Primers and probe used for real-time fluorescent PCR assay of apricot chlorotic leafroll phtoplasma and method thereof

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, so as to improve the detection rate and increase the The level of quarantine technology and the effect of rapid detection

Inactive Publication Date: 2010-12-01
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 phytoplasma chlorophyll rolls in apricot usually adopts the method of immunoelectron microscopy, which is c

Method used

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  • Primers and probe used for real-time fluorescent PCR assay of apricot chlorotic leafroll phtoplasma and method thereof
  • Primers and probe used for real-time fluorescent PCR assay of apricot chlorotic leafroll phtoplasma and method thereof
  • Primers and probe used for real-time fluorescent PCR assay of apricot chlorotic leafroll phtoplasma and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Synthesis of primers and probes for real-time fluorescent PCR detection of Phytoplasma chlorophyllum in apricot:

[0042] Firstly, the 16srDNA nucleic acid sequences of all phytoplasmas and representative bacteria were collected in Genebank, and DNASTAR software was used for sequence comparison to find out the genetic difference sites between Phytoplasma chlorophyllum and other phytoplasma, and real-time fluorescent PCR was designed. Cycling probe (XTLJY Probe) and primers (XTLJY Primer-F, XTLJY Primer-R), and synthesized in a biological company, synthesize a specific Cycling probe and a pair of primers:

[0043] (1) Probe (XTLJY Probe): 5'-(FAM) ATTCTGACTGTA-3'

[0044] (2) Primer (XTLJY Primer-F): 5'-ACTCTGACCGAGCAACGCC -3'

[0045] (XTLJY Primer-R): 5’-GATAACGCTTGCCCCCTATG-3’

[0046] Note: RNA bases are in the box.

[0047]

[0048] Uniformity and stability test:

[0049] 1. Specificity test

[0050] 2×Cycleave PCR Reaction Mix

12.5 μl

...

Embodiment 2

[0065] Real-time fluorescent PCR detection method for apricot chlorotic leaf roll:

[0066] According to the method described in Example 1, the primers and probes for the real-time fluorescent PCR detection of Phytoplasma chlorophyllum were synthesized, (1) Probe (XTLJY Probe): 5'-(FAM) ATTCTGACTGTA-3'

[0067] (2) Primer (XTLJY Primer-F): 5'-ACTCTGACCGAGCAACGCC -3'

[0068] (XTLJY Primer-R): 5’-GATAACGCTTGCCCCCTATG-3’

[0069] Note: RNA bases are in the box.

[0070] After the XTLJY Probe specificity verification is passed, it is used for detection.

[0071] 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

[0072] 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, 45 cycles; extension at 72°C for 15 sec.

[0073] T...

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Abstract

The invention belongs to the technical field of plant quarantine, and relates to primers and a probe used for real-time fluorescent PCR assay of apricot chlorotic leafroll phtoplasma, and a detection method thereof. The primers and the probe used for real-time fluorescent PCR assay of apricot chlorotic leafroll phtoplasma are characterized by comprising a specificity Cycling probe and a pair of primers: (1) the probe (XTLJYProbe): 5'-(FAM)ATTCTGACTGTA-3'; (2) the primers: (XTLJYPrimer-F): 5'-ACTCTGACCGAGCAACGCC-3' and (XTLJYPrimer-R): 5'-GATAACGCTTGCCCCCTATG-3', wherein an RNA basic group is in the box. By comparing ITS sequences of a large amount of phtoplasma and bacteria, a set of Cycling probe and primers for real-time fluorescent PCR are designed, have the sensitivity of 0.2pg/l<-1>, and can quickly and accurately detect the apricot chlorotic leafroll phtoplasma from samples. The method has the advantages of simple operation, easy control and wide application to daily detection work in labs.

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 apricot chlorotic leaf roll phytoplasma, and also relates to a detection method thereof. Background technique [0002] Apricot chlorotic phytoplasma is one of the fruit tree quarantine phytoplasmas listed in the "List of Imported Plant Quarantine Pests" newly issued by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. It is a disease with high risk to my country's fruit tree industry . For a long time, the detection of phytoplasma chlorophyll rolls in apricot usually adopts the method of immunoelectron microscopy, which is complicated and has low detection sensitivity. These problems have always troubled the quarantine personnel. Meet the needs of quarantine work. Contents of the invention [0003] The object of the present invention is to overcome...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 王有福姜丽李鑫刘卉秋
Owner 王有福
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