Triple RT-PCR method capable of detecting three viruses causing potato tuber necrosis simultaneously and primer combination thereof

A potato block and potato technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of high cost and long time consumption, and achieve improved detection efficiency, high sensitivity, and high specificity Effect

Inactive Publication Date: 2015-11-11
HUNAN AGRICULTURAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For PVY NTN , TSWV, and PMTV viruses, a single-plex RT-PCR method has been established at home and abroad (HuX, HeC, XiaoY, XiongX, NieX. and Cubeta MA. Tomatos spotted wilt virus in Potato in Eastern North Carolina. Amer Jof Potato Res 2005, 82: 255-261; PCR detection method, one RT-PCR can only detect one virus, and three viruses need three RT-PCRs to complete, which takes a long time and costs high. The conserved sequences of the whole genome or partial sequences of these three viruses, and the design of universal primers, aim to establish a simple, efficient and sensitive triple RT-PCR detection technology, and simultaneously detect PVY that can cause necrosis in potato tubers NTN , TSWV and PMTV three viruses

Method used

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  • Triple RT-PCR method capable of detecting three viruses causing potato tuber necrosis simultaneously and primer combination thereof
  • Triple RT-PCR method capable of detecting three viruses causing potato tuber necrosis simultaneously and primer combination thereof
  • Triple RT-PCR method capable of detecting three viruses causing potato tuber necrosis simultaneously and primer combination thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0029] Embodiment 1 Triple RT-PCR detects PVY simultaneously NTN , TSWV, PMTV method establishment

[0030] 1. Primer design

[0031] Due to the need to detect TSWV and PVY at the same time NTN , PMTV these three kinds of viruses, to establish a multiple RT-PCR detection method with the characteristics of many types of viruses detected, simple detection steps, accuracy, and economy. The establishment of this multiplex PCR system has high requirements on primers, which need both specificity and compatibility to ensure that the triplex PCR can work normally and establish an optimal system. According to the nucleocapsid gene sequence of TSWV in Genbank, use Primer5.0 to design primers to design a universal primer pair (forward primer TSWV-F, reverse primer TSWV-R), and the amplified target fragment size is 251bp; according to PVY in Genbank NTN The unique recombination site sequence was designed with Primer5.0 universal primer pair (forward primer PVY NTN -F, reverse prime...

Embodiment 2 3

[0046] Example 2 Optimization of Triple RT-PCR Detection Method and Its Detection Sensitivity (Influence of Different Annealing Temperatures and Different Template Dilution Factors on Triple RT-PCR Detection Effect)

[0047] Since the Tm values ​​of each pair of primers are not the same, it is necessary to select a suitable annealing temperature so that triple RT-PCR can obtain three target fragments and improve detection sensitivity. The annealing temperature is set to 8 temperature gradients of 51.7, 53.6, 55.7, 57.9, 60.1, 62.3, 64.4, and 66.3°C. The electrophoresis results of PCR products are shown in figure 2 . The results showed that the annealing temperature was the most suitable at 57.9°C-62.3°C, and three clear target fragments could be obtained by single-plex and triple-plex RT-PCR.

[0048] In order to test the sensitivity of the triple RT-PCR detection, the initial RNA was diluted 10, 100, 1000 and 10000 times, respectively, that is, the RNA concentration was 10 ...

Embodiment 3 3

[0049] Example 3 triple RT-PCR detection of potato plant samples

[0050] Laboratory samples infected with TSWV and PVY respectively NTN , PMTV potato plants or tubers, a total of 13 samples. After grinding with liquid nitrogen, extract the total RNA of the plant, and use the established triple RT-PCR system to detect the virus infection. The test results are shown in Figure 4 . The results show that the target bands amplified by this system are very clear and specific, and these three viruses can be detected 100%, indicating that the established triple RT-PCR system can be well used in TSWV, PVY NTN , PMTV detection.

[0051] Hunan Agricultural University

[0052] A Triple RT-PCR Method and Primer Combination for Simultaneous Detection of Three Viruses Causing Potato Tuber Necrosis

[0053] 6

[0054] 1

[0055] 20

[0056] DNA

[0057] Artificial sequence

[0058]

[0059] Description of Artificial Sequences: Artificially Synthesized Sequences

[0060] 1

[...

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Abstract

The invention discloses a triple RT-PCR method capable of detecting three viruses causing potato tuber necrosis simultaneously and a primer combination thereof. Particularly, three pairs of primers and one RT-PCR are used for detecting a potato virus Y tuber necrosis strain (PVYNTN) injuring potato tubers, a tomato spotted wilt virus (TSWV) and a potato mop-top virus (PMTV) simultaneously. Due to the fact that the TSWV, the PVYNTN and the PMTV can all cause potato tuber necrosis, symptoms are difficult to distinguish. When a traditional single RT-PCR method is used, detection can only be completed through three reactions, and time and labor are consumed. Accordingly, the three viruses can be detected simply through a multiple RT-PCR reaction, the detection efficiency is greatly improved, and the detection cost is greatly lowered.

Description

technical field [0001] The invention belongs to the technical field of crop virus detection, and in particular relates to a simultaneous detection of potato virus Y tuber necrosis strain (PVY) that can cause potato tuber necrosis. NTN ), tomato spotted wilt virus (TSWV), potato broom top virus (PMTV) and their primer combinations. Background technique [0002] Potatoes, also known as potatoes, artichokes, etc., are common food and vegetable crops in people's daily life. As food crops, they are the fourth largest food crops in the world after wheat, corn and rice. China is the largest producer of potatoes in the world. In 2011, the sown area was about 5.425 million hectares and the total output was about 88.29 million tons, ranking first in the world, accounting for about 1 / 4 and 1 / 5 of the world respectively. Although my country's potato planting area and total output rank first in the world, the per unit area yield is far behind the advanced potato production countries and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/686
Inventor 胡新喜雷艳熊兴耀周华兰
Owner HUNAN AGRICULTURAL UNIV
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