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One-step method real-time fluorescent reverse transcription PCR detection of bean pod mottle virus

A mottled virus and real-time fluorescence technology, applied in the field of bean pod mottled virus, can solve the problems of soybean production loss and achieve the effect of rapid detection, strong specificity and high sensitivity

Inactive Publication Date: 2011-02-02
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Recently, my country's entry-exit ports have repeatedly detected vegetable pod mottle virus from imported American soybeans. Since 2005, the Tianjin Entry-Exit Inspection and Quarantine Bureau has detected the virus through ELISA more than a dozen times. It has constituted a serious threat to my country's soybean production. There is a real threat, if the virus is introduced into the country along with imported soybeans and colonized, it will cause immeasurable losses to domestic soybean production

Method used

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  • One-step method real-time fluorescent reverse transcription PCR detection of bean pod mottle virus
  • One-step method real-time fluorescent reverse transcription PCR detection of bean pod mottle virus
  • One-step method real-time fluorescent reverse transcription PCR detection of bean pod mottle virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: RNA Extraction of Soybean Pod Mottle Virus

[0023] The strains involved in the experiment included 10 soybean-hosted virus positive controls purchased from Agdia in the United States, and 4 BPMV isolates intercepted from American soybeans in recent years by our laboratory. The relevant information is shown in Table 1.

[0024] Table 1

[0025]

[0026] From the soybean seeds, the seeds with stains around the hilum were visually selected, and the seed coats with stains were carefully torn off with tweezers, and placed in a dry 1.5mL centrifuge tube treated with DEPC water, and the positive quality control powder was carefully placed in the treated In a dry 1.5mL centrifuge tube treated with DEPC water, add liquid nitrogen to the centrifuge tube and grind thoroughly according to the Simply P Total RNA of Bioer Technology Co., Ltd.

[0027] Extraction Kit (number: BSC52S2) was used to extract viral RNA.

Embodiment 2

[0028] Example 2: The establishment of a one-step real-time fluorescent RT-PCR detection method for BPMV

[0029] Probes and primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd. Fluorescence PCR reagent is ExTaq from TaKaRa Company TM R-PCR version 2.1 kit, real-time fluorescent PCR instrument is ABI7000 type, and fluorescent PCR tubes and caps are purchased from ABI Company. The reverse transcription reagent is the BioRT One Step RT-PCR kit from Hangzhou Bioer Technology Co., Ltd.

[0030] (1) Preparation of reaction mixture

[0031]The reaction system is 25 μL, including: 10×RT-PCR Buffer 2.5 μL (which contains MgCl with a final concentration of 3 mmol / L 2 ), 10×Ex Taq Buffer 2.5μL (which contains MgCl with a final concentration of 1.5mmol / L 2 ), a total of 1 μL of four dNTPs with a concentration of 2.5 mmol / L, 1 μL of primers with a concentration of 10 μmol / L, 1 μL of probes with a concentration of 20 μmol / L, 1 μL RNase inhibitor (40 U / μL), and 1 μL temp...

Embodiment 3

[0044] Embodiment 3: Sensitivity test of one-step real-time fluorescent RT-PCR detection method

[0045] The concentration of the cloning plasmid solution of BPMV cp gene was measured and set at 200ng / μL, and then diluted 10 times in equal proportions, and the sensitivity test of the one-step real-time fluorescent RT-PCR detection method was carried out.

[0046] (1) Preparation of reaction mixture

[0047] The reaction system is 25 μL, including: 10×Ex Taq Buffer 2.5 μL (which contains MgCl at a final concentration of 1.5 mmol / L 2 ), a total of 1 μL of four dNTPs with a concentration of 2.5 mmol / L, 1 μL of primers with a concentration of 10 μmol / L, 1 μL of probes with a concentration of 20 mmol / L, 1 μL of plasmid solution, and 0.5 μL of Ex Taq DNA polymerase (5 U / μL), add sterile water to 25μL.

[0048] (2) The real-time fluorescent PCR reaction program is

[0049] Pre-denaturation at 95°C for 10 minutes;

[0050] Denaturation at 95°C for 15 seconds;

[0051] 60°C annea...

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Abstract

The invention relates to a method for detecting bean pod mottle virus (BPMV for short) by use of one-step real-time fluorescence RT-PCR technology, belonging to the virus detecting field of leguminous crops. The invention designs and synthesizes one set of Taqman probe and a primer thereof against the BPMV cp gene sequence in GenBank and establishes the method for detecting the BPMV by use of theone-step real-time fluorescence RT-PCR technology. The method is characterized by convenience, fastness, strong specificity and high sensitivity and has a detecting lower limit of 20fg / microliter anda detecting time of 3h to 4h.

Description

technical field [0001] The invention relates to a method for detecting bean pod mottle virus (Bean pod mottlevirus, referred to as BPMV) parasitic on soybeans by using one-step real-time fluorescence reverse transcriptase PCR (Real-time fluorescence reverse transcriptase PCR, referred to as real-time fluorescent RT-PCR) technology, belonging to Field of virus detection in leguminous crops. Background technique [0002] Soybean originates in China and is an important economic crop in my country. Bean pod mottle virus is an important seed-borne virus that harms soybeans and other leguminous plants. It is also a quarantine pest that is prohibited from entering the country. The loss of yield can also lead to the danger of germplasm transfer and the spread of the virus. At the same time, the virus may also have an adverse effect on the viability and genetic integrity of the preserved seeds. At present, there is no distribution of this virus in our country. Bean pod mottle virus ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/70
Inventor 廖芳郭京泽张裕君黄国明黄庆林刘鹏
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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