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Nested RT-PCR primer group, detection method and kit for CYVCV

A technology of RT-PCR and citrus yellow vein virus, which is applied in the field of detection method and kit, nested RT-PCR primer set of citrus yellow vein virus, can solve the problem of unsatisfactory detection effect of early diseased trees in the field, and the sensitivity needs to be determined. Improvement and other issues to achieve the effect of high accuracy, easy operation and sensitive detection

Inactive Publication Date: 2014-11-19
CITRUS RES INST SOUTHWEST UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although conventional RT-PCR has begun to be used in the detection of CYVCV, its detection effect on early diseased trees in the field is not ideal, and the sensitivity needs to be improved
The detection sensitivity of nested RT-PCR is much higher than that of conventional RT-PCR, but this technology has not been applied to the detection of CYVCV so far

Method used

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  • Nested RT-PCR primer group, detection method and kit for CYVCV
  • Nested RT-PCR primer group, detection method and kit for CYVCV
  • Nested RT-PCR primer group, detection method and kit for CYVCV

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Effect test

Embodiment 1

[0025] Nested RT-PCR detection specific verification of embodiment 1 citrus yellow vein virus

[0026] 1 Primer set for detecting citrus yellow vein virus

[0027] According to the genome sequence of CYVCV (JX040635.1) in GenBank, the outer nest primer pair (CYVCV-1f and CYVCV-1r) required by the CYVCV nested RT-PCR detection method was designed for the conserved region at its 3' end, and the inner nest primer pair Primer pair (CYVCV-2f and CYVCV-2r). And through the Blast comparison in GenBank, the specificity of the primers was guaranteed. The primer sequences are listed in Table 1.

[0028] Table 1 Primer sequences used for nested RT-PCR detection of CYVCV

[0029]

[0030] 2 Extract the total RNA of the sample to be tested

[0031] Select 15-30 mg each of the leaves showing typical symptoms of CYVCV and the leaves of virus-free seedlings as positive and negative controls, respectively. Grind the leaves in liquid nitrogen and add 1mL RNAiso Plus, place at room tempe...

Embodiment 2

[0042] The nested RT-PCR detection sensitivity detection of embodiment 2 citrus yellow vein virus

[0043] right figure 1 The total RNA template of sample 4 infected with citrus yellow vein disease in China was diluted to 10 times in a 10-fold gradient. 8 Times, using conventional RT-PCR and nested RT-PCR in Example 1 to carry out amplification detection respectively, routine RT-PCR obtains the specific amplification fragment of 612bp, electrophoresis result is as follows image 3 As indicated, total RNA was diluted to 10 5 The conventional RT-PCR cannot be detected at times, and the total RNA is diluted to 10 6 Nested RT-PCR can still detect CYVCV when double times, so the detection sensitivity of nested RT-PCR is 100 times that of conventional RT-PCR. The primers used for conventional RT-PCR of citrus yellow vein virus are: upstream primer: 5'-TACCGCAGCTATCCATTTCC-3'; downstream primer: 5'-GCAGAAATCCCGAACCACTA-3'.

[0044]

[0045]

[0046]

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Abstract

The invention relates to a nested RT-PCR (reverse transcription-polymerase chain reaction) primer group, a detection method and a kit for CYVCV (Citrus Yellow Vein Clearing Virus) and belongs to the technical field of molecular biology. The invention provides nested RT-PCR external nest primer pairs CYVCV-1f and CYVCV-1r and nested RT-PCR internal nest primer pairs CYVCV-2f and CYVCV-2r for CYVCV. The external nest primer pairs are utilized to carry out a first round of RT-PCR amplification; then a product obtained in the first round of RT-PCR amplification is used as a template and the internal nest primer pairs are utilized to carry out a second round of RT-PCR amplification; a PCR product obtained in the second round is detected to obtain a sample to be detected of the 469bp PCR product, i.e. the sample infected by CYVCV. The nested RT-PCR primer group is simple and convenient to operate and has good repeatability and high accuracy; sensitivity of the nested RT-PCR primer group is 100 times of that of common RT-PCR; and the nested RT-PCR primer group also can timely detect viruses in the incubation period.

Description

technical field [0001] The invention relates to the field of plant epidemic disease monitoring, in particular to a nested RT-PCR primer set, detection method and kit for citrus yellow vein virus. Background technique [0002] Citrus yellow vein disease caused by Citrus yellow vein clearing virus (CYVCV) is an important disease that seriously threatens citrus production. It is mainly distributed in India, Pakistan, Turkey and other countries. caused extremely serious losses. In recent years, the disease has also been found in Yunnan, Sichuan and other major lemon producing areas in my country, and its occurrence is constantly expanding and aggravating. Since there is currently no medicament that can effectively control citrus yellow vein disease, the use of virus-free seedlings and the eradication of diseased trees in the field are important means of prevention and control. Therefore, it is necessary to establish a rapid, accurate and sensitive detection method of CYVCV to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/70C12Q1/6848C12Q2531/113C12Q2549/119
Inventor 周彦陈洪明王雪峰李中安周常勇
Owner CITRUS RES INST SOUTHWEST UNIV
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