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Method for detecting burkholderia cepacia flora on fruits or vegetables and identifying seeds

A Burkholderia cepacia, identification method technology, applied in the field of molecular biology, can solve the problems of long period, cumbersome process, unreliable detection results, etc.

Inactive Publication Date: 2010-09-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention provides a method for detection and species identification of Burkholderia cepacia on vegetables or fruits, which solves the problems of long cycle, cumbersome process and unreliable detection results of traditional methods

Method used

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  • Method for detecting burkholderia cepacia flora on fruits or vegetables and identifying seeds
  • Method for detecting burkholderia cepacia flora on fruits or vegetables and identifying seeds
  • Method for detecting burkholderia cepacia flora on fruits or vegetables and identifying seeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Take a little tissue from the junction between disease and health of fruit-rotten apricot in an orchard in Deqing, mash it in 40 μL of sterile water, spread the suspension on a PCAT plate with a sterilized wave stick, and incubate at 28 °C. After 2 days, 23 milky white single colonies were picked from the PCAT plate and purified, numbered Bca01-Bca23.

[0028] Extract 1 to 2 purified bacterial colonies, place them in cell lysate (including 0.25% by weight SDS, 0.05mol / L NaOH), heat at 95°C for 15min, and then add 180 μL bismuth to the cell lysate Distilled in water and stored at -20°C, the resulting solution was used as a DNA template.

[0029] Using the extracted DNA as a template, PCR amplification was performed using specific primers BCR1 and BCR2 of the recA gene of Burkholderia cepacia.

[0030] Primer BCR1: 5'-TGACCGCCGAGAAGAGCAA-3'

[0031] Primer BCR2: 5'-CTCTTCTTCGTCCATCGCCTC-3'

[0032] The 20μL reaction system for PCR amplification includes:

[0033] Prim...

Embodiment 2

[0039] Digest the PCR products of 15 strains determined to belong to the Burkholderia cepacia group with HaeIII, and the 15 μl enzyme digestion system includes:

[0040] PCR product 10μL

[0041] 10×buffer 1.5μL

[0042] HaeIII endonuclease 1 μL

[0043] wxya 2 O 2.5 μL.

[0044] Digestion as figure 2 As shown, compared with the genotype restriction map reported by Mahenthiralingam et al. (2000) and Dalmastri et al. (2005), it was preliminarily determined that 7 strains Bca01-Bca07 belonged to B. cenocepacia IIIA, and 8 strains Bca08-Bca015 belonged to B. cenocepacia IIIB .

Embodiment 3

[0046] Utilize the specific primers of B.cenocepacia IIIA and IIIB, carry out PCR amplification with the DNA of 7 identifications as B.cenocepacia IIIA and 8 identifications as B.cenocepacia IIIB bacterial strains that embodiment 1 extracts respectively, PCR amplifies The amplification system and reaction conditions are the same as in Example 1.

[0047] Take 5 μL of the amplified PCR product and electrophoresis on 1% agarose gel. After electrophoresis, the gel was stained in EB solution for 20 minutes, and then placed on a UV gel imager to observe the results. The results are as follows: image 3 shown. Bc01~Bc07 showed positive amplification to the specific primers of B.cenocepacia IIIA, and Bc08~Bc13 showed positive amplification to the specific primers of B.cenocepacia IIIB, which was consistent with the enzyme digestion results; Bc14 and Bc15 showed positive amplification to B.cenocepacia IIIB The specific primers did not show positive amplification, which was inconsist...

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Abstract

The invention discloses a method for detecting burkholderia cepacia flora on fruits or vegetables and identifying seeds. The method comprises the following steps: separating flora on sick-healthy borderlines, extracting DNA, utilizing specific primers to perform PCR amplification, separating amplification products through gel electrophoresis, use an indicator to develop color and identifying whether a strain belongs to burkholderia cepacia flora according to band situations; performing enzyme digestion on PCR products of each strain, obtaining a restriction enzyme digestion map and preliminarily determining seed of each strain; utilizing the specific primers of corresponding seeds to perform PCR amplification and judging the seeds of the strains after gel electrophoresis separation according to the band situations; and sequencing last PCR products of the strain if gel shows no band, obtaining a recA sequence, constructing a molecular phylogenetic tree and determining the seed of the strain. The method can accurately detect the existence of Bcc bacteria on fruits and vegetable with pathological changes and confirm every seed in Bcc flora in order to take effective measures to dangerous bacteria.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for detecting and identifying species of Burkholderia cepacia on fruits or vegetables. Background technique [0002] Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria widely present in soil, water, plant surfaces and hospital environments, and it is also a group of bacteria with similar phenotypes but different genotypes ,由17个种组成,分别为B.cepacia,Burkholderia multivorans,Burkholderia cenocepacia,Burkholderiastabilis,Burkholderia vietnamiensis,Burkholderia dolosa,Burkholderiaambifaria,Burkholderia anthina,Burkholderia pyrrocinia,Burkholderiaubonensis,Burkholderia lateens,Burkholderia diffusa,Burkholderiaarboris,Burkholderia seminalis,Burkholderia metallica , Burkholderia contaminans and Burkholderia lata. The bacterium was originally found on onions to cause onion acid skin disease. Since the 1980s, the bacterium has been widely reported as a human oppo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
Inventor 方媛谢关林楼妙苗朱勃李斌
Owner ZHEJIANG UNIV
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