Citrus huanglongbing bacterium asiatic species detection primer and application thereof

Abstract

The application discloses a detection primer for citrus huanglongbing bacterium Asian species and application thereof. The application takes a homologous gene site of a Type 4 type phage and CLas in the citrus huanglongbing bacterium as a target, and designs qPCR primers hg4-5_F / hg4-5_R. The primers can significantly improve the detection sensitivity of CLas for huanglongbing samples containing the Type 4 type phage, and are also suitable for CLas sample detection without the phage. In addition, the application also provides double real-time fluorescent PCR primers composed of the primer pair and CLas-4G / HLBr primers. The double primers can significantly improve the detection sensitivity when detecting huanglongbing samples containing or not containing the Type 4 type phage, and are suitable for low-concentration CLas detection. The primers provided by the application provide technical support for accurate and efficient detection of CLas, especially CLas containing the Type 4 type phage.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostic technology for plant pathology, and more specifically, to a primer for detecting Asian species of Huanglongbing fungus in citrus and its application. Background Technology

[0002] Citrus Huanglongbing (HLB) is a devastating disease that severely restricts the development of the global citrus industry and poses a significant threat to citrus production. In my country, HLB is caused by Candidatus Liberibacter asiaticus (CLas), a species of α-Proteobacteria. CLAs has the widest distribution and causes the most damage, and is currently the only pathogen species prevalent in my country.

[0003] Citrus plants are susceptible to Huanglongbing (HLB) throughout their entire growth cycle, but the symptoms and severity of infection vary depending on the plant's resistance, growth stage, and environmental conditions. Typical symptoms of infected plants include: uniform yellowing or zinc deficiency-like chlorosis in new leaves; irregular mottling on older leaves; and deformed leaf development (uneven or curled). Flowering occurs prematurely, with small, deformed flowers that easily fall off. In the later stages of the disease, the root system rots extensively, with the xylem turning dark brown and separating from the bark. Diseased fruit is smaller, asymmetrical, and has a bland taste, with distinct characteristics between varieties: in varieties like Satsuma mandarin, diseased fruit begins to color from the stem end at maturity, forming a "red-nosed fruit"; in oranges and other varieties, "green fruit" is more likely to appear, maturing earlier than healthy fruit but easily falling off.

[0004] Currently, Huanglongbing (HLB) lacks effective treatments and superior resistant varieties. Control relies heavily on early detection and quarantine, removal of infected plants (sources of infection), and blocking transmission vectors – key strategies to prevent the pathogen from invading disease-free areas. However, field diagnosis of HLB presents significant challenges: firstly, the symptoms are complex and easily confused with nutrient deficiency symptoms and citrus decline disease, leading to misdiagnosis; secondly, traditional detection methods have inherent flaws: indicator plant identification is time-consuming and cannot meet the need for rapid detection; electron microscopy requires sophisticated sample preparation and is complex to operate; serological testing is prone to missed detections and has a low detection rate, and relies on expensive instruments and reagents, limiting its applicability. Furthermore, although HLB is a systemic disease, the distribution of CLas within the plant is extremely uneven, and the inoculum is very low in the early infection stage, directly resulting in a significant decrease in the accuracy and reliability of early diagnosis.

[0005] Currently, Huanglongbing (HLB) detection primarily relies on molecular biology methods, with real-time quantitative PCR being the mainstream technique. Existing detection methods often target multi-copy conserved genes in the CLas genome (such as the 16S rRNA gene), but the sensitivity of these targets is insufficient to meet the detection needs of early infection stages in fruit trees (when pathogen content is low), resulting in unsatisfactory practical application. In addition to multi-copy sites on the CLas genome, early research has also developed primers for CLas detection based on related phage sites. This is mainly due to the fact that CLas-related phages often exist in multi-copy form, thus improving the sensitivity of early detection. For example, patent CN 116083610A discloses a specific real-time fluorescent PCR primer GP04-F / GP04-R designed based on the homologous gene sites of the bacteriophage CLasMV1 (a multi-copy bacteriophage in CLas) and CLas. Compared with conventional detection primers, this primer can improve sensitivity. When used in conjunction with CLas-4G / HLBr, it further improves and enhances the detection sensitivity, enabling preliminary diagnosis at the early stage of Huanglongbing infection in fruit trees when the concentration of the pathogen "Ca.Liberibacter spp." is low, allowing for timely removal of diseased plants to eliminate the source of infection or the implementation of other control measures. However, as can be seen from the examples, the detection sensitivity of the GP04-F / GP04-R primers in detecting Huanglongbing samples containing CLasMV1 is 3.18 times higher than that of the CLas-4G / HLBr primers and 1.35 times higher than that of the RNRf / RNRr primers. The sensitivity of using the CLas-4G / HLBr and GP04-F / GP04-R double primers is approximately 40 times and 11.5 times higher than that of using the CLas-4G / HLBr and RNRf / RNRr primers, respectively. In samples of Huanglongbing fungus lacking CLasMV1, the Ct values ​​obtained by GP04f / GP04r primers were 1.59 (±1.13) and 2.62 (±1.29) higher than those obtained by CLas-4G / HLBr and RNRf / RNRr primers, respectively. The sensitivity of using the CLas-4G / HLBr and GP04-F / GP04-R double primers was approximately 5.9 times and 3.1 times higher than that of using the CLas-4G / HLBr and RNRf / RNRr primers, respectively. However, the sensitivity and stability of this primer for detecting Asian species of citrus Huanglongbing still need further improvement. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a primer for detecting Asian species of Huanglongbing fungus in citrus.

[0007] The second objective of this invention is to provide the application of the above-mentioned primers for detecting citrus Huanglongbing fungus in the detection of Asian species of citrus Huanglongbing fungus or in the preparation of a kit for detecting Asian species of citrus Huanglongbing fungus.

[0008] The third objective of this invention is to provide a detection kit for Asian species of Huanglongbing fungus in citrus.

[0009] The fourth objective of this invention is to provide a method for detecting the Asian species of Huanglongbing fungus in citrus.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention provides a primer for detecting Asian species of Huanglongbing fungus in citrus, comprising primer pair hg4-5_F / hg4-5_R; the nucleotide sequence of hg4-5_F is shown in SEQ ID NO.1; the nucleotide sequence of hg4-5_R is shown in SEQ ID NO.2.

[0012] This invention targets the homologous gene (ORF4_5, the sequence of which is shown in SEQ ID NO.7) of the multi-copy Type 4 phage in citrus Huanglongbing (CLas) with CLAs, and designs specific real-time fluorescent PCR primers hg4-5_F / hg4-5_R to establish a relatively complete and highly sensitive molecular detection system. For Huanglongbing samples containing Type 4 phage, the detection sensitivity of the hg4-5_F / hg4-5_R primer was 3.61 times higher than that of the CLas-4G / HLBr primer and 1.87 times higher than that of the RNRf / RNRr primer, which can significantly improve the detection sensitivity of CLas. For Huanglongbing samples without Type 4 phage, the Ct value obtained by the hg4-5_F / hg4-5_R primer was 1.02 (±0.08) and 1.48 (±0.14) higher than that of the CLas-4G / HLBr and RNRf / RNRr primers, respectively. Although the detection sensitivity of the hg4-5_F / hg4-5_R primer was lower than that of the CLas-4G / HLBr and RNRf / RNRr primers, it was still suitable for detecting CLas samples without this phage.

[0013] Meanwhile, this invention combines hg4-5_F / hg4-5_R with CLas-4G / HLBr primers to form a double primer, further optimizing the detection system. In two groups of samples, one containing Type 4 phage and the other not containing Type 4 phage, calculations showed that the sensitivity of detection using the CLas-4G / HLBr and hg4-5_F / hg4-5_R double primers was approximately 40 times and 5.9 times higher than using the CLas-4G / HLBr primers, respectively, and 11.5 times and 3.1 times higher than using the RNRf / RNRr primers, respectively. Therefore, regardless of whether Type 4 bacteriophages are present, the effectiveness of real-time quantitative PCR detection using dual primers has been significantly improved. Specifically, the combination of hg4-5_F / hg4-5_R and CLas-4G / HLBr primers to form dual primers can greatly improve the detection sensitivity of Huanglongbing samples with or without Type 4 bacteriophages. This technology can achieve accurate preliminary diagnosis in the early stage of Huanglongbing infection in citrus trees, when the concentration of pathogen CLas is low. This provides key technical support for timely removal of diseased trees to eliminate the source of infection or for taking other targeted control measures.

[0014] Furthermore, the primers also include the primer pair CLas-4G / HLBr; the nucleotide sequence of CLas-4G is shown in SEQ ID NO.3; and the nucleotide sequence of HLBr is shown in SEQ ID NO.4. Simultaneous detection using dual primers under low CLas concentration conditions significantly improves detection sensitivity, providing further technical support for laboratory detection of CLas.

[0015] Therefore, the present invention also provides the application of the above-mentioned primers for detecting citrus Huanglongbing fungus in the detection of Asian citrus Huanglongbing fungus or in the preparation of a kit for detecting Asian citrus Huanglongbing fungus.

[0016] The present invention also provides a detection kit for Asian species of citrus Huanglongbing fungus, the kit comprising any of the above-mentioned primers for detecting citrus Huanglongbing fungus.

[0017] Furthermore, the kit also includes reagents required for real-time quantitative PCR reactions.

[0018] The present invention also provides a method for detecting the Asian species of Huanglongbing fungus in citrus, the method comprising the following steps:

[0019] S1. Extract genomic DNA from the sample to be tested;

[0020] S2. Using the DNA from step S1 as a template, perform single or double real-time quantitative PCR using any of the above-mentioned primers for detecting Asian species of citrus Huanglongbing. If the Ct value obtained by real-time quantitative PCR is greater than 32, it indicates that the sample to be tested is not infected with Asian species of citrus Huanglongbing. If the Ct value is less than 32, it indicates that the sample to be tested is infected with Asian species of citrus Huanglongbing.

[0021] Furthermore, the reaction conditions for the single or double real-time quantitative PCR reaction in step S2 are: 94–96℃, 3 min; 94–96℃, 10 s; 59–61℃, 30 s; 40 cycles; fluorescence signal reading is performed at the end of each cycle at 60℃.

[0022] Preferably, the reaction conditions for the single or double real-time quantitative PCR reaction in step S2 are: 95℃, 3 min; 95℃, 10 s, 60℃, 30 s, 40 cycles; and fluorescence signal reading is performed at the end of each cycle at 60℃.

[0023] Furthermore, the melting curve program for the single or double real-time quantitative PCR reaction in step S2 is as follows: 95℃, 2 min; 40℃, 2 min; 65℃~95℃ with a temperature increase of 0.1℃ every 5 s and acquisition of fluorescence signals.

[0024] Preferably, the melting curve program for the single or double real-time quantitative PCR reaction in step S2 is as follows: 95℃, 2 min; 40℃, 2 min; 75℃~90℃, increasing the temperature by 0.1℃ every 5 s and collecting fluorescence signals.

[0025] Furthermore, the amplification system for the single or double real-time quantitative PCR reaction in step S2 also includes Supermix.

[0026] Further, the amplification system for the singlet real-time quantitative PCR reaction in step S2 is as follows: ddH2O 8.0 μL, Supermix 10.0 μL, 10 μmol / L hg4-5_F 0.5 μL, 10 μmol / L hg4-5_R 0.5 μL, DNA template 1.0 μL, totaling 20 μL; the amplification system for the dualt real-time quantitative PCR reaction is as follows: ddH2O 7.0 μL, Supermix 10.0 μL, 10 μmol / L CLas-4G 0.5 μL, 10 μmol / L HLBr 0.5 μL, 10 μmol / L hg4-5_F 0.5 μL, 10 μmol / L hg4-5_R 0.5 μL, DNA template 1.0 μL, totaling 20 μL.

[0027] Preferably, the aforementioned Asian species of citrus Huanglongbing (HLB) is a HLB containing Type 4 phage. Due to the high copy number and wide geographical distribution of Type 4 phage in HLB pathogens, validation of the detection system with numerous field samples revealed that the single or double real-time quantitative PCR primers of this invention significantly improve the detection sensitivity for samples simultaneously carrying both Type 4 phage and HLB pathogens. Simultaneously, these primers can also effectively detect HLB pathogen samples lacking Type 4 phage. Therefore, this invention not only opens up new target sites for the molecular detection of HLB but also provides crucial technical support for its early diagnosis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention provides primers for detecting the Asian species of *Citrus citrus Huanglongbing Fever* (CLas). Based on the homologous gene sites of the multiple copies of Type 4 bacteriophage in *Citrus citrus Huanglongbing Fever* (CLas) and *Citrus citrus Huanglongbing Fever* (CLas), this invention designs specific real-time quantitative PCR primers hg4-5_F / hg4-5_R. For Huanglongbing samples containing Type 4 bacteriophage, this primer significantly improves the detection sensitivity of *Citrus citrus Huanglongbing Fever*; simultaneously, it is also applicable to the detection of *Citrus citrus Huanglongbing Fever* samples without this bacteriophage, demonstrating broad applicability. Furthermore, this invention also provides a dual real-time quantitative PCR primer set composed of hg4-5_F / hg4-5_R and *Citrus citrus Huanglongbing Fever*-4G / HLBr primers. This dual primer set significantly improves the detection sensitivity for Huanglongbing samples with or without Type 4 bacteriophage, and is suitable for low *Citrus citrus Huanglongbing Fever* conditions, i.e., the detection of low *Citrus citrus Huanglongbing Fever* pathogen concentrations in the early stages of Huanglongbing infection in fruit trees. The primer pairs provided by this invention offer further technical support for the accurate and efficient detection of CLas, especially CLas containing Type 4 phages. Attached Figure Description

[0030] Figure 1 For different primer pairs CLas + Type 4 + The detection sensitivity of DNA samples (the numbers in the bar represent the mean and standard error).

[0031] Figure 2 Real-time PCR amplification curves and melting curves for different primers are shown. Figure 2 Figure a shows the melting curve of the CLas-4G / HLBr primer; figure b shows the melting curve of the RNRf / RNRr primer; and figure c shows the melting curve of the hg4-5_F / hg4-5_R primer.

[0032] Figure 3The melting curves of the hg4-5_F / hg4-5_R+CLas-4G / HLBr double primers at high resolution (0.1℃ increase per cycle).

[0033] Figure 4 Comparison of detection sensitivity for CLas4G / HLBr primers, RNRf / RNRr primers, and CLas-4G / HLBr+hg4-5_F / hg4-5_R double primers (the numbers in the bars represent the mean and standard error). Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] Example 1: Primer design for the detection of Huanglongbing (HLB)

[0037] Type 4 phages are the Huanglongbing bacteriophages previously discovered by the inventors (Fang, F., Chen, T., Wang, C., Liang, J., Li, Z., Zheng, Y., Wallis, C., Chen, J., Deng, X., Zheng, Z. (2025). Genomic Diversity of Microviridae Phage CLasMV1 in “Candidatus Liberibacter asiaticus”. Phytopathology).

[0038] Homologous gene regions were obtained by aligning the genomes of Type 4 bacteriophages with the chromosome genome of Huanglongbing (CLas). Primers were then designed based on the common homologous gene ORF4_5 (its sequence is shown in SEQ ID NO.7) shared by the Type 4 bacteriophage genome and CLAs. The primer Tm value was approximately 60℃. After screening, one pair of primers, hg4-5_F / hg4-5_R, was obtained (Table 1). The primer sequences were then compared with the NCBINucleotide Database using NCBI web BLASTn (expect threshold=10, word size=11) to evaluate primer specificity before being used in the next step of Real-time PCR.

[0039] Table 1. Primer Information for hg4-5_F / hg4-5_R

[0040]

[0041] Example 2 Primer sensitivity assessment

[0042] 1. Method

[0043] Citrus leaf samples with typical mottling symptoms were collected from nine provinces in China, including Guangdong, Guangxi, Fujian, Jiangxi, Hunan, Zhejiang, Guizhou, and Sichuan. Each sample was taken from the same diseased branch of a single tree, and one sample was taken from each tree for DNA extraction, serving as candidate samples for subsequent CLAs molecular testing. Healthy citrus leaves were also collected as negative control DNA samples.

[0044] The detection performance of hg4-5_F / hg4-5_R obtained in Example 1 was compared with that of commonly used primers. The samples used were collected from nine provinces in China, including Guangdong, Guangxi, Fujian, Jiangxi, Hunan, Zhejiang, Guizhou, and Sichuan, and included 658 Huanglongbing bacteria (CLas) containing Type 4 phages. + Type 4 + ) samples, 118 Huanglongbing bacteria (CLas) that do not contain Type 4 bacteriophages + Type 4 - The samples were compared with 35 healthy citrus samples. The primers used were CLas-4G / HLBr (F: 5'-AGTCGAGCGCGTATGCGAAT-3' (SEQ ID NO.3) / R: 5'-GCGTTATCCCGTAGAAAAAGGTAG-3' (SEQ ID NO.4)) based on 3 copies of the 16S rRNA gene, RNRf / RNRr (F: 5'-CATGCTCCATGAAGCTACCC-3' (SEQ ID NO.5) / R: 5'-GGAGCATTTAACCCCACGAA-3' (SEQ ID NO.6)) based on 5 copies of the nrdB gene, and hg4-5_F / hg4-5_R based on homologous genes of CLas and Type 4 phages.

[0045] Real-time PCR was performed using the SYBR Green method. The total reaction volume of hg4-5_F / hg4-5_R was 20 μL, and the amplification system is shown in Table 2. All preparation and subsequent operations were performed on an ice box. The reaction conditions were: 95℃ for 3 min; (95℃ for 10 s; 60℃ for 30 s) for 40 cycles. Fluorescence signals were read at the end of each cycle at 60℃. Each reaction included negative, positive, and water controls.

[0046] Table 2 SYBR Green amplification system

[0047]

[0048] The reaction system and conditions for CLas-4G / HLBr with RNRf / RNRr are the same as those for the hg4-5_F / hg4-5_R reaction system described above.

[0049] 2. Results

[0050] Sensitivity analysis compared with CLas-4G / HLBr and RNRf / RNRr revealed that, among 658 Huanglongbing samples containing Type 4 phages, the Ct values ​​obtained by hg4-5_F / hg4-5_R were lower than those obtained by CLas-4G / HLBr. P <0.001) and RNRf / RNRr ( P <0.05)( Figure 1 Furthermore, after conversion, the primer sensitivity increase value R can be obtained (calculated using the ΔCt method, i.e., R=2). -ΔCt The detection sensitivity of the hg4-5_F / hg4-5_R primers was 3.61 times higher than that of the CLas-4G / HLBr primers and 1.87 times higher than that of the RNRf / RNRr primers. This indicates that when Huanglongbing samples contain Type 4 bacteriophages, the hg4-5_F / hg4-5_R primers can significantly improve the detection sensitivity of Huanglongbing bacteria. In 104 Huanglongbing samples that did not contain Type 4 bacteriophages, the Ct values ​​obtained by hg4-5_F / hg4-5_R primers were 1.02 (±0.08) and 1.48 (±0.14) higher than those of the CLas-4G / HLBr and RNRf / RNRr primers, respectively. This indicates that when Huanglongbing samples do not contain Type 4 bacteriophages, the detection sensitivity of hg4-5_F / hg4-5_R primers is lower than that of the CLas-4G / HLBr and RNRf / RNRr primers, but it can still be used for detection.

[0051] Example 3 Evaluation of the effect of simultaneous detection with two pairs of primers

[0052] 1. Method

[0053] DNA was extracted from 776 citrus samples collected from nine provinces, including Guangdong, Guangxi, Yunnan, Hunan, Guizhou, Hainan, and Jiangxi, and tested using SYBR Green Real-time PCR.

[0054] Following the reaction system and conditions in Example 2, Real-time PCR was performed using CLas-4G / HLBr primers, RNRf / RNRr primers, and hg4-5_F / hg4-5_R primers. Dual Real-time PCR was also performed using two primer pairs, CLas-4G / HLBr and hg4-5_F / hg4-5_R. The SYBR Green real-time fluorescent PCR dual primer amplification system is shown in Table 3, and the reaction conditions were the same as in Example 2. Melting curves were added after the reaction conditions. The standard program for the melting curve was: 95℃, 2 min; 40℃, 2 min; 65℃~95℃, increasing the temperature by 0.5℃ every 5 s and acquiring the fluorescence signal. The high-resolution program for the melting curve was: 95℃, 2 min; 40℃, 2 min; 75℃~90℃, increasing the temperature by 0.1℃ every 5 s and acquiring the fluorescence signal. Amplification and melting curves for dual qPCR were plotted using Bio-Rad CFXManager.

[0055] Table 3 SYBR Green Real-Time Quantitative PCR Dual Primer Amplification System

[0056]

[0057] Increase in sensitivity (R): Calculated using the ΔCt method, i.e., the increase in sensitivity of the hg4-5_F / hg4-5_R primer relative to the CLas-4G / HLBr primer is calculated as follows: ΔCt = Ct(hg4-5_F / hg4-5_R) - Ct(CLas-4G / HLBr).

[0058] Samples with a Ct value greater than 32 obtained by real-time quantitative RCP detection are considered negative samples, and samples with a Ct value less than 32 are considered positive samples.

[0059] 2. Results

[0060] Using standard primers, the melting curves of the products obtained from qPCR amplification with CLas-4G / HLBr primers, RNRf / RNRr primers, and hg4-5_F / hg4-5_R primers are shown below. Figure 2 As shown, the Tm values ​​of the amplification products of the CLas-4G / HLBr primers were 82.0–82.75℃, and the Tm values ​​of the amplification products of the hg4-5_F / hg4-5_R primers were 79.85–80.30℃. The melting curves of the amplification products were obtained using the CLas-4G / HLBf / r and hg4-5_F / hg4-5_R double primers. Figure 3As shown, two peaks appeared, at 77.5℃ and 83.5℃, indicating that the simultaneous use of the CLas-4G / HLBr and hg4-5_F / hg4-5_R primers did not result in nonspecific amplification.

[0061] like Figure 4 As shown, in the two groups of Huanglongbing bacteria samples containing Type 4 bacteriophage and those not containing Type 4 bacteriophage, the primer sensitivity increase value R was calculated using the ΔCt method, i.e., R=2. -ΔCt The results showed that the sensitivity of detection using the CLas-4G / HLBr and hg4-5_F / hg4-5_R double primers was approximately 40-fold and 5.9-fold higher than that using the CLas-4G / HLBr primers, respectively, and 11.5-fold and 3.1-fold higher than that using the RNRf / RNRr primers, respectively. Therefore, regardless of the presence of Type 4 phages, the effectiveness of real-time quantitative PCR detection using double primers was significantly improved.

[0062] Example 4: Using serially diluted samples as templates, a comparative system was used to evaluate the detection sensitivity of different primers.

[0063] 1. Method

[0064] The detection sensitivity of different primers was evaluated using serially diluted samples as templates in a comparative system, specifically in CLas. + Type 4 + Groups and Classes + Type 4 - DNA samples with low Ct values ​​detected by CLas-4G / HLBr primers were selected from the group and diluted 10-fold, resulting in a total of 8 dilutions (10-10). -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Real-time PCR detection was performed using either the CLas-4G / HLBr primers and the RNRf / RNRr primers alone, or by using both CLas-4G / HLBr and hg4-5_F / hg4-5_R primers simultaneously.

[0065] 2. Results

[0066] The results are shown in Table 4. + Type 4 + In this group, the endpoint was detected using both primers CLas-4G / HLBr and hg4-5_F / hg4-5_R, reaching 10. -8(2.0 copies / μL), which is higher than the detection endpoint of primers CLas-4G / HLBr (200 copies / μL) and primers RNRf / r (20 copies / μL).

[0067] In CLas + Type 4 - In this group, the simultaneous use of primers CLas-4G / HLBr and hg4-5_F / hg4-5_R, compared to primers CLas-4G / HLBr (420 copies / μL) and RNRf / RNRr (420 copies / μL), still improved the detection endpoint by two orders of magnitude, reaching 4.2 copies / μL, even under high dilution conditions. Therefore, the aforementioned dual primers CLas-4G / HLBr+hg4-5_F / hg4-5_R significantly improve the detection sensitivity for Huanglongbing samples, whether containing or not containing Type 4 phages, and are suitable for low CLas concentration conditions, i.e., the early stage of Huanglongbing infection in fruit trees. Ca. The lower concentration stage of Liberibacterspp. provides further technical support for laboratory detection of CLas.

[0068] Table 4. Ct values ​​of different primers at different DNA samples and template concentrations.

[0069]

[0070] Note: N / A indicates no amplification during real-time fluorescence PCR detection.

Claims

1. A primer for detecting Asian species of Huanglongbing fungus in citrus, characterized in that, It includes primer pair hg4-5_F / hg4-5_R and primer pair CLas-4G / HLBr; the nucleotide sequence of hg4-5_F is shown in SEQ ID NO.1; the nucleotide sequence of hg4-5_R is shown in SEQ ID NO.2; the nucleotide sequence of CLas-4G is shown in SEQ ID NO.3; and the nucleotide sequence of HLBr is shown in SEQ ID NO.

4.

2. The use of the detection primers for Asian species of citrus Huanglongbing as described in claim 1 in the detection of Asian species of citrus Huanglongbing or in the preparation of a detection kit for Asian species of citrus Huanglongbing.

3. A detection kit for Asian species of Huanglongbing fungus in citrus, characterized in that, Includes the detection primers for Asian species of Huanglongbing fungus of citrus as described in claim 1.

4. A method for detecting Asian species of Huanglongbing fungus in citrus, characterized in that, The method includes the following steps: S1. Extract genomic DNA from the sample to be tested; S2. Using the DNA from step S1 as a template, perform a dual real-time quantitative PCR reaction using the primers for detecting Asian species of citrus Huanglongbing as described in claim 1. If the Ct value obtained by real-time quantitative PCR is greater than 32, it indicates that the sample to be tested is not infected with Asian species of citrus Huanglongbing; if the Ct value is less than 32, it indicates that the sample to be tested is infected with Asian species of citrus Huanglongbing.

5. The method according to claim 4, characterized in that, The reaction conditions for the dual real-time quantitative PCR reaction in step S2 are as follows: 94–96℃, 3 min; 94–96℃, 10 s; 59–61℃, 30 s; 40 cycles; fluorescence signal reading is performed at the end of each cycle at 60℃.

6. The method according to claim 4, characterized in that, The melting curve program for the dual real-time quantitative PCR reaction in step S2 is as follows: 95℃, 2 min; 40℃, 2 min; 65℃~95℃, with a temperature increase of 0.1℃ every 5 s and fluorescence signal acquisition.

7. The method according to claim 6, characterized in that, The melting curve program for the dual real-time quantitative PCR reaction in step S2 is as follows: 95℃, 2 min; 40℃, 2 min; 75℃~90℃, with a temperature increase of 0.1℃ every 5 s and fluorescence signal acquisition.

8. The method according to claim 4, characterized in that, The amplification system for the dual real-time quantitative PCR reaction in step S2 is as follows: ddH2O 7.0 μL, Supermix 10.0 μL, 10 μmol / L CLas-4G 0.5 μL, 10 μmol / L HLBr 0.5 μL, 10 μmol / L hg4-5_F 0.5 μL, 10 μmol / L hg4-5_R 0.5 μL, DNA template 1.0 μL, totaling 20 μL.

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