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Quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and method capable of simultaneously detecting multiple chili viruses

A technology of RT-PCR and capsicum, which is applied in the field of simultaneous detection of multiple capsicum virus quadruple RT-PCR detection primers, can solve the problems of aggravating the degree of harm, widening the occurrence range of capsicum virus disease, affecting the yield and quality of capsicum, and achieving The cost of detection is reduced, the types of viruses are increased, and the detection efficiency is improved

Inactive Publication Date: 2015-12-23
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the 1970s, due to frequent exchanges between varieties in various places, and the scope of exchanges has continued to expand, as well as farming systems and other reasons, the occurrence of pepper virus diseases in my country has become wider and the degree of damage has increased, seriously affecting the yield and quality of peppers.
A variety of multiple detection methods have been developed for common viruses such as TMV, CMV, PVY, and ToMV at home and abroad. However, no corresponding multiple detection methods have been found for domestically reported and newly discovered pepper viruses.

Method used

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  • Quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and method capable of simultaneously detecting multiple chili viruses
  • Quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and method capable of simultaneously detecting multiple chili viruses
  • Quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and method capable of simultaneously detecting multiple chili viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0031] 1. Primer design

[0032] Due to the need to detect 4 kinds of pepper viruses, in order to establish a multiplex RT-PCR detection method with the characteristics of many types of viruses, simple, accurate and economical detection steps, Primer6. 0 Design primers Design primer pair (forward primer BBWV2-F and reverse primer BBWV2-R), the target fragment size of amplification is 693bp; According to the conservative sequence of pepper vein yellowing virus (PeVYV), design primer pair (forward Primer PevYV-F and reverse primer PevYV-R), the amplified target fragment size is 383bp; also design primer pairs (forward primer ChiVMV-F and reverse primer ChiVMV-R, and forward primer PMMoV-F and reverse primer PMMoV-R), the amplified target fragment sizes are 513bp and 236bp respectively; the primer sequences are shown in Table 1. The sizes of the fragments amplified by each pair of primers are 693, 383, 513 and 236bp respectively. The size of the fragments is quite different, the...

Embodiment 2

[0047] Example 2 Effects of different annealing temperatures and different template dilution factors on the detection effect of quadruple RT-PCR

[0048] 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 6 temperature gradients of 60°C, 58°C, 56°C, 54°C, 52°C and 50°C. The electrophoresis results of PCR products are shown in figure 2 . The results showed that when the annealing temperature was 58-54°C, four clear target fragments could be obtained by quadruple RT-PCR.

[0049] In order to test the sensitivity of the quadruple RT-PCR detection, the initial RNA concentration was 125ng / μl, which were diluted 10, 100, 1000 and 10000 times and 100000 respectively, that is, the RNA concentration was 10 times the initial concentration. -1 、10 -2 、10 -3 and 10 -4 and 10 -5 times. The ...

Embodiment 3 4

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

[0051] Field surveys in Furong District, Changsha City, Hunan Province and main pepper producing areas in Liuyang City, and collected pepper plants with yellowing and mosaic symptoms, brought the plants back to the laboratory with soil, selected young leaves, and extracted RNA after grinding with liquid nitrogen. A good quadruple RT-PCR detects its virus infection, and the test results are shown in Figure 4 . The results showed that quadruple RT-PCR can detect the viruses in the pepper samples from the main production areas of Hunan very well, and the pepper plant samples from the two main production areas of Hunan, Changsha and Liuyang, were commonly infected by 3-4 kinds of viruses.

[0052]

[0053]

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Abstract

The invention discloses quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and a method capable of simultaneously detecting multiple chili viruses. The method adopts four pairs of primers disclosed as SEQ ID NO:1-SEQ ID NO:8; and one RT-PCR process can simultaneously detect four chili viruses: Broad bean wilt virus 2 (BBWV 2), Pepper vein yellows virus (PeVYV), Chilli veinal mottle virus (ChiVMV) and Pepper mildmottle virus (PMMoV). Compared with the existing single RT-PCR and duplex RT-PCR detection methods, the method disclosed by the invention can simultaneously detect the four chili viruses by one RT-PCR process, and is capable of detecting more virus varieties, greatly enhancing the detection efficiency and obviously lowering the detection cost.

Description

technical field [0001] The invention belongs to the technical field of crop virus detection, and in particular relates to a quadruple RT-PCR detection primer and a detection method for simultaneous detection of multiple pepper viruses. Background technique [0002] Capsicum (Capsicumannuum L.) is a plant of the genus Capsicum in the family Solanaceae. It is an annual or limited perennial herbaceous plant. The fruit is usually conical or oblong, green when immature, and bright red, green or purple when mature. Most common, its original range was from Mexico to Colombia; it is now cultivated all over the world. Capsicum has been cultivated in my country for hundreds of years and is an important vegetable and condiment. It is currently one of the main types of vegetables grown in large areas in my country. In addition to being an important vegetable crop, pepper has high nutritional value. The content of vitamin C occupies the first place in vegetables, which is 7-15 times tha...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q1/70C12Q2600/16
Inventor 唐前君戴良英刘湘宁刘茂炎李迅刘双清李魏
Owner HUNAN AGRICULTURAL UNIV
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