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Polymerase chain replacement reaction detection method of candidatus liberibacter asiaticus

A citrus Huanglongbing and substitution reaction technology is applied in the field of polymerase chain displacement reaction detection of citrus Huanglongbing Asian species, which can solve the problems of difficult separation and identification of amplified products, and achieves the improvement of molecular diagnosis accuracy and false positives. Effects with low probability and broad market prospects

Active Publication Date: 2016-06-01
广西特色作物研究院
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Problems solved by technology

However, the loop-mediated isothermal amplification technology has certain defects because the amplification product is a series of nucleic acid fragments ranging from large to small without identification of one or a few nucleic acid fragments of a specific length: (1) Amplified products are not easy to separate; (2) If there is non-specific amplification, it is difficult to distinguish and prone to false positives [6][9][10]

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  • Polymerase chain replacement reaction detection method of candidatus liberibacter asiaticus
  • Polymerase chain replacement reaction detection method of candidatus liberibacter asiaticus
  • Polymerase chain replacement reaction detection method of candidatus liberibacter asiaticus

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[0034] The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

[0035] 1. Principle explanation

[0036] Such as figure 1 As shown, on the genome of H. citriensis Asiatic species, there are two upstream primer binding sites, which are two tandem repeat sequence units, and only one downstream primer binding site, which is a sequence-specific nucleic acid region. Since the DNA polymerase (SD enzyme) used in the reaction has dual characteristics of heat resistance and strand displacement activity, in the first round of reaction, the target nucleic acid fragment (double-stranded DNA) on the genome of Huanglongbing citri Denatured, unzipped into two DNA single strands, and then at 62°C annealing / extension temperature: the two upstream primers simultaneously bind to the DNA single-st...

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Abstract

The invention discloses a polymerase chain replacement reaction detection method of candidatus liberibacter asiaticus and belongs to the technical field of molecular biology. The method includes the following steps that firstly, DNA of citrus plant leaf midrib tissue is extracted; secondly, primers are designed and synthesized, the sequence of the upstream primer is 5'-TGTCCATATGATCTTCGATAATGCGA-3', and the sequence of the downstream primer is 5'-AGGGTGGATTAGATCGTTTTCTCTCAA-3'; thirdly, a polymerase chain replacement reaction is performed; fourthly, a result is judged. The detection method is high in detection sensitivity, good in specificity, low in false positive rate, easy to operate, broad in market prospect and suitable for large-scale application.

Description

technical field [0001] The invention relates to a polymerase chain displacement reaction detection method for citrus huanglongbing bacteria Asiatic species, belonging to the technical field of molecular biology. Background technique [0002] Huanglongbing (huanglongbing) is a worldwide destructive bacterial disease of citrus, and its pathogen is phloem-restricted hard-to-culture bacteria. So far, three species have been discovered: Asian species (Candidatus Liberibacterasiaticus), African species (Candidatus Liberibacter africanus) and American species (Candidatus Liberibacter americanus), among which the Asian species is the most widespread, and the Asian species is also popular in my country. The economic loss caused by Huanglongbing to my country's citrus industry is as high as several billion yuan every year. Timely diagnosis and removal of Huanglongbing-infected trees in the field is an important part of the comprehensive prevention and control of Huanglongbing. There...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6844C12Q2531/119C12Q2565/125
Inventor 娄兵海宋雅琴邓崇岭
Owner 广西特色作物研究院
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