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CN-115820886-B - Method, reagent and application for identifying or tracing detection of citrus canker pathogens

CN115820886BCN 115820886 BCN115820886 BCN 115820886BCN-115820886-B

Abstract

The application discloses a method, a reagent and application of citrus canker pathogen identification or traceability detection. The method comprises the steps of obtaining seven gene sequences of citrus canker to be detected, carrying out multi-site sequence typing analysis on the seven gene sequences, determining alleles and numbering, connecting the allele numbers of the seven genes in series as sequence types, comparing the sequence types with sequence types of reference strains to obtain tracing information of the strains to be detected, wherein the reference strains are citrus canker with known tracing information, and the seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA and pliY. The method adopts seven housekeeping genes suitable for citrus canker multi-site sequence typing to carry out traceability detection, can distinguish different geographical source strains with high resolution, has important significance for strain area research, evolution, propagation, disease epidemic and prevention and control, and can be used for identification and traceability of citrus canker.

Inventors

  • XU XIAOLI
  • GAO RUIFANG
  • ZHANG GUIMING
  • ZHANG DANDAN
  • LI NA
  • WANG YING

Assignees

  • 深圳海关动植物检验检疫技术中心

Dates

Publication Date
20260512
Application Date
20221021

Claims (13)

  1. 1. The method for identifying or tracing the bacterial strain of the citrus canker is characterized by comprising the steps of obtaining the sequences of seven genes of the bacterial strain of the citrus canker to be detected, carrying out multi-site sequence typing analysis on the sequences of the seven genes, determining alleles and numbering of each gene, connecting the numbers of the alleles of the seven genes in series in sequence to be used as the sequence type of the bacterial strain of the citrus canker to be detected, and comparing the sequence type of the bacterial strain of the citrus canker to be detected with the sequence type of a reference bacterial strain to obtain identification or tracing information of the bacterial strain of the citrus canker to be detected; The reference strain is a citrus canker strain with known traceability information, and the sequence type of the reference strain is a sequence type obtained by serially connecting the allele numbers of seven genes of the reference strain according to the same sequence of the citrus canker strain to be detected; The seven genes are xac_rs07980, xac_rs21550, copB, egl, fliL, hrpA, and pliY.
  2. 2. The method of claim 1, wherein the sequence type is obtained by concatenating the allele numbers of the seven genes in the order XAC_RS07980-XAC_RS 21550-copB-egl-fliL-hrpA-pliY.
  3. 3. The method according to claim 1, further comprising a population structure analysis including treating a typing group comprising 2 or more sequence types as1 clone complex, defining an ancestral sequence type in the 1 clone complex during clustering, using the ancestral sequence type as a basis for evolution of other sequence types, connecting each sequence type with the ancestral sequence type by a straight line, wherein the length of the straight line indicates the distance of homology, and obtaining the population structure of the citrus canker strain to be tested and the reference strain.
  4. 4. The method according to claim 3, wherein the sequence types are defined as monomers without dividing the sequence types in any 1 clone complex, wherein the sequence types have only 1 allele-number difference and the sequence types have 2 allele-number differences.
  5. 5. The method of claim 1, further comprising splicing the sequences of the seven genes in the order XAC_RS07980-XAC_RS21550-copB-egl-fliL-hrpA-pliY to obtain an analysis sequence, and constructing a phylogenetic tree using the analysis sequences of the test strain of Leuconostoc citrulli and the reference strain.
  6. 6. The method of claim 5, wherein the phylogenetic tree is constructed by maximum likelihood and/or adjacency.
  7. 7. The method of claim 1 to 6, further comprising performing genome-wide SNP locus cluster analysis on the strain of Leptospira citri to be tested and the reference strain.
  8. 8. The method of claim 7, wherein the whole genome SNP locus cluster analysis comprises the steps of respectively concatenating SNP loci of the to-be-detected citrus canker strain and the reference strain in the same sequence as an analysis sequence of each strain, and constructing a phylogenetic tree based on the analysis sequence of the whole genome SNP loci of each strain.
  9. 9. A reagent for identifying or tracing and detecting citrus canker pathogens is characterized by comprising a primer combination for amplifying seven genes of the citrus canker pathogens, wherein the seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA and pliY.
  10. 10. The reagent according to claim 9, wherein the primer set comprises a first primer set for amplifying the XAC-RS 07980 gene, a second primer set for amplifying the XAC-RS 21550 gene, a third primer set for amplifying the copB gene, a fourth primer set for amplifying the egl gene, a fifth primer set for amplifying the fliL gene, a sixth primer set for amplifying the hrpA gene, and a seventh primer set for amplifying the pliY gene; the upstream and downstream primers of the first primer group are respectively sequences shown as a Seq ID No.1 and a Seq ID No. 2; the upstream and downstream primers of the second primer group are respectively sequences shown as a Seq ID No.3 and a Seq ID No. 4; The upstream and downstream primers of the third primer group are respectively sequences shown as a Seq ID No.5 and a Seq ID No. 6; The upstream and downstream primers of the fourth primer group are respectively sequences shown as a Seq ID No.7 and a Seq ID No. 8; the upstream and downstream primers of the fifth primer group are respectively sequences shown as a Seq ID No.9 and a Seq ID No. 10; the upstream and downstream primers of the sixth primer group are respectively sequences shown as a Seq ID No.11 and a Seq ID No. 12; The upstream and downstream primers of the seventh primer group are respectively sequences shown as a Seq ID No.13 and a Seq ID No. 14; Seq ID No.1:5’-CTGCGTACCGAACTTAAGACCCTCA-3’ Seq ID No.2:5’-ACGCTCGAACACGTCGGACT-3’ Seq ID No.3:5’-GCAGCATTGACGCCACACCCT-3’ Seq ID No.4:5’-GGTTTCCAGCGCCAACTGTTC-3’ Seq ID No.5:5’-TCTCAGGCCGCACCCATCGAC-3’ Seq ID No.6:5’-CGGTTGGTCAGCAGTACCTCGTA-3’ Seq ID No.7:5’-CCAATGGTGCTGCTGACACAGGT-3’ Seq ID No.8:5’-TCCGCCTGCCGATCCTGTGGGAA-3’ Seq ID No.9:5’-CACTTCTTGCCGGTCTCGCTGGT-3’ Seq ID No.10:5’-AGCATTCCCCTGGAGCAGACT-3’ Seq ID No.11:5’-GCAAGATCACCCCGGACAACGAGGA-3’ Seq ID No.12:5’-TTGCTGCACAAGCGCTCCGAT-3’ Seq ID No.13:5’-CGGACACAAAGCTTGCCTGAAA-3’ Seq ID No.14:5’-GGTGCGAACAACAATAAGACGATT-3’。
  11. 11. The reagent according to claim 9 or 10, further comprising a primer specific for citrus canker, wherein the primer upstream and downstream of the primer specific for citrus canker is the sequence shown in Seq ID No.15 and Seq ID No.16, respectively; Seq ID No.15:5’-CGCCATCCCCACCACCACCACGAC-3’ Seq ID No.16:5’-AACCGCTCAATGCCATCCACTTCA-3’。
  12. 12. Use of seven genes, or seven gene-based DNA barcodes, in the identification or traceability detection of canker citrus, the seven genes being xac_rs07980, xac_rs21550, copB, egl, fliL, hrpA and pliY.
  13. 13. The application of a reagent for detecting seven genes or a DNA bar code based on seven genes in the preparation of a citrus canker bacteria identification reagent or a citrus canker bacteria traceability detection reagent, wherein the seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA and pliY.

Description

Method, reagent and application for identifying or tracing detection of citrus canker pathogens Technical Field The application relates to the field of citrus canker pathogen detection, in particular to a method, a reagent and application for identifying or tracing detection of citrus canker pathogen. Background Citrus is an important tropical and subtropical fruit, widely distributed in many countries and regions of the world 140, the first largest fruit in the world with the greatest yield. The citrus canker is one of the diseases with the biggest harm to citrus production worldwide, and is an important detection object for quarantine of plants at home and abroad. The pathogenic bacteria of citrus canker, namely citrus canker (Xanthomonas citri subsp. Citri). The citrus canker pathogens have a plurality of bacterial systems and subspecies, and are divided into 5 bacterial systems, namely A, B, C, D, E bacterial systems according to geographic distribution and pathogenicity differences. The strain A originates from Asia and is the strain with the strongest invasiveness and the largest damage range to citrus varieties. Studies have shown that there are also variations in the a lines themselves, for example the a line and the a W line, both of which originate in different regions. In addition, the conventional research on the differentiation of the bacterial strain shows that pathogenic bacteria in different areas, such as the bacterial strain A, have a common differentiation phenomenon. At present, various methods such as microscopic diagnosis, serological diagnosis, PCR diagnosis, DNA probe diagnosis and the like are available for detecting and diagnosing the citrus canker pathogens of different bacterial systems, but the traceable detection and research on the citrus canker pathogens are relatively lacking. Disclosure of Invention The application aims to provide a novel method, a reagent and application for traceable identification or traceable detection of citrus canker pathogens. The application adopts the following technical scheme: The application discloses a method for identifying or tracing detection of citrus canker, which comprises the steps of obtaining sequences of seven genes of a citrus canker strain to be detected, carrying out multi-site sequence typing analysis on the sequences of the seven genes, determining alleles of each gene and numbering, connecting the alleles of the seven genes in series in sequence to be used as the sequence type of the citrus canker strain to be detected, comparing the sequence type of the citrus canker strain to be detected with the sequence type of a reference strain to obtain identification or tracing information of the citrus canker strain to be detected, wherein the reference strain is the citrus canker strain with known tracing information, the sequence type of the reference strain is the sequence type obtained by connecting the alleles of the seven genes of the reference strain in series according to the same sequence of the citrus canker strain to be detected, and the seven genes are XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA and pliY. The identification or tracing information of the bacterial strain of the citrus canker to be detected is obtained by comparing the sequence type of the bacterial strain of the citrus canker to be detected with the sequence type of a reference bacterial strain, for example, the identification or tracing information of the reference bacterial strain closest to the sequence type of the bacterial strain of the citrus canker to be detected is endowed to the bacterial strain of the citrus canker to be detected. The application adopts seven genes of XAC_RS07980, XAC_RS21550, copB, egl, fliL, hrpA and pliY to carry out multi-site sequence typing (MLST) analysis on the citrus canker, has higher resolution, can identify the citrus canker, trace the geographical source of the citrus canker, study the regional diversity of the citrus canker, and know and master the evolution, the transmission and the epidemic occurrence of diseases of the citrus canker. The method provided by the application is used for identifying and tracing analysis of the citrus canker, and can effectively improve the control effect of the citrus canker. In one implementation of the application, the sequence type is obtained by concatenating the allele numbers of the seven genes in the order XAC-RS 07980-XAC-RS 21550-copB-egl-fliL-hrpA-pliY. In one implementation mode, the citrus canker pathogen traceability detection method further comprises population structure analysis, wherein the population structure analysis comprises the steps of regarding a parting group containing 2 or more sequence types as 1 clone complex, defining an ancestral sequence type in the 1 clone complex during clustering, taking the ancestral sequence type as the basis of evolution of other sequence types, connecting each sequence type with the ancestral sequence type through a straight li