Rapid Nucleic Acid Detection Technology Of Botrytis Cinerea In Ginseng

Ginseng gray mold is one of the main diseases of ginseng which is caused by the infection of Botrytis cinerea.Ginseng gray mold is mostly prevented and controlled by chemical methods in production,and this traditional plant disease prevention method always lead to excessive pesticide residues in both ginseng and soil.Conventional pathogen testing methods often take a long time and it is also cannot achieve early detection of the disease.Therefore,to conduct an efficient,rapid and accurate detection method used for the early identified of Botrytis cinerea in ginseng is of great important in prevention and control of the ginseng diseases.Based on the wide application of molecular detection technology,this study attempted to establish a newly pathogenic bacteria nucleic acid extraction technology,combined with a PCR-Nucleic acid sensor(PCR-NAS)rapid detection system to achieve rapid detection of Botrytis cinerea,it may providing a new detection technology for the early control of ginseng gray mold.The research results are as follows:1.As a sample pretreatment process,nucleic acid extraction plays a decisive role in the accuracy of the final analytical results.In this study,based on the study of nucleic acid extraction methods,a fungal genomic DNA extraction technique was established.The method obtained DNA samples with high yield and high purity,and it can effectively remove some PCR inhibitors such as humic acid,and it can better suit for the downstream PCR detection.2.In this study,the newly developed nucleic acid extraction method and automatic nucleic acid extractor was used for DNA extraction of real samples,the results showed that the average DNA concentration extracted by nucleic acid extraction procedure R1 was 416.97ng/μL,and a Ct Mean of real time PCR detection was 17.9;the average DNA concentration extracted by nucleic acid extraction procedure R2 was 277.01ng/μL,with a Ct Mean of real time PCR was 19.9.Therefore,Program R1 was used in this study.Using the automatic nucleic acid extractor,a total of 96 DNA samples can simultaneous extracted within 35 min.3.In this study,a PCR-NAS rapid detection technique was established.The method was specific,sensitive and reproducible,with a lower limit of detection of1ng/μL for Botrytis cinerea genome and no cross-reactivity with other ginseng pathogens.The CV of intra-and inter-batch reproducibility of the test was less than11.4%.4.In this study,PCR-NAS and qPCR assay techniques were used to detect artificial mock samples,and the results showed that both methods were able to detect the B.cinerea in ginseng leaves at 6 h of inoculation,and in ginseng soil at 12 h of inoculation.For in field detection of 50 real samples using PCR-NAS,the results showed that 20 diseased soils were all detected as positive,besides that 5 samples were also detected positive in 30 healthy soils.Besides that,in other 50 real sampls,20 diseased ginseng leaves were all detected as positive,11 samples were also detected as positive in 30 healthy leaves in total real samples.These results showed that the detection technique developed in this study has a high specificity and high sensitivity.