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TC (Test tube Capturing)-RT (Reverse Transcription)-nested-PCR (Polymerase Chain Reaction) detection kit for cordate telosma mosaic virus and detection method of cordate telosma mosaic virus

A technology of evening primrose flower and leaf virus, applied in the direction of microbial determination/inspection, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve unseen problems

Pending Publication Date: 2018-09-21
POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

About TC-RT-nested PCR (test tube capture nested RT-PCR) technology to detect tuberose mosaic virus, there is no report at home and abroad so far, and there is no TC-RT-nested PCR specially used for the detection of tuberose mosaic virus Detection kit

Method used

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  • TC (Test tube Capturing)-RT (Reverse Transcription)-nested-PCR (Polymerase Chain Reaction) detection kit for cordate telosma mosaic virus and detection method of cordate telosma mosaic virus
  • TC (Test tube Capturing)-RT (Reverse Transcription)-nested-PCR (Polymerase Chain Reaction) detection kit for cordate telosma mosaic virus and detection method of cordate telosma mosaic virus
  • TC (Test tube Capturing)-RT (Reverse Transcription)-nested-PCR (Polymerase Chain Reaction) detection kit for cordate telosma mosaic virus and detection method of cordate telosma mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Embodiment 1, the complete set of specific primers and TC-RT-nested PCR detection kit of tuberose mosaic virus

[0094] 1. A complete set of specific primers for the detection of tuberose mosaic virus

[0095] According to the coat protein gene of the tuberose mosaic virus, the present invention designs two pairs of specific primers on the outside and inside, wherein the amplification region of the inside specific primer is located inside the amplification region of the outside specific primer. The primer sequences are as follows:

[0096] Outer forward primer: 5'-TACAAGCCTAACCAAACGGAT-3' (SEQ ID NO: 1);

[0097] Outer reverse primer: 5'-TGATTTACATCTCGTGCAGT-3' (SEQ ID NO: 2);

[0098] Inner forward primer: 5'-TTCAACACAAGGGCAACCAA-3' (SEQ ID NO: 3);

[0099] Internal reverse primer: 5'-CCTTTCAGTATCTTCGCCAGT-3' (SEQ ID NO: 4).

[0100] 2. TC-RT-nested PCR kit for detecting tuberose mosaic virus (10 detections)

[0101] The TC-RT-nested PCR kit for detecting tuberose...

Embodiment 2

[0116] The detection method of the TC-RT-nested PCR detection kit of embodiment 2, tuberose mosaic virus

[0117] 1. Establishment of TC-RT-nested PCR detection system for tuberose mosaic virus and optimization of reaction conditions

[0118] 1. Preparation of the sample extract: take the sample containing the tuberose mosaic virus as the sample to be tested, add the virus extraction buffer in a ratio of 1g:10mL according to the mass volume ratio of the sample to the virus extraction buffer, fully grind, 4 Centrifuge at 10,000 g for 10 min, and collect the supernatant as sample extract.

[0119] 2. Reverse transcription: Add 50 μL of sample extract into the PCR tube, incubate at 37°C for 4 hours; pour off the sample extract, wash the tube 3 times with PBST buffer, and then use RNase-free ddH 2 Wash the tube once, and perform reverse transcription directly in the PCR tube; add RNase-free ddH to the PCR tube first 2 O 10 μL and outer reverse primer 1 μL at a concentration of 1...

Embodiment 3

[0139] Embodiment 3, the specificity determination of the TC-RT-nested PCR detection kit of tuberose mosaic virus

[0140] Take healthy passionflower leaves, cucumber mosaic virus (CMV) samples, beet mosaic virus (BtMV) samples, soybean mosaic virus (SMV) samples, tiger eye perennial mosaic virus (OrMV) samples, East Asian passionflower Virus (EAPV) samples, passionflower latent virus (PLV) samples, and evening primrose mosaic virus (TeMV) samples were used as samples to be tested. The TC-RT-nested PCR detection kit for the tuberose mosaic virus in Example 1 was used to detect the tuberose mosaic virus in each sample to be tested. At the same time, the sample not containing tuberose mosaic virus (TeMV) was used as a negative control. Specific steps are as follows:

[0141] 1. Preparation of sample extract: the following samples to be tested: healthy passionflower leaves, cucumber mosaic virus (CMV) samples, beet mosaic virus (BtMV) samples, soybean mosaic virus (SMV) samples...

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Abstract

The invention discloses a TC (Test tube Capturing)-RT (Reverse Transcription)-nested-PCR (Polymerase Chain Reaction) detection kit for cordate telosma mosaic virus and a detection method of the cordate telosma mosaic virus. The detection kit for the cordate telosma mosaic virus, disclosed by the invention, is prepared from a virus extracting buffering solution, a PBST (Phosphate Buffered Saline with Tween-20) buffering solution, an outer-side forward primer, an outer-side backward primer, an inner-side forward primer, an inner-side backward primer, RT Buffer, an RNA (Ribonucleic Acid) enzyme inhibition factor, reverse transcriptase, dNTPs (deoxyribonucleotide triphosphates), PCR Mix, positive control and negative control. According to the detection kit disclosed by the invention, two pairsof specific primers are designed according to a coat protein gene of the cordate telosma mosaic virus; the cordate telosma mosaic virus is detected by adopting a TC-RT-nested PCR technology. The detection method does not need an RNA extraction step, has the advantages of simplicity in operation, low cost, strong specificity, high sensitivity and good accuracy and is applicable to rapid detectionand monitoring of the cordate telosma mosaic virus in agricultural production and entry and exit ports.

Description

technical field [0001] The invention relates to a TC-RT-nested-PCR detection kit for the tuberose mosaic virus and a detection method thereof, which belong to the technical field of plant quarantine and are suitable for rapid detection and detection of the tuberose tuberculosis virus in agricultural production and entry and exit ports. monitor. Background technique [0002] Passiflora edulia Sims (Passiflora edulia Sims) is a perennial herbaceous vine of the Passiflora family (Passifloraceae), native to Brazil and Argentina in South America, widely distributed in tropical and subtropical regions, and mainly distributed in Taiwan in my country , Guangdong, Fujian, Guangxi, Zhejiang, Sichuan and other regions. As a high-grade natural beverage crop, passionflower is often harmed by diseases and insect pests during the cultivation and production process. Among them, viral disease is the second largest disease in the production of passionflower, which seriously affects the yield ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6848C12N15/11
CPCC12Q1/6848C12Q1/701C12Q2549/119
Inventor 谢丽雪李韬张立杰郑姗张小艳
Owner POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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