Multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for synchronously detecting five watermelon viruses and application of method

An RT-PCR, watermelon virus technology, applied in the field of crop disease diagnosis and molecular biology, can solve the problems of high cost of small RNA deep sequencing, high bioinformatics analysis skills, and difficulty in large-scale application in production practice.

Inactive Publication Date: 2017-05-24
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the high cost of small RNA deep sequencing and the need for high bio...

Method used

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  • Multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for synchronously detecting five watermelon viruses and application of method
  • Multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for synchronously detecting five watermelon viruses and application of method
  • Multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for synchronously detecting five watermelon viruses and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Extraction of watermelon leaf tissue total RNA

[0053] Total RNA was extracted from healthy watermelon plants and watermelon plants co-infected by CiLCV, CGMMV, ZYMV, MABYV and WMV using TRIzol reagent from Invitrogen, USA. The operation steps are as follows: 1) After taking fresh or frozen watermelon leaves and adding liquid nitrogen to fully grind, quickly transfer 100 mg of dry powder tissue to a 1.5 ml sterile centrifuge tube treated with DEPC water; 2) Add 1 mL of TRIzol reagent, shake well, Stand at room temperature for 5 minutes; 3) Add 0.2 mL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes; centrifuge at 12000g / 4°C for 15 minutes; 4) Take the supernatant, add an equal volume of isopropanol, mix well, Ice bath for 10 minutes; centrifuge at 12000g / 4°C for 15 minutes, and remove the supernatant; 5) add 1 mL of pre-cooled 75% ethanol (prepared with sterile DEPC water) to the precipitate for washing, centrif...

Embodiment 2

[0054] Embodiment 2. Design of five kinds of virus-specific primers

[0055] According to the nucleotide sequences of CiLCV, CGMMV, ZYMV, MABYV and WMV published by NCBI, the Vector NTI software was used to design specific primer pairs for each virus, and the annealing temperature of each pair of primers was similar (51.9°C-59.9°C) ), has a certain degree of conservatism, the amplified product does not form a secondary structure, and the GC content is between 40% and 60%; the bases between the five pairs of primers are not complementary, and the primers are synthesized by Shanghai Bioengineering Co., Ltd. (5 kinds of virus See Table 1 for specific primers.

[0056] Table 1. The specific primers of CiLCV, CGMMV, ZYMV, MABYV and WMV five kinds of viruses

[0057]

Embodiment 3

[0058] Embodiment 3. The specificity analysis of five kinds of viral primers

[0059] Five kinds of viruses including CiLCV, CGMMV, ZYMV, MABYV and WMV can be detected by RT-PCR using the five pairs of primers designed in Example 2. The remaining 5 viruses belonging to the same genus Deltapartitivirus as CiLCV are Beet cryptic virus 2 (BCV2), Beet cryptic virus 3 (BCV3), Fig Fig cryptic virus (FCV), Pepper cryptic virus 1 (PCV1) and Pepper cryptic virus 2 (PCV2) have not been reported to infect watermelons. The other 16 viruses belonging to the same genus Polerovirus as MABYV have not been reported to infect watermelon. However, both ZYMV and WMV belong to the genus Potyvirus, and the primers of the two did not amplify the band of the other party in this system, indicating that the primers of the two are unique. However, TMV belonging to the genus Tobamovirus (Tobamovirus) with CGMMV can infect watermelon, and is a common virus on watermelon. The following method of the pres...

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Abstract

The invention belongs to the technical field of crop disease diagnosis and molecular biology and discloses a multiple RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for synchronously detecting five watermelon viruses. A PCR system contains five pairs of specific primers (SEQ ID NO1-10), and the five viruses are synchronously multiplied in one PCR system, so as to obtain specific bands 1464bp, 1023bp, 810bp, 600bp and 327bp and establish and optimize a multiple RT-PCR reaction system capable of synchronously detecting the viruses CiLCV, CGMMV, ZYMV, MABYV and WMV. A detection result indicates that the optimized multiple RT-PCR reaction system can detect a field sample quickly, efficiently and accurately. The method has a significant meaning for forcasting of occurrence and disease epidemiology of five common viruses such as CiLCV and the like on monitored watermelons.

Description

technical field [0001] The invention belongs to the technical field of crop disease diagnosis and molecular biology, and in particular relates to a system of reverse transcription polymerase chain reaction (RT-PCR) and its application, which can detect five kinds of infested watermelons by one PCR RNA virus, to achieve the purpose of accurate, rapid and efficient detection. Background technique [0002] Watermelon (Citrullus lanatus) is a plant of the genus Watermelon in the family Cucurbitaceae. It is a summer fruit with sweet and juicy flesh that can cool down the heat; its seeds contain oil and can be used as a recreational food; Since 1978, the world's watermelon production has continued to develop, and the planting area, total output and per unit yield have all increased significantly. According to the data of the United Nations Food and Agriculture Organization Statistical Database (FAOSTAT) in 2013, there are 117 countries in the world that grow watermelon. No. 1; my...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2521/107
Inventor 李莉冯耿王锡锋刘文文
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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