Multiplex PCR (Polymerase Chain Reaction) detection kit for garlic virus

A technology for detection kits and viruses, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, and methods based on microorganisms, etc., can solve the problems of small interval between bands and the influence of stray bands, so as to ensure accuracy and reduce detection. Cost, specific effect

Pending Publication Date: 2022-01-28
XUZHOU INST OF AGRI SCI IN JIANGSU XUHUAI DISTRICT (JIANGSU XUZHOU SWEETPOTATO CENT)
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  • Abstract
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AI Technical Summary

Problems solved by technology

However, when the primers reported in the existing literature are amplified in the same system, they may have the effect of heterogeneous bands, or the interval between some target bands is too small, which may easily cause misjudgment

Method used

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  • Multiplex PCR (Polymerase Chain Reaction) detection kit for garlic virus
  • Multiplex PCR (Polymerase Chain Reaction) detection kit for garlic virus
  • Multiplex PCR (Polymerase Chain Reaction) detection kit for garlic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Design and verification of multiplex PCR primer pairs

[0048] 1. Design and synthesis of primers

[0049] Specific primers were designed according to the nucleotide sequence of the garlic virus coat protein (CP) gene published in Genebank and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences of each primer pair are as follows:

[0050] SLV-F: 5'-GAGCGAAAGTAGATTCAACAAAC-3' (SEQ ID NO: 1),

[0051] SLV-R: 5'-CCTTATCAGACCCTCAAGTGGT-3' (SEQ ID NO: 2);

[0052] GCLV-F: 5'-GCTAAGCGGTTTTTGGAGCG-3' (SEQ ID NO: 3),

[0053] GCLV-R: 5'-AAAGCCCCTCAGGGTCGTAAC-3' (SEQ ID NO: 4);

[0054] LYSV-F: 5'-ACGACCAGTTGTAGAGCACG-3' (SEQ ID NO: 5),

[0055] LYSV-R: 5'-TCCGTGCATTCACGTCATGT-3' (SEQ ID NO: 6);

[0056] OYDV-F: 5'-TAATGGCACATTTCAGTGATGC-3' (SEQ ID NO: 7),

[0057] OYDV-R: 5'-TCCGTGTCCTTCTTCCGTTGT-3' (SEQ ID NO: 8);

[0058] Allexi-F: 5'-CYGCTAAGCTATATGCTGAARGG-3' (SEQ ID NO: 9),

[0059] Allexi-R: 5'-TGTTRCAARGTAAGTTTAGYAATATCAACA-3' (SEQ ID NO: 1...

Embodiment 2

[0071] Example 2 Optimization of annealing temperature and primer ratio

[0072] 1. Viral plasmid template preparation

[0073] Measure SLV, GCLV, LYSV, OYDV and Allexiviruses The initial concentration of the positive clone plasmid was calculated to be 1.64×10 10 , 1.56×10 10 , 1.69×10 10 , 1.76×10 10 , and 2.80×10 10 copies / μL. Dilute and mix the 5 plasmids in proportion to each plasmid concentration is 1.0 × 10 8 Copy / μL plasmid mixture, store at -20°C for future use.

[0074] 2. Optimization of annealing temperature

[0075] The annealing temperature was screened according to the following multiplex PCR reactions:

[0076] Reaction system: 2×Taq PCR MasterMix 25 μL, cDNA template 2 μL, reagent P 2 (SLV, GCLV, LYSV, OYDV and Allexiviruses Primer pair molar ratio 1:1:1:1:1) 20 μL, ddH 2 Make up to 50 μL with O; reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 54.0, 54.2, 54.5, 55.1, 55.9, 56.6, 57.4, 58.1, 58.9, 59....

Embodiment 3

[0090] The preparation of embodiment 3 garlic virus multiplex PCR detection kit

[0091] SLV, GCLV, LYSV, OYDV, Allexiviruses Primer pairs were mixed into powder form according to the molar ratio of 10:8:3:3:16. Reagent P was placed in a 1mL EP tube, mixed with 2× Taq PCR MasterMix and ddH 2 O constitutes a multiplex PCR detection kit for garlic virus.

[0092] Application Example 1 Application of Garlic Virus Multiplex PCR Detection Kit to Detect Garlic Tube Seedlings

[0093] According to the following methods to detect the poisonous situation of garlic test-tube seedlings:

[0094] (1) The sampling time of the test-tube plantlets of garlic was in June 2018. The variety "Xusuan No. 6" was cultivated from the shoot tips with two leaf primordia. Selected 24 robust test-tube plantlets and cut the pseudostems of the above-mentioned garlic test-tube plantlets. Tissue above 1.5-2 cm from the base, stored at -80°C for later use;

[0095] (2) Total RNA was extracted using the co...

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Abstract

The invention belongs to the technical field of plant virus detection, and provides a multiplex PCR (Polymerase Chain Reaction) detection kit for a garlic virus. The kit comprises five groups of primer pairs including SLV, GCLV, LYSV, OYDV and Allexiviruses, and the nucleotide sequences of the five groups of primer pairs are as shown in SEQ ID NO: 1-10. The garlic virus is at least one of an SLV (shallot latent virus), a GCLV (garlic common latent virus), an LYSV (leek yellow stripe virus), an OYDV (onion yellow dwarf virus) and an Allexivirus. The garlic virus detection kit provided by the invention can be used for simultaneously detecting 12 kinds of viruses infecting garlic in China, such as an SLV, a GCLV, an LYSV, an OYDV and a shallot X virus through one-time PCR reaction, so that the detection efficiency is improved, and the detection cost is reduced. According to the present invention, the used primers have characteristics of strong specificity and reasonable target band size distribution, and can effectively identify the type of the infected virus while the accuracy of the detection result is ensured.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, and in particular relates to a primer for detecting garlic virus and a kit composed thereof. Background technique [0002] Garlic is an important traditional characteristic vegetable and foreign exchange earning vegetable in my country, accounting for 70% of the world's total garlic trade. Garlic belongs to asexually propagated crops, which can only be reproduced by splitting or forming aerial bulbs, which leads to a very serious accumulation of viral diseases. Zhang Wei et al. and Ming Yanlin et al. respectively investigated the incidence of garlic virus diseases in Heilongjiang Province and Xiamen City, and found that garlic is generally infected by viruses. From 2016 to 2017, the inventor investigated the occurrence of garlic viruses in Xuzhou, Jiangsu and some areas of Henan, and found that the incidence of garlic virus diseases in production was 100%, and they were generally i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/94
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 赵永强刘灿玉张碧薇葛洁杨青青杨峰樊继德陆信娟史新敏李勇
Owner XUZHOU INST OF AGRI SCI IN JIANGSU XUHUAI DISTRICT (JIANGSU XUZHOU SWEETPOTATO CENT)
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