A primer probe combination, kit and method for detecting palm yellowing phytoplasma based on real-time fluorescent quantitative PCR technology

Abstract

The application provides a probe AM-Prode and specific primers AM-f / AM-r designed based on a 16S rDNA gene sequence of Arecaflavescence phloemast, a kit and a detection method. The application has high specificity for detecting Arecaflavescence phloemast, and Areca genome, diseases, pathogens and endophytes cannot cause non-specific interference to the detection technology; the application has high accuracy and good repeatability, and in a sensitivity test, 1.16x10 1 copies / μL is detected, and in application, the minimum detection concentration is up to 1.50 copy / μL; the application has wide applicability, and detection of adult trees and seedlings has good stability, and can be used for detecting phloemast carried by Areca pests in the field, and can be used for detecting plant diseases caused by phloemast infection, such as guayule witches broom, bamboo witches broom, vinca minor leaf disease, khayaflavescence, tiny round witches broom, pepper yellowing disease, Corchorus capsularis witches broom and peanut witches broom.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogen detection technology, specifically relating to a primer and probe combination, kit, and method for detecting areca etiolated phytoplasma based on real-time quantitative PCR technology. Background Technology

[0002] Areca catechu (Areca cathecu L.) is a perennial tropical cash crop belonging to the Arecaceae family and is the leading medicinal herb in southern China. It is mainly cultivated in tropical and subtropical countries and regions such as India, China, Myanmar, Bangladesh, and Indonesia. In China, areca catechu is cultivated in Hainan, Taiwan, and Yunnan provinces, with Hainan Province accounting for over 95% of the national planting area and ranking second in the world in terms of output (Yang Lianzhen et al., 2018). According to the "Hainan Statistical Yearbook 2021," the planting area of ​​areca catechu reached 124,686 hectares, making it the second largest tropical cash crop after natural rubber. In 2019, the annual output value of my country's areca catechu industry reached 70-80 billion yuan, of which the total output value of Hainan Province's areca catechu industry was approximately 26 billion yuan, and the deep processing and related industries of areca catechu in Hunan Province reached 50-60 billion yuan. Currently, areca nut is an important tropical economic crop in Hainan Province and one of the "three trees" (rubber, areca nut, and coconut) that the Hainan Provincial Government focuses on developing. It is the main source of income for 2.3 million farmers in the central and eastern parts of Hainan Province and an important pillar industry in the central region (Dai Wenting et al., 2021).

[0003] With the increasing economic benefits of areca nut, farmers' enthusiasm for planting has greatly increased, and the planting area has continued to expand. However, plant protection problems are becoming increasingly serious. Areca palm yellow leaf disease (YLD), caused by phytoplasma, is one of the most serious and fatal systemic diseases affecting areca nut. This disease first appeared in the Muvattupuzha region of Ernakualm, India in 1914 (Fan Haikuo et al., 2007), and first appeared in Tunchang County, Hainan Province, my country in 1981 (Zhao Jiansheng, 1985). Currently, the disease has spread to almost all areca nut planting areas in India and Hainan Province, my country (Tang Qinghua et al., 2021; Cao Xueren et al., 2016), and has also caused damage in some parts of Sri Lanka (Silva et al., 2015). The disease has been causing increasing damage in Hainan Province year by year. Mild cases result in a 10% to 20% reduction in yield, while severe cases result in a 50% to 60% reduction. In some areas, the disease has even led to complete crop failure. According to incomplete statistics, the annual losses caused by yellowing disease exceed 2 billion yuan, which seriously affects the wealth creation of farmers and the growth of the rural economy in Hainan Province (Che Haiyan et al., 2018).

[0004] Accurate detection is crucial for the diagnosis of YLD. Currently, the main detection techniques for phytoplasma include nested PCR (Meng Xiuli et al., 2021), real-time quantitative PCR (Che Haiyan, 2010), loop-mediated isothermal amplification (Yu et al., 2020), and droplet PCR (Yu Shaoshuai et al., 2020). Nested PCR is the most commonly used technique. However, the specificity or sensitivity of current detection techniques is not sufficient for the large-scale detection of YLD. Therefore, we need to establish an accurate and efficient detection technique, namely real-time quantitative PCR. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology, study the specific primers AM-f / Am-r and AM-Prode probes designed based on the 16S rDNA gene sequence of phytoplasma, and the real-time fluorescence quantitative PCR detection method, and verify the sensitivity, specificity, stability and application effect of the method, in order to provide a reliable technical means for the accurate diagnosis of YLD, pathogen monitoring and molecular epidemiology research.

[0006] The first aspect of the present invention is to provide a probe designed based on the 16S rDNA gene sequence of Areca etiolated phytoplasma, which is probe AM-Prode, with the nucleotide sequence: 5'-CCTACCAAGACTATGATGTG-3', the 5' end is labeled with a fluorescent group and the 3' end is labeled with a quenching group;

[0007] Preferably, the fluorescent group labeled at the 5' end is FAM, and the quenching group labeled at the 3' end is MGB.

[0008] A second aspect of the present invention is to provide a primer pair designed based on the 16S rDNA gene sequence of Phytoplasma arecae, including an upstream primer AM-f and a downstream primer AM-r, the nucleotide sequences of which are shown below:

[0009] Upstream primer AM-f: 5'-CCCAATGTGGCCGTTCAAC-3';

[0010] Downstream primer AM-r: 5'-GAAACGACTGCTAAGACTGGATAGGA-3'.

[0011] A third aspect of the present invention is to provide a primer-probe combination for detecting areca etiolated phytoplasma based on real-time quantitative PCR technology, comprising the probe described in the first aspect of the present invention and the primer pair described in the second aspect of the present invention.

[0012] A fourth aspect of the present invention is to provide a kit containing the probe described in the first aspect of the present invention.

[0013] Preferably, the kit comprises the probe described in the first aspect of the present invention and the primer pair described in the second aspect of the present invention.

[0014] Preferably, the kit further includes a positive control;

[0015] Preferably, the positive control is a plasmid standard containing the 16S rDNA amplification sequence of Areca catechu yellow phytoplasma.

[0016] Preferably, the areca nut yellowing phytoplasma 16S rDNA amplification sequence plasmid standard is a recombinant vector constructed by ligating the product (amplification product length is 173bp) obtained by PCR amplification using the primer pair described in the second aspect of the present invention with a vector, using the areca nut yellowing disease positive sample as a template.

[0017] The fifth aspect of the present invention is to provide a method for detecting areca yellowing phytoplasma, using the kit described in the fourth aspect of the present invention.

[0018] Preferably, the detection method includes the following steps: using the total DNA of the areca nut to be tested as a template, using the primer pair described in the second aspect of the present invention as primers, and using the probe described in the first aspect of the present invention as a probe, real-time fluorescent PCR detection is performed; if the fluorescent signal is enhanced during the PCR process, then the plant is infected with phytoplasma.

[0019] Preferably, the PCR reaction system consists of 0.6 μL of upstream primer (10 μM), 0.6 μL of downstream primer (10 μM), 0.4 μL of probe (10 μM), 10 μL of 2×SuperRealPreMix, 2 μL of DNA template, 0.2 μL of 50×ROX Reference Dye*3, and ddH2O added to a final volume of 20 μL.

[0020] Preferably, the PCR amplification reaction conditions are: pre-denaturation at 95°C for 15 min, denaturation at 95°C for 3 sec, annealing / extension at 60°C for 30 sec, for a total of 45 cycles.

[0021] The sixth aspect of the present invention is to provide a method for detecting Areca catechu phytoplasma using real-time quantitative PCR technology, employing the kit described in the fourth aspect of the present invention.

[0022] Preferably, the method includes the following steps: using the total DNA of the areca nut to be tested and positive controls of different concentrations as templates, using the primer pair described in the second aspect of the present invention as primers, and using the probe described in the first aspect of the present invention as probes, a real-time quantitative PCR amplification reaction is performed; the positive or negative sample is determined by reading the real-time quantitative PCR amplification curve and the corresponding Ct value.

[0023] Preferably, the criteria for judging the test results are as follows: if the Ct value of the sample to be tested is ≤39, it is positive for phytoplasma; if the Ct value of the sample to be tested is 39 < Ct value <45, it is considered suspicious; if the Ct value of the sample to be tested is >45, or shows Undetermined, it is negative for phytoplasma.

[0024] Preferably, a healthy areca nut sample is used as a negative control, and the total DNA from the healthy areca nut sample is used as a template. The primer pair described in the second aspect of the present invention is used as primers, and the probe described in the first aspect of the present invention is used as probes to perform a real-time fluorescence quantitative PCR amplification reaction.

[0025] Preferably, the positive control includes: standard 8, with a concentration of 1.16 × 10⁸. 0 copies / μL; Standard 7, concentration 1.16×10⁻⁶ 1 copies / μL; Standard 6, concentration 1.16×10 2 copies / μL; 5 standard samples, concentration 1.16 × 10⁻⁶. 3 copies / μL; 4 standard samples, concentration 1.16×10⁻⁶. 4 copies / μL; Standard 3, concentration 1.16×10 5 Copies / μL; Standard 2, concentration 1.16×10⁻⁶ 6 copies / μL; Standard 1, concentration 1.16×10 7 copies / μL.

[0026] Preferably, the PCR reaction system consists of 0.6 μL of upstream primer (10 μM), 0.6 μL of downstream primer (10 μM), 0.4 μL of probe (10 μM), 10 μL of 2×SuperRealPreMix, 2 μL of DNA template, 0.2 μL of 50×ROX Reference Dye*3, and ddH2O added to a final volume of 20 μL.

[0027] Preferably, the PCR amplification reaction conditions are: pre-denaturation at 95°C for 15 min, denaturation at 95°C for 3 sec, annealing / extension at 60°C for 30 sec, for a total of 45 cycles.

[0028] The sixth aspect of the invention is to provide the use of the probe as described in the first aspect, or the primer pair as described in the second aspect, or the primer-probe combination as described in the third aspect, or the kit as described in the fourth aspect, in detecting Areca catechu yellowing phytoplasma, and / or Acer palmatum thorny phytoplasma, and / or Bamboo thorny phytoplasma, and / or Vinca minor phytoplasma, and / or Melia azedarach yellowing phytoplasma, and / or Vine slender vine thorny phytoplasma, and / or Capsicum yellowing phytoplasma, and / or Jute thorny phytoplasma, and / or Peanut thorny phytoplasma.

[0029] The beneficial effects of this invention are as follows:

[0030] 1. Due to the low concentration of Areca catechu xanthophylloma DNA template, high sensitivity is required for its detection, which presents a technical challenge in this application. This invention provides a real-time quantitative PCR primer-probe combination, kit, and method for detecting Areca catechu xanthophylloma, characterized by high sensitivity; the sensitivity test can detect up to 1.16 × 10⁻⁶. 1 With copies / μL levels, the minimum detectable concentration in applications can reach 1.50 copies / μL, meeting the needs of most detection applications. Its sensitivity is 10,000 times higher than that of nested PCR detection technology and about 1,000 times more sensitive than the currently reported real-time fluorescence quantitative PCR detection technology for areca yellow phytoplasma.

[0031] 2. Due to the high homology (76%) between the 16S rDNA gene and some areca chloroplast genome sequences, the primer-probe combination exhibits high specificity for areca etiolated phytoplasma, thus avoiding non-specific amplification. However, currently reported nested PCR and real-time quantitative PCR technologies have failed to achieve this in practical applications, leading to false positives in the aforementioned two techniques. Furthermore, the currently reported droplet digital PCR technology has not undergone specificity verification. Therefore, designing highly specific primers / probes is the biggest technical challenge in this application. Through extensive innovative experiments, this application has screened a highly specific primer-probe combination from the 16S rDNA double strand. Experiments have demonstrated that the areca nut genome (including the chloroplast genome), areca nut endophytic bacteria, and the genomes of major areca nut diseases do not interfere with the specificity of this technology. This effectively solves the technical challenge of accurately detecting areca etiolated phytoplasma.

[0032] 3. This invention provides a real-time fluorescence quantitative PCR primer and probe combination, kit, and method for detecting areca etiolated phytoplasma. It has broad-spectrum detection of other phytoplasmas, can detect phytoplasmic diseases of different plants, can rapidly diagnose whether suspected intermediate hosts are damaged by phytoplasmas, and is applicable to the detection of insects carrying phytoplasmas. However, the currently reported droplet digital PCR technology has not demonstrated its effectiveness in detecting phytoplasmic diseases of other plants and insects carrying phytoplasmas.

[0033] 4. This invention provides primer pairs, probes, kits, and methods for real-time fluorescence quantitative PCR detection of areca etiolated phytoplasma. Compared with the currently reported droplet digital PCR technology, the instruments and reagents used in this invention are less expensive, reducing detection costs; the dynamic range is wide and the application range is broad; the operation is simple, reducing pollution, and it has potential application value. Attached Figure Description

[0034] Figure 1This is the result of verifying the accuracy of the real-time quantitative PCR primer / probe combination of this invention.

[0035] Figure 2 Amplification curves for positive standards of Areca catechu yellowing phytoplasma. The concentration of standard 8 is 1.16 × 10⁻⁶. 0 copies / μL; the concentration of standard 7 is 1.16×10⁻⁶. 1 copies / μL; the concentration of standard 6 is 1.16 × 10⁶. 2 copies / μL; Standard 5 concentration is 1.16×10 3 copies / μL; Standard 4 concentration is 1.16×10 4 copies / μL; the concentration of standard 3 is 1.16×10⁻⁶. 5 copies / μL; the concentration of standard 2 is 1.16×10⁻⁶. 6 copies / μL; the concentration of standard 1 is 1.16 × 10⁻⁶. 7 copies / μL.

[0036] Figure 3 The results are for nested PCR sensitivity testing, where the concentration of standard 3 is 1.16 × 10⁻⁶. 5 copies / μL; the concentration of standard 2 is 1.16×10⁻⁶. 6 copies / μL; the concentration of standard 1 is 1.16 × 10⁻⁶. 7 copies / μL.

[0037] Figure 4 The standard curve is for the concentration of positive standard of areca yellow phytoplasma.

[0038] Figure 5 This is the specificity detection result of the primer-probe combination of the present invention.

[0039] Figure 6 Amplification curves for samples of different plant phytoplasma diseases.

[0040] Figure 7 Results of phytoplasm detection for areca nut pests.

[0041] Figure 8 This is the verification result for positive seedlings. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0043] 1. Materials

[0044] 1.1 Test Sample

[0045] Genomic DNA of *Arecanut yellow leaf phytoplasma* (AYL), cDNA of *Areca palm velarivirus 1* (APV1), and cDNA of *Areca palmnecrotic ringspot virus* (ANRSV) were provided by the Coconut Research Institute of the Chinese Academy of Tropical Agricultural Sciences and tested positive by RT-PCR and nested PCR, respectively. *Areca anthracnose* and endophytic or pathogenic bacteria isolated from areca leaves, including *Burkholderia andropogonis*, *Pantoea ananatis*, *Sphingomonas yantingensis*, *Curtobacterium albidum*, *Bacillus cereus*, and *Frondihabitans*, were also detected. The genomic DNA of *Australicus* was provided by the Coconut Research Institute of the Chinese Academy of Tropical Agricultural Sciences; healthy areca leaf samples were collected from the areca nursery of the Coconut Research Institute; DNA from *Aristolochia debilis* phyllotropis (16SrⅡ-A) and *Catharanthus roseus* phyllotropis (16SrⅠ-B) was provided by the Institute of Environment and Plant Protection of the Chinese Academy of Tropical Agricultural Sciences; DNA from *Bambusa textilis* phyllotropis (16SrⅡ-B), *Melia azedarach* phyllotropis (16SrⅡ-B), *Vitis thunbergii* phyllotropis (16SrⅠ-B), and *Heliotropium indicum* phyllotropis (16SrⅠ-B) were also provided. DNA from 16SrXXXⅡ-D and peanut arbuscular phytoprotoplasm (16SrⅡ-B) and corresponding healthy sample DNA were provided by the Coconut Research Institute of the Chinese Academy of Agricultural Sciences. Suspected YLD samples were collected from 17 cities and counties in Hainan Province, including 240 samples from Wanning, 100 from Qionghai, 100 from Tunchang, 30 from Sanya, 45 from Ledong, 65 from Qiongzhong, 50 from Baoting, 60 from Ding'an, 51 from Lingshui, 40 from Wenchang, 40 from Chengmai, 20 from Wuzhishan, 15 from Baisha, 20 from Haikou, 30 from Danzhou, 10 from Dongfang, and 10 from Lingao, totaling 926 samples.

[0046] 1.2 Instruments

[0047] The KZ-II high-speed tissue homogenizer was purchased from Wuhan Saiweier Biotechnology Co., Ltd.; the Eppendorf 5810R benchtop refrigerated centrifuge; the DYY-6C electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory; the Bio-Rad T100 PCR instrument was purchased from Jena GmbH, Germany; the QuantStudio 3 Real-time PCR instrument was purchased from Thermo Fisher Scientific, USA; the Thermo ND2000 ultra-micro spectrophotometer was purchased from Thermo Fisher Scientific; and the HH-4 constant temperature water bath was purchased from Jintan Kexing Instrument Factory.

[0048] 1.3 Reagents

[0049] Plant total DNA extraction kit, 2×Taq PCR mix, agarose gel extraction kit, plasmid miniprep kit, and real-time quantitative PCR (probe method) kit were purchased from Tiangen Biotech (Beijing) Co., Ltd. pMD TM 18-T Vector cells were purchased from TaKaRa; DH5α competent cells were purchased from Beijing TransGen Biotech Co., Ltd.; other reagents were domestically produced analytical grade.

[0050] 2. Methods and Results

[0051] 2.1 Primer and probe design and synthesis

[0052] Based on the 16S rDNA gene sequence of *Phytoplasma* (GenBank accession number: KF728948.1), real-time quantitative PCR specific primers and probes were designed using PrimerExpress 3.0 software for the conserved region of this gene. The primer AM-f / AM-r and probe AM-Prode sequence information is as follows:

[0053] AM-f: 5'-CCCAATGTGGCCGTTCAAC-3';

[0054] Am-r: 5'-GAAACGACTGCTAAGACTGGATAGGA-3';

[0055] AM-Prode: 5'-CCTACCAAGACTATGATGTG-3';

[0056] The 5' end of the probe AM-Prode was modified with FAM, and the 3' end was modified with MBG. The primers and probe were synthesized by Sangon Biotech Co., Ltd., and purified by HPLC.

[0057] 2.2 DNA Extraction

[0058] Take 0.1g of sample leaf, extract DNA according to the instructions of the plant total DNA extraction kit, and store at -20℃.

[0059] 2.3 Validation of the accuracy of real-time quantitative PCR primer / probe combinations

[0060] Real-time quantitative PCR was performed on five sequenced positive samples using primers AM-f / AM-r and probe AM-Prode. Healthy areca nut samples served as negative controls, and sterile, enzyme-free ddH2O served as a blank control. The reaction mixture consisted of 10 μL of 2×SuperRealPreMix (Probe), 0.6 μL each of forward and reverse primers (10 μM), 0.4 μL of fluorescent probe (10 μM), 2 μL of DNA template, 0.2 μL of 50×ROX Reference Dye*3, and ddH2O to a final volume of 20 μL. The reaction program was as follows: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 3 s; 60℃ annealing / extension for 30 sec; 45 cycles.

[0061] The results are as follows Figure 1 As shown, all five positive samples were detected, exhibiting an "S"-shaped amplification curve with smooth curves, while the negative samples and blank control did not show any amplification curves, achieving an accuracy of 100%. This indicates that the primer / probe combination for this real-time quantitative PCR meets the detection requirements.

[0062] 2.4 Preparation of Positive Plasmid Standards

[0063] Using primers AM-f / AM-r, PCR amplification was performed with DNA from positive areca nut yellowing disease samples as templates, DNA from healthy areca nut samples as negative controls, and ddH2O as a blank control. The PCR products were detected by agarose gel electrophoresis. The gel containing the target band was excised and purified using an agarose gel extraction kit. The purified product was then ligated into pMD... TM 18-TVector was transformed into DH5α competent cells, and plasmids of positive clones were screened and extracted, then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The concentration of correctly sequenced plasmids was determined using an ultra-micro spectrophotometer and stored at -20℃ for later use. The copy number of recombinant plasmids was calculated using Shirima's method (Shirima RR et al, 2017): Recombinant plasmid copy number (copies / μL) = (Avogadro's constant × C) / (10^6)^2. 9 × number of recombinant plasmid base pairs × 660 dalton / bp). The Avogadro constant is 6.02 × 10⁻⁶. 23 C represents the concentration of the recombinant plasmid (ng / μL). The final calculated copy number of the recombinant plasmid is 1.16 × 10⁻⁶. 10Copies / μL. PCR reaction system: 10 μL of 2×SuperReal PreMix, 0.6 μL each of forward and reverse primers (10 μM), 2.0 μL of DNA template, 0.2 μL of 50×ROX Reference Dye*3, and ddH2O to a final volume of 20 μL. PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 45 s, 58℃ annealing for 45 s, 72℃ extension for 30 s, 40 cycles; 72℃ extension for 10 min.

[0064] 2.5 Sensitivity Test and Establishment of Standard Curve

[0065] Using the positive plasmid standard as the initial template, the plasmid was serially diluted to a concentration of 1.16 × 10⁻⁶. 7 1.16×10 6 1.16×10 5 1.16×10 4 1.16×10 3 1.16×10 2 1.16×10 1 1.16×10 0 The above eight gradient concentrations of positive plasmid standards were used as templates, and healthy areca nut samples were used as negative controls for real-time quantitative PCR. The reaction system consisted of 10 μL of 2×SuperReal PreMix (Probe), 0.6 μL each of forward and reverse primers (10 μM), 0.4 μL of fluorescent probe (10 μM), 2 μL of DNA template, 0.2 μL of 50×ROX Reference Dye*3, and ddH2O to a final volume of 20 μL. The reaction program was: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 3 s, 60℃ annealing / extension for 30 sec, for 45 cycles. Kinetic amplification curves and PCR amplification cycle thresholds (Ct values) were obtained. Using Excel 2020 software, a standard curve was plotted with Ct value on the ordinate and the logarithm of the plasmid copy number (base 10) on the abscissa. The regression line equation was obtained, and the amplification efficiency of this system was calculated: Amplification efficiency (%) = 10^ (-1 / 斜率)-1. Using the above-mentioned positive plasmid standards at eight different concentrations as templates and healthy areca nut samples as negative controls, nested PCR was performed using primers F4 / R1 and F2 / R2 (Meng Xiuli et al., 2021). The PCR products were verified by gel electrophoresis and compared with the results of real-time quantitative PCR. Nested PCR reaction program: First round of amplification: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 45 s, 50℃ annealing for 1 min, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min; Second round of amplification: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 45 s, 45℃ annealing for 1 min, 72℃ extension for 45 s, 35 cycles; 72℃ extension for 10 min.

[0066] The results showed that the combination of primers AM-f / AM-r and probe AM-Prode could detect 1.16 × 10⁻⁶. 1 copies / μL level ( Figure 2 Nested PCR can detect as low as 1.16 × 10⁻⁶. 5 copies / μL ( Figure 3 This real-time quantitative PCR method showed a sensitivity 10,000 times higher than nested PCR for detecting YLD. The standard curve showed a linear relationship between the Ct value and the logarithm of the copy number, with the equation y = -3.4185x + 43.624. The amplification efficiency was 96.12%, and the correlation coefficient R0 was [missing value]. 2 =0.9833 ( Figure 4 ).

[0067] 2.6 Specificity test

[0068] Using DNA from areca xanthophytum as a positive control and DNA from healthy areca nuts as a negative control, real-time quantitative PCR was performed using cDNA from ANRSV and APV1, and DNA from areca anthrax bacteria, Burkholderia andropogonis, Pantoea ananatis, Sphingomonasyantingensis, Curtobacterium albidum, Bacillus cereus, and Frondihabitans australicus as templates to detect their specificity. The reaction system and procedure were as described in "2.5 Sensitivity Test and Establishment of Standard Curve".

[0069] The results are as follows Figure 5As shown, only *Areca catechu* showed an amplification curve, while healthy areca nuts (negative control), APV1, ANRSV, *Areca anthracis*, *Burkholderia andropogonis*, *Pantoea ananatis*, *Sphingomonasyantingensis*, *Curtobacterium albidum*, *Bacillus cereus*, and *Frondihabitans australicus* showed no amplification curve. This indicates that the real-time quantitative PCR method has good specificity for the detection of *Areca catechu*, and other areca genomes, pathogens, and endophytes do not cause non-specific interference to this detection method.

[0070] 2.7 Repeatability Test

[0071] Select 1.16×10 5 1.16×10 4 1.16×10 3 Using positive plasmid standards at three concentrations (copies / μL) as templates, real-time quantitative PCR was performed. Each concentration was repeated three times for a total of three tests. Intra-group repeatability tests were conducted, and the coefficient of variation (Ct) was calculated. Simultaneously, the positive plasmid standards at the above concentrations were stored at -20℃ for 3, 6, and 9 days, and inter-group tests were performed under the same reaction conditions. The coefficient of variation (Ct) was calculated to verify the repeatability and stability of the method. The reaction system and procedure were as described in "2.5 Sensitivity Test and Establishment of Standard Curve".

[0072] The results are shown in Table 1. The coefficient of variation within groups was less than 1%, and the coefficient of variation between groups was less than 2%, indicating that the real-time quantitative PCR method had good repeatability for the detection of areca nut yellow phytoplasma.

[0073] Table 1 Repeated Experiments of Quantitative Real-Time PCR

[0074]

[0075] 2.8 Applications on different plant phytoplasms

[0076] Real-time quantitative PCR was used to detect phytoplasma from eight different plants, including *Cynanchum aegyptium*, *Cynanchum basilica*, *Catharanthus roseus*, *Melia azedarach*, *Vitis thunbergii*, *Polygonum hydropiper*, *Jute arborescens*, and *Peanut arborescens*. DNA from healthy plant samples served as negative controls, and enzyme-free and sterile ddH2O served as blank controls. The reaction system and procedure followed the guidelines in section 2.5, "Sensitivity Test and Establishment of Standard Curve".

[0077] The results are shown in Table 2. Figure 6As shown, the tested phytoplasma species, including *Cymbidium faberi*, *Cymbidium baicalensis*, *Cinnamomum camphora*, *Melia azedarach*, *Vitis thunbergii*, *Cymbidium ensifolium*, *Cymbidium lancifolium*, *Cymbidium lancifolium*, *Cymbidium lancifolium*, and *Cymbidium oleracea*, were all positive for phytoplasma by this real-time quantitative PCR method. The corresponding healthy negative controls showed no amplification curve, indicating that this invention has a broad spectrum of phytoplasma detection and can detect multiple phytoplasmic diseases.

[0078] Table 2. Results of Real-time Quantitative PCR Detection of Different Plant Phytoplasms

[0079]

[0080] 2.9 Application of major piercing-sucking pests in areca palm fields

[0081] Real-time quantitative PCR was used to detect the DNA of four common piercing-sucking pests in areca nut fields: *Dictyophora indica*, *Citrus spiny mealybug*, *Citrus coccinea*, and *Citrus pulvinata*. Enzyme-free and sterile ddH2O was used as a blank control. The reaction system and procedure followed the guidelines in "2.5 Sensitivity Test and Establishment of Standard Curve". The amplified target bands were recovered, cloned, and sequenced to verify the detection results in insects.

[0082] The results are as follows Figure 7 As shown, three types of piercing-sucking insects tested positive for phytoplasma: *Dystomata chinensis*, *Dystomata citrus*, and *Dystomata coccinea*. Sequencing confirmed that these insects were indeed phytoplasmas, indicating that this invention is suitable for detecting venomous insects in areca nut.

[0083] 2.10 Application of Areca Nut Seedlings in Nursery

[0084] Real-time quantitative PCR was performed on four seedling samples that were positive for areca nut yellowing disease according to sequencing. Healthy seedlings served as negative controls, and enzyme-free and sterile ddH2O served as blank controls. The reaction system and procedure were as described in "2.5 Sensitivity Test and Establishment of Standard Curve".

[0085] The results are as follows Figure 8 As shown, all four positive seedlings were amplified, while no amplification curves were observed in the negative samples and the blank control, indicating that this invention is suitable for seedling detection.

[0086] 2.11 Field Sample Testing

[0087] Genomic DNA was extracted from 926 suspected YLD disease samples collected from areca palm orchards (10-15 years old) in 17 cities and counties of Hainan Province. The real-time quantitative PCR method established in this study was used for detection, with enzyme-free and sterile ddH2O as a blank control. The detection rate of YLD in these 17 cities and counties was obtained. Based on the standard curve regression equation in "2.5 Sensitivity Test and Standard Curve Establishment," the highest, lowest, and average copy numbers and standard deviations of phytoplasma in the field samples were calculated. The reaction system and procedure were as described in "2.5 Sensitivity Test and Standard Curve Establishment."

[0088] The results are shown in Table 3. From the application effect, the minimum detection concentration of this invention can reach 1.50 copy / μL. In terms of detection rate, the detection rates in the 17 cities and counties of Hainan Province are as follows: Qionghai = Ding'an = Wenchang > Tunchang > Qiongzhong > Wanning > Wuzhishan = Haikou > Ledong > Lingshui > Danzhou > Baoting > Sanya > Chengmai > Baisha = Dongfang = Lingao. Areca nut yellowing disease mainly occurs in the main areca nut growing areas of Hainan Province, distributed in cities and counties along the central and eastern coastlines of Hainan. Looking at the phytoplasm content in each of the 17 cities and counties, the standard deviation value of phytoplasm content in each city and county is relatively large, indicating that the content distribution of areca nut phytoplasm varies significantly among different trees.

[0089] Table 3. Detection of field samples by real-time quantitative PCR

[0090]

[0091]

[0092] Note: The average value has been calculated by removing the maximum and minimum values.

[0093] The specific embodiments of the present invention have been described in detail above, but these are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this utility model are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A primer-probe combination for detecting areca etiolated phytoplasma based on real-time quantitative PCR technology, characterized in that, The kit includes a probe and a primer pair. The probe is AM-Prode, with the nucleotide sequence 5'-CCTACCAAGACTATGATGTG-3', labeled with a fluorescent group at the 5' end and a quencher group at the 3' end; the fluorescent group labeled at the 5' end is FAM, and the quencher group labeled at the 3' end is MGB. The primer pair includes an upstream primer AM-f and a downstream primer AM-r, with the nucleotide sequences shown below: Upstream primer AM-f: 5'-CCCAATGTGGCCGTTCAAC-3'; Downstream primer AM-r: 5'-GAAACGACTGCTAAGACTGGATAGGA-3'.

2. A reagent kit, characterized in that, The kit comprises the probes and primer pairs of the primer-probe combination as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes a positive control; the positive control is a plasmid standard for the 16S rDNA amplification sequence of Areca catechu yellowing phytoplasma; the plasmid standard for the 16S rDNA amplification sequence of Areca catechu yellowing phytoplasma is a recombinant vector constructed by ligating the product obtained by PCR amplification using the primer pair described in claim 2 with a vector, using a positive sample of Areca catechu yellowing disease as a template.

4. A method for detecting areca nut yellowing phytoplasma, characterized in that, The kit described in claim 2 or 3 is used.

5. The detection method according to claim 4, characterized in that, Using the total DNA of the areca nut to be tested as a template, the primer pair described in claim 2 as primers, and the probe described in claim 1 as probe, real-time fluorescent PCR detection was performed. If the fluorescence signal was enhanced during the PCR process, then the plant phytoplasma was infected.

6. A method for detecting areca nut etiolated phytoplasma using real-time quantitative PCR, characterized in that, The kit described in claim 3 was used.

7. The method as described in claim 6, characterized in that, Includes the following steps: Using the total DNA of the areca nut to be tested and positive controls of different concentrations as templates, the primer pair described in claim 2 was used as primers and the probe described in claim 1 was used as probes to perform real-time fluorescence quantitative PCR amplification reaction; By analyzing the amplification curve of real-time quantitative PCR and reading the corresponding Ct value, the positive or negative result of the sample can be determined. The criteria for judging the test results are as follows: if the Ct value of the sample to be tested is ≤39, it is positive for phytoaplasma; if the Ct value of the sample to be tested is 39 < Ct value <45, it is considered suspicious. If the Ct value of the sample to be tested is >45, or if it shows "Undetermined", it is negative for phytoplasma.

8. The method as described in claim 6, characterized in that, A healthy areca nut sample was used as a negative control. The total DNA from the healthy areca nut sample was used as a template. The primer pair described in claim 2 was used as primers, and the probe described in claim 1 was used as probes to perform real-time quantitative PCR amplification.

9. The method as described in claim 6, characterized in that, The positive control includes: Standard 8, with a concentration of 1.16 × 10⁸. 0 Copies / μL; Standard 7, concentration 1.16×10⁻⁶ 1 Copies / μL; Standard 6, concentration 1.16×10⁶ 2 Copies / μL; Standard 5, concentration 1.16×10⁻⁶ 3 copies / μL; 4 standard samples, concentration 1.16×10⁻⁶. 4 Copies / μL; Standard 3, concentration 1.16×10⁻⁶ 5 Copies / μL; Standard 2, concentration 1.16×10⁻⁶ 6 copies / μL; Standard 1, concentration 1.16×10 7 copies / μL.

10. The application of the primer-probe combination as described in claim 1 or the kit as described in claim 2 or 3 in the detection of Areca catechu yellowing phytoplasma, and / or Acer rubrum phytoplasma, and / or Bambusa phytoplasma, and / or Vinca minor phytoplasma, and / or Melia azedarach yellowing phytoplasma, and / or Vine arborescens phytoplasma, and / or Capsicum yellowing phytoplasma, and / or Jute arborescens phytoplasma, and / or Peanut arborescens phytoplasma.

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