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Internal reference genes of fluorescence quantitative analysis of candidatus liberibacter asiaticus and application thereof

A technology of citrus huanglongbing and citrus huanglongbing, which is applied in the field of molecular plant pathology and can solve the problems of lack of applicable internal reference genes and the like

Active Publication Date: 2019-05-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the RT-qPCR minimum standard (minimum information for publication of quantitative real-time PCR experiments, MIQE) shows that there are no internal reference genes suitable for all experimental conditions

Method used

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  • Internal reference genes of fluorescence quantitative analysis of candidatus liberibacter asiaticus and application thereof
  • Internal reference genes of fluorescence quantitative analysis of candidatus liberibacter asiaticus and application thereof
  • Internal reference genes of fluorescence quantitative analysis of candidatus liberibacter asiaticus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Screening of embodiment 1 internal reference gene

[0090] 1. Experimental method

[0091] 1. Preparation of test samples

[0092] Huanglongbing was inoculated onto plants of different species using the Huanglongbing disease bud grafting technique: October orange (Citrus reticulata Blanco cv.ShiyueJu), pomelo (Citrus maxima), year orange (Citrusreticulata Blancocv.Nian Ju) and lemon (Citrus limon( L.) Burm.F.), 3 biological replicates for each species, and the materials were cultivated under natural conditions in a net room. The leaves of typical symptoms and leaves of healthy plants were randomly collected 3 to 18 months after grafting, and each sample was collected from a different plant. After cutting a part of each leaf and freezing it in liquid nitrogen, store it at -80°C for future use, and store the other part at 4°C for future use.

[0093] 2. DNA extraction and RT-qPCR detection

[0094]Plant tissue DNA was extracted according to the instructions of the OME...

Embodiment 2

[0143] Example 2 A kit for RT-qPCR analysis of citrus Huanglongbing

[0144] 1. Composition

[0145] Includes amplification primers for internal reference genes gyrA and ftsZ, SYBR Green qPCR Master Mix and ddH 2 O; wherein, the nucleotide sequence of the amplification primer of the internal reference gene gyrA is as shown in SEQ ID NO.2~3; the nucleotide sequence of the amplification primer of the internal reference gene ftsZ is as shown in SEQ ID NO.5~6, The primer concentration was 20 μmol / L.

[0146] 2. How to use

[0147] 1. Extraction of total RNA from plant tissue: Take 80mg-100mg of the midrib of each sample, grind it into powder in liquid nitrogen, and extract the plants of each sample with reference to the OMEGA Plant Tissue Total RNA Extraction Kit (OMEGA Abio-tek, Plant RNAKit R6827) Tissue total RNA.

[0148] 2. Total RNA quality detection: 1.0% agarose gel electrophoresis, TBE×5 buffer solution, 130V, 35min; concentration and purity were determined by NanoDro...

Embodiment 3

[0155] Application of embodiment 3 fluorescence quantitative kit and its specificity detection

[0156] In order to further prove the reliability of the internal reference gene, 16S rRNA and gyrA+ftsZ combination were respectively used as internal reference genes to analyze the expression pattern of Las△5313, an important pathogenic gene of H. citri, in leaves of plants infected with HLB.

[0157] 1. Experimental method

[0158] 1. Sample preparation: Take pomelo, Nianju, and tangerine plants grafted with diseased buds for 6 months, and pomelo plants grafted with diseased buds for 12 months as samples, and refer to Example 2 to extract total RNA and synthesize the first strand of cDNA.

[0159] 2. Specific detection: refer to the fluorescent quantitative PCR and detection steps in Example 2.

[0160] 2. Experimental results

[0161] The result is as Figure 10 As shown, the results show that the expression pattern of Las△5313 obtained by using 16S rRNA as an internal refere...

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Abstract

The present invention discloses internal reference genes of fluorescence quantitative analysis of candidatus liberibacter asiaticus and an application thereof. The internal reference genes are gyrA and ftsZ, and nucleotide sequences thereof are respectively shown in SEQ ID NO.9 and SEQ ID NO.10. An internal reference gene combination of the gyrA and ftsZ is firstly screened to be suitable for geneexpression analysis of candidatus liberibacter asiaticus infected with different hosts and after infected with the hosts in different periods, specific primers are further provided, and the internalreference gene combination provides a reliable guarantee for accurate and quantitative detection of the candidatus liberibacter asiaticus gene, and at the same time can be used to detect whether samples are infected with the candidatus liberibacter asiaticus. The selected internal reference gene combination of the gyrA and ftsZ has advantages of high reliability, good stability and wide applicability. A detection method and a detection kit for the candidatus liberibacter asiaticus constructed by using the internal reference gene combination have great advantages compared with single or traditional internal reference genes, and are worthy of promotion and application in a large area.

Description

technical field [0001] The invention belongs to the technical field of molecular plant pathology. More specifically, it relates to an internal reference gene for fluorescence quantitative analysis of citrus Huanglongbing bacteria and its application. Background technique [0002] Citrus Huanglongbing (HLB) is one of the most devastating diseases in citrus production, which seriously restricts the development of citrus industry in China. Citrus huanglongbing is now generally believed to be caused by a Gram-negative bacterium confined to the sieve tube cells of the phloem. The bacterium belongs to the genus Candidatus Liberibacter spp. of the α-Proteobacteriacea class (Proteobacteriacea); it can be divided into heat-resistant Asian species (Candidatus Liberibacter asiaticus, CLas), American species (Candidatus Liberibacter spp. Liberibacter americanus, CLam) and the African species (Candidatus Liberibacter africanus, CLaf) that occur in South Africa can infect almost all cit...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11
Inventor 许美容郑永钦郑正邓晓玲
Owner SOUTH CHINA AGRI UNIV
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