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Microdroplet type digital PCR (Polymerase Chain Reaction) kit for detecting fusarium solani

A technology of Fusarium solani and a detection kit, which is applied to the determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., and can solve the lack of uniformity in the quality control of DNA extraction methods and the detection limit value of detection equipment Standards, detection sensitivity, precision limitations, lack of detection implementation standards, etc., to achieve the effects of good result stability, high microbial diversity, and easy operation

Pending Publication Date: 2022-06-21
JILIN AGRICULTURAL UNIV
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AI Technical Summary

Problems solved by technology

However, due to the lack of uniform testing implementation standards among PCR platforms in different laboratories, there is a lack of unified standards for sample types, DNA extraction methods, DNA fragments detected, quality control, testing equipment, detection limit values, etc., resulting in the same sample being used in different laboratories. There are large differences in the test results of different laboratories, and the experimental results cannot be repeated, and even the results are contradictory.
And relying on the Cq value is still the biggest technical bottleneck of fluorescent quantitative PCR at present. In this sense, the so-called "quantitative" is only relative, and under the conditions of low copy target molecules and slight differences in template concentration, its detection sensitivity, Accuracy is limited

Method used

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  • Microdroplet type digital PCR (Polymerase Chain Reaction) kit for detecting fusarium solani
  • Microdroplet type digital PCR (Polymerase Chain Reaction) kit for detecting fusarium solani
  • Microdroplet type digital PCR (Polymerase Chain Reaction) kit for detecting fusarium solani

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: Identification of pathogenic bacteria of ginseng root rot

[0055] 1.1 Morphological identification of pathogenic bacteria of ginseng root rot

[0056] Culture medium was used to isolate and purify the fungus carried by ginseng root rot diseased strains from different production areas. The pathogenic strains isolated and purified from soil samples were inoculated on PDA plates, cultured at 25±3°C for about 3 days, and their morphological characteristics were observed. The result is as figure 1 shown. Depend on figure 1 It can be seen that the mycelium is flocculent, and the back side is light yellow. The hyphae of Fusarium solani strains are white, the large conidia of the pathogenic strain are sickle-shaped or cylindrical, and the small conidia are oval. Strain identification was carried out by observing the colony morphology, hyphae and spore morphological characteristics of the strain according to the "Fungi Identification Manual". The pathogen was...

Embodiment 2

[0063] Example 2. Establishment of Fusarium solani molecular detection method

[0064] 2.1 Design of specific primers for ginseng root rot

[0065] The sequencing results of Fusarium solani in Example 1 were compared with the gene of Fusarium solani ITS and its related species sequences in the GenBank database for multiple homology comparisons, and based on the comparison results, sequence difference sites were selected to design specificity Primers and probes (3 pairs), the 5' end of the probe is labeled with the FAM fluorescent signal group, and the 3' end is labeled with the fluorescent quencher group BHQ1. NCBI-Blast was used to preliminarily verify the theoretical specificity of the primers. The primers and probes were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The laboratory finally confirmed that primer 1 (as shown in Table 2) had good specificity, and primer 2 or 3 had good specificity. Expansion of closely related species.

[0066] Table 2 Specific prim...

Embodiment 3

[0090] Embodiment 3. The dynamic detection of the quantity of pathogenic bacteria in the rhizosphere soil of suffering from ginseng

[0091] Ginseng root rot is a fungal disease that mainly harms the roots of 3-6-year-old ginseng. Both soil and seedlings with bacteria can transmit the disease. The droplet digital PCR method established in 2.3 of Example 2 was used to study the quantitative dynamics of Fusarium solani in soil. Three-year-old ginseng seedlings were transplanted into flowerpots (Φ=23.5cm), and 3 plants per pot were placed in the shade shed of the medicine planting garden of Jilin Agricultural University, and the rest of the field management was the same as that of the field. After the ginseng seedlings were adapted to the potted environment, ginseng with the same size was selected for the experiment.

[0092] Inducing group: collect the mycelium of Fusarium solani cultured by PDA, shake and filter to make spore suspension, and adjust the concentration to 3×10 4...

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Abstract

The invention discloses a microdroplet type digital PCR (Polymerase Chain Reaction) kit for detecting fusarium solani. The kit comprises an amplification primer and a probe of a target fragment, the sequence of the amplification primer of the target fragment is as shown in SEQ ID No.3 and SEQ ID No.4, and the sequence of the probe of the target fragment is as shown in SEQ ID No.5. The kit provided by the invention has high sensitivity, high accuracy and high precision, can be used for distinguishing main fungal diseases (inter-family) of ginseng under the background of complex soil and plant microorganisms, and can also realize good distinguishing of fusarium solani and sibling species, and the specific identification capability of the kit can reach the inter-species level. Therefore, early prevention and control of diseases are facilitated.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a droplet digital PCR kit for detecting root rot pathogen Fusarium solani. Background technique [0002] The supply of ginseng medicinal materials in my country mainly depends on intensive artificial planting, and ginseng planted in farmland is often accompanied by a variety of fungal diseases, which is also an important reason for the reduction in yield and quality of cultivated ginseng. Among them, the root disease has a high incidence rate, is difficult to detect, is easy to infect, and is harmful, and the yield reduction rate reaches more than 80%. Fusarium solani is the main pathogen of plant root rot. At present, disease monitoring and detection has become an important link in the ginseng cultivation process. Effective disease detection methods and early warning systems can not only prevent the development and spread of diseases in time, but also have great significance for re...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6851C12Q1/06C12N15/11
CPCC12Q1/6895C12Q1/6851C12Q2600/166C12Q2563/159C12Q2563/107C12Q2545/113
Inventor 杨莉王春雨刘翠晶林红梅聂丹丹于俐杜立财杨利民
Owner JILIN AGRICULTURAL UNIV
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