Table 1, two. People often appear together, similar to morphology, both yellow, bulge, smooth round colonies, it is difficult to distinguish. However, after a series of identification and Kakhwhwide law verified, Pantoea sp. It is not a pathogenic bacteria, but it has caused great troubles to diagnose bacterial perforation.

[0051] Table 1 peach bacterial perforated disease pattern separation strain

[0052]

[0053]

[0054] The CRISPR / Cas12A spectrophotomy identifies a T nucleotide (5'-TTTV-3 ') in the present invention. Based on this, 3 pairs of RPA primers are designed according to the FTSX target gene sequence, and each primer targeting sequence is rich in one to more PAM sites (Table 2), the primer is synthesized by Wuhan Tianyi Huiyuan...

Example Embodiment

[0060] Example 2 Optimization of RPA Reaction Parameters

[0061] 1. Optimization of the reaction time: The incubation temperature was maintained at 37 ° C, and the reaction time was 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, respectively, RPA reaction (Example 1). The results showed that the XAP strain can be amplified from 5 min to 30 min, and the amplification efficiency is gradually enhanced. When 15 minutes, the saturation is reached, so it is selected as the optimal reaction time (see figure 1 B picture).

[0062] 2. Optimization of the reaction temperature: set 30 ° C; 33 ° C; 35 ° C; 37 ° C; 39 ° C; 42 ° C Total 6 temperature gradient, the reaction time is an optimum value of 15 min, respectively, RPA reaction (Example 1) . The results indicate that the six temperature gradients set can be successfully activated, and the amplification efficiency continuously increases with temperature, 37 ° C can achieve high amplification efficiency, select 37 ° C as a subsequent reac...

Example Embodiment

[0063] Example 3 Screening of CrRNA by XAP Bacteria RPA-Cas12A

[0064] The PAM site contained in the targeting sequence contained in the optimal RPA1F / 1R primer is designed and synthesized from 3 crrnas, and the CrRNA guidance sequence is shown in Table 3. The DSDNA substrate exotic reactions and RPA-Cas12 preliminary fluorescence detection were verified to screen DSDNA and SSDNA cleavage activity for DSDNA and SSDNA.

[0065] DSDNA substrate outer cleavage reaction system:

[0066]1.DSDNA substrate synthesis (FTSX gene): XAPY17-F and XAPY17-R were used using primers. PCR amplification system (total length is 25 μl): 2 × HIEFF TM PCR MASTER MIX (Yeasen, Shanghai) 12.5 μl, PRIMER F (upstream primer) 1 μl, PRIMER R (downstream primer) 1 μl, DNA template 1 μl, DD H 2 O 9.5 microliters. PCR reaction procedure: 94 ° C predetermitability 4min; 94 ° C degeneration 30s, 55 ° C until 30s, 72 ° C, 35 cycles; 72 ° C. The PCR product was detected by 1% agarose gel electrophoresis. After co...

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Abstract

The invention relates to a kit for rapidly diagnosing peach bacterial shot hole, and belongs to the technical field of plant protection. The kit comprises an RPA primer pair, a specific crRNA and a single-stranded DNA probe; the sequence of the RPA primer pair is as shown in SEQ ID NO.1-2, the sequence of the specific crRNA is as shown in SEQ ID NO.3, and the sequence of the single-stranded DNA probe is as shown in SEQ ID NO.4 and/or SEQ ID NO.5. The kit is used for detecting the main pathogenic bacteria Xap of the peach bacterial shot-hole disease on the basis of an RPA technology and a Crispr/Cas system, visual detection is realized through a transverse flow detection test strip and a miniature ultraviolet flashlight, the kit is simple and flexible, low in cost, high in sensitivity and high in specificity, a user can freely select the kit, the problem that the kit cannot be used in some scenes is effectively solved, and the method has a good application prospect in the field of peach disease diagnosis and monitoring.

Description

technical field [0001] The invention relates to a kit for rapidly diagnosing peach bacterial perforation disease, which belongs to the field of combined use of plant protection and RPA-Crispr / Cas technology. Background technique [0002] In recent years, peach bacterial perforation disease caused by Xanthomonas arboricola pv.pruni (also known as Xap) is one of the main diseases with high incidence and serious damage on peach trees. In the early stage of the disease, small water-soaked gray-brown spots formed on the fruit surface and leaves, which continued to extend and aggravate in the later stage, forming large-scale perforation or cracking. Anthracnose and other symptoms are confused, making it difficult for grassroots agricultural technology extension personnel and fruit farmers to distinguish, and bringing technical difficulties to accurate diagnosis. In addition, the pathogen will quickly transfer and colonize new tissues after causing leaf necrosis, and the survival ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/64
CPCC12Q1/689C12Q1/6844
Inventor 罗朝喜罗梅孟凡珠周扬曾哲政尹良芬阴伟晓王丽谭钦
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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