Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus candidatus liberobacter asiaticum Jagoueix and testing process thereof

Abstract

This invention relates to oligonucleotide prime, mandarin orange yellow dragon germ real time fluorescence PCR detecting probe, solid phase reagent and its detecting method of mandarin orange yellow dragon germ detecting. It is a special quick detecting method direct towards mandarin orange yellow dragon disease, there is only 3 hours from sampling to detection, and detecting sensitivity is high, specificity is strong, stable and reliable. Misjudging in existing normal diagnosis method, missed diagnosis in electrical glass by asymmetry of germ and hard antibody preparation in single clone antibody and lack of specific reaction are conquered by this method, and false negative and false positive of normal PCR are avoided. Operation of solid phase kit is convenient, and its using is simple, and detecting sensitivity is high, specificity is strong, stable and reliable. So it is propitious to port detection of mandarin orange of import and export. Also it is propitious to allocation and transportation of mandarin orange inoculation, scion and other asymptomatic germ material, and early stage warning detection of non epidemic-stricken area orange yellow dragon disease.

Description

technical field [0001] The invention relates to a biotechnology, in particular to a simple, fast, specific and sensitive solid-phase molecular detection kit for Candidatus Liberibacter asiaticus, which is suitable for port quarantine, agricultural production, plant protection and other departments use. Background technique [0002] Citrus Huanglongbing Disease (HLB) is a devastating plant disease, which is listed as an important quarantine disease at home and abroad. The disease mainly occurs in Asia and more than 40 countries in Africa. It is mainly distributed in Southeast Asia, South Africa, East Africa, Arabian Peninsula, Reunion Island, Mauritius, Madagascar and South America in the Indian Ocean abroad; in April 2004, Sao Paulo, Brazil, In September 2005, HLB was discovered successively in Florida, USA. In the late 1990s, among the 17 provinces and cities producing citrus in my country, 11 provinces and cities were infected by Huanglongbing. [0003] Citrus huanglong...

AI Technical Summary

Problems solved by technology

Due to the small number of pathogenic bacteria in the diseased tree tissue in the early stage, the distribution is uneven, the detection rate is low, and it is easy to miss the diagnosis

(3) Immunological methods, because the preparation of pathogenic bacteria antibodies is difficult, the titer is low, and it is accompanied by non-specific reaction interference; most of them are suitable for fluorescence detection, but it is difficult to be popularized and applied

The representative of the former category is the fluorescence-labeled probe method, which has high specificity and can effectively avoid non-specific amplification and false positive phenomena that are difficult to avoid in conventional PCR, but its detection cost is relatively high; One type of representative is the SYBR Green I fluorescent dye method, which is relatively cheap. Since each double-stranded DNA molecule can non-specifically bind to multiple SYBR Green I dye molecules, the fluorescent signal is stronger than the former, so the sensitivity is also higher. High, but this method also has high requirements for the specificity of the primer itself, and it is necessary to avoid the formation of primer dimers, resulting in false positive detection, so the design and optimization work must be carefully completed