A primer composition and its use in pathogen detection

Abstract

The application discloses a primer probe composition which is composed of a forward primer, a reverse primer and a probe primer. The sequence of the forward primer is shown as SEQ ID No. 1, the sequence of the reverse primer is shown as SEQ ID No. 2, and the sequence of the probe is shown as SEQ ID No. 3. The application establishes a detection method for Pseudomonas syringae pv. actidii, which comprises the following steps: extracting genomic DNA of a sample to be detected, taking the genomic DNA as a template, and performing RAA detection by using the primer composition; and determining whether the sample to be detected contains Pseudomonas syringae pv. actidii according to a strip of a LFD test paper. The detection method provided by the application has a minimum detection sensitivity of 1 pg / µL for Pseudomonas syringae pv. actidii DNA, and has a minimum detection limit of 1×10 3 cfu / mL for a bacterial suspension, and can provide a new technical means for early diagnosis and disease prevention and control of bacterial canker disease of kiwifruit.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a detection method of kiwifruit pathogenic bacteria, in particular to a primer composition and detection method for detecting Pseudomonas syringae pv. actinidiae by RAA-LFD technology. BACKGROUND

[0002] Kiwifruit bacterial canker is a devastating disease caused by Pseudomonas syringae pv. actinidiae (Psa). Pseudomonas syringae pv. Actinidiae Psa has strong pathogenicity, and kiwifruit bacterial canker has spread to major kiwifruit producing areas in the world. Once the kiwifruit orchard plants are infected with canker, it may lead to yield reduction, or even destroy the orchard, causing serious economic losses.

[0003] The traditional identification (detection) method of Pseudomonas syringae pv. actinidiae mainly includes pathogenic bacteria isolation from diseased tissues, morphological identification, pathogenicity determination, etc. Due to the long time consumption, low sensitivity, and interference of human and environmental factors, the traditional classification and identification method cannot make a quick diagnosis in the latent period and early stage of the disease, so it is difficult to make timely monitoring and effective control of the disease occurrence.

[0004] The development of molecular biology provides a rapid, sensitive and accurate technical support for the diagnosis of kiwifruit bacterial canker, and various PCR, hybridization probe, RAPD, AFLP and other molecular detection methods have been widely used in the detection and identification of Pseudomonas syringae pv. actinidiae. Although PCR method has great improvement in detection specificity and sensitivity, the diagnosis process is complicated, the cycle is long, the cost is high, and expensive instruments and reagents are needed, which cannot meet the demand of rapid detection. Although loop-mediated isothermal amplification (LAMP) is used for the detection and identification of Pseudomonas syringae pv. actinidiae, it does not need PCR instrument and other precision instruments, but the primer design is more complex than PCR, and the product analysis uses turbidity, metal indicator and other colorimetric methods for detection, which has the problem of difficult to distinguish false positive.

[0005] Recombinase Aided Amplification (RAA) is a new type of isothermal (37-42℃) nucleic acid amplification technology, which has the significant advantage that only one pair of primers is needed to achieve the amplification of template nucleic acid at a constant temperature of 39℃. Compared with ordinary PCR, RAA technology has the advantages of simplicity, rapidity, high efficiency, high sensitivity and strong specificity, and can obtain detection amplification products within 30 min. The reaction process does not require high-temperature denaturation and low-temperature annealing, which reduces the requirement for precise temperature control equipment and significantly shortens the reaction time. The RAA amplification result is combined with the lateral flow detection (LFD) test strip to realize visual reading, and the result can be judged by naked eye, which truly realizes the rapid on-site detection of pathogenic bacteria and provides a rapid and convenient technical means for plant pathogen detection and disease control. SUMMARY

[0006] The technical problem to be solved by the present application: The technical means including chemical pesticide control is difficult to effectively curb the spread of kiwifruit bacterial canker disease, and the rapid on-site detection of Pseudomonas syringae pv. actinidiae has important significance that cannot be ignored. For the RAA detection of Pseudomonas syringae pv. actinidiae, there is no related application report at present.

[0007] The present application aims to establish a method for conveniently, rapidly and sensitively detecting Pseudomonas syringae pv. actinidiae. In order to effectively solve the technical problems of the present application and achieve the technical purpose of the present application, the present application provides a primer composition for detecting Pseudomonas syringae pv. actinidiae based on RAA-LFD technology. The primer composition is composed of a forward primer, a reverse primer and a probe primer, and the target sequence of the primer composition is the sequence of a hypothetical gene (WP_017704201.1) in the genome of Pseudomonas syringae pv. actinidiae.

[0008] Specifically, the sequence of the forward primer is shown in SEQ ID No. 1. In the present application, the forward primer is RAA-Psa-F:

[0009] 5'-CACCATGATGCGGAGCTACTACGGCGACTG-3' (SEQ ID NO. 1).

[0010] Specifically, the sequence of the reverse primer is shown in SEQ ID No. 2, and in the present application, the reverse primer is RAA-Psa-R:

[0011] 5'-Biotin-AAGTAATTTCCGCAGGAGGGAGGCTGTTAG-3' (SEQ ID NO. 2). It is known from the sequence of SEQ ID No. 2 that the sequence of the 5' end of the reverse primer is labeled with biotin (Biotin). As a preferred embodiment, the biotin used is vitamin B7.

[0012] Specifically, the sequence of the probe primer is as shown in SEQ ID No. 3. In the present application, the probe primer (sequence) is RAA-Psa-P:

[0013] 5'-FAMACGGCGACTGGTGGCGAGTATTTGAAAGCG / THF / TGGGGAAGTGGGTG / C3-spacer (SEQ ID NO. 3). It is known from the sequence of SEQ ID No. 3 that the 5' end of the probe primer is labeled with fluorescein (FAM), and tetrahydrofuran (THF) is used as a spacer at a position 31 bases away from the 5' end dSpacer Instead of one base, the 3' end is blocked with C3-spacer. As a preferred embodiment, the fluorescein used is 6-carboxyfluorescein.

[0014] Based on the above-mentioned primer composition designed, the present application claims to protect a detection composition. The detection composition claimed in the present application contains the primer composition. This composition is used for the detection of Pseudomonas syringae pv. actinidiae and can be prepared into a preparation or reagent suitable for the detection of Pseudomonas syringae pv. actinidiae.

[0015] Based on the above-mentioned primer composition designed, as a preferred embodiment, the present application claims to protect a detection kit. The constituent components of the detection kit contain the primer composition, which is used for the detection of Pseudomonas syringae pv. actinidiae. Further, the constituent components of the detection kit also include a reaction unit tube containing RAA lyophilized enzyme powder, A Buffer, magnesium acetate solution (B Buffer), deionized water, running buffer, and lateral flow chromatographic test strips. The present application provides a method for detecting Pseudomonas syringae pv. actinidiae, which comprises: using the primer composition to perform RAA amplification with the DNA of the sample to be tested as a template; using lateral flow chromatographic test strips to detect the RAA amplification product, if two brown bands appear on the lateral flow chromatographic test strips, one in the quality control area and one in the detection area, then the sample to be tested contains Pseudomonas syringae pv. actinidiae; if only one brown band appears on the lateral flow chromatographic test strips in the quality control area and no band appears in the detection area, then the sample to be tested does not contain Pseudomonas syringae pv. actinidiae.

[0016] The method for detecting Pseudomonas syringae pv. actinidiae comprises the following specific steps:

[0017] 1) Extracting the genomic DNA of the sample to be tested;

[0018] 2) Using the primer composition to perform RAA amplification with the genomic DNA of the sample to be tested as a template;

[0019] 3) Using a lateral flow chromatographic test strip to detect the RAA amplification product. When two brown bands (Control line and Test line) appear on the lateral flow chromatographic test strip, one in the control area and one in the detection area, the result is positive, indicating that the sample contains P. syringae pv. actinidiae; when only one brown band (Control line) appears in the control area and no band appears in the detection area, the result is negative, indicating that the sample does not contain P. syringae pv. actinidiae.

[0020] In the above method, the reaction system of the RAA-LFD detection in step 2) is as follows: 25 μL of A buffer, 12.9 μL of ultrapure water, 10 μM of forward primer RAA-Psa-F 2 μL, 10 μM of reverse primer RAA-Psa-R 2 μL, 10 μM of probe primer RAA-Psa-P 0.6 μL, 2 μL of DNA template, and 2.5 μL of magnesium acetate (B buffer) are added to the RAA freeze-dried enzyme powder, and the total reaction system is 50 μL.

[0021] Among them, the specific components of A buffer are 100 mM KCl, 1.2 M Tris-HCl (pH 8.0), 25 mM MgCl2, 60 mM (NH4)2SO4, and 5% glycerol. The concentration of B buffer (magnesium acetate solution) is 280 mmol / L.

[0022] Further, the amplification reaction is performed in a 39°C water bath for 30 min under the RAA-LFD detection system. In the above method, 10 μL of RAA amplification product is removed and detected on the sample pad, and the sample pad end is placed in 200 μL of running buffer. When two brown bands appear on the test strip, one in the control area and one in the detection area, the result is positive, indicating that the sample contains P. syringae pv. actinidiae; when only one brown band appears in the control area and no band appears in the detection area, the result is negative, indicating that the sample does not contain P. syringae pv. actinidiae.

[0023] In another method for detecting P. syringae pv. actinidiae, the RAA amplification is performed using the detection kit.

[0024] The Pseudomonas syringae pv. actidii detection method provided by the application has extremely high detection sensitivity, and the detection sensitivity of Pseudomonas syringae pv. actidii DNA is at least 1 pg / μL, and the minimum detection limit of the bacterial suspension of Pseudomonas syringae pv. actidii is 1×10 3 cfu / mL.

[0025] In addition, the application provides the application of the Pseudomonas syringae pv. actidii detection method in Pseudomonas syringae pv. actidii detection or auxiliary detection.

[0026] Compared with the prior art, the “primer composition and application thereof in pathogen detection” has the following technical advantages or beneficial effects:

[0027] 1) The detection method provided by the application has high specificity. The primer composition is obtained through a large number of test verification and screening, and can specifically detect Pseudomonas syringae pv. actidii, and has no cross reaction with other pathogenic bacteria.

[0028] 2) The detection method provided by the application has extremely high detection sensitivity. The method can detect Pseudomonas syringae pv. actidii genomic DNA with a minimum limit of 1 pg / μL and bacterial suspension with a minimum concentration of 1.0×10 3 cfu / mL, and the minimum detection limit is consistent with the detection limit of ordinary PCR.

[0029] 3) The detection method provided by the application can complete amplification within 30 min at 39℃, and compared with ordinary PCR detection, the detection speed is faster, and no precise instruments and equipment are needed, so that the detection efficiency is greatly improved.

[0030] 4) The RAA detection system applied by the application, combined with a lateral flow chromatographic test strip (LFD), can realize visual reading without any equipment, and the detection result can be directly judged by naked eyes.

[0031] 5) The bacterial canker disease of kiwifruit has a fast field incidence rate, and the application can be suitable for rapid detection of Pseudomonas syringae pv. actidii in primary laboratories and fields. The application can be used for rapid detection of Pseudomonas syringae pv. actidii in the field, and the detection result can be obtained in 30 min, which has great significance for early diagnosis of diseases, determination of the best prevention period and establishment of effective disease prevention strategies.

[0032] 6) The application first adopts the RAA-LFD technology to establish a method for rapidly detecting Pseudomonas syringae pv. actidii, which has strong specificity, high sensitivity and fast detection speed, and can be used for field sample detection, thereby providing a new method for field detection of Pseudomonas syringae pv. actidii. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Figure 1 is a result chart of the detection of the strains in Table 1 by the method of the present application. 1 represents Pseudomonas syringae pv. actinidiae M228, 2 represents Pseudomonas syringae pv. actinidiae SXZZF4-8, 3 represents Pseudomonas syringae pv. actinidiae SXZZ-Y-6, 4 represents Pseudomonas syringae pv. actinidiae SBJM-FL-2, 5 represents Erwinia amylovora (SXER-T-1), 6 represents Enterobacter asburiae (SXLA-Y-1), 7 represents Bacillus sp. (SXBA-4), 8 represents Pantoea agglomerans (BJWG-4), 9 represents Flavobacterium sp. (BJM-2), 10 represents Acinetobacter sp. (SXZZ-Ac-2). Erwinia rhapontici Lelliottia amnigena Bacillus sp. Pantoea agglomerans Flavobacterium sp. Acinetobacter sp.

[0034] Figure 2 Figure 2 is a result chart of the sensitivity test of Pseudomonas syringae pv. actinidiae by the method of the present application (genomic DNA).

[0035] Figure 3 Figure 3 is a result chart of the sensitivity test of Pseudomonas syringae pv. actinidiae by the method of the present application (genomic DNA). C represents control line, and T represents test line.

[0036] Figure 4 Figure 4 is a result chart of the sensitivity test of Pseudomonas syringae pv. actinidiae by the method of the present application (bacterial suspension concentration).

[0037] Figure 5 Figure 5 is a result chart of the detection of field samples by the method of the present application. 1-8 represent suspected samples of bacterial canker disease of kiwifruit, and 9-10 represent healthy samples. DETAILED DESCRIPTION

[0038] The present application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0039] In the examples, the experimental methods are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified. EXAMPLES​​​​​​

[0040] The test strains of this embodiment are shown in Table 1, and all 10 test strains were isolated and preserved by the Fruit Tree Disease Pathogen Biology and Comprehensive Prevention Research Team Laboratory of Northwest A&F University.

[0041] Table 1, test strain information

[0042] The strain genomic DNA extraction kit was purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and the genomic DNA of the strain was extracted according to the instructions. The RAA-nfo nucleic acid amplification reagent (test strip type) was purchased from Hangzhou Zhongce Biological Technology Co., Ltd. The primer composition was synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.; the LFD measurement chromatography colloidal gold test strip was purchased from Germany Milenia Geneline Company.

[0043] According to a partial sequence of a hypothetical gene (NCBI accession number: WP_017704201.1) in the genome of Pseudomonas syringae var. actinidiae (Psa) Pseudomonas syringae pv . actinidiae ), a forward primer RAA-Psa-F, a reverse primer RAA-Psa-R and a probe primer RAA-Psa-P were designed. The reverse primer RAA-Psa-R sequence 5' end was labeled with biotin (Biotin); the probe RAA-Psa-P sequence 5' end was labeled with fluorescein (FAM), and at a position 31 bases away from the 5' end, tetrahydrofuran (THF) was used as a substitute for one base, and the 3' end was blocked with C3-spacer. The specific sequences are as follows: dSpacer

[0044] RAA-Psa-F: 5'-CACCATGATGCGGAGCTACTACGGCGACTG-3' (SEQ ID NO. 1);

[0045] RAA-Psa-R: 5'-Biotin-AAGTAATTTCCGCAGGAGGGAGGCTGTTAG-3' (SEQ ID NO. 2);

[0046] RAA-Psa-P:

[0047] 5'-FAMACGGCGACTGGTGGCGAGTATTTGAAAGCG / THF / TGGGGAAGTGGGTG / C3-spacer (SEQ ID NO. 3).

[0048] ​The extracted genomic DNA was used as a template, and the RAA reaction was performed in the reaction system using the designed primers: 25 μL of A buffer, 12.9 μL of ultrapure water, 10 μM of primer RAA-Psa-F 2 μL, 10 μM of primer RAA-Psa-R 2 μL, 10 μM of probe primer RAA-Psa-P 0.6 μL, 2 μL of DNA template, and 2.5 μL of magnesium acetate were added to the RAA lyophilized enzyme powder, and the total reaction system was 50 μL.

[0049] The above reaction system was subjected to amplification reaction in a 39°C water bath for 30 min. The amplification product was detected by using a lateral flow chromatographic test strip (LFD). 10 μL of the amplification product was taken to the sample pad, and the sample pad end was placed in 200 μL of running buffer. When two brown bands appeared on the lateral flow chromatographic test strip, one in the quality control zone and one in the detection zone, the result was positive, indicating that the sample contained P. syringae pv. actinidiae; when only one brown band appeared in the quality control zone and no band appeared in the detection zone, the result was negative, indicating that the sample did not contain P. syringae pv. actinidiae. Example

[0050] The specificity of the P. syringae pv. actinidiae detection method was verified.

[0051] Four strains of P. syringae pv. actinidiae and six strains of P. syringae endophytic bacteria were selected, and genomic DNA was used as a template to verify the specificity of the detection method. The results are shown in Figure 1 The LFD lateral flow chromatographic test strip results showed that only P. syringae pv. actinidiae (strain numbers M228, SXZZF4-8, SXZZ-Y-6, SBJM-FL-2) were positive results, i.e., two brown bands appeared on the LFD test strip, one in the quality control zone and one in the detection zone. The DNA detection results of the remaining six strains were negative, i.e., only one brown band appeared on the LFD test strip, which was in the quality control zone, and the result was negative. This indicates that the designed RAA-LFD reaction system has high specificity. Example

[0052] The sensitivity of the P. syringae pv. actinidiae detection method was verified.

[0053] The sensitivity of the detection method was determined using extracted P. syringae pv. actinidiae genomic DNA and bacterial suspensions of different concentrations. The extracted genomic DNA was serially diluted by 10 times to obtain eight gradients with mass concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL, respectively. The template DNA was replaced with an equal amount of ultrapure water as a blank control (CK). The P. syringae pv. actinidiae bacterial suspension was diluted by 10 times with sterile water, and the concentrations were 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 , and 1.0×10 8 cfu / mL, respectively, and ultrapure water was used as a blank control (CK).

[0054] The results are shown in Table 1. Figure 2 , Figure 3 The RAA-LFD detection was performed using P. syringae pv. actinidiae genomic DNA with concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL, respectively. Two brown bands appeared on the LFD flow chromatography test strip, indicating a positive reaction. The LFD flow chromatography test strip containing 100 fg / μL, 10 fg / μL, and 1 fg / μL of P. syringae pv. actinidiae DNA in the reaction system only showed one brown band, indicating a negative reaction. The color development results showed that the sensitivity of the P. syringae pv. actinidiae detection method reached 1 pg / μL.

[0055] As shown in Table 2, the RAA-LFD detection was performed using P. syringae pv. actinidiae bacterial suspensions with concentrations of 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , and 1.0×10 3 cfu / mL, respectively. Two brown bands appeared on the test strip, indicating a positive reaction. The color development results showed that the sensitivity of the P. syringae pv. actinidiae detection method reached 1.0×10 cfu / mL. Example

[0056] From the kiwi fruit production areas of Zhouzhi, Mei County, Wugong, etc. in Shaanxi Province, 8 suspected samples of kiwi fruit bacterial canker disease and 2 healthy kiwi fruit samples were collected for RAA-LFD detection. After washing with clean water and surface disinfection with 75% alcohol, the plant tissues were cut into 1 cm long and wide with a sterilized scalpel, chopped and placed in a 1.5 mL centrifuge tube, 1 mL of sterile water was added, and after standing for 10 min, 100℃ water bath for 10 min, the bacterial suspension was used as a template for RAA-LFD detection. The results are shown in Figure 5 Figure 5 As can be seen from

[0057] In summary, the present application establishes a visual detection method for Pseudomonas syringae pv. actinidiae based on RAA-LFD technology, and evaluates its specificity and sensitivity for detecting Pseudomonas syringae pv. actinidiae. It has been verified that the method has high detection sensitivity, and the detection limit can reach 1 pg / μL, and has good specificity and no cross reaction with other genus pathogens. In addition, the present application further verifies the detection effect of the established method (RAA-LFD detection system) on actual samples, and the method can quickly identify suspected samples and healthy samples of kiwi fruit bacterial canker disease, and the detection results are accurate and reliable. The detection method for Pseudomonas syringae pv. actinidiae of the present application has the characteristics of rapidness, specificity, sensitivity and simplicity, and provides a new technical means for plant pathogen detection and diagnosis and disease control.

[0058] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement within the technical range disclosed by the present application, which is within the spirit and principles of the present application, should be covered within the protection scope of the present application.​

Claims

1. A primer-probe composition, characterized in that, The primer-probe composition comprises a forward primer, a reverse primer, and a probe. The sequence of the forward primer is shown in SEQ ID No. 1, the sequence of the reverse primer is shown in SEQ ID No. 2, and the sequence of the probe is shown in SEQ ID No.

3. The 5′ end of the reverse primer is labeled with biotin; The 5′ end of the probe is labeled with fluorescein, the position 31 bases from the 5′ end is replaced with tetrahydrofuran, and the 3′ end is blocked with a C3-spacer.

2. A detection composition, characterized in that, The detection composition comprises the primer-probe composition of claim 1.

3. A test kit, characterized in that, The detection kit comprises the primer and probe composition of claim 1; The test kit also includes reaction unit tubes containing RAA lyophilized enzyme powder and lateral flow chromatography test strips.

4. The application of the primer-probe composition of claim 1, or the detection composition of claim 2, or the detection kit of claim 3 in the detection of pathogenic species of *Pseudomonas syringae* in *Actinidia kiwifruit*.

5. A method for detecting pathogenic strains of *Pseudomonas syringae* in kiwifruit, characterized in that, include: Using the DNA of the sample to be tested as a template, RAA amplification is performed using the primer and probe composition of claim 1, the detection composition of claim 2, or the detection kit of claim 3. The RAA amplification products were detected using lateral flow chromatography strips. If two brown bands appeared on the lateral flow chromatography strip, one in the control area and one in the detection area, the sample contained *Pseudomonas syringae* pathogenic species in kiwifruit. If only one brown band appeared in the control area and no band appeared in the detection area, the sample did not contain *Pseudomonas syringae* pathogenic species in kiwifruit. The limit of detection (LOD) for *Pseudomonas syringae* pathogenic DNA in kiwifruit is 1 pg / μL, and the LOD for *Pseudomonas syringae* pathogenic suspension in kiwifruit is 1 × 10⁻⁶. 3 cfu / mL.

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