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Application of candidatus liberibacter asiaticus PAP protein antibody in preparation of citrus yellow shoot early infection detection kit

A technology for citrus Huanglongbing and citrus Huanglongbing, which is applied in the field of molecular biology, can solve the problem that there is no commercial protein product for early infection detection of citrus Huanglongbing, and achieves the effects of low detection cost, simple kit and high sensitivity

Inactive Publication Date: 2018-12-28
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are currently no commercial protein products for early detection of H. citri infection

Method used

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  • Application of candidatus liberibacter asiaticus PAP protein antibody in preparation of citrus yellow shoot early infection detection kit
  • Application of candidatus liberibacter asiaticus PAP protein antibody in preparation of citrus yellow shoot early infection detection kit
  • Application of candidatus liberibacter asiaticus PAP protein antibody in preparation of citrus yellow shoot early infection detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Screening of early infection samples of H. citri:

[0027] 1. Screening of samples infected with H. citrus early stage

[0028] The leaves of 15 green orange plants without obvious symptoms were collected from the green orange orchard infected by HLB in Qiongzhong Li and Miao Autonomous County, Hainan Province, and the total DNA was extracted by the CATB method (refer to Wei, S.H. and Meng, N., 2011), through fluorescent quantitative PCR detection, it was determined that 10 strains were infected by HLB citrus, but did not show symptoms such as yellow flowers and mottled, so they were identified as early samples of HLB infection.

Embodiment 2

[0030] Target protein screening for early detection of citrus Huanglongbing:

[0031] 1. Preparation of total RNA from early infection samples of Huanglongbing citrus

[0032] The 10 strains of HLB infection early samples screened in Example 1 were used to extract the total RNA of the samples according to the conventional protocol.

[0033] 2. Gene screening for the detection of target proteins of Huanglongbing citrus

[0034] The total RNA of the sample was synthesized into the first strand of cDNA with PrimeScript reverse transcriptase (TAKARA). Using 16S RNA primers as internal reference (refer to Yan J.et al, 2012 for methods and 18S RNA primers), multiple secreted protein genes include: ATPase (ACT56858.2), MFP (ACT56859.1), Serralysin (ACT56857.1) , 408(ACT57211.2), 24A(ACT57202.1), PAP(ACT57200.1), MSr(ACT57168.1), MSP(ACT57161.1), fATP(ACT57157.1), 377(ACT57577.1), etc. Real-time quantitative PCR screening was carried out, and the target gene with high expression le...

Embodiment 3

[0037] Application of PAP protein antibody against citrus huanglongbing in the preparation of citrus huanglongbing early detection kit:

[0038] In this example, the preparation of polyclonal antibody is taken as an example for illustration. The monoclonal antibody prepared by using PAP protein as an antigen is also applicable to the early detection of citrus Huanglongbing.

[0039] 1. Preparation of PAP purified protein:

[0040] 1) Determination of the base sequence of the PAP protein gene

[0041] According to the PAP protein gene, the nucleotide sequence of the gene is obtained by PCR amplification, and the base sequence shown in SEQ ID No.1 is obtained.

[0042] 2) Construction of recombinant plasmids

[0043] Primers were designed according to the base sequence of the multiple cloning site of pET32a and the PAP protein gene:

[0044] Upstream primer: 5'-CCGGATATCTTGCATCGTAAGCGCCAAAGAG-3'

[0045] Downstream primer: 5'-CCGCTCGAGTCCTGACGGGAGGAGAGGAGG-3'

[0046] Ampli...

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Abstract

The invention belongs to the field of molecular biology and discloses application of a candidatus liberibacter asiaticus PAP protein antibody in preparation of a citrus yellow shoot early infection detection kit. An operating titer of a PAP protein multiclonal antibody prepared by utilizing the conventional way is determined to be 1:(500-1000) by virtue of an enzyme-linked immunosorbent assay (ELISA) method. The PAP multiclonal antibody is utilized for performing ELISA detection, and the lowest PAP protein content which can be detected is about 0.005 [mu]g. In the actual use, the prepared PAPmulticlonal antibody can be utilized and applied to ELISA detection on an HLB early infected sample, and 7 pathogenic bacteria can be detected at least.

Description

technical field [0001] The application belongs to the field of molecular biology, and specifically relates to the application of PAP protein antibody of citrus huanglongbing bacteria in the preparation of citrus huanglongbing early infection detection kit. Background technique [0002] Citrus Huanglongbing (HLB) is one of the devastating diseases that seriously endanger the world's citrus industry. At present, the disease has endangered and spread in more than 50 countries in Asia, Africa, Oceania, and North and South America. Among the 19 major citrus producing areas in China, 11 have been affected by HLB. Citrus huanglongbing is caused by the infection of citrus huanglongbing bacteria. At present, there are three main types of pathogenic bacteria, namely, Asian type (Candidatus Liberibacter asiaticus), African type (Candidatus Liber ibacter africanus) and American type (Candidatus Liberibacter americanus). H. citri is a Gram-negative bacterium belonging to the genus Cand...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56911
Inventor 余乃通刘志昕李宾宾杨毅黎维丽
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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