Unlock instant, AI-driven research and patent intelligence for your innovation.

Nested PCR detection method for candidatus liberobacter asiaticum

A technology for citrus Huanglongbing and citrus Huanglongbing is applied in the detection field of common citrus Huanglongbing, which can solve the problems of low sensitivity, false positive sensitivity, missed detection, etc., so as to reduce the false negative rate and false positive rate, and improve the accuracy. Effect

Pending Publication Date: 2018-10-02
HUNAN AGRICULTURAL UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two methods all have flaws, and ordinary PCR often misses detection due to its low sensitivity, while fluorescent quantitative PCR detects a large number of false positives due to its high sensitivity (Kunta, M., J.V. da Graça, N.Malik, E. S. Louzada, and M. Sétamou. 2014. Quantitative distribution of Candidatus Liberibacter asiaticus in the aerial parts of the HLB-infected citrus trees in Texas. HortScience 49: 65-68.)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Nested PCR detection method for candidatus liberobacter asiaticum
  • Nested PCR detection method for candidatus liberobacter asiaticum
  • Nested PCR detection method for candidatus liberobacter asiaticum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] primer specificity signature

[0037] With these two pairs of primers screened, the inventor's laboratory has isolated and identified Huanglongbing citrus (HLB), Bacteria sativa L. oryzae ( Xanthomonas oryzae pv. oryzicola ), Canker sorrel citrus ( Xanthomonas axonopodis pv. citri ), Cucumber bacterial angular leaf spot ( Pseudomonas syringae PV . lachrymans ) and Bacillus pumilus ( Bacillus pumilus ) at the same time for nested PCR, only the 447bp target band was amplified by H. citri (see image 3 ).

[0038]It shows that the two pairs of specific primers of the present invention are only specific to the pathogen of citrus huanglongbing, and can be well distinguished from the pathogen of citrus canker.

Embodiment 2

[0040] Use these two pairs of specific primers of the present invention to carry out common PCR, fluorescence quantitative PCR and nested PCR to more than 1200 suspected samples of citrus Huanglongbing disease in Hunan citrus Huanglongbing high-incidence area, and it is found that common PCR-positive samples are tested by fluorescence quantitative PCR and nested PCR. Nested PCR tests are all positive, and some ordinary PCR negative samples are also tested positive by fluorescent quantitative PCR and nested PCR, but some samples that are positive by fluorescent quantitative PCR are negative by nested PCR, and then combined with symptoms Analysis found nested PCR to be the most accurate (see Figure 4 ).

[0041] It shows that the specific primers of the present invention are specific to the Asian citrus Huanglongbing bacteria, and the early monitoring of the citrus Huanglongbing in my country can be carried out accurately through the nested PCR.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a nested PCR detection method for candidatus liberobacter asiaticum. The method is characterized in that two pairs of specific primers F3 / B3 and F1 / B1 are designed by using theprinciple of high conservation of an outer membrane protein gene of the candidatus liberobacter asiaticum; PCR amplification of a to-detected sample is performed by applying the two pairs of primers;an amplification product is subjected to clone sequencing to obtain a gene segment with the length of 447bp; by NCBI online comparison, the segment can completely cover an outer membrane protein synthetase gene of the candidatus liberobacter asiaticum, and the consistency is 100 percent. The pairs of primers used by the detection method disclosed by the invention are different from other detection technologies and can be used for quickly and accurately identifying the candidatus liberobacter asiaticum.

Description

technical field [0001] The invention relates to a detection method for common citrus huanglongbing bacteria, in particular to a nested PCR detection method for detecting citrus huanglongbing bacteria Asian species. Background technique [0002] Citrus huanglongbing is a worldwide citrus disease that occurs in the phloem of citrus and causes devastating damage to the citrus industry. Once the disease occurs, there is no cure, so it is called "citrus cancer". It is a non-culturable Gram-negative bacterium of the α-Proteobacteria (α-Proteobacteria) Cadidatus Caused by Liberibacter, they have three subspecies, which are Cadidatus Liberibacter asiaticus (abbreviated as CLas), Cadidatus Liberibacter africanus (abbreviated as CLaf) Cadidatus Liberibacter americanus (abbreviated as CLam) causes citrus huanglongbing in Asia, citrus huanglongbing in Africa and citrus huanglongbing in America, respectively. Citrus huanglongbing has no specific symptoms, and there is no specific dru...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12R1/01
CPCC12Q1/686C12Q1/689C12Q2549/119
Inventor 洪艳云易图永罗永阳戴良英谭小平
Owner HUNAN AGRICULTURAL UNIV