CN-121272110-B - Specific molecular target, primer group, detection method and application of ginseng colletotrichum gloeosporioides

Abstract

The invention relates to the technical field of molecular detection, and particularly discloses a molecular target for detecting ginseng discoium spinosum. Aiming at the technical problems of long time consumption, poor specificity, difficulty in realizing accurate quantification of pathogenic bacteria in soil and the like of the existing detection method of the ginseng radix ophiopogonis, the invention provides a section of long nucleotide sequence special for the ginseng radix ophiopogonis as a molecular target, and the nucleotide sequence is shown as SEQ ID NO. 1. The specific primer group designed based on the molecular target, the kit containing the primer group and the detection method for non-diagnosis purpose form a complete detection system together, can realize rapid, sensitive, specific qualitative and accurate quantitative detection of the ginseng colletotrichum in ginseng plant tissues, seeds and soil samples, and provides a reliable technical tool for early warning of ginseng anthracnose, dynamic monitoring of pathogenic bacteria and scientific prevention and control.

Inventors

• GAO JIE
• FENG SHI
• DU HAN
• CHEN YUTONG
• SHI YUE
• SONG YUEJIA
• FENG SHUANG
• LIU LIPING
• YANG LINA
• HE RONGLIN
• CHEN CHANGQING
• LU BAOHUI
• LI XIN
• YIN NAN

Assignees

• 吉林农业大学

Dates

Publication Date

20260512

Application Date

20251210

Claims (8)

1. 1. The application of the molecular target in detecting the ginseng discodermia spinosa is that the nucleotide sequence of the molecular target is shown as SEQ ID NO. 1.
2. 2. A primer set for detecting a colletotrichum gloeosporioides, characterized in that the primer set comprises at least one of the following primer pairs: Primer pair a consists of a primer pair with sequences shown in SEQ ID NO. 2 and SEQ ID NO. 3; primer pair b consists of primer pairs shown in SEQ ID NO. 4 and SEQ ID NO. 5.
3. 3. A kit for detecting ginseng radix echinococcus, which is characterized in that, the kit comprises the primer set of claim 2.
4. 4. The detection method of the ginseng raw disc spore is characterized by comprising the following steps of: S1, extracting genome DNA of a sample to be detected; S2, using the genome DNA obtained in the step S1 as a template, and performing PCR amplification by using the primer group of claim 2; S3, detecting PCR amplified products, wherein when the primer pair a is used, a DNA band with the length of 484 bp is detected through gel electrophoresis, and/or when the primer pair b is used, the value of C t is detected to be less than 40 through real-time fluorescence quantitative PCR, and the fact that the sample contains the ginseng-induced septoria is indicated.
5. 5. The method according to claim 4, wherein the PCR amplification is real-time fluorescent quantitative PCR, and the step of constructing a standard curve comprises the steps of: Performing real-time fluorescent quantitative PCR amplification using a known copy number of a standard comprising the molecular target sequence of claim 1, obtaining a series of C t values; drawing a standard curve by taking the logarithmic value of the copy number of the standard sample as an abscissa and the corresponding C t value as an ordinate; And after the step S2, substituting the C t value of the sample to be tested into the standard curve, and calculating to obtain the copy number of the ginseng colletotrichum.
6. 6. The method according to claim 5, wherein the linear regression equation of the established standard curve is y= -4.29x+44.03, where x is the logarithmic value of the copy number of the standard substance and y is the corresponding C t value.
7. 7. The method according to any one of claims 4 to 6, wherein the sample to be tested is derived from ginseng plant, ginseng seed or soil.
8. 8. Use of the primer set according to claim 2 for preparing a preparation or a kit for detecting the ginseng radix et caulis Opuntiae Dillenii.

Description

Specific molecular target, primer group, detection method and application of ginseng colletotrichum gloeosporioides Technical Field The invention relates to the technical field of molecular detection. More specifically, the invention relates to a ginseng discodermia acuminata specific molecular target, a primer group, a detection method and application. Background Ginseng (Panax ginsengC.A. Mey) is known as a 'Baicaowang' as a traditional rare Chinese medicinal material in China, and the yield and the quality of the ginseng are directly related to huge economic value and medicinal value. However, during ginseng cultivation, it is subject to attack by a variety of diseases, of which anthrax caused by the colletotrichum (Colletotrichum panacicola) is a serious foliar disease. The disease can cause the ginseng leaves to have necrotic spots and premature senility and fall off, and even the whole plant is dead when serious, thereby obviously threatening the yield and quality of the ginseng and causing great economic loss for ginseng farmers. Currently, identification and detection of the ginseng colletotrichum gloeosporioides mainly depend on traditional tissue separation and morphological identification methods. The method comprises the steps of firstly separating and purifying pathogenic bacteria from pathogenic tissues, then culturing the pathogenic bacteria on a proper culture medium for 5-7 days, and then observing the colony morphology, the microcosmic discs, the bristles, the conidium and other microstructure characteristics through a microscope to identify species. However, this approach has a number of limitations: 1. the time consumption is long, the efficiency is low, more than one week is usually required from sample collection to identification result acquisition, the requirements of disease rapid monitoring and early warning cannot be met, and the screening of large-scale field samples is difficult to deal with. 2. The accuracy of the method is highly dependent on the professional experience and subjective judgment of operators, and the ginseng discodermia spinosa is difficult to distinguish from the closely related species (such as C. destructivum, C. gloeosporioides and the like) in morphology, and misjudgment is easy to cause. 3. The accurate quantification cannot be realized, the traditional method can only carry out qualitative judgment, and the capacity analysis of the latent trace or dormant pathogenic bacteria in the environmental samples such as soil, seeds, irrigation water and the like cannot be carried out, so that the epidemic risk and the infection source of the diseases are difficult to accurately evaluate. 4. The sensitivity is limited, and for asymptomatic latent infection or early infection, the traditional method often has missed detection due to the separation culture of pathogenic bacteria. Although molecular biology techniques such as conventional PCR have been widely used for detection of plant pathogenic bacteria, a specific and high-sensitivity molecular detection system for the ginseng discorea spinosa has not been reported yet. The prior art lacks a fully verified specific molecular target which can accurately distinguish the ginseng colletotrichum gloeosporioides from other related species and other common ginseng pathogenic bacteria. In addition, the conventional PCR method is still insufficient in sensitivity, and absolute quantification of pathogenic bacteria cannot be realized, so that the application of the conventional PCR method in accurate monitoring of trace pathogenic bacteria in complex matrixes such as soil is limited. Therefore, the development of a rapid, sensitive and specific molecular detection method capable of realizing accurate quantification is urgently needed in the field, and the method is used for early monitoring and risk assessment of the ginseng colletotrichum in ginseng plants, propagation materials and planting soil, so that a reliable technical tool is provided for green prevention and control of ginseng anthracnose and safe production of traditional Chinese medicinal materials. Disclosure of Invention It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later. The invention firstly provides a section of DNA sequence special for ginseng discodermia acuminata as a core molecular target (SEQ ID NO: 1). Based on the analysis of the long sequence, two pairs of specific primers are designed and are respectively used for conventional PCR qualitative detection and real-time fluorescence quantitative PCR (qPCR) qualitative and absolute quantitative detection. To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a molecular target for detecting a colletotrichum gloeosporioides, the nucleotide sequence of which is shown in SEQ ID NO. 1. There is provided a primer set for detecting a colletotrichum gloeosporioi