Vigna radiata specific PCR (polymerase chain reaction) primer pair and method for detecting vigna radiata in phytophagous insect bodies

A primer pair and specificity technology, which is applied in the field of mung bean-specific PCR primer pair and its detection in herbivorous insects, can solve the problems of difficult success, high cost, and laboriousness, and achieve high accuracy, high sensitivity, and easy operation. simple effect

Inactive Publication Date: 2015-12-09
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods all have defects such as time...

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  • Vigna radiata specific PCR (polymerase chain reaction) primer pair and method for detecting vigna radiata in phytophagous insect bodies
  • Vigna radiata specific PCR (polymerase chain reaction) primer pair and method for detecting vigna radiata in phytophagous insect bodies
  • Vigna radiata specific PCR (polymerase chain reaction) primer pair and method for detecting vigna radiata in phytophagous insect bodies

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Experimental program
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Embodiment 1

[0025] The amplification effect of specific primer LraF1 / LraR1 of embodiment 1 to mung bean

[0026] 1. Preparation of mung bean genomic DNA

[0027] The mung bean leaves were ground in a mortar, and the mung bean genome was extracted with the DNAquick Plant System Quick Plant Genomic DNA Extraction Kit (non-spin column type) (Tiangen Company). The DNA solution was stored at -20°C for later use, and 1 μL of the solution was taken as a DNA template during PCR amplification.

[0028] 2. Synthesis of specific primers for mung bean detection

[0029] According to the mung bean rbcL sequence (AP014692.1) published in GenBank, the following specific PCR primer pairs were designed:

[0030] LraF1: 5'-TCACATCGAACCTGTTCCTGGG-3' (SeqIDNo.1)

[0031] LraR1: 5'-CCACCACGAAGACATTCATAAA-3' (SeqIDNo.2)

[0032] 3. PCR amplification

[0033] Reaction system 20μL:

[0034]

[0035] PCR amplification program: 95°C pre-denaturation for 10 minutes; 95°C for 30 seconds, 58°C for 30 seconds...

Embodiment 2

[0045] Example 2 Primer LraF1 / LraR1 Determination of Mung Bean Minimum Detection Amount

[0046] 1. For the preparation of the mung bean template genome, refer to step 1 in Example 1.

[0047] 2) Preparation of mung bean plasmid

[0048] The mung bean genomic DNA was amplified according to the method in step 3 in Example 1, and detected by 1.5% agarose gel electrophoresis after the amplification was completed. Use the Axygen Agarose Gel DNA Purification Kit to recover the PCR product, the PCR product is cloned on the pEasy-T3 vector after being recovered by gel (operate according to the kit instructions), the recombinant plasmid is transformed into Trans1-T1 competent cells, and then spread on the medium containing Ampicillin / X-gal / IPTG LB plates were cultured upside down at 37°C for 15 hours. After blue-white screening, 10 positive clones were picked and cultured overnight in LB liquid medium containing Amp, and the next day, the plasmid was extracted with the Axygen plasmi...

Embodiment 3

[0050] Example 3 Using primers LraF1 / LraR1 to detect trace amounts of mung beans in insects

[0051] 1. Starvation treatment of 10 Lygus green bugs for 24 hours, feeding with mung bean leaves for 3 hours, and immediately killing them by freezing with liquid nitrogen.

[0052] / 2. Put the single-headed Lygus chinensis in a 1.5ml centrifuge tube and grind it, and extract DNA by CTAB method.

[0053]Add 40 μL of PBS (150 mM NaCl, 150 mM NaCl, 3 PO 4 , pH7.2,), followed by adding 450 μL of TES buffer (0.1MTris, pH8, 10mM EDTA, 2% SDS), 10 μL of proteinase K (10mg / ml) and 10mg of PVP, placed in a metal bath at 58°C for 12h. After taking it out from the metal bath, add 150 μL of 5M NaCl and 65 μL of 10% CTAB, place the sample in the metal bath at 65°C for 10 minutes, add chloroform / isoamyl alcohol (24:1) for extraction after taking it out, add 225 μL after standing still 5MNH 4 Ac, put it on ice for more than 30 minutes, take it out and centrifuge at 12000g for 20 minutes, disca...

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Abstract

The invention provides a vigna radiata specific PCR (polymerase chain reaction) primer pair which is a pair of specific primers designed according to a vigna radiata rbcL sequence (AP014692.1) published in GenBank. The primer pair which is high in specificity has an amplification function for vigna radiata only and has no amplification products for other sympatric species. The invention further provides a method of using the primer pair for detecting trace amount of vigna radiata in phytophagous insect bodies. By adoption of the method and the primers for detecting vigna radiata, high accuracy and simplicity, quickness and high efficiency in operation are realized, detection can be generally finished within five hours, sensitivity is high, and plasmid concentration detection limit is 4.49E+03. By DNA (deoxyribose nucleic acid) amplification of apolygus lucorum eating the vigna radiata, the detection rate reaches 100%, so that feasibility of the method is proved.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to mung bean-specific PCR primer pair and its detection method in herbivorous insects. Background technique [0002] Mung bean (Vignaradiata (L.) Wilczek), belongs to the bean family. Also known as Qingxiaodou, Ludou, Zhidou, etc., it has been cultivated in China for more than two thousand years. Its seeds and stems are widely eaten. Mung bean soup is a summer drink that is often prepared by the family. It is refreshing and appetizing, suitable for all ages. [0003] Mung bean is also a preferred host plant for many insects, such as the green Lygus, three-spotted Lygus, and mung bean elephant. The relationship between insects and plants has always been a hot research topic. The research on the feeding of insects on plants and the transfer and spread of insects among plants is traditionally reflected indirectly through the dynamics of insect population changes, or through...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2565/125
Inventor 王倩杨帆陆宴辉杨益众
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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