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Method for detecting quarantine Xanthomonas and special primers thereof

A Xanthomonas and primer pair technology, applied in the field of microbiology, can solve the problems of not meeting the requirements of rapid customs clearance for plant quarantine at ports, time-consuming, complicated operations, etc.

Active Publication Date: 2019-01-08
CHINESE ACAD OF INSPECTION & QUARANTINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The damage caused by these plant pathogenic bacteria is very serious, so it is of great significance to strengthen the research on detection and identification technology. Identification by traditional means such as reaction, serological reaction, etc. is generally time-consuming, complicated to operate, and low in sensitivity, which cannot meet the rapid customs clearance requirements of plant quarantine at ports; at the same time, existing molecular detection methods include conventional PCR, real-time fluorescent PCR, etc. It is also difficult to effectively distinguish these quarantine Xanthomonas at the same time

Method used

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  • Method for detecting quarantine Xanthomonas and special primers thereof
  • Method for detecting quarantine Xanthomonas and special primers thereof
  • Method for detecting quarantine Xanthomonas and special primers thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, design and synthesis of primers

[0072] The type III secretion system of pathogenic bacteria plays a crucial role in the interaction between pathogens and hosts. The hrp (hypersensitive response & pathogenicity) gene encoding type III secretion system widely exists in Gram-negative plant pathogenic bacteria, and its gene cluster is composed of hrc (hypersensitive response & conserved), hpa (hypersensitive response & associated), hrp, and some transposons. Through preliminary experiments and sequence comparison, it was found that the hpaA gene sequence is relatively conservative within the species, but has certain variability among species, which is a good tool for distinguishing and identifying different species and pathogenic variants of Xanthomonas barcoded genes.

[0073] According to the hpaA gene of Xanthomonas quarantineis, two primer pairs were designed, named primer pair A and primer pair B respectively. The primer pair A is composed of primer X...

Embodiment 2

[0080] Embodiment 2, establishment of the method for detecting quarantine Xanthomonas

[0081] The established method is as follows:

[0082] 1. Extract the genomic DNA of the bacterium to be tested and use it as a template to carry out PCR amplification using the synthetic primer pair A or primer pair B in Example 1 to obtain a PCR amplification product.

[0083] Reaction system: Genomic DNA (6—50) ng of the sample to be tested, Taq PCR Mastermix (product of Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number KT201-02) 12.5 μL, primer pair A (primer XHpaAT-F and primer XHpaAT-R each 10 pmol) or primer pair B (primer XHpaAH-F and primer XHpaAH-R each 10 pmol), make up to 25 μL with deionized water.

[0084] Reaction program: 94°C for 5min; 94°C for 40S, 55°C for 40S, 72°C for 1min, 35 cycles; 72°C for 10min.

[0085] 2. After step 1 is completed, the PCR amplification product is sequenced, and the following judgment is made according to the sequencing result: ...

Embodiment 3

[0098] Embodiment 3, identify whether 58 kinds of test bacterial strains are quarantine Xanthomonas according to the method established in Example 2

[0099] The basic information of the 58 tested strains is shown in Table 1. Among them, there were 11 kinds of quarantine Xanthomonas, 3 strains of each; 10 kinds of non-quarantine Xanthomonas, 25 strains.

[0100] The bacterial strain numbers LMG497, LMG578, LMG576, LMG539, LMG550, LMG553, LMG555, LMG693, LMG547, LMG671, LMG672, LMG673, LMG630 and LMG655 are deposited in BCCM / LMG BacteriaCollection (website: http: / / bccm.belspo.be / index.php ), the public can obtain from this repository.

[0101] 菌株编号为NCPPB4412、NCPPB4372、NCPPB2375、NCPPB2376、NCPPB1925、NCPPB485、NCPPB2972、NCPPB211、NCPPB577、NCPPB1160、NCPPB1834、NCPPB301、NCPPB302、NCPPB796、NCPPB181、NCPPB2059、NCPPB2061、NCPPB942、NCPPB3078、NCPPB4386、NCPPB2597、NCPPB2567、NCPPB3168、NCPPB1822 , NCPPB3743, NCPPB2095, NCPPB3242, NCPPB793, NCPPB1154, NCPPB2921 and NCPPB1585 are preserved in the...

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Abstract

The invention discloses a method for detecting quarantine xanthomonas and a special primer thereof. The primer is composed of a first primer pair and a second primer pair. The first primer pair is composed of a primer XHpaAT-F and a primer XHpaAT-R. The second primer pair is composed of a primer XHpaAH-F and a primer XHpaAH-R. The nucleotide sequence of the primer XHpaAT-F, the nucleotide sequence of the primer XHpaAT-R, the nucleotide sequence of the primer XHpaAH-F and the nucleotide sequence of the primer XHpaAH-R are shown as the sequence 1, the sequence 2, the sequence 3 and the sequence 4 in the sequence list. Experiments show that by means of the method for detecting quarantine xanthomonas and the special primers thereof, whether a strain to be detected is quarantine xanthomonas or not can be identified simply, fast and effectively, accuracy is 100%, and the method has significant application to detection or identification of different kinds of quarantine pathogenic bacteria in xanthomonas.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a method for detecting quarantine Xanthomonas and special primers thereof. Background technique [0002] Xanthomonas belongs to the Bacteria domain (Bacteria), Proteobacteria (Proteobacteria), Gammaproteobacteria (Gammaproteobacteria), Xanthomonadales (Xanthomonadales), Xanthomonadaceae (Xanthomonadaceae), is Gram Negative bacteria. Most members of this genus are plant pathogens, causing plant diseases all over the world, infecting at least 124 species of monocotyledonous plants and 268 species of dicotyledonous plants, causing significant losses to agricultural production. More than 100 species of Xanthomonas have been identified as different plant pathogens. [0003] Quarantine Xanthomonas refers to the phytopathogenic bacteria in the genus Xanthomonas that are defined as entry plant quarantine pests by my country, such as Xanthomonas axonopodis pv. betlicola and Xanthomonas axo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/689C12Q1/04
CPCC12Q1/686C12Q1/6895C12Q2545/113
Inventor 田茜赵文军冯建军卢松玉
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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