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RT-PCR kits and detection methods for detecting eight cherry viruses

A RT-PCR, kit technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to save time, avoid cross-contamination, and reduce the number of operations

Active Publication Date: 2021-01-26
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no reports on the simultaneous detection of the above eight cherry viruses

Method used

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  • RT-PCR kits and detection methods for detecting eight cherry viruses
  • RT-PCR kits and detection methods for detecting eight cherry viruses
  • RT-PCR kits and detection methods for detecting eight cherry viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059]The 8 pairs of specific primers (SEQ ID NO.1-SEQ ID NO.15) designed in this experiment that can be used to detect cherry virus have the characteristics of high specificity and sensitive response. A single band of the target size can be amplified from the sample.

[0060]Use RT-PCR for nucleic acid level virus detection. RT reaction system (25.0μl) (reagents purchased from TakaRa): total plant RNA, 20-50ng, reverse primer (100mM), 5.0μl, 10mM dNTP, 4.0μl, dd H2O, 5.0μl; 80°C, 3min, 5min on ice; 5×Reverse Transcriptase M-MLV Buffer, 5.0μl, ReverseTranscriptase M-MLV, 0.5μl, Recombinant RNase Inhibitor, 0.5μl; 42°C, 90min.

[0061]PCR system (20.0μl): RT product, 2.0μl, forward primer, 0.5μl, reverse primer, 0.5μl, 2×TagMaster Mix (Kangwei company), 10μl, dd H2O, 7.0 μl. Reaction conditions: 94℃, 5min, 94℃, 40s, 50~54℃, 40s, 72℃, 20~35s, 30Cycles, 72℃, 10min. Note: The annealing temperature is the Tm value of the primer with the lower Tm value -5°C.

[0062]The amplified product is subjec...

Embodiment 2

[0064]The purified amplified product is used as a template for multiplex PCR amplification.

[0065]PCR system (100.0μl): template, 50ng, forward primer CVA-5838-F: CGRM-7474-F: LCh1-15849-F: LCh-2-5670-F: PBN-13568-F: PNRS-R3- 115-F: PD-CP-135-F: CM-R3-119-F is 1:1:1:1:1:1:1:1, 16.0μl in total, reverse primer Q0-dT: LCh1-16827-R: LCh-2-6141-R: PBN-14139-R: PNRS-R3-1152-R: PD-CP-564-R: CM-R3-768-R is 2:1:1 :1:1:1:1, 16.0μl in total, 2×Tag Master Mix (Kangwei Company), 50μl, add dd H2O to 100μl. Reaction conditions: 94°C, 5min, 94°C, 40s, 50°C, 40s, 72°C, 35s, 30Cycles, 72°C, 10min.

[0066]The products amplified by multiplex PCR were detected by gel electrophoresis. Sample 1 detected bands at 1100 bp, 1000 bp, 900 bp, 750 bp, 650 bp, 540 bp, 470 bp, and 430 bp, respectively, containing the viruses CVA, CGRMV, LChV-1, LChV- 2. PBNSPaV, PNRSV, PDV and CMV; sample two detected bands at 1100bp, 1000bp, 900bp, 750bp, 650bp, 540bp and 430bp, respectively, containing the viruses CVA, CGRMV, LChV...

Embodiment 3

[0068]Multiplex PCR was used to detect cherry virus disease types from samples collected in the field. Extract total plant RNA: Use TransZol Plant reagent (TransGen company) to extract RNA. Take a small part of all leaves in each sample, grind it with liquid nitrogen in a mortar, and grind to powder. Refer to the extraction instructions for the extraction procedure. Place the total RNA of the plant at -80°C for use. RT-PCR: RT reaction system (50.0μl) (reagents purchased from TakaRa Company): total plant RNA, 20-50ng, reverse primer Q0-dT: LCh1-16827-R: LCh2-6141-R: PBN-14139-R: PNRS-R3-1152-R: PD-CP-564-R: CM-R3-768-R is 2:1:1:1 :1:1:1, total 16.0μl, 10mM dNTP, 5.0μl, add dd H20 to 48.0μl; 80°C, 3min, 5min on ice; 5×Reverse Transcriptase M-MLV Buffer, 10.0μl, Reverse Transcriptase M-MLV, 1.0μl, Recombinant RNase Inhibitor, 1.0μl; 42°C, 90min.

[0069]PCR system (100.0μl): RT product, 50ng, CVA-5838-F: CGRM-7474-F: LCh1-15849-F: LCh2-5670-F: PBN-13568-F: PNRS-R3-115-F: PD-CP-135-F: CM-...

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Abstract

The invention discloses an RT-PCR kit and a detecting method for detecting eight cherry viruses. The RT-PCR kit contains eight pairs of primers which are independently packaged or mixed, wherein the sequences of the eight pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO.15. The RT-PCR kit disclosed by the invention can be used for field detection and seedling detection subjected to virus-free treatment, provides technical support for virus-free cultivation of the cherries and cultivation of virus-free nursery stock and prevents and treats spread and popularity of cherry virus disease; inaddition, the RT-PCR kit can provide field abundant original resource of cherry virus and lays a foundation for researching molecular mechanisms such as copy and translation of the cherry viruses.

Description

Technical field[0001]The invention relates to the technical field of plant virus detection, in particular to an RT-PCR kit for detecting eight cherry viruses and a detection method.Background technique[0002]Cherry (Prunus avium L.) is a subgenus of Prunus in the Rosaceae family. It is native to Europe and has been introduced into China for more than 100 years. The cherry fruit has bright appearance, unique flavor and rich nutrition, and is known as the "first branch of spring fruit". China has introduced and cultivated sweet cherries since the 1870s, and the current cultivated area has reached 140,000 ha2, The output is about 500,000 tons. Because of its good drought resistance, high yield, simple management and low investment, cherries are one of the fruit trees with the highest cultivation efficiency. With the improvement of the logistics and transportation industry in recent years, the market has expanded year by year, and cherry has gradually become the second largest fruit indu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/70C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 原雪峰曹欣然于成明
Owner SHANDONG AGRICULTURAL UNIVERSITY