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Primer composition for detecting pepper mottle potyvirus as well as application of primer composition, kit consisting of primer composition and application of kit

A technology of capsicum mottle virus and primer composition, which is applied in the field of agricultural science, can solve the problems of cumbersome operating instruments, cumbersome electrophoresis and ultraviolet observation process, etc., and achieve the effects of high sensitivity, convenient and fast detection method, and strong specificity

Active Publication Date: 2016-01-27
HUNAN PLANT PROTECTION INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2011, the occurrence of this virus disease was reported in Taiwan Province of my country, but there is no report of the harm of PepMoV in the inland of my country
[0003] At present, the detection methods for PepMoV mainly include electron microscopy, RT-PCR and qRT-PCR, but these methods require cumbersome operations and expensive instruments. observation process

Method used

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  • Primer composition for detecting pepper mottle potyvirus as well as application of primer composition, kit consisting of primer composition and application of kit
  • Primer composition for detecting pepper mottle potyvirus as well as application of primer composition, kit consisting of primer composition and application of kit
  • Primer composition for detecting pepper mottle potyvirus as well as application of primer composition, kit consisting of primer composition and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] According to the nucleotide sequence of pepper mottle virus, the relatively conserved and specific region 4180-4378 bp in the sequence was selected, and the corresponding region on LN832375 was used to design primers through the online primer explorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index. html) to design primers, and finally select a set of most suitable primers by comparing the stability of the corresponding 3' or 5' ends of the primers and the primer dimer parameters, specifically the outer primer F3, the outer primer B3, and the inner primer FIP , Internal primer BIP.

[0037]Wherein, the DNA sequence of the outer primer F3 is the DNA sequence described in SEQ ID NO.1, specifically: CTTCAATGGCATTTAGAAGCT.

[0038] The DNA sequence of the outer primer B3 is the DNA sequence described in SEQ ID NO.2, specifically: CCGTATTGGATCATGTCACAA.

[0039] The DNA sequence of the internal primer FIP is the DNA sequence described in SEQ ID NO.3, specifically: GTGAACTCCAC...

Embodiment 2

[0042] A detection kit, comprising:

[0043] Outer primer F3 (concentration 10μM): 0.5μl;

[0044] Outer primer B3 (concentration 10μM): 0.5μl;

[0045] Internal primer FIP (concentration 40μM): 1μl;

[0046] Internal primer BIP (concentration 40μM): 1μl;

[0047] 2×RM reaction buffer: 12.5 μl;

[0048] BstDNA polymerase: 1 μl.

[0049] Supplement DEPC water to: 25 μl.

[0050] An application method of the detection kit in this embodiment, specifically comprising the following steps:

[0051] (1) Extraction of total plant RNA: Collect diseased plants from Fujian, Hunan and other places, and use RT-PCR to screen pepper mottle virus samples, refer to TRIzol-A + Instructions for Reagents (TIANGEN) to extract total RNA from capsicum mottle virus samples. Refer to instructions for Hiscript??? 1ststrandcDNAsynthesisKit (Vazyme), use Randomhexamers to reverse transcribe total RNA into cDNA.

[0052] (2) LAMP reaction:

[0053] The cDNA concentration obtained in step (1) was m...

Embodiment 3

[0064] Embodiment 3: Investigate the influence of reaction temperature on detection effect during RT-LAMP reaction process.

[0065] (1) Extract the total RNA of the plant and reverse transcribe it into cDNA, as in Example 1.

[0066] (2) LAMP reaction:

[0067] Measure the concentration of cDNA obtained in step (1) with a microplate reader: 0.147 μg / μl; then dilute 10 times with DEPC water and store at -20°C as a template. Take 2 μL of the above template and add it into the detection kit of Example 1 to obtain a detection system. Divide the detection system into 7 parts on average, react at 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, and 66°C for 60 minutes, and then react at 80°C for 5 minutes to terminate the reaction to obtain the amplification product .

[0068] (3) Detection:

[0069] Take 2.5 μL of the amplification product obtained in step (2), 2 μL of loading buffer, and 3 μL of SYBRGreenI (1:5000) for loading, and electrophoresis on 2% agarose gel.

[0070] Figure 4 i...

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Abstract

The invention discloses a primer composition for detecting pepper mottle potyvirus as well as application of the primer composition, a kit consisting of the primer composition and application of the kit. The primer composition comprises an outer primer and an inner primer, wherein the outer primer comprises an outer primer F3 and an outer primer B3; the DNA sequence of the outer primer F3 is as shown in SEQ ID NO.1; the DNA sequence of the outer primer B3 is as shown in SEQ ID NO.2; the inner primer comprises an inner primer FIP and an inner primer BIP; the DNA sequence of the inner primer FIP is as shown in SEQ ID NO.3; the DNA sequence of the inner primer BIP is as shown in SEQ ID NO.4. The primer composition and the kit consisting of the primer composition can be applied to rapid diagnosis and identification of pepper mottle potyvirus, and have the advantages of good detection sensitivity, good specificity, simplicity, convenience and rapidness in operation, and the like.

Description

technical field [0001] The invention relates to the field of agricultural science and technology, in particular to a primer composition for detecting capsicum mottle virus and its application, a kit composed of the primer composition and the application of the kit. Background technique [0002] pepper mottle virus ( PepperMottleVirus , PepMoV) is a serious virus disease on peppers, PepMoV is a (+) single-stranded RNA virus, and belongs to the genus Potatovirus ( Potyvirus ), Potatoviridae ( Potyviridae ) definite species. The virus is non-persistently transmitted by aphids and mainly infects important crops of the Solanaceae family, including peppers and tomatoes. The field symptoms of PepMoV are mainly dwarfed plants, leaf mosaic, spots, atrophy, fruit deformity, resulting in reduced yield, and more seriously, reduced fruit commerciality, resulting in huge economic losses. The virus was first reported in the United States, followed by reports of harmful effects in Sou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107C12Q2565/125
Inventor 刘勇张松柏张德咏刘健罗香文郑立敏彭静杜娇
Owner HUNAN PLANT PROTECTION INST
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