Primer pair set for East Asian passiflora virus whole-genome determination and application of primer pair set

A whole genome and primer pair technology, applied in the field of molecular biology detection and identification, can solve the problem of inability to accurately identify viruses

Active Publication Date: 2020-02-28
POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are great limitations in the detection and identification of East Asian Passiflora virus by traditional identification host inoculation, serological detection and electron microscope observation methods, which cannot be quickly and accurately identified for the virus, and false negative or false positive results are prone to occur

Method used

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  • Primer pair set for East Asian passiflora virus whole-genome determination and application of primer pair set
  • Primer pair set for East Asian passiflora virus whole-genome determination and application of primer pair set
  • Primer pair set for East Asian passiflora virus whole-genome determination and application of primer pair set

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Experimental program
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Effect test

Embodiment 1

[0127] Embodiment 1, be used for the optimization of the design and the amplification condition of the complete set of primers that are used to amplify the whole genome of Passionflower virus in East Asia

[0128] One, be used for the design of the complete set of primer pair that is used for amplifying the full genome of East Asia passionflower virus

[0129] Utilizing the sequences amplified by general primers of Potyvirus and the gene sequence of East Asian Passiflora virus published so far, primers for whole genome amplification and sequencing of East Asian Passiflora virus were designed. The primer sequences are shown in Table 1.

[0130] Table 1. The whole genome amplification and sequencing primers of East Asian passionflower virus

[0131]

[0132]

[0133]

[0134] Note: F means upstream primer, R means downstream primer

[0135] After repeated design, testing and optimization, 7 pairs of primer pairs for cloning the whole genome of Passionflower virus in Ea...

Embodiment 2

[0149] Embodiment 2, East Asia Passiflora virus China Fujian isolate whole genome sequencing method

[0150] 1. Extraction of total RNA from samples

[0151] Take 0.1 g of passion flower leaves (Fujian, China) infected with East Asian passion flower virus, add TrizoL reagent, extract total RNA according to conventional methods, and detect by agarose gel electrophoresis, and obtain 50 μL of good quality total RNA.

[0152] 2. Whole genome RT-PCR amplification

[0153] 1) Reverse transcription: Take 3 μL of extracted total RNA as a template, add 1 μL of Random Primer, ddH 2 After 7 μL of O, first keep warm at 70°C for 10 minutes, then quickly ice-bath for 5 minutes, then add 5×RT Buffer 5 μL, dNTPs (10 mmol L -1 )2μL, RNasin (40U·μl -1 ) 1 μL and M-MuLV reverse transcriptase (200U·μl -1 ) 1 μL, and finally, cDNA was synthesized by incubating at 42°C for 1h and at 70°C for 10min.

[0154] 2) PCR amplification: with the cDNA obtained by step 1) reverse transcription as a templa...

Embodiment 3

[0164] Embodiment 3, the detection and the identification of Chinese Taiwan Passiflora sample East Asia Passiflora virus

[0165] 1. Extraction of total RNA from samples

[0166] Take 2 samples (numbers TW-1 and TW-2) suspected of being infected with East Asian passionflower virus from Taiwan, China, as materials, weigh 0.1g respectively, add TrizoL reagent, and extract total RNA according to conventional methods. And detected by agarose gel electrophoresis, each 50 μL of total RNA with good quality was obtained.

[0167] 2. Whole genome RT-PCR amplification

[0168] 1) Reverse transcription: Take 3 μL of extracted total RNA as a template, add 1 μL of Random Primer, ddH 2 After 7 μL of O, first keep warm at 70°C for 10 minutes, then quickly ice-bath for 5 minutes, then add 5×RT Buffer 5 μL, dNTPs (10 mmol L -1 )2μL, RNasin (40U·μl -1 ) 1 μL and M-MuLV reverse transcriptase (200U·μl -1 ) 1 μL, and finally, cDNA was synthesized by incubating at 42°C for 1h and at 70°C for 1...

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Abstract

The invention discloses a primer pair set for East Asian passiflora virus whole-genome determination and application of the primer pair set. The primer pair set comprises primer pairs 1-7, and nucleotide sequences of the seven primer pairs are shown as sequences 1-14 as shown in the specification. East Asian passiflora virus whole-genome sequencing includes that RNA of an East Asian passiflora virus infected sample is taken as a template, and RT-PCR amplification and sequencing are realized by virtue of the seven primer pairs. The method not only can be used for East Asian passiflora virus whole-genome amplification and sequencing but also can be used for rapid detection and identification of the East Asian passiflora virus and has advantages of simplicity and convenience in operation, high specificity, high sensitivity and high accuracy. In addition, powerful technical means is also provided for evolution analysis, molecular epidemiologic study and scientific prevention and control ofthe East Asian passiflora virus, and important significances to prevention and control of the East Asian passiflora virus and protection of passion flower production safety are achieved.

Description

technical field [0001] The invention relates to the field of molecular biology detection and identification, in particular to a set of primer pairs for the determination of the whole genome of East Asian passionflower virus and its application, which is suitable for the detection and identification of East Asian passionflower virus in agricultural production and port plant quarantine . Background technique [0002] Passionflower (Passiflora edulia Sims) is a kind of fragrant and delicious fruit, which not only has high nutritional value, but also has high economic value. As the second largest disease in the production of passionflower, virus disease has a great impact on the yield and quality of passionflower. East Asian passionflower virus belongs to Potyviridae family (Potyviridae), a member of Potyvirus genus (Potyvirus), and is a single-stranded positive-sense RNA virus. The virus is one of the main pathogens that have been clearly reported to cause passionflower ligni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/94
CPCC12Q1/701C12Q1/686C12Q2600/158C12Q2521/107C12Q2565/125Y02A50/30
Inventor 谢丽雪李韬张立杰郑姗张小艳
Owner POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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