Nested PCR (Polymerase Chain Reaction) detection kit for canna yellow mottle virus and detection method thereof

A technology for detecting kits and mottled viruses, which is applied in biochemical equipment and methods, and microbial measurement/inspection. It can solve the problems of unseen nested PCR detection kits and nested PCR detection methods, and achieve The effect of fast time, short detection time and high sensitivity

Active Publication Date: 2014-02-12
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

But so far, there is no nested PCR detection method specially used for CaYMV detection, let alone a nested PCR detection kit specially used for the detection of this virus

Method used

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  • Nested PCR (Polymerase Chain Reaction) detection kit for canna yellow mottle virus and detection method thereof
  • Nested PCR (Polymerase Chain Reaction) detection kit for canna yellow mottle virus and detection method thereof
  • Nested PCR (Polymerase Chain Reaction) detection kit for canna yellow mottle virus and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Configuration of Canna Yellow Mottle Virus Nested PCR Detection Kit (10 detections)

[0027] 1) Outer upstream primer: 10 μmol / L, 1 tube (30 μL);

[0028] 2) Outer downstream primer: 10 μmol / L, 1 tube (30 μL);

[0029] 3) Inner upstream primer: 10 μmol / L, 1 tube (30 μL);

[0030] 4) Inner downstream primer: 10 μmol / L, 1 tube (30 μL);

[0031] 5) dNTPs: 10mmol / L, 1 tube (20μL);

[0032] 6) PCR Buffer: 10×, 1 tube (60μL);

[0033] 7) Mgcl 2 : 25mmol / L, 1 tube (50μL);

[0034] 8) Taq DNA polymerase: 2.5U / μL, 1 tube (10μL);

[0035] 9) Canna yellow mottle virus positive control sample, 1 tube (50 μL);

[0036] 10) Negative control sample without canna yellow mottle virus, 1 tube (50 μL);

[0037] 11) ddH 2 O, 1 tube (1 mL).

Embodiment 2

[0038] Embodiment 2: the detection method of canna yellow mottle virus nested PCR detection kit

[0039] The detection method of above-mentioned canna yellow mottle virus nested PCR detection kit comprises the following steps:

[0040] 1) The first round of PCR reaction: Add 4 μL of the sample DNA to be tested in the PCR tube, add 0.5 μL of Taq DNA polymerase at a concentration of 2.5 U / μL, 0.5 μL of dNTPs at a concentration of 10 mmol / L, and 2.5 μL of 10×PCR Buffer for each tube , the concentration is 25mmol / L MgCl 2 2 μL, concentration of 10 μmol / L outer upstream primer 2 μL, concentration of 10 μmol / L outer downstream primer 2 μL, ddH 2 O11.5 μL, so that the total reaction volume is 25 μL; the mixed reaction solution is pre-denatured at 94°C for 3 minutes, then denatured at 94°C for 30 s, annealed at 53°C for 45 s, and extended at 72°C for 1 min, so a total of 35 cycles, after the end of the last cycle Continue to extend at 72°C for 10 minutes, and the reaction ends;

[00...

Embodiment 3

[0043] Example 3: Specificity determination of canna yellow mottle virus nested PCR detection kit

[0044] 1) DNA extraction: Canna yellow mosaic virus (CaYMV), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), tomato infertility virus (Tomato aspermy virus) Virus, TAV) samples as materials, take 50-100mg of each sample and thoroughly grind it with liquid nitrogen, transfer to a 1.5mL centrifuge tube, add 500μL TES, 50-100μg proteinase K, incubate at 56°C for 0.5-1h, and shake gently during the Mix well; add a certain volume of 10mol / L NaCl (to make the final concentration 1.4mol / L), 1 / 10 times the volume of 10% CTAB, and incubate at 65°C for 10 minutes; add 1 times the volume of chloroform / isoamyl alcohol (24:1 ), shake gently to mix, put on ice for 30min, centrifuge at 12000r / min at 4℃ for 10min; take the supernatant, add 225μL NH 4 AC (5mol / L), mix well, place on ice for 1h, centrifuge at 12000r / min at 4°C for 10min; take the supernatant, add 3μL RNase, incubat...

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Abstract

The invention relates to a nested PCR (Polymerase Chain Reaction) detection kit for canna yellow mottle virus and a detection method thereof, which are specially used for detecting the canna yellow mottle virus. The kit comprises an outer-side upstream primer, an outer-side downstream primer, an inner-side upstream primer, an inner-side downstream primer, PCR Buffer, dNTPs, Mgcl2, Taq DNA polymerase, positive control, negative control and ddH2O. According to the invention, two pairs of specific primers are designed according to the gene sequence of the canna yellow mottle virus, extracted DNA is adopted as a template to carry out first-round PCR amplification, first-round PCR products are adopted as a template to carry out second-round PCR amplification, amplification products are detected by gel electrophoresis of agarose, and the detected virus is judged to be the canna yellow mottle virus if specific target fragments with the size being 325bp occur. The kit detects the canna yellow mottle virus by utilizing a nested PCR technology, has the advantages of strong specificity, high sensitivity and short detection time, and can meet the need of entry and exit port quarantine and agricultural production.

Description

technical field [0001] The invention relates to a canna yellow mottle virus nested PCR detection kit and a detection method thereof, which belong to the technical field of plant quarantine and are suitable for rapid detection and monitoring of canna yellow mottle virus in entry and exit and agricultural production. Background technique [0002] Canna yellow mottle virus (CaYMV) belongs to the family Caulimo Viridae and a member of the genus Badnavirus, and was first discovered on canna in Japan. CaYMV virus particles are rod-shaped (120-130nm×28nm), and the viral genome is circular double-stranded DNA (containing 3 ORFs). The host range of CaYMV is relatively narrow, and the reported natural host is canna. The virus can cause various symptoms after infecting canna. Typical symptoms include chlorotic spots on leaves, yellowing of veins, streaks on stems and flowers, etc. Harm the normal growth of canna, affect its ornamental value and economic value. CaYMV is mainly distrib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6848C12Q1/686C12Q1/70C12Q2549/119C12Q2531/113
Inventor 沈建国虞赟蔡伟陈智明张永江闫诚
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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