Method and special primer for simultaneously detecting cherry whether to be infected by apple chlorotic leaf spot virus and prunus necrotic ringspot virus

A technology for ringspot virus and apples, applied in the field of simultaneously detecting whether cherries are infected with apple chlorotic leaf spot virus and Prunus necrotic ringspot virus and special primers, can solve the problems of being vulnerable to viruses, save time, and be easy to operate Effect

Inactive Publication Date: 2013-04-10
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Cherry is one of the fruits with high eco

Method used

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  • Method and special primer for simultaneously detecting cherry whether to be infected by apple chlorotic leaf spot virus and prunus necrotic ringspot virus
  • Method and special primer for simultaneously detecting cherry whether to be infected by apple chlorotic leaf spot virus and prunus necrotic ringspot virus
  • Method and special primer for simultaneously detecting cherry whether to be infected by apple chlorotic leaf spot virus and prunus necrotic ringspot virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The amplification of ACLSV and PNRSV virus in the blade of embodiment 1, ' red light '

[0046] Take 0.5 μl of 'Red Lantern' leaf RNA and detect it with a UV spectrophotometer, the OD260 / 280 is between 1.8-2.0 (Figure 1), and the cDNA after reverse transcription is used as a template. Through the PCR amplification of ACLSV and PNRSV viral primers AF1 / AR1 and PF / PR, 357bp and 455bp were obtained respectively ( figure 2 ) electrophoresis band, after gel cutting and recovery, connected to the T vector, transformed and sequenced by Escherichia coli DH5α, compared with the CP gene sequences of ACLSV and PNRSV registered on NCBI, the homology was 94% and PNRSV respectively. 98%. Therefore, the harvested 'Red Lantern' leaves are infected with both ACLSV and PNRSV, and the above-mentioned 'Red Lantern' leaves can be selected as a positive control.

Embodiment 2

[0047] Embodiment 2, establishment of multiple RT-PCR detection system

[0048] The leaves of 'Red Lantern' were used as experimental materials.

[0049] Annealing temperature selection: explore the annealing temperature of ACLSV virus primer AF2 / AR2 and PNRSV virus primer PF / PR, by image 3 It can be seen that the range of primer AF2 / AR2 is 45°C-57°C, and the annealing temperature of primer PF / PR is 45°C-60°C. In order to reduce the generation of primer dimers, we choose 57°C as the annealing temperature of the multiplex PCR reaction .

[0050] Only add ACLSV viral primer AF2 / AR2: only a single target band 632bp ( Figure 5 middle lane 1).

[0051] Only add PNRSV virus primer PF / PR: only get a single target band 455bp ( Figure 5 middle lane 4).

[0052] Adding primers AF1 / AR1 and PF / PR at the same time: no two target bands were obtained at the same time.

[0053] First add primer AF1 / AR1 and then add primer PF / PR: no two target bands were obtained at the same time.

...

Embodiment 3

[0059] Embodiment 3, sensitivity analysis

[0060] The total RNA amount of the 'red light' compound infected in the reverse transcription system, in order of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 μg, after PCR amplification, agarose gel electrophoresis, scanning gel imaging, to get Image 6 . As the total RNA concentration decreased, the template of ACLSV virus in the system also gradually decreased, resulting in the darker and darker target bands; while the target bands of PNRSV virus tended to become brighter first. It shows that increasing the concentration of the template can reduce the effect of the primer with strong template binding ability on the reaction. When the total RNA amount of the diseased plants in the reverse transcription system is 10 -4 At the time of μg, after PCR amplification, two viruses can still be detected at the same time.

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Abstract

The invention discloses a method and a special primer for simultaneously detecting whether a cherry to be infected by an apple chlorotic leaf spot virus and a prunus necrotic ringspot virus. The primer used for detecting plant viruses comprises a primer pair A and a primer pair B, wherein the primer pair A is used for detecting the apple chlorotic leaf spot virus; a primer sequence is shown as SEQ ID No.1 (sequence identification number 1); the other primer sequence is shown as SEQ ID No.2; the primer pair B is used for detecting the prunus necrotic ringspot virus; a primer sequence is shown as SEQ ID No.3; the other primer sequence is shown as SEQ ID No.4. A multi-RT-PCR (reverse transcription-polymerase chain reaction) established in the primer can simultaneously detect ACLSV (apple chlorotic leaf spot virus) and PNRSV (prunus necrotic ringspot virus) compounded infection of the cherry in production, and saves time compared with a single RT-PCR; the operation is simpler; in addition, consumed drugs in a test are reduced; and economy is facilitated. When a total amount of RNA (ribonucleic acid) of an infected plant in a reverse transcription system ranges from 10 micrograms to 4 micrograms, the two viruses can still be detected simultaneously after PCR (polymerase chain reaction) amplification is performed.

Description

technical field [0001] The invention relates to a method and special primers for simultaneously detecting whether cherries are infected with apple chlorotic leaf spot virus and plum necrotic ringspot virus. Background technique [0002] Cherry is one of the fruits with high economic value, but it is easily damaged by viruses. So far, 68 viruses infecting cherries have been discovered, among which Apple chlorotic leaf spot virus (ACLSV) and Prunus necrotic ringspot virus (PNRSV) are more serious. In recent years, due to the continuous introduction of a large number of new strains of cherry seeds and seedlings from abroad and the frequent transfer of seedlings and seeds from different regions in China, the spread of the virus has been accelerated. Li Chunmin et al. (1997) used PAS-ELISA to detect the infection rate of sweet cherry ACLSV in Changli area was 47.1%. Niu Jianxin et al. (2003) detected ACLSV on Korla pears by RT-PCR. Li Qing et al. (1996) used the ELISA method t...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
Inventor 李天忠马国玲张开春
Owner CHINA AGRI UNIV
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