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Nested RT-PCR detection kit of bean pod mottle virus ELISA and detection method of nested RT-PCR detection kit

A technology of RT-PCR and detection kit, which is applied in the direction of biochemical equipment and methods, microbe determination/inspection, etc., to achieve good accuracy, improve specificity and accuracy, and strong specificity

Inactive Publication Date: 2016-10-12
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no ELISA-nested RT-PCR detection kit specially used for BPMV detection that combines ELISA and RT-PCR.

Method used

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  • Nested RT-PCR detection kit of bean pod mottle virus ELISA and detection method of nested RT-PCR detection kit
  • Nested RT-PCR detection kit of bean pod mottle virus ELISA and detection method of nested RT-PCR detection kit
  • Nested RT-PCR detection kit of bean pod mottle virus ELISA and detection method of nested RT-PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Configuration of Bean Pod Mottle Virus ELISA-Nested RT-PCR Detection Kit (10 detections)

[0041] 1) PBST buffer: 1×, 1 tube (300mL);

[0042] 2) Coating buffer: 1×, 1 tube (50mL);

[0043] 3) BPMV antibody, 1 tube (500 μL);

[0044] 4) Enzyme-labeled antibody buffer: 1×, 1 tube (50mL);

[0045] 5) BPMV enzyme-labeled antibody, 1 tube (500 μL);

[0046] 6) pNPP, 1 tube (0.3g);

[0047] 7) Outer forward primer: 10 μmol / L, 1 tube (100 μL);

[0048] 8) Reverse primer: 10 μmol / L, 1 tube (100 μL);

[0049] 9) Inner forward primer: 10 μmol / L, 1 tube (100 μL);

[0050] 10) RT Buffer: 5×, 1 tube (100μL);

[0051] 11) RNase inhibitor: 40U / μL, 1 tube (100μL);

[0052] 12) Reverse transcriptase: 200U / μL, 1 tube (100μL);

[0053] 13) dNTPs: 10mmol / L, 1 tube (100μL);

[0054] 14) Taq PCR Mix: 2×, 1 tube (200μL);

[0055] 15) Positive control sample of bean pod mottle virus, 1 tube (1 mL);

[0056] 16) Negative control sample without bean pod mottle virus, 1 tu...

Embodiment 2

[0058] Embodiment 2: the detection method of bean pod mottle virus ELISA-nested RT-PCR detection kit

[0059] Utilize the detection method of bean pod mottle virus ELISA-nested RT-PCR detection kit, comprise the following steps:

[0060] 1) ELISA detection: Dilute the BPMV antibody 200 times with coating buffer, add 100 μL to each well of the microplate, and bathe in water at 37°C for 2 hours; drain the liquid in the microplate, wash the plate 3 times with PBST buffer, 3 minutes each time; dilute the BPMV enzyme-labeled antibody 200 times with the enzyme-labeled antibody buffer and mix it with the sample extract in equal volumes, then add 200 μL of the mixed solution to each well of the enzyme-labeled plate, coat at 4°C overnight, and set Positive control group and negative control group; pour out the liquid in the microplate, wash the plate with PBST buffer 3 times, each time for 3 minutes; add 100 μL of freshly prepared pNPP solution with a concentration of 1 mg / mL to each w...

Embodiment 3

[0065] Example 3: Specificity determination of bean pod mottle virus ELISA-nested RT-PCR detection kit

[0066] 1) Preparation of virus sample extracts: bean pod mottle virus (BPMV), soybean mosaic virus (SMV), tobacco ringspot virus ( Tobacco ringsport virus , TRSV), tomato ringspot virus ( Tomato ringspot virus , ToRSV), Arabidopsis mosaic virus ( Arabis mosaic virus , ArMV) and Southern Bean Mosaic Virus ( Southern bean mosaic virus , SBMV) and other 6 virus samples as materials, add PBST buffer at a ratio of 1:20 (W / V), grind thoroughly, centrifuge at 10 000 rpm for 10 min at 4°C, and take the supernatant as virus sample extract;

[0067] 2) ELISA detection: Dilute the BPMV antibody 200 times with coating buffer, add 100 μL to each well of the microplate, and bathe in water at 37°C for 2 hours; drain the liquid in the microplate, wash the plate 3 times with PBST buffer, 3 minutes each time; dilute the BPMV enzyme-labeled antibody 200 times with the enzyme-labeled an...

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Abstract

The invention relates to a nested RT-PCR detection kit of bean pod mottle virus ELISA and a detection method of the nested RT-PCR detection kit. The kit comprises a PBST buffer solution, a coating buffer solution, a BPMV antibody, an enzyme-labeled antibody buffer solution, a BPMV enzyme-labeled antibody, pNPP, an outside forward primer, a reverse primer, an inside forward primer, an RT buffer, an RNA enzyme inhibitor, reverse transcriptase, dNTPs, Taq PCR Mix, a positive control, a negative control and RNase-free ddH2O. ELISA and nested RT-PCR are combined, firstly, ELISA detection is performed on a sample extracting solution, then reverse transcription is directly conducted in a hole of an enzyme-labeled plate to synthesize cDNA, cDNA is taken as a template to perform the first turn of PCR, then, a product of the first turn of PCR is taken as a template to perform the second turn of PCR, amplification products are subjected to agarose gel electrophoresis detection, and if specific target fragments with the sizes of 296 bp appear, then the detected virus is determined as bean pod mottle virus. The nested RT-PCR detection kit of bean pod mottle virus ELISA is high in specificity, excellent in accuracy, high in sensitivity, simple and convenient to operate, and suitable for quick detection and determination of bean pod mottle virus on import and export and agricultural production.

Description

technical field [0001] The invention relates to an ELISA-nested RT-PCR detection kit for vegetable pod mottle virus and a detection method thereof, belonging to the technical field of plant quarantine, and suitable for rapid detection and identification of vegetable pod mottle virus entering and exiting the country and agricultural production. Background technique [0002] Bean pod mottle virus ( Bean pod mottle virus , BPMV) belongs to the cowpea mosaic virus family ( Comoviridae ), cowpea mosaic virus ( Comovirus ) member, is one of the main seed-borne viruses that harm soybeans, and the yield loss caused by infecting soybeans is serious. The earlier the infection time is, the greater the losses are, and soybean mosaic virus ( Soybean Mosaic Virus , SMV) co-infection caused soybean yield losses of more than 85%. In addition, soybeans infected with BPMV were susceptible to Phomopsis ( Phomopsis ) fungus damage, resulting in a further reduction in the quality of soyb...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6804C12Q1/6848C12Q1/701C12Q2527/127C12Q2549/119C12Q2521/107
Inventor 沈建国廖富荣蔡伟高芳銮
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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