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Bean pod mottle virus fluorescent quantitative RT-PCR kit and detection method thereof

A mottled virus, fluorescent quantitative technology, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of unseen bean pod mottled virus detection, unreported, etc., and achieve false positives The effect of low probability, high detection sensitivity and accuracy, and high sensitivity and accuracy

Inactive Publication Date: 2009-03-11
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] At present, TaqMan-MGB fluorescent quantitative RT-PCR technology has been successfully applied to the rapid detection of some plant viruses, but so far, TaqMan- MGB fluorescent RT-PCR quantitative detection method has not been reported so far, let alone a fluorescent quantitative RT-PCR kit specially used for the detection of bean pod mottle virus

Method used

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  • Bean pod mottle virus fluorescent quantitative RT-PCR kit and detection method thereof
  • Bean pod mottle virus fluorescent quantitative RT-PCR kit and detection method thereof
  • Bean pod mottle virus fluorescent quantitative RT-PCR kit and detection method thereof

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Embodiment 1

[0032] Embodiment 1: the configuration of the common bean pod mottle virus fluorescent quantitative RT-PCR kit (this embodiment provides 40 times of detection, and the specific quantity configured in the kit can be adjusted as required during specific implementation)

[0033] 1) Reverse transcriptase: 200U / μL, 1 tube (10μL);

[0034] 2) RNase inhibitor: 40U / μL, 1 tube (20μL);

[0035] 3) Hot start Taq DNA polymerase: 5U / μL, 1 tube (20μL);

[0036] 4) Fluorescent quantitative reaction solution: 0.2 μmol / L forward primer, 0.2 μmol / L reverse primer, 0.2 μmol / L fluorescent probe, 5 mmol / L Mgcl 2 , 0.2mmol / L dNTP, 1 tube (40μL);

[0037] Wherein, the sequence of the forward primer is: 5'-ATCAGCCAAATTTCCAAATGCTA-3';

[0038] The sequence of the reverse primer is: 5'-CTATTCTCATGAATATGCCAAATGC-3';

[0039] The sequence of the fluorescent probe is: 5'-FAM-CTCCACATTGAAAGAAC-MGB-3', the fluorescent reporter group labeled at the 5' end of the fluorescent probe is FAM, and the fluorescen...

Embodiment 2

[0043] Example 2: The detection method of the common bean pod mottle virus fluorescent quantitative RT-PCR kit

[0044] The detection method of above-mentioned bean pod mottle virus fluorescence quantitative RT-PCR kit comprises the following steps:

[0045] 1) Add 8 μL of total RNA of the sample to be tested, 12.5 μL of 2×Buffer, 0.5 μL of RNase inhibitor, 0.25 μL of reverse transcriptase, 0.5 μL of hot-start Taq DNA polymerase, and 1 μL of fluorescence quantitative reaction solution into the PCR tube, and then add 2.25 μL of RNase-free sterile double-distilled water to make the total reaction volume 25 μL;

[0046] 2) RT-PCR reaction: take the mixed reaction solution in step 1, first reverse transcribe at 42°C for 30 minutes, then denature at 94°C for 2 minutes, and then enter the following cycle: denaturation at 94°C for 5 seconds, annealing and extension at 60°C for 30 seconds, a total of 40 cycles ;

[0047] 3) Judgment result: After the RT-PCR reaction is completed, ju...

Embodiment 3

[0048] Example 3: Detection of pod mottle virus on imported soybean samples

[0049] 1) Extraction of total RNA from soybean samples: Take 100mg of susceptible tissue and grind it into powder with liquid nitrogen, quickly transfer it to a 1.5mL centrifuge tube, add 1mL Trizol, shake vigorously (fully homogenate), centrifuge at 12000g for 10min at 4°C Remove the insoluble matter, transfer the supernatant to a new 1.5mL centrifuge tube, let stand at room temperature for 5min, add 0.2mL chloroform, shake vigorously for 15s, let stand at room temperature for 2-10min, centrifuge at 12000g for 15min at 4°C, and dilute the water The phase was transferred to a new 1.5mL centrifuge tube, 0.5mL isopropanol was added, the liquid in the tube was gently inverted and mixed, and after standing at room temperature for 15min, centrifuged at 12000g for 10min at 4°C, and the supernatant was discarded. The precipitate was discarded with 75% ethanol (DEPC treated ddH 2 O water configuration), gen...

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Abstract

The invention relates to a bean pod mottle virus fluorescence quantitative RT-PCR reagent kit and a detection method thereof. The reagent kit comprises reverse transcriptase, an RNA enzyme inhibitor, hot start Taq DNA polymerase, fluorescence quantitative reaction solution, 2 x Buffer, sterilized double distilled water containing no RNA enzyme and positive contrast. The reagent kit and the detection method thereof use a conservative fragment of coat protein CP gene of bean pod mottle virus as a target, design a specificity primer and a TaqMan-MGB fluorescent probe, utilize the fluorescence quantitative RT-PCR technology for detecting the bean pod mottle virus, have the advantages of high sensitivity, strong specificity, rapidness, simplicity, accuracy and reliability, need no PCR posttreatment step, can effectively prevent cross contamination and false positive, are suitable for entry and exit plant quarantine, species introduction and rapid detection and diagnosis of the bean pod mottle virus in agricultural production, and can be used in real-time monitoring simultaneously, prewarning and forecasting of the epidemic situation of the bean pod mottle virus and have wide application and prospect.

Description

technical field [0001] The invention relates to a fluorescent quantitative RT-PCR kit for the detection of the bean pod mottle virus and a detection method thereof, which belongs to the field of plant quarantine technology and is specially used for the rapid detection of the bean pod mottle virus, and is not only suitable for entry and exit The detection and diagnosis of vegetable pod mottle virus in plant quarantine, introduction and agricultural production can also be used for real-time monitoring and early warning of the epidemic situation of vegetable pod mottle virus disease. Background technique [0002] The bean pod mottle disease caused by bean pod mottle virus (BPMV) infecting soybean is one of the main viral diseases that harm soybean, and it causes serious losses to soybean production. According to statistics, the average yield loss caused by BPMV infection alone ranges from 3% to 52%, and the yield loss is between 10% and 40% in most areas. The impact of BPMV on...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 沈建国郭琼霞虞赟王念武黄可辉翁瑞泉
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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