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Method for detecting Psa in kiwi fruit pollens

A detection method and kiwifruit technology, applied in the directions of microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc., to achieve the effect of improving detection efficiency, enhancing specificity, and enhancing sensitivity

Inactive Publication Date: 2016-09-14
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The kiwifruit canker caused by Psa is a devastating bacterial disease. Once the fruit tree gets sick, there is no effective medicine to treat it

Method used

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  • Method for detecting Psa in kiwi fruit pollens
  • Method for detecting Psa in kiwi fruit pollens
  • Method for detecting Psa in kiwi fruit pollens

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Establishment of nested PCR detection method for kiwifruit Psa

[0036] (1) Cultivation of kiwifruit Psa and Pseudomonas syringae pv.tomato, Pseudomonas syringaepv.Theae, Pseudomonas avellanae: kiwifruit Psa was cultured with King’B liquid medium,

[0037] (2) DNA extraction of pathogenic bacteria: Ezup column genomic DNA extraction kit (bacteria) was used to extract target DNA.

[0038] (3) Design of primers: two pairs of primers were designed according to the hrpw gene of Psa, the dominant pathogen of kiwifruit canker in Shaanxi.

[0039] The enrichment method of kiwi pollen Psa is as follows: Weigh 0.01 g of kiwi pollen, add it to a glass test tube filled with 15 mL of high-pressure sterilized and cooled King’s B liquid medium, seal it with a cotton plug, and place it in a constant temperature shaking incubator for 24 After culturing at 180r / min for 12 hours, store at 4°C until use.

[0040] The method of extracting the DNA of Psa in the pollen suspension is as fol...

Embodiment 2

[0050] Sensitivity of kiwifruit Psa PCR detection

[0051] Pick a single colony of Psa and place it in 100 mL of sterile King’s B liquid medium, seal it with kraft paper, and culture it at 24°C and 200 r / min for 16 hours to obtain a bacterial suspension. Use a hemocytometer to measure the concentration of the bacterial suspension, and adjust the concentration gradient of the bacterial suspension to 10 with sterile normal saline. 7 cfu / mL, 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2 cfu / mL, 10cfu / mL, extract DNA respectively, carry out PCR amplification, study the detection limit of Psa in the bacterial suspension, and determine the detection sensitivity of this method.

[0052] Depend on figure 2 It can be seen that: Swimming lanes 1-7: the concentration of the detected bacterial suspension is 10 in sequence 7 cfu / mL, 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2 cfu / mL, 10cfu / mL, the target band becomes darker with the decrease of the conc...

Embodiment 3

[0054] Detection of Psa PCR in kiwifruit pollen

[0055] (1) Cultivation of kiwifruit pollen Psa: Take 0.01 g of prepared kiwifruit pollen, respectively, and add them into glass test tubes numbered 1-23 containing 15 mL of autoclaved and cooled King’B liquid medium, and seal with cotton plugs. After culturing in a constant temperature shaking incubator at 24°C and 180r / min for 12h, a mixed bacterial suspension was obtained. Store at 4°C until use (short-term storage only).

[0056] (2) Extraction of Psa DNA from the bacterial suspension: Take 1 mL of the overnight cultured bacterial suspension in a 1.5 mL centrifuge tube, centrifuge at room temperature at 11,000 rpm for 1 min, discard the supernatant, and collect the bacterial cells. Add 1mLddH 2 O Pipette repeatedly, wash the cells, centrifuge at 11,000rpm for 1min, and discard the supernatant. Repeat wash once. Take 100 μL ddH 2 O, pipette repeatedly to dissolve the precipitate, put it in a water bath at 100°C for 15 mi...

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Abstract

The invention provides a method for detecting Psa in kiwi fruit pollens. The method comprises the following steps: extracting DNA from pollen mixed bacterial suspension; designing two pairs of specific primers according to hrpW gene of kiwi fruit Psa, carrying out nested-PCR, amplifying a pollen sample to obtain an outer primer amplified band with a size of about 590 bp and an inner primer amplified band with a size of about 240 bp; and finally carrying out sequencing to verify the Psa. The detection sensitivity is 103 cfu / mL. The provided Psa rapid detection method has the advantages of stronger specificity, higher stability, and higher sensitivity, can differentiate pathogenic bacteria having close ties of consanguinity with Psa such as pseudomonas syringae pv.Theae, pseudomonas avellanae, and the like, and is capable of reducing the false positive results.

Description

technical field [0001] The invention relates to the technical field of fruit and vegetable disease detection, in particular to a method for detecting Psa in kiwi pollen. Background technique [0002] In recent years, the large-scale outbreak of kiwifruit bacterial canker has led to a decline in kiwifruit production, seriously affecting the development of the industry. Kiwifruit canker is a bacterial disease caused by Pseudomonas syingae pv.actinidiae (Psa). With the help of natural conditions such as insects and wind and rain, the pathogens independently infect the shoots of kiwifruit plants, leaf marks of branches, new forks, lenticels of leaves, stomata, etc., or the above tissues with poor nutrition, and then spread into each branch of the fruit tree to cause The whole plant is infected; or spread through pruning wounds and frost cracked wounds. [0003] Gallelli.A et al. (2011) found that canker pathogens existed in kiwifruit pollen in Italy through molecular detection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/38
CPCC12Q1/6848C12Q2531/113C12Q2549/119C12Q2565/125
Inventor 高贵田曹凡杜莹琳朱丹
Owner SHAANXI NORMAL UNIV
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