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RT-PCR used for detecting rice black streaked dwarf virus (RBSDV) and southern rice black-streaked dwarf virus (SRBSDV) and application of RT-PCR

A rice black-streaked dwarf and black-streaked dwarf virus technology, applied in the field of PCR detection, can solve the problems of difficult to accurately identify pathogens and complicated operation methods, and achieve the effects of improving detection efficiency, accurate detection results, and convenient detection costs

Active Publication Date: 2017-09-05
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above detection methods are difficult to accurately identify the pathogen, and the operation method is relatively complicated

Method used

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  • RT-PCR used for detecting rice black streaked dwarf virus (RBSDV) and southern rice black-streaked dwarf virus (SRBSDV) and application of RT-PCR
  • RT-PCR used for detecting rice black streaked dwarf virus (RBSDV) and southern rice black-streaked dwarf virus (SRBSDV) and application of RT-PCR
  • RT-PCR used for detecting rice black streaked dwarf virus (RBSDV) and southern rice black-streaked dwarf virus (SRBSDV) and application of RT-PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The selection of embodiment 1 rice black-streaked dwarf virus conserved sequence and the design of specific primer

[0040] Through the sequencing and comparison of the rice black-streaked dwarf virus gene nucleotide sequence at multiple points for many years, the sequence of the S5 segment of the conserved region was determined, and its nucleotide sequence is shown in SEQ ID NO.5. The target sequence is to detect rice black-streaked dwarf virus. Genetic markers of dwarf virus.

[0041] The present invention was applied in Beijing (I), Hebei Tangshan (II) and Baoding (III), Shandong Jinan (IV) and Jining (V), Henan Zhengzhou (VI) and Xinyang (IX), Jiangsu Yancheng (VII) in 2012-2014 ) and Nanjing (VIII) collected a total of 127 samples of maize rough dwarf and rice black-streaked dwarf with typical symptoms, and amplified with 3 pairs of RBSDV genome S5-specific primers. The results showed that the 2398-2832bp of RBSDV-S5 was the most conserved, and it was the overlapp...

Embodiment 2

[0061] The specificity evaluation of the RT-PCR detection method of embodiment 2 rice black-streaked dwarf virus and southern rice black-streaked dwarf virus

[0062] Take the plants with obvious symptoms of corn dwarf mosaic disease and corn rough dwarf disease in the field, extract leaf RNA, and use the preferred primers RS5-F, RS5-R and SS5-F, SS5-R in Example 1 to detect after reverse transcription , the results showed that the diseased plants of corn dwarf mosaic disease were all negative, while the diseased plants of corn rough dwarf disease were all positive, such as image 3 shown. It shows that the preferred primer pairs of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus screened in Example 1 of the present invention have good specificity and can specifically detect the pathogen of maize rough dwarf diseased plants.

Embodiment 3

[0063] Example 3 Rice black-streaked dwarf virus of the present invention and southern rice black-streaked dwarf virus RT-PCR detection of maize rough dwarf pathogen

[0064] The preferred detection primers RS5-F, RS5-R and SS5-F, SS5-R of rice black-streaked dwarf virus and southern rice black-streaked dwarf virus screened in Example 1 were used to identify the corn rough dwarf pathogen.

[0065] The plants with typical symptoms of rough dwarf disease of maize collected in my country's Huanghuaihai region (R1, R2, R3) and Hainan province (S1, S2, S3) were selected, and the diseased leaves were collected and stored in a -80°C refrigerator. TransZolTM Up (Transgen Biotech) method was used to extract total RNA from a single maize leaf, and reverse transcription was performed using the Fast Quant RT Kit (With gDNase) (Tiangen) kit, using the preferred primers RS5-F / RS5-R and SS5-F / SS5- R was amplified separately. The results show that samples R1, R2 and R3 can detect the target ...

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Abstract

The invention provides RT-PCR used for detecting rice black streaked dwarf virus (RBSDV) and southern rice black-streaked dwarf virus (SRBSDV) and an application of the RT-PCR and belongs to the technical field of PCR detection. Through comparison of genomic sequences of RBSDV with SRBSDV, a conservative-area target sequence is determined, a specific primer pair used for detecting the RBSDV and the SRBSDV is designed, and the nucleotide sequences are as shown in SEQ ID NO. 1-4. The invention further provides a method for detecting the RBSDV and the SRBSDV and a detection kit. The method has the advantages of accurate detection, high flexibility, high specificity, convenience and rapidness, crop infection of the RBSDV or the SRBSDV can be distinguished, low cost is achieved, and good economic value and application prospect are realized.

Description

technical field [0001] The invention relates to the technical field of PCR detection, in particular to target sequences and RT-PCR primers for detecting rice black-streaked dwarf virus and southern rice black-streaked dwarf virus. Methods and kits for detection of adenovirus and southern rice black-streaked dwarf virus. Background technique [0002] Maize rough dwarf disease (MRDD) is one of the worldwide viral diseases that endanger maize production, and it occurs seriously in the maize production areas of my country. From 2008 to 2013, maize rough dwarf disease caused serious yield loss, generally reaching 30% to 50%, and in severe areas it could reach more than 80%, or even no harvest. [0003] There are four main sources of maize rough dwarf disease, all of which belong to the Fijivirus genus of the family Reoviridae. In Europe, the source of disease is maize rough dwarf virus (Maize roughdwarf virus, MRDV); in South America, the source of disease is Mal de Río Cuarto ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/701
Inventor 周羽王振华李新海翁建峰邸宏张泓祖洪月张晓明
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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