A PCR And Real-time Fluorogenic Quantitative-PCR For Rapid Identification Of Bee Chalkbrood Disease

The bee chalkbrood is a new malignant infectious disease affecting Apis meilifera L.Apathogenic Ascosphaera apis was separated from bee larva died of chalkbrood. It has been reported that the Ascosphaera apis spores can remain infective for at least 15 years. The disease spreads when spores are transported on drifting bees, hive parts, clothing, and contaminated pollen or honey .This kind of disease is very difficult to control. It is the most serious disease of honeybee broods around the world and causes considerable economic losses to beekeepers. In the field the disease is detected by inspection, and a positive diagnosis is based on clinical symptoms. At present, there is not a standardized norm for the disease detection and prevention in countries both at home and abroad.Along with PCR technology development, people used the PCR technology to carry on a series research which had the significance exploration. Now molecular biology detection technologies(e.g.PCR) diagnose the aspect in the human and the animal infectious disease widely to apply, gets sick the diagnosis research aspect in overseas in the bee also to have some research report, but in domestic does not have this aspect research report. The PCR examination method has the high sensitivity, the strong specificity and so on the merits, available in fast diagnosis honeybee infectious disease, the mix infection distinction diagnosis which causes regarding viral disease and many kinds of causes of disease is especially more effective.A polymerase chain reaction was developed to simultamneous detect bee chalkbrood disease. The internal transcribed spacers, ITS1 and ITS2, and 5.8S region of ribosomal DNA(rDNA) reported and registered in Genebank of different strains of Ascosphaera apis(A.apis) was respectively and compared with each other.One set of specific primers of Ascosphaera apis was designed.The primers can be amplified 471-bp,the designed primers were used to PCR reaction.The result showed that the design of the one set of primers was right. Then the optimal condition of PCR for A.apis was determined. This research has established the PCR best response condition and the best reacting system through the series experiment optimization, and through the sensitivity test, the specificity experiment and the duplicated experiment indicated establishes the PCR method has the good sensitivity, the specificity and the stability. The establishment PCR method may examine 7CFU/ml the cause of disease sample, the entire experiment operating process 5 hours, compared the conventional clinical diagnosis or the ELISA diagnosis method enhanced the sensitivity also reduced the diagnosis time, which can examine the honeybee chalkbrood disease germ fast and accurately. Real-time fluorogenic quantitative-PCR(RT-FQ-PCR) assay was developed for monitoring and detecting A.apis DNA.Using PCR assay, the FQ-PCR was based on a 530-bp region of the internal transcribed spacers,ITS1 and ITS2, and 5.8S region of ribosomal DNA gene. One set of specific primers and a fluorescene probe were designed and synthesized, a 82-bp sequence was amplified. The probe for TaqMan was labeled with a fluorescent reporter dye(FAM) at 5' end and a quencher dye (TAMRA) at 3' end. Then the sensibility t, specificity and reproducibility test were carried out, the results showed that the method could detect the minimum limit was 1ng. After the experimental proof, this experimental application primer, the probe to be possible to expand increases 1ng of the Ascosphaera apis fungus DNA sample, the examination of specificity assumes the negative, explained this research establishment fluorescence real-time PCR has highly sensitive and the specificity; This research uses the instrument is ABI 7000, may simultaneously examine 96 samples, and in real-time monitor reaction system fluorescence intensity change situation, and further because does not use the electrophoresis, reduces the PCR product pollution greatly, therefore this experimental establishment method namely has the high flux, available does not have the PCR product pollution in the massive sample long-term examination, enhanced the method specificity, but also has the fast examination characteristic, generally makes the examination result in 4 hours. If has the false positive, but very quickly implements the fast examination with the backup sample to carry on the correction, makes compared to the conventional PCR more objective test result, more objective determination masculine gender, negative and suspicious. The duplicated experiment proved this experimental establishment the method is stable, which can apply in the practice to identify Ascosphaera apis.