Kit and method for detecting Candidatus Liberibacter asiaticum based on enzyme-mediated double-amplification nucleic acid amplification

Abstract

The invention discloses a kit and a method for detecting Candidatus Liberibacter asiaticum on the basis of enzyme-mediated dual-amplification nucleic acid amplification. The kit comprises an RNA (Ribonucleic Acid) probe and a DNA (Deoxyribonucleic Acid) amplification primer, nucleic acid amplification group enzyme system isothermal amplification is firstly carried out on DNA of a sample to be detected, target nucleic acid is amplified to 109 times within 10-30 minutes under the condition of constant temperature of 42 DEG C, and one nucleic acid amplification product can generate more than 10,000 fluorescence signals under the condition of constant temperature of 42 DEG C by integrating a signal amplification group enzyme system. According to the present invention, the Candidatus liberobacter asiaticum is taken as the target nucleic acid, the fluorescence is released through the RNA probe circulation combination-shedding, the signal secondary amplification is achieved, the one-tube completion is achieved, the accumulation efficiency from the target nucleic acid to the fluorescence signal is substantially improved, the specificity is strong, the sensitivity can be as low as 1.61 copy / [mu] L, the plants infected with the Candidatus liberobacter asiaticum can be early found, and the diseased plants can be eradicated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant pathology molecular diagnosis, and particularly relates to a kit and method for detecting Candidatus Liberibacter asiaticus based on enzyme-mediated double amplification of nucleic acid amplification. BACKGROUND

[0002] Citrus Huanglongbing is a devastating citrus disease, which causes serious economic losses to the world citrus industry. The pathogen of Citrus Huanglongbing is three bacteria in Candidatus Liberibacter, namely, Asian species (Ca. L. asiaticus, CLas), African species (Ca. L. africanus, CLaf) and American species (Ca. L. americanus, CLam), and the citrus Huanglongbing in China is Candidatus Liberibacter asiaticus. Citrus Huanglongbing is mainly transmitted by citrus psyllids in the field, and the transportation of diseased seedlings and scions is the main way of long-distance transmission. Since there is no disease-resistant / tolerant variety in current commercial citrus planting, and there is no "special drug" for preventing and controlling Huanglongbing, the "three hammers" for preventing and controlling Huanglongbing, that is, "using virus-free seedlings, digging diseased trees, and killing psyllids", become the most effective prevention and control measures. In order to implement the use of virus-free seedlings and digging of diseased trees, the prerequisite is to accurately diagnose the diseased trees.

[0003] The field diagnosis of Citrus Huanglongbing mainly uses the typical symptoms of leaf mottling yellowing and "red nose fruit" as the basis for judgment, but in addition to the "red nose fruit" in the fruiting stage, the leaf symptoms may also be caused by lack of nutrients, etc., so the field diagnosis can only be used as a preliminary basis for judgment. Citrus plants in the latent and early stages of Huanglongbing without symptoms need to be accurately diagnosed using plant pathogen molecular detection. At present, conventional PCR, nested PCR, semi-nested PCR and fluorescent quantitative PCR technologies have been established to detect citrus Huanglongbing, which continuously improves the sensitivity and accuracy of detection. However, the laboratory molecular detection process is time-consuming and complicated, and it is seriously dependent on precision instruments, so the growers often cannot accurately diagnose the diseased trees or virus-free seedlings in the field, resulting in missing the best treatment time for the diseased trees. Therefore, it is the most effective way to fundamentally cut off the source of Citrus Huanglongbing to invent a field molecular rapid detection technology and apply it to various citrus orchards and virus-free seedling breeding centers. Specifically, it is urgent to provide a rapid detection kit and method for Citrus Huanglongbing, which has the advantages of accuracy comparable to professional laboratories and rapid, simple and portable. SUMMARY

[0004] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and to provide a kit suitable for rapid detection of Candidatus Liberibacter asiaticus in the field.

[0005] The second object of the present application is to provide the use of the kit in on-site rapid detection of Candidatus Liberibacter asiaticum.

[0006] The third object of the present application is to provide a method for on-site rapid detection of Candidatus Liberibacter asiaticum.

[0007] The above objects of the present application are achieved by the following technical solutions:

[0008] The kit for detecting Candidatus Liberibacter asiaticum based on enzyme-mediated double amplification of nucleic acid comprises an RNA fluorescent probe, a DNA upstream primer and a DNA downstream primer.

[0009] The present application is directed to the conserved region of 16S rDNA sequence in the genome of Candidatus Liberibacter asiaticum, and an RNA fluorescent probe, a DNA upstream primer and a DNA downstream primer are designed based on enzyme-mediated double amplification of nucleic acid rapid detection technology.

[0010] Preferably, the kit further comprises a nucleic acid amplification enzyme system and a signal amplification enzyme system, and is integrated into one reaction tube.

[0011] Preferably, the 5' end and 3' end of the RNA fluorescent probe are connected with a fluorescent reporter group FAM and a fluorescent quenching group BHQ1, respectively.

[0012] Preferably, the kit further comprises a 16S rDNA plasmid standard of Candidatus Liberibacter asiaticum.

[0013] Preferably, the kit further comprises a high-efficiency lysis solution for extracting DNA of Candidatus Liberibacter asiaticum detection sample.

[0014] Preferably, the kit comprises a nucleic acid amplification detection reagent, which is composed of an RNA fluorescent probe (5'-UCCAUGCGUUAUCCCGUAGAAAAAGGUA-3'), a DNA upstream primer (5'-AAGCTAATACGACTCACTATAGGGTAACACATGCAAGTCGAGCGCGTATGCG-3'), a DNA downstream primer (5'-TCCAACGCAGGCTCATCTCTCTCCAATA-3'), a nucleic acid amplification enzyme system, a signal amplification enzyme system, NTP (nucleoside triphosphate), a buffer, RNase-free water, and a freeze-drying protective agent; and the nucleic acid amplification detection reagent system is freeze-dried to form pre-packaged freeze-dried microspheres.

[0015] Preferably, the nucleic acid amplification enzyme system is composed of a recombinase, a single-strand binding protein, a polymerase, and an ATP energy regeneration enzyme; and the signal amplification enzyme system is composed of a positioning enzyme and a cleavage enzyme.

[0016] The application also provides the use of the above-mentioned kit in the detection of Candidatus Liberibacter asiaticus.

[0017] The application also provides a method for detecting Candidatus Liberibacter asiaticus based on enzyme-mediated double amplification of nucleic acid amplification, which comprises the following steps:

[0018] S1. Extracting DNA from a sample to be tested, using the extracted DNA as a template, adding pre-packaged freeze-dried microspheres in the kit, and using a portable fluorescence detector to perform isothermal amplification reaction;

[0019] S2. Taking the fluorescence signal Ct value of the step S1 reaction as the detection result of the Huanglongbing bacteria, if there is no Ct value in the cycle, it indicates that the sample to be tested is not infected with Candidatus Liberibacter asiaticus, and the detection result is negative; if there is a Ct value, it indicates that the sample to be tested is infected with Candidatus Liberibacter asiaticus, and the detection result is positive.

[0020] Preferably, the step of extracting DNA from the sample to be tested is: collecting the midrib of the sample to be tested, adding the high-efficiency lysis solution in the kit, and lysis at 95℃ for 10 minutes.

[0021] Preferably, the condition of the isothermal amplification reaction is 42℃ for 10-30 minutes.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] (1) The DNA extraction step of the citrus leaf provided by the application is simplified, and only lysis solution and heat treatment for 10 minutes are needed.

[0024] (2) The enzyme-mediated double amplification nucleic acid amplification technology for detecting the Asian species of citrus Huanglongbing bacteria in the application can amplify the target nucleic acid to 10 9 times within 10-30 minutes under the constant temperature condition of 42 DEG C through the integration of nucleic acid amplification group enzymes.

[0025] (3) The molecular detection for detecting the Asian species of citrus Huanglongbing bacteria in the application has high specificity, and the sensitivity can be as low as 1.61 copies per muL, so that the plant infected with citrus Huanglongbing disease can be found early.

[0026] (4) The application can be used with a palm constant temperature fluorescence instrument, and can be carried to any place such as a field to carry out detection, so as to meet the application scenes of various citrus orchards and disease-free seedling breeding centers.

[0027] (5) The operation process of the application is simple, suitable for anyone to use, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is the flow chart of the application for detecting citrus Huanglongbing bacteria based on enzyme-mediated double amplification nucleic acid amplification.

[0029] Figure 2 It is the RNA fluorescence probe screening result (RNA3-F3R2, RNA3-F3R3, RNA4-F3R2, RNA4-F3R3) of example 1 of the application.

[0030] Figure 3 It is the RNA fluorescence probe screening result (RNA1-F3R2, RNA1-F3R3, RNA2-F3R2, RNA2-F3R3) of example 1 of the application.

[0031] Figure 4 It is the RNA fluorescence probe screening result (RNA5-F3R2, RNA5-F3R3, RNA6-F3R2, RNA6-F3R3) of example 1 of the application.

[0032] Figure 5 It is the DNA downstream primer screening result of example 2 of the application.

[0033] Figure 6 It is the DNA upstream primer screening result of example 3 of the application.

[0034] Figure 7 It is the specific experiment result (citrus Huanglongbing bacteria vs. four kinds of non-target bacteria) of example 4 of the application.

[0035] Figure 8 Specificity results for Example 4 of the present application (Huanglongbing of citrus vs. 3 non-target viruses).

[0036] Figure 9 Sensitivity results for Example 5 of the present application (qPCR amplification curves of different concentrations of templates).

[0037] Figure 10 Sensitivity results for Example 5 of the present application (qPCR amplification curves of different copy numbers).

[0038] Figure 11 Physical diagram of the kit.

[0039] Figure 12 Comparison of the results of the present method and the qPCR detection method.

[0040] Figure 13 On-site detection results of the portable detector. DETAILED DESCRIPTION

[0041] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0042] Example 1

[0043] The citrus Huanglongbing positive samples used in the experiment were collected from the Guangzhou Subtropical Fruit Tree Major Epidemic Control Laboratory of Zhongkai Agricultural Engineering College. The citrus variety was Shatangju (Citrus reticulata cv. Shatangju). After 3 months of infection through bud grafting of 1-year-old disease-free Shatangju, continuous detection by fluorescent quantitative PCR was positive. The Shatangju used in this study was 2 years old.

[0044] Total DNA was extracted from leaves using FastPure Plant DNA Isolation Mini Kit (Vazyme, DC104-01). The extracted DNA was verified by agarose electrophoresis imaging and detected by microspectrophotometer (Nanodrop) for concentration, and then stored in a -80℃ refrigerator.

[0045] Reaction system: 1 μL DNA upstream primer (10 μM), 1 μL DNA downstream primer (10 μM), 1 μL RNA fluorescent probe (1 μM), 7 μL DNA template were added into eight-tube containing basic dry powder (5-100 nM recombinant enzyme, 10-200 nM single-chain binding protein, 5-100 nM polymerase, 1-2 μM ATP energy regenerating enzyme, 0.05-2.5 μM positioning enzyme and 0.05-2.5 μM cleavage enzyme), and then centrifuged instantaneously, and left at room temperature for 2 min. 10 μL of activating solution (NTP, nucleotide triphosphate) and buffer solution were added along the wall of the eight-tube, and then centrifuged instantaneously. After shaking for 10 s, the mixture was centrifuged instantaneously and then detected on a machine. Fluorescence quantitative PCR instrument (LightCycler 480 II (Roach)), reaction temperature 42℃, signal acquisition once per cycle, a total of 30 cycles, reaction time 30 min, reporter group FAM.

[0046] The leaf DNA of CLas+ verified by the previous qPCR was used as a template, the leaf DNA of CLas- was used as a negative control, and RNase-free Water (Takara, 9012) was used as a blank control to perform RNA fluorescent probe screening. The primers F2 and F3 were cross combined with the primers R2 and R3 to test, respectively F2R2, F2R3, F3R2 and F3R3, and the RNA fluorescent probes RNA1, RNA2, RNA3, RNA4, RNA5 and RNA6 were added to each group of primers. The same primer groups were compared, and the RNA fluorescent probe with the minimum Ct value and the highest end-point fluorescence value was selected. Meanwhile, the DNA primer screening results of the corresponding four groups were determined according to the RNA fluorescent probe, and the primer group with the minimum Ct value, the highest end-point fluorescence value and no amplification signal in the negative control was selected. The results showed that the four groups of primers F2R2, F2R3, F3R2 and F3R3 and the six RNA fluorescent probes RNA1, RNA2, RNA3, RNA4, RNA5 and RNA6 were combined, a total of 24 combinations, and 12 combinations had better amplification effect. Among them, RNA1-F3-R2 had the lowest Ct value of 6.87, relatively high fluorescence value, and no amplification signal in the negative control, and was determined to be selected as the RNA1 fluorescent probe, and the RNA1-F3-R2 combination was used to carry out subsequent experiments. Figures 2-4

[0047] Example 2

[0048] ​The DNA of CLas+ leaf was used as a template, and the RNA1 fluorescent probe and the upstream primer F3 were fixed according to the sample loading system of Example 1. The downstream primers R1, R2, R3, R4, R5 and R6 were taken for screening, respectively. The primer group with the lowest Ct value and the highest end-point fluorescence value was selected, and the negative control had no amplification signal. The results showed that in the downstream primer screening results of DNA, the Ct value of RNA1-F3-R1 group was lower, which was 5.89, and the fluorescence value was relatively high, indicating that the downstream primer R1 was better, and the negative control had no amplification signal. It was determined to select R1 primer, and the RNA1-F3-R1 combination was taken for follow-up experiments. Figure 5

[0049] Example 3

[0050] The DNA of CLas+ leaf was used as a template, and the RNA1 fluorescent probe and the downstream primer R1 were fixed according to the sample loading system of Example 1. The upstream primers F1, F2, F3, F4, F5 and F6 were taken for screening, respectively. The primer group with the lowest Ct value and the highest end-point fluorescence value was selected, and the negative control had no amplification signal. The results showed that in the upstream primer screening results of DNA, the Ct value of RNA1-F1-R1 group was lower, which was 5.45, and the fluorescence value was higher, indicating that the upstream primer F1 was better, and the negative control had no amplification signal. It was determined to select F1 primer, and the RNA1-F1-R1 combination was used for experiments. Figure 6

[0051] Table 1. Primer screening table for the present embodiment (SEQ ID NO. 1-18)

[0052]

[0053]

[0054] Example 4

[0055] The TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (Tkara, 9763) was used to extract the genomic DNA of Gram-negative bacteria (G - ): Escherichia coli, Pseudomonas aeruginosa and Gram-positive bacteria (G + ​​Staphylococcus aureus, Bacillus subtilis were extracted DNA as template, according to the sample loading system of Example 1, with CLas-leaf DNA as negative control, with RNase-free Water as blank control, with the optimal primer set RNA1-F1-R1 screened according to the reaction system, time and temperature of Example 1 for specificity detection. The results showed that 2 kinds of gram-negative bacteria (G - ) and 2 kinds of gram-positive bacteria (G + ) were selected for specificity experiment, and the 4 tested bacteria were not detected, and the citrus Huanglongbing bacteria were detected; Similarly, citrus tristeza virus, citrus yellow vein phlperovirus, and citrus leaf fall virus were selected for specificity experiment, and the 3 tested viruses were not detected, and the citrus Huanglongbing bacteria were detected, indicating that the primer and probe combination screened in this study had high specificity ( Figures 7-8 ).

[0056] Example 5

[0057] Reaction system: the system containing basic dry powder (5-100 nM recombinant enzyme, 10-200 nM single-strand binding protein, 5-100 nM polymerase, 1-2 μM ATP energy regenerating enzyme, 0.05-2.5 μM positioning enzyme and 0.05-2.5 μM cutting enzyme), 10 μM DNA upstream primer (F1), 10 μM DNA downstream primer (R1), 1 μM RNA fluorescent probe (RNA1), activation liquid NTP (nucleoside triphosphate), buffer (10-30 mM Tris-HAc, pH 7.2-7.8; 20-80 mM potassium acetate; 2-5 mM magnesium acetate; 1-4 mM mercaptoethanol; 0.5-1 mM ATP), RNase-free Water, and freeze-drying protectant were prepared into pre-packaged freeze-dried balls. Figure 11 ). 50 μL of template DNA was added to the freeze-dried ball, the lid was closed and shaken for 10 s, and then centrifuged for 10 s before detection. The fluorescence quantitative PCR instrument was used with reaction temperature of 42℃, signal collection once per cycle, a total of 30 cycles, reaction time of 30 minutes, and reporter group set as FAM.

[0058] The 16S rDNA plasmid standard of Asian species of citrus Huanglongbing bacteria was used as the sample to be tested, and the plasmid copy number was 1.29×10 11 . The plasmid was gradient diluted with RNase-free Water, and the copy number was selected as 1.29×10 7 , 1.29×10 6 , 1.29×10 5 , 1.29×10 4 , 1.29×103 1.29 x 10 2 1.29 x 10 1 6.45, 3.225, 1.6125 Figures 9-10

[0059] Example 6

[0060] The leaves of the laboratory grafted citrus Huanglongbing infected sugar oranges and healthy sugar oranges were used as the samples to be detected. The sample DNA was rapidly extracted using the lysis solution. The leaf DNA of CLas+ was used as the positive control, the leaf DNA of CLas- was used as the negative control, and the RNase-free water was used as the blank control. The optimal primer set RNA1-F1-R1 screened was used to determine the effect of the method in the actual samples according to the sample addition system, time and temperature of Example 5. Fourteen samples of CLas+ sugar oranges and healthy sugar oranges were randomly selected from the laboratory greenhouse for detection. The sample DNA was rapidly extracted using the lysis solution (100 mM Tris-HCl, pH 8, 10 mM NaCl, 1 mM EDTA, 0.01% (w / v) SDS, 5% (w / v) PVP-40 and 1 μM proteinase K) after punching with a puncher, and the extraction condition was lysis at 95 °C for 10 minutes. The remaining leaves after punching were used for qPCR detection. The results showed that the detection results were consistent with the qPCR detection results, and the accuracy was 100% (Table 2 and Figure 12

[0061] Table 2. Comparison of the results of the present method and the qPCR detection method

[0062]

[0063] Example 7

[0064] The 16S rDNA plasmid standard of the Asian species of citrus Huanglongbing bacteria was used as the positive control, and the leaf DNA of CLas- was used as the negative control to determine the effect of the method in the field detection of actual samples. The sample addition system of Example 5 was used, and a portable qPCR instrument was used. The reaction temperature was 42 °C, signal acquisition was performed once per cycle, there were 30 cycles in total, the reaction time was 30 minutes, and the reporter group was set to FAM. Three samples were randomly selected in the laboratory greenhouse for in-situ detection. The sample DNA was rapidly extracted using the lysis solution after punching with a puncher, and the remaining leaves after punching were used for qPCR detection. The results showed that the effect of rapid detection in the field was consistent with the detection results of qPCR, and the accuracy was 100% (Table 3 and​​Figure 13 ).

[0065] Table 3. Comparison of results from field testing with qPCR testing method

[0066]

[0067] The above-described embodiments are merely illustrative for the preferred embodiments of the present application and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A kit for detecting Candidatus Liberibacter asiaticum based on enzyme-mediated dual amplification of nucleic acid amplification, characterized in that, The RNA fluorescent probe has a nucleotide sequence of 5'-UCCAUGCGUUAUCCCGUAGAAAAAGGUA-3', the DNA upstream primer has a nucleotide sequence of 5'-AAGCTAATACGACTCACTATAGGGTAACACATGCAAGTCGAGCGCGTATGCG-3', and the DNA downstream primer has a nucleotide sequence of 5'-TCCAACGCAGGCTCATCTCTCTCCAATA-3'.

2. The kit of claim 1, wherein The 5' end and the 3' end of the RNA fluorescent probe are connected with a fluorescent reporter group FAM and a fluorescent quencher group BHQ1, respectively.

3. The kit of claim 1, wherein The kit further comprises a 16S rDNA plasmid standard of Asian species of Candidatus Liberibacter asiaticus.

4. The kit of claim 1, wherein The kit further comprises a high-efficiency lysis solution for extracting DNA of a sample to be detected, and the high-efficiency lysis solution comprises pH 8 Tris-HCl, NaCl, EDTA, SDS, PVP-40 and proteinase K.

5. The kit of claim 1, wherein The kit comprises a nucleic acid amplification detection reagent, which is composed of the RNA fluorescent probe, the DNA upstream primer, the DNA downstream primer and a nucleic acid amplification enzyme system, a signal amplification enzyme system, NTP, a buffer, RNase-free water and a freeze-drying protective agent according to claim 1; and the reagent system is freeze-dried to form pre-packaged freeze-dried microspheres.

6. The kit of claim 5, wherein The nucleic acid amplification enzyme system is composed of a recombinase, a single-strand binding protein, a polymerase and an ATP energy regeneration enzyme; and the signal amplification enzyme system is composed of a positioning enzyme and a cleavage enzyme.

7. Use of the kit according to any one of claims 1-6 in detecting Asian species of Candidatus Liberibacter asiaticus.

8. Use according to claim 7, characterized in that, The kit is used in combination with a palm metal bath and a palm fluorescent detector to realize rapid nucleic acid detection of Candidatus Liberibacter asiaticus on site.

9. A method for on-site rapid detection of Candidatus Liberibacter asiaticum in citrus based on enzyme-mediated dual-amplification nucleic acid amplification, characterized by, The kit comprises the following steps: S1. Extracting DNA of a sample to be detected, using the extracted DNA as a template, adding the pre-packaged freeze-dried microspheres in the kit according to claim 5, and using a palm fluorescent detector to perform isothermal amplification reaction; S2. Taking the fluorescence signal Ct value of the reaction in step S1 as a detection result of Candidatus Liberibacter asiaticus, and if there is no Ct value in the cycle, it indicates that the sample to be detected is not infected with Candidatus Liberibacter asiaticus, and the detection result is negative; if there is a Ct value, it indicates that the sample to be detected is infected with Candidatus Liberibacter asiaticus, and the detection result is positive.

10. The method of claim 9, wherein, The isothermal amplification reaction is performed at 42 DEG C for 10-30 minutes.

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