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Primer, probe and method for real-time fluorescence polymerase chain reaction (PCR) detection of pear decline phytoplasma

A real-time fluorescence and phytoplasma technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of low detection sensitivity and complicated detection methods, achieve rapid methods and improve quarantine technology Level, the effect of improving the detection rate

Inactive Publication Date: 2011-01-19
王有福 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For a long time, the detection of pear decay phytoplasma has been detected by immunoelectron microscopy. The detection method is complicated and the detection sensitivity is low. These problems have always plagued the quarantine personnel. Therefore, a set of fast, accurate and easy-to-operate methods can be developed to meet the needs of quarantine work.

Method used

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  • Primer, probe and method for real-time fluorescence polymerase chain reaction (PCR) detection of pear decline phytoplasma
  • Primer, probe and method for real-time fluorescence polymerase chain reaction (PCR) detection of pear decline phytoplasma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Probe (LST Probe): 5'-(FAM) ATTCTGACTGTA-3'

[0040] (2) Primer (LST Primer-F): 5'-ACTCTGACCGAGCAACGCC -3'

[0041] (LST Primer-R): 5’-GATAACGCTTGCCCCCTATG-3’

[0042] Wherein: RNA bases are in the box.

[0043] Uniformity and Stability Test

[0044] 1. Specificity test

[0045] 8 phytoplasma and 5 kinds of bacteria of different genera (see attached table 1) were used to verify the specificity of LST Probe (the appropriate type and quantity of phytoplasma and bacteria can be selected according to the actual situation), and its real-time fluorescent PCR reaction system is :

[0046] 2×Cycleave PCR Reaction Mix

12.5 μl

Cycleave Primer Mix (10uM)

1 μl

Cycleave Probe (3uM)

1 μl

dna

1 μl

dH 2 o

9.5 μl

Total

25 μl

[0047] The PCR reaction conditions are: pre-denaturation at 95°C for 10 sec; denaturation at 95°C for 5 sec, annealing at 55°C for 10 sec, extension at 72°C for 15 sec, and 45 cy...

Embodiment 2

[0064] (2) Primer (LST Primer-F): 5'-ACTCTGACCGAGCAACGCC -3'

[0065] (LST Primer-R): 5’-GATAACGCTTGCCCCCTATG-3’

[0066] Wherein: RNA bases are in the box.

[0067] After the specificity verification is passed, it is used for detection.

[0068] Extract the total DNA of the sample to be tested (using TIANGEN Plant Total DNA Extraction Kit), set up positive control, healthy plant total DNA control and clean water control respectively, use LST Probe and primers for daily detection, real-time fluorescent PCR reaction system:

[0069] 2×Cycleave PCR Reaction Mix

12.5 μl

Cycleave Primer Mix (10uM)

1 μl

Cycleave Probe (3uM)

1 μl

Sample DNA to be tested

1 μl

dH 2 o

9.5 μl

Total

25 μl

[0070] PCR reaction conditions: pre-denaturation at 95°C for 10 sec; denaturation at 95°C for 5 sec, annealing at 55°C for 10 sec, extension at 72°C for 15 sec, 45 cycles.

[0071] Table 1 List of tested phytoplasmas and ...

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Abstract

The invention belongs to the technical field of plant quarantine and relates to a primer and a probe for a real-time fluorescence polymerase chain reaction (PCR) detection of pear decline phytoplasma. The primer and the probe for the real-time fluorescence PCR detection of the pear decline phytoplasma contain a specific cycling probe and a pair of primers: (1) probe LSTProbe: 5'-(FAM) ATTCTGACTGTA (Eclipse)-3'; and (2) primer LSTPrimer-F:5'-ACTCTGACCGAGCAACGCC-3'; LSTPrimer-R:5'-GATAACGCTTGCCCCCTATG-3', wherein a ribonucleic acid (RNA) alkali base exists in a square frame. Through sequence comparison of a large number of phytoplasmas and bacteria ITS, a set of real-time fluorescence PCR cycling probe and primer is designed, wherein the sensitivity is up to 0.5pgl-1; and the pear decline phytoplasma can be rapidly and correctly detected from a sample. The method has the advantages of simple operation, easy grasping and wide application to daily detection work of laboratories.

Description

technical field [0001] The invention belongs to the technical field of plant quarantine, and relates to primers and probes for real-time fluorescent PCR detection of pear decaying phytoplasma, and also relates to a real-time fluorescent PCR detection method for pear decaying phytoplasma. Background technique [0002] Pear decay phytoplasma is one of the fruit tree quarantine phytoplasmas listed in the "List of Imported Plant Quarantine Pests" newly released by the General Administration of Quality Supervision, Inspection and Quarantine, and is a disease with high risk to my country's fruit tree industry. For a long time, the detection of pear decay phytoplasma has been detected by immunoelectron microscopy. The detection method is complicated and the detection sensitivity is low. These problems have always plagued the quarantine personnel. Therefore, a set of fast, accurate and easy-to-operate methods can be developed to meet the needs of quarantine work. . Contents of the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 王有福李鑫曹冬梅胡强
Owner 王有福
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