LAMP primer composition and detection method for detecting ganoderma pseudoferreum

Abstract

The present invention discloses a LAMP primer composition and a detection method for detecting ganoderma pseudoferreum. The LAMP primer composition for detecting the ganoderma pseudoferreum consists of DNA shown in SEQ ID NO:1 of a sequence Listing, DNA shown in SEQ ID NO:2 of the sequence listing, DNA shown in SEQ ID NO:3 of the sequence listing and DNA shown in SEQ ID NO:4 of the sequence listing. In the established visual detection method for the ganoderma pseudoferreum based on the LAMP primer composition, green fluorescence or trapezoidal bands showing LAMP characteristics can be observedby a chromogenic reaction or an agarose gel electrophoresis detection after LAMP constant temperature amplification. The LAMP primer composition and the detection method can realize the rapid, sensitive and accurate detection and identification of the ganoderma pseudoferreum in the fields, provide the rapid, simple and low-cost detection technology for scientific research and production practice,and provide reliable technical and theoretical basis for early diagnosis and prevention and control of rubber tree red root diseases caused by the ganoderma pseudoferreum in agricultural production.

Description

technical field

[0001] The invention relates to the field of crop disease detection and identification technology and molecular biology technology, in particular to a LAMP primer composition, kit and detection method for detecting Ganoderma pseudoferreum, which can realize the disease caused by the infection of Ganoderma pseudoferreum. Early diagnosis of rubber red root disease, detection and identification of pathogens. Background technique

[0002] Red root disease is the most widespread and most influential worldwide root infectious disease on rubber trees. It is mainly distributed in tropical areas such as Southeast Asia from Indonesia to Papua New Guinea and New Caledonia, Malaysia, the Philippines, and Cote d'Ivoire. It generally occurs in all rubber planting areas in China, and the incidence rate of severe forest segments reaches 40%. If it is not prevented and controlled in time, the mortality rate is 100%.

[0003] The damage part of rubber red root disease is the...

Examples

Embodiment 1

[0048] Embodiment 1: the design of the LAMP detection primer composition of Ganoderma pseudoferreum

[0049] According to the mitochondrial Large rDNA sequence of the Ganoderma pseudoferreum strain that causes rubber tree red root disease obtained in our laboratory and other genera or species of Ganoderma lucidum downloaded from the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) The large rDNA homologous sequence of the mitochondrial large rDNA of the fungus was compared multiple times on the MultAlin website to obtain the polymorphic (specific) region of the ganoderma lucidum rDNA sequence, and then use the online software primer explorer4.0 (http: / / primerexplorer.jp / ) to perform primers Design, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP), such as figure 1 Shown, its nucleotide sequence is respectively:

[0050] F3: 5'-TCGGAATGACATAAATTGATGA-3' (sequence 1 in the sequence listing);

[0051] B3: 5'-GGTGTAATATTTCTATTGTTTAAGGC-3' (sequence 2 in the seq...

Embodiment 2

[0054] Example 2. Establishment of a visual detection method for Ganoderma pesudoferreum LAMP.

[0055] 1. Establishment of LAMP amplification system

[0056] The primer composition obtained in Example 1 was used to prepare the obtained primer composition, and the LAMP amplification was carried out using the DNA of Hevea rhizome strain DNA as a template. The LAMP amplification system is 25 μL, including 2 μL each of 2.5 μmol / L F3 and B3; 1.5 μL each of 20 μmol / L FIP and BIP; 3.5 μL of 10 mmol / L dNTPs; 25 mmol / L MgSO 4 4 μL; 10mol / L betaine 2 μL; 10×Bst-DNA polymerase buffer 2.5 μL; 8U Bst 3.0 DNA polymerase 1 μL; 10~100ng / μL DNA template 1 μL; ddH 2 O 4 μL.

[0057] The above 25 μL LAMP reaction system was quickly placed at different temperatures (57°C, 58°C, 59°C, 60°C, 61°C, 63°C, 64°C, 65°C) for LAMP reaction, and after 1 hour of reaction, it was extinguished at 85°C. After 8 min, the reaction was terminated.

[0058] 2. Judgment of LAMP reaction result

[0059] Three...

Embodiment 3

[0061] Embodiment 3, Ganoderma pseudoferreum (Ganoderma pseudoferreum) LAMP reaction specificity detection

[0062] In this experiment, 4 strains of Ganoderma pseudoferreum from different sources and 11 strains of other pathogenic bacteria were used. Other pathogens include: 3 strains of Phellinus noxius, Rigidoporus lignosus, and purple root of rubber tree. Helicobasidium compactum, Colletotrichum gloeosporioides, Mango anthracnose (Collectotrichungloeosporioides), Stylized anthracnose (Collectotrichung gloeosporioides), Sisal zebra nicotianae (Phytophora nicotianae), Rubber tree stinkroot (Sphaerostilbe repens), Rubber tree Powdery mildew bacteria (Oidium heveae Steinm) each 1 strain uses fungal DNA extraction kit (Fungal DNA Kit, U.S. Omega company) to extract the DNA of above-mentioned bacterial strain as template, utilizes the reaction system that the primer set that obtains prepares in embodiment 1 and embodiment 2 establishes, LAMP amplification was performed separately...

technical field

[0001] The invention relates to the field of crop disease detection and identification technology and molecular biology technology, in particular to a LAMP primer composition, kit and detection method for detecting Ganoderma pseudoferreum, which can realize the disease caused by the infection of Ganoderma pseudoferreum. Early diagnosis of rubber red root disease, detection and identification of pathogens. Background technique

[0002] Red root disease is the most widespread and most influential worldwide root infectious disease on rubber trees. It is mainly distributed in tropical areas such as Southeast Asia from Indonesia to Papua New Guinea and New Caledonia, Malaysia, the Philippines, and Cote d'Ivoire. It generally occurs in all rubber planting areas in China, and the incidence rate of severe forest segments reaches 40%. If it is not prevented and controlled in time, the mortality rate is 100%.

[0003] The damage part of rubber red root disease is the...

Examples

Embodiment 1

[0048] Embodiment 1: the design of the LAMP detection primer composition of Ganoderma pseudoferreum

[0049] According to the mitochondrial Large rDNA sequence of the Ganoderma pseudoferreum strain that causes rubber tree red root disease obtained in our laboratory and other genera or species of Ganoderma lucidum downloaded from the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) The large rDNA homologous sequence of the mitochondrial large rDNA of the fungus was compared multiple times on the MultAlin website to obtain the polymorphic (specific) region of the ganoderma lucidum rDNA sequence, and then use the online software primer explorer4.0 (http: / / primerexplorer.jp / ) to perform primers Design, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP), such as figure 1 Shown, its nucleotide sequence is respectively:

[0050] F3: 5'-TCGGAATGACATAAATTGATGA-3' (sequence 1 in the sequence listing);

[0051] B3: 5'-GGTGTAATATTTCTATTGTTTAAGGC-3' (sequence 2 in the seq...

Embodiment 2

[0054] Example 2. Establishment of a visual detection method for Ganoderma pesudoferreum LAMP.

[0055] 1. Establishment of LAMP amplification system

[0056] The primer composition obtained in Example 1 was used to prepare the obtained primer composition, and the LAMP amplification was carried out using the DNA of Hevea rhizome strain DNA as a template. The LAMP amplification system is 25 μL, including 2 μL each of 2.5 μmol / L F3 and B3; 1.5 μL each of 20 μmol / L FIP and BIP; 3.5 μL of 10 mmol / L dNTPs; 25 mmol / L MgSO 4 4 μL; 10mol / L betaine 2 μL; 10×Bst-DNA polymerase buffer 2.5 μL; 8U Bst 3.0 DNA polymerase 1 μL; 10~100ng / μL DNA template 1 μL; ddH 2 O 4 μL.

[0057] The above 25 μL LAMP reaction system was quickly placed at different temperatures (57°C, 58°C, 59°C, 60°C, 61°C, 63°C, 64°C, 65°C) for LAMP reaction, and after 1 hour of reaction, it was extinguished at 85°C. After 8 min, the reaction was terminated.

[0058] 2. Judgment of LAMP reaction result

[0059] Three...

Embodiment 3

[0061] Embodiment 3, Ganoderma pseudoferreum (Ganoderma pseudoferreum) LAMP reaction specificity detection

[0062] In this experiment, 4 strains of Ganoderma pseudoferreum from different sources and 11 strains of other pathogenic bacteria were used. Other pathogens include: 3 strains of Phellinus noxius, Rigidoporus lignosus, and purple root of rubber tree. Helicobasidium compactum, Colletotrichum gloeosporioides, Mango anthracnose (Collectotrichungloeosporioides), Stylized anthracnose (Collectotrichung gloeosporioides), Sisal zebra nicotianae (Phytophora nicotianae), Rubber tree stinkroot (Sphaerostilbe repens), Rubber tree Powdery mildew bacteria (Oidium heveae Steinm) each 1 strain uses fungal DNA extraction kit (Fungal DNA Kit, U.S. Omega company) to extract the DNA of above-mentioned bacterial strain as template, utilizes the reaction system that the primer set that obtains prepares in embodiment 1 and embodiment 2 establishes, LAMP amplification was performed separately...

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