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Primer and probe combination for detecting candidatus Liberibacter asiaticus and detection method

A technology of citrus huanglong and primer probes, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve problems such as long incubation period and uncontrollable

Inactive Publication Date: 2017-11-17
CHINESE ACAD OF INSPECTION & QUARANTINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The length of the incubation period varies depending on the species, tree age, health status, planting environment, etc., but compared with other pathogens, the incubation period of Huanglongbing is longer
And there is currently no resource of genetic resistance in citrus, once the tree is infected, it is impossible to control

Method used

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  • Primer and probe combination for detecting candidatus Liberibacter asiaticus and detection method
  • Primer and probe combination for detecting candidatus Liberibacter asiaticus and detection method
  • Primer and probe combination for detecting candidatus Liberibacter asiaticus and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050]Example 1, Design and synthesis of RPA primers and probes for identification of Huanglongbing citrus

[0051] According to the sequence of the 16S ITS region of H. citriensis Asiatica, multiple sets of theoretically feasible RPA primers (including three upstream primers and three downstream primers) were designed using primer design software, as shown in Table 1.

[0052] Table 1 RPA candidate primers and probe sequences (5'-3')

[0053]

[0054] Different combinations of upstream primers and primers (F1 / R1, F1 / R2, F1 / R3, F2 / R1, F2 / R2, F2R3) in Table 1 were used for effect verification, sensitivity analysis, and specificity analysis (see the implementation for specific operation methods) Example 2-4), the result is as follows figure 1 As shown, F2 / R1 has the best sensitivity and amplification efficiency. Using F2 / R1 to detect citrus chlorophyll DNA, phytoplasma phytoplasma and phytoplasma papaya microphylla did not produce positive results, which met the specificity...

Embodiment 2

[0062] Embodiment 2, establishment of RPA detection method

[0063] 1. Use the magnetic bead method plant genomic DNA extraction kit (Tiangen Biochemical Technology DP342) to extract the total DNA of the citrus plant leaf veins to be tested. The specific operations are as follows:

[0064] Take about 100 mg of the leaf vein or root bark tissue of the citrus plant to be tested, add liquid nitrogen and grind it thoroughly. Quickly transfer the ground powder to a centrifuge tube pre-filled with 400 microliters of buffer GPM and 5 microliters of RNase A (10mg / ml), quickly invert and mix well, then place the centrifuge tube in a 70°C water bath for 10 minutes, Invert the centrifuge tube several times during the water bath to mix the sample. Centrifuge at 12,000 rpm (~13,400×g) for 4 minutes, and transfer 300 microliters of the supernatant to a new centrifuge tube. Add 300 μl buffer GHB, 300 μl isopropanol and 15 μl magnetic bead suspension G, shake and mix for 5 minutes. Place t...

Embodiment 3

[0070] Embodiment 3, the specificity of RPA detection method

[0071] Samples for testing: citrus plants infected with citrus H. asiatica; citrus plants not infected with citrus H. chinensis; bamboo plants infected with Phytoplasma chinensis; Papaya plants infected with Phytoplasma papaya .

[0072] The detection method established in Example 2 was used to detect the RPA of each sample.

[0073] The result is as figure 2 shown. figure 2 Among them, curves 1-3 are RPA real-time fluorescence curves obtained by template amplification of leaf vein DNA extracted from three citrus Huanglongbing bacteria-infected citrus plants, respectively. Curve 4 is the DNA of uninfected citrus leaves, curve 5 is the DNA of leaves infected with Phytoplasma papaya, and curve 6 is the DNA of leaves infected with Phytoplasma papaya.

[0074] The results showed that only the vein DNA samples (curves 1, 2, 3) of plants that had been infected with citrus huanglongbing had fluorescent signals to ri...

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Abstract

The invention discloses a primer and probe combination for quickly detecting candidatus Liberibacter asiaticus and a detection method. A primer pair for detecting candidatus Liberibacter asiaticus provided by the invention is composed of a) or b): a) a primer pair composed of two single-stranded DNA molecules shown in sequence 1 and sequence 2; and b) a prime pair which is composed of two single-stranded DNA molecules shown in sequences 1 and 2 substituted by one or more nucleotides and / or deleted and / or added and is as same as the primer pair in function. The probe provided by the invention is as follows from 5' end to 3' end: sequence 3-(dT-fluorophore)-tetrahydrofuran residual radical-(dT-quenching radical)-sequence 4; modification is performed on the 3' end of the probe. The whole detection process is within 1h, and the primer and probe combination which has the characteristics of being quick, specific and sensitive can meet quick primary inspection of field and forestry citrus samples and quick identification of bacterial DNA.

Description

technical field [0001] The invention belongs to the technical field of agriculture and plant quarantine, and relates to a combination of primers and probes and a detection method for detecting citrus Huanglongbing bacteria. Background technique [0002] Citrus Huanglongbing (HLB), also known as yellow tip disease, yellow wilt, green fruit disease, was first reported in South China in 1919, caused by a Gram-negative bacterium (Candidatus liberobacter asiaticum, Las) can infect a variety of Rutaceae plants, including Citrus, Citrus, Kumquat and Murata. Citrus huanglongbing is a devastating disease of citrus production in the world. The disease is mainly distributed in nearly 50 countries and regions in Asia, Africa, Oceania, South America and North America. Typical symptoms of infected fruit are abnormal colouration, small, misshapen fruit. The fruits of broad-skinned citrus such as Shatangju often appear to fade to green and turn color around the fruit pedicle and shoulder,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2521/507C12Q2563/107
Inventor 雷荣杨毅孔君陈乃中朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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