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A Quadruple RT-PCR Method for Simultaneous Detection of Multiple Garlic Viruses and Its Primer Combination

A technology of RT-PCR and virus, which is applied in the field of simultaneous detection of a variety of garlic viruses and its primer combination, can solve the problems of few types of viruses and difficulty in distinguishing fragments, and achieve high sensitivity, improved detection efficiency, and high detection efficiency. The cost reduction effect of

Active Publication Date: 2015-11-18
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For virus detection of a large number of garlic species propagated through the shoot tip, RT-PCR detection technology is required. It has the characteristics of many types of viruses, simple, accurate and economical detection steps. The existing garlic virus RT-PCR detection technology exists to detect virus types less, fragments are difficult to distinguish, and because there are too many types of viruses, it is difficult to establish a ten-fold RT-PCR to detect these 10 viruses

Method used

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  • A Quadruple RT-PCR Method for Simultaneous Detection of Multiple Garlic Viruses and Its Primer Combination
  • A Quadruple RT-PCR Method for Simultaneous Detection of Multiple Garlic Viruses and Its Primer Combination
  • A Quadruple RT-PCR Method for Simultaneous Detection of Multiple Garlic Viruses and Its Primer Combination

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Primer design

[0031] Since it is necessary to detect 10 kinds of garlic viruses, 7 of which are shallot virus genus viruses, in order to establish a multiple RT-PCR detection method with the characteristics of many types of viruses to be detected, simple, accurate and economical detection steps, according to the Genbank shallot virus genus ( Allexivirus ) The conserved sequences of 7 viruses including GarV-A, GarV-B, GarV-C, GarV-D, GarV-E, GarV-X and ShV-X were designed using Primer5.0 to design primers to design universal primer pairs (forward primers Allexivirus -F, reverse primer Allexivirus -R)( figure 1 ), the size of the amplified target fragment is 180bp, which is a common characteristic band of 7 kinds of viruses; according to the conserved sequence of garlic latent virus (GLV, also known as onion latent virus SLV), a universal primer pair is designed (forward primer SLV- F, reverse primer SLV-R), the size of the amplified target fragment is 592bp; ...

Embodiment 2

[0046] Example 2 Effects of different annealing temperatures and different template dilution factors on the detection effect of quadruple RT-PCR

[0047] Since the Tm values ​​of each pair of primers are not the same, it is necessary to select a suitable annealing temperature to obtain four target fragments by quadruple RT-PCR and improve detection sensitivity. The annealing temperature is set to 8 temperature gradients of 42.5, 43.7, 45.6, 47.7, 49.9, 52.1, 54.3 and 56.4°C, and the electrophoresis results of PCR products are shown in image 3 . The results showed that when the annealing temperature was 42.5-45.6°C, four clear target fragments could be obtained by quadruple RT-PCR.

[0048] In order to test the sensitivity of the quadruple RT-PCR detection, the initial RNA was diluted 10, 100, 1000 and 10000 times respectively, that is, the RNA concentration was 10 times the initial concentration. -1 、10 -2 、10 -3 and 10 -4 times.

[0049] The results of electrophoresis ...

Embodiment 3 4

[0050] Embodiment 3 quadruple RT-PCR detection field garlic plant sample

[0051] Field surveys in Furong District, Changsha City, Hunan Province, and Chaling County, Zhuzhou City, the main garlic production areas, collected garlic plants with yellowing and mosaic symptoms, brought the plants back to the laboratory with soil, selected young leaves, ground them with liquid nitrogen, and extracted them. RNA, using the established quadruple RT-PCR to detect the virus infection, the test results are shown in Figure 5 . The results showed that quadruple RT-PCR can detect viruses in garlic samples from the main production areas of Hunan very well, and the garlic plant samples from the two main production areas of Liling and Furong districts in Hunan were commonly infected by 3-4 kinds of viruses.

[0052] Hunan Agricultural University

[0053] A Quadruple RT-PCR Simultaneous Detection of Multiple Garlic Viruses and Its Primer Combination

[0054] 8

[0055] 1

[0056] 20

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Abstract

The invention discloses a method of simultaneously detecting a plurality (10) of garlic viruses by quadruple RT-PCR and a primer composition thereof. In particular, four pairs of primers are adopted. RT-PCR for one time can simultaneously detect the main viruses of garlic as follows: onion yellow dwarf viruses (OYDV), leek yellow stripe viruses (LYSV), shallot latent viruses (SLV), allexivirus (comprising GarV-A, GarV-B, GarV-C, GarV-D, GarV-E, GarV-X and ShV-X), and the like. Accordingly, the method and the primer composition have characteristics of increased detected virus varieties in one detection step, largely increased detection efficiency and largely reduced detection cost.

Description

technical field [0001] The invention belongs to the technical field of crop virus detection, and in particular relates to a method for simultaneously detecting multiple garlic viruses and a primer combination thereof. Background technique [0002] garlic( Allium sativum L.) belongs to Liliaceae Allium, and is a vegetative propagation crop. Garlic has high nutritional value and unique flavor. It is a condiment that consumers like to eat. It is widely planted all over the world. The global planting area is about 1.1 million hectares 2 , the total output is about 14 million tons. With the in-depth research and application of garlic's medicinal value, garlic has been favored by the international medical community and consumers. Modern medical research has proved that garlic and natural garlic preparations have very good effects in preventing and curing diseases. Garlic has antibacterial and anti-inflammatory properties, anti-cancer and anti-cancer effects, improves liver dysf...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/70C12Q2531/113C12Q2537/143
Inventor 胡新喜熊兴耀雷艳汪沛何长征宋勇汤琳菲
Owner HUNAN AGRICULTURAL UNIV
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