Specific primer pairs used for auxiliary identification of potato viruses and application thereof

A specific primer pair, potato virus technology, applied in microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of limitation, reduced sensitivity, time-consuming and laborious, and achieve good specificity and sensitivity. Widely popularized and applied , the effect of high sensitivity

Inactive Publication Date: 2014-12-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the indicator plant method has a long period and requires strict environmental conditions, so it is difficult to use for large-scale detection
The ELISA method is economical, simple, fast and intuitive, but it also has certain disadvantages: firstly, it takes a long time to prepare the antibody, which is time-consuming and laborious; secondly, it can only detect one virus at a time, and the sensitivity decreases when detecting multiple viruses
In addition, this method often has false positive reactions in the detection of viruses, which brings difficulties to the detection of virus-free vaccines.
At present, most of the kits used for potato virus detection use enzyme immunoassay, which is limited by the sensitivity and false positives of serological methods, and cannot diagnose viruses with high sensitivity and accuracy.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Specific primer pairs used for auxiliary identification of potato viruses and application thereof
  • Specific primer pairs used for auxiliary identification of potato viruses and application thereof
  • Specific primer pairs used for auxiliary identification of potato viruses and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The design of embodiment 1, potato virus primer

[0047] According to the conserved sequences of three potato viruses, 20 pairs of specific primers for potato virus X (PVX), 20 pairs of specific primers for potato virus S (PVS), and 18 pairs of specific primers for potato leafroll virus (PLRV) were designed. After screening and optimization, one pair of specific primers for PVX, PVS, and PLRV were obtained, as follows:

[0048] Specific primer pair for PVX (also known as specific primer pair A, the target sequence is 391bp):

[0049] Upstream primer F1: 5'-ACTGCAGGCGCAACTCCTGCCACAG-3' (sequence 1 of the sequence listing);

[0050] Downstream primer R1: 5'-GTGCTTGCCAGTTAGCAGGTGGAC-3' (SEQ ID NO: 2 in the Sequence Listing).

[0051] Specific primer pair for PVX (also known as specific primer pair B, the target sequence is 231bp):

[0052] Upstream primer F2: 5'-ATCGATGAGCTGTTCAAGATGG-3' (sequence 3 of the sequence listing);

[0053] Downstream primer R2: 5'-GATCGAGTCC...

Embodiment 2

[0057] Embodiment 2, the specificity of primer pair (singleplex RT-PCR)

[0058] 1. Specificity of specific primers to formazan

[0059] Potato virus X, potato S virus and potato leafroll virus are carried out as follows respectively:

[0060] 1. Extract the total RNA of the virus and reverse transcribe it into cDNA.

[0061] 2. Use the cDNA in step 1 as a template, and use specific primers to perform PCR amplification on A to obtain a PCR amplification product.

[0062] PCR amplification system: template 2 μl, 2×PCR buffer 10 μl, upstream primer (20 μM) 1 μl, downstream primer (20 μM) 1 μl, ddH 2 O 6 μl.

[0063] PCR amplification conditions: 94°C for 3min; 30 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 30s; 72°C for 10min.

[0064] 3. Perform 1.5% agarose gel electrophoresis on the PCR amplification product, the results are shown in figure 1 ( figure 1 Among them, lane 1 is molecular weight marker, lane 2 is potato virus X, lane 3 is potato virus S, and lane 4 i...

Embodiment 3

[0077] Embodiment 3, the sensitivity of primer pair

[0078] 1. Sensitivity of specific primers to formazan

[0079] 1. Extract the total RNA of Potato virus X and reverse transcribe it into cDNA, and measure the cDNA concentration with a nucleic acid protein analyzer. After determination, the cDNA concentration of PVX is 501.5 ng / μl.

[0080] 2. Dilute the cDNA with sterile water to 400ng / μl, 300ng / μl, 200ng / μl, 100ng / μl, 50ng / μl, 25ng / μl and 10ng / μl with seven concentration gradient dilutions (according to the concentration from high to Low, named dilution 1 to dilution 7 in sequence).

[0081] 3. Using each dilution as a template, PCR amplification is carried out on armor with specific primers to obtain PCR amplification products.

[0082] The PCR amplification system and PCR amplification conditions are the same as Step 1 of Example 2.

[0083] 4. Perform 1.5% agarose gel electrophoresis on the PCR amplification product, see the results Figure 4 ( Figure 4 In, lane ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses specific primer pairs used for auxiliary identification of potato viruses and application thereof. The invention provides a primer pair composition which comprises a primer pair I composed of DNA molecules respectively represented by a sequence 1 and a sequence 2, a primer pair II composed of DNA molecules respectively represented by a sequence 3 and a sequence 4 and a primer pair III composed of DNA molecules respectively represented by a sequence 5 and a sequence 6. The primer pair composition can be used for auxiliary identification of potato viruses and for detection that whether a to-be-detected sample is infected by a potato virus. With the primer pair composition provided by the invention, the potato virus X, the potato virus S and the potato leaf curl virus can be rapidly, accurately and sensitively detected at the same time; and the primer pair composition has the characteristics of high stability, high sensitivity, low mutability, hypotoxicity and the like and can be widely popularized and applied.

Description

technical field [0001] The invention relates to a pair of specific primers for auxiliary identification of potato virus and its application. Background technique [0002] Potato is the fourth largest crop in the world after rice, wheat and corn. my country is the world's largest potato producer. According to statistics, during the "Tenth Five-Year Plan" period, the average annual potato planting area was 70.159 million mu, accounting for 1 / 4 of the world's total area, and the annual output was 14.156 million tons, accounting for 1% of the world's total annual output. / 5. [0003] Due to the characteristics of drought resistance, cold resistance, barren resistance and wide adaptability of potatoes, the soil and climatic conditions in most parts of the country can meet its production. China is a country with a large population. The contradiction between the decrease of cultivated land and the increase of population is irreversible. Water resources are increasingly scarce and ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/686C12Q1/70C12Q2531/113
Inventor 仲乃琴武晓敏马银平董彦旭马琼蒙静沈振荣
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap