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Sensitive and Rapid Method for Candidatus Liberibacter Species Detection

a technology of candidus liberibacter and detection method, applied in combinational chemistry, biochemistry apparatus and processes, library screening, etc., can solve the problem that pcr detection methods can miss the targeted dna for amplification, and the etiology of hlb remains large, and the detection accuracy is poor

Inactive Publication Date: 2016-06-02
US SEC AGRI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a sensor and rapid real-time PCR method using a specific primer, which can detect the presence of a specific nucleic acid in a sample. The method can also involve boiling the sample to obtain the nucleic acid. The detector molecule used can be a fluorescence reporter dye or a quencher dye. Additionally, a probe can be used with the detector molecule on one end and a quencher dye on the other end of the probe. Overall, the method allows for sensitive and rapid detection of nucleic acids.

Problems solved by technology

Although HLB presents systemically, low titer and uneven distribution of the HLB bacteria within infected plants (Tatineni et al, 2008, supra; Teixeira et al., Mol. Cell. Probes, Volume 22, 139-150, 2008; Li et al., Phytopathology, Volume 99, 139-144, 2009) can make reliable detection difficult.
However, these detection methods are typically diagnostic only after HLB associated phenotypic symptoms are observable.
Furthermore, the etiology of HLB remains, to a large extent, undefined.
However, current PCR detection methods can miss the targeted DNA for amplification because the Ca.

Method used

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  • Sensitive and Rapid Method for Candidatus Liberibacter Species Detection
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  • Sensitive and Rapid Method for Candidatus Liberibacter Species Detection

Examples

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example 1

[0058]The hyvI and hyvII genes were identified by analyzing the PCR amplicons form psyllid 62 which was the genomic DNA source used to produce the Ca. L. asiaticus genomic sequence, was generated during the gap closing process of the Ca. L. asiaticus genome sequence study (Duan et al., Mol. Plant. Microb Interact., 2009, supra, herein incorporated by reference in its entirety) and verified using BAC clones of Ca. L. asiaticus genome. Partial hyvI amplicons were subsequently cloned into the pCR® 2.1-TOPO® cloning vector in accordance with manufacture protocol (Invitrogen, Carlsbad, Calif.) and sequenced. Clone LH153.1 was shown to contain a single repeat of a complete 132 bp segment.

[0059]New primers LJ900f (forward), LJ900r (reverse) and LJ900p TaqMan® probe (Table 1), were designed based on the tandem-repeats of hyvI / hyvII using OLIGO 7 Primer Analysis Software version 7.23 (Molecular Biology Insights, Cascade, Colo.) targeting an integral 100 bp core sequence of the approximately ...

example 2

[0060]A series of in silico evaluations of both LJ900f and LJ900r primers and LJ900p probe were done using the NCBI BLAST megablast algorithm parameters for highly similar sequence alignment against the nucleotide (nr / nt) database having either the Ca. L. asiaticus genome that either included or excluded in separate searches respectively for each. Additional specificity of these primers and probe was evaluated by real-time PCT against a variety of DNA extracts from plant pathogens, Xanthomonas citri subsp. citri, X. axonopodis pv. citrumelo, Ralstonia solanacearum, Escherichia coli DH5α, soil bacteria in USHRL (United State Horticulture Research Laboratory) Picos farm and the USHRL facility greenhouse maintained citrus varieties and from plants of proximal and distal locations indicating specificity for the LJ900 primers (f, r, p) to Ca. L. species bacteria.

[0061]The specificity of the LJ900 primers and probe was evaluated in real-time PCR reactions, as stated above, that returned n...

example 3

[0062]Primer sets LJ900fr (with SYBR Green 1) and LJ900fpr (with TaqMan®) final optimal primer concentrations are approximately 600 and 900 nanomolar (nM) of LJ900f and LJ900r respectively, with an addition of approximately 500 nM of LJ900p to the LJ900fpr. These ratios were determined via gridded-paired primer concentrations against the single repeat pLJ153.1 (having a minimum of three technical replicates per pairing). The optimal annealing temperature is 62 degrees C. for maximum efficiency for both LJ900fr and LJ900frp methods as determined via gradient temperature experiments.

[0063]Amplification settings for LJ900fr are, initial denaturation (one cycle) at approximately 95 degrees C. for approximately 3 minutes, followed by approximately 40 cycles at approximately 95 degrees C. for approximately 3 seconds, then approximately 62 degrees C. for approximately 30 seconds, with fluorescence signal capture at the end of each 62 degree C. step followed by a default melt (disassociatio...

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Abstract

DNA amplification methods using novel primers obtained from the novel genes for hyvI and hyvII s from the Candidatus Liberibacter asiaticus genome and useful for detecting Ca. L. species in plants and insect hosts.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]This invention relates DNA amplification methods including improved real-time PCR detection methods, for the detection of Candidatus Liberibacter species from citrus and psyllid hosts. It also relates to novel DNA sequences, novel primers and probes made from the novel DNA sequences, and to kits containing said primers and reagents for the DNA amplification methods for the detection of Candidatus Liberibacter species.[0003]2. Description of the Related Art[0004]Citrus huanglongbing (HLB), also known as citrus greening, is a destructive disease that was first noted in the early 20th century in China (Zhao, Proc. Intl. Soc. Citriculture I: 466-469, 1981). This disease has spread throughout the global citrus producing regions, and has recently invaded North America, with first detection in Florida in 2005 (Knighten et al., USDA Departmental Release, Sep. 2, 2005). Three fastidious α-proteobacteria species of Candidatus Lib...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q2600/16C12Q1/689
Inventor DUAN, YONGZHOU, LIJUANMORGAN, JOHN KENT
Owner US SEC AGRI
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