Method for analyzing and mining activity detection target of maize bacterial wilt germs based on comparative genomics

Abstract

The invention relates to the technical field of microbiological detection, and discloses a method for analyzing and mining an activity detection target of maize bacterial wilt on the basis of comparative genomics, which comprises the following steps: S1, comparing and analyzing genomes of maize bacterial wilt PSS and an approximate species thereof to obtain an active target gene sequence; s2, designing a specific primer according to the active target gene sequence, and carrying out real-time fluorescent RT-PCR specific amplification to obtain a candidate target gene; and S3, evaluating the stability of the candidate target gene killed by heat treatment to obtain an activity detection target. The live bacterium RT-qPCR detection system for the maize bacterial wilt, which is established according to the screened mRNA target gene, can solve the problem that dead bacteria and live bacteria cannot be distinguished in molecular detection, and provides technical support for quarantine and prevention and control of the maize bacterial wilt.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and specifically relates to a method for mining target genes for detecting the viability of Pantoea stewartii subsp. stewartii (Pss) based on comparative genomics analysis. Background Art

[0002] Maize bacterial wilt is one of the devastating bacterial diseases, and its pathogen is Pantoea stewartii subsp. stewartii (Pss). Maize bacterial wilt is a vascular disease. Infected plants are dwarfed or wilted, which can cause huge economic losses to maize production, especially sweet maize production. Maize bacterial wilt is mainly spread over long distances by infected seeds. For major maize-producing and consuming countries that need to import a large amount of maize, once the pathogen is introduced, it will inevitably cause significant economic losses. Therefore, establishing an efficient and sensitive detection method for Pantoea stewartii subsp. stewartii is very important for the early diagnosis of the disease and the formulation of scientific and effective prevention and control measures.

[0003] Currently, molecular biology techniques such as ordinary PCR, nested PCR, real-time fluorescence quantitative PCR, etc. are mostly used for the detection of Pantoea stewartii subsp. stewartii at home and abroad. However, the above methods are all detection techniques based on genomic DNA as the target, and none of them can distinguish whether Pantoea stewartii subsp. stewartii is a live bacterium or a dead bacterium, and false positives are likely to occur. Therefore, there is an urgent need to establish a method for quickly detecting live Pantoea stewartii subsp. stewartii in samples.

[0004] In recent years, the RT-PCR technique with RNA as the detection target has shown attractive application prospects in the field of detecting viable bacteria in medical diseases and food microorganisms, but there is no reported method for detecting viable plant pathogenic bacteria based on RNA as the target. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for mining target genes for detecting the viability of Pantoea stewartii subsp. stewartii based on comparative genomics analysis. By comparing and analyzing the genomes of maize bacterial wilt and its closely related species, differentially expressed genes unique to the species are screened out, and their signal pathways and functions are predicted and analyzed. Suitable mRNA target genes are screened out, and the linear relationship between the disappearance of the viability of the pathogen after heat treatment and the degradation time of mRNA is evaluated. A real-time fluorescence quantitative PCR (RT-qPCR) detection system for live maize bacterial wilt is established, aiming to solve the problem of being unable to distinguish dead bacteria and live bacteria in molecular detection, and provide technical support for the quarantine and prevention and control of maize bacterial wilt.

[0006] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0007] The present invention provides a method for mining active detection targets of *Xanthomonas stewartii* subsp. *stewartii* based on comparative genomics analysis, comprising the following steps:

[0008] S1. Perform genomic alignment analysis on *Xanthomonas stewartii* subsp. *stewartii* (PSS) and its closely related species to obtain active target gene sequences;

[0009] S2. Design specific primers according to the active target gene sequences, and perform real-time fluorescence RT-PCR specific amplification to obtain candidate target genes;

[0010] S3. Evaluate the stability of the candidate target genes after heat treatment-induced death to obtain active detection targets.

[0011] Preferably, the method of the alignment analysis is specifically to perform orthologous clustering analysis on the whole-genome protein sequences of the PSS strain and its closely related strains, and further screen according to the GO functional annotation results to obtain active target gene sequences.

[0012] Preferably, the active detection targets are atpB, rpoE, MET, and IDH1.

[0013] The present invention also provides a primer for TaqMan fluorescence quantitative RT-PCR detection of viable *Xanthomonas stewartii* subsp. *stewartii*, and the primer is any one of primer pair atpB-1 for detecting the above target atpB, primer pair rpoE-2 for detecting the above target rpoE, primer pair MET-3 for detecting the above target MET, and primer pair IDH1-4 for detecting the above target IDH1;

[0014] The primer pair atpB-1 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 2;

[0015] The primer pair rpoE-2 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 4 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 5;

[0016] The primer pair MET-3 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 8;

[0017] The primer pair IDH1-4 includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 10 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 11;

[0018] The present invention also provides a probe for TaqMan fluorescence quantitative RT-PCR detection of viable cells of Xanthomonas stewartii subsp. stewartii, and the nucleotide sequence of the probe used in combination with the above primer pair atpB-1 is shown in SEQ ID NO: 3; the nucleotide sequence of the probe used in combination with the above primer pair rpoE-2 is shown in SEQ ID NO: 6; the nucleotide sequence of the probe used in combination with the above primer pair MET-3 is shown in SEQ ID NO: 9; the nucleotide sequence of the probe used in combination with the above primer pair IDH1-4 is shown in SEQ ID NO: 12.

[0019] The present invention also provides a kit for TaqMan fluorescence quantitative RT-PCR detection of viable cells of Xanthomonas stewartii subsp. stewartii, which at least includes the above primers for TaqMan fluorescence quantitative RT-PCR detection of viable cells of Xanthomonas stewartii subsp. stewartii and the above probes for TaqMan fluorescence quantitative RT-PCR detection of viable cells of Xanthomonas stewartii subsp. stewartii.

[0020] The present invention also provides a method for TaqMan fluorescence quantitative RT-PCR detection of viable cells of Xanthomonas stewartii subsp. stewartii using the above primers, probes or kits. The reaction system for the fluorescence quantitative RT-PCR detection consists of the following components: 12.5 μL of One Step PrimeScript III RT-qPCR Mix, 0.6 μL of the upstream primer at 10 μmol / L, 0.6 μL of the downstream primer at 10 μmol / L, 0.4 μL of the probe at 10 μmol / L, 2.0 μL of the RNA template, and RNase Free ddH2O is added to make up to 25 μL.

[0021] Preferably, the reaction program for the fluorescence quantitative RT-PCR detection is: reverse transcription at 42 °C for 5 min; pre-denaturation at 95 °C for 10 s; denaturation at 95 °C for 5 s, annealing at 60 °C for 60 s, for 40 cycles.

[0022] The present invention also provides the application of the primers, probes, kits, reaction systems and / or reaction programs of the above method for TaqMan fluorescence quantitative RT-PCR detection of viable cells of Xanthomonas stewartii subsp. stewartii in detecting viable cells of Xanthomonas stewartii subsp. stewartii.

[0023] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0024] Through the genomic alignment analysis of Pantoea stewartii subsp. stewartii and its closely related species, the present invention can screen out suitable mRNA target genes, and the viable bacteria RT-qPCR detection system for Pantoea stewartii subsp. stewartii established based on the screened mRNA target genes can solve the problem of inability to distinguish dead bacteria from viable bacteria in molecular detection, providing technical support for the quarantine and prevention and control of Pantoea stewartii subsp. stewartii. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the analysis result of PSS specific genes based on comparative genomics in the present invention;

[0026] Figure 2 is the screening result diagram of PSS specific primers in the present invention;

[0027] Figure 3 is the electrophoresis diagram for evaluating the stability of target genes after treatment and death in the present invention;

[0028] Figure 4 is the amplification result of detecting PSS by 4 groups of primers and probes in the present invention (S1 is atpB; S2 is rpoE; S3 is MET; S4 is IDH1);

[0029] Figure 5 is the sensitivity analysis of TaqMan real-time fluorescence RT-PCR method with S1 group primers and probes in the present invention (1-7 are 10 8 CFU / mL, 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 CFU / mL and 10 0 CFU / mL of Pantoea stewartii subsp. stewartii). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the protection scope of the present invention.

[0031] Embodiment:

[0032] I. Mining the key genes of the metabolic activity of Pantoea stewartii subsp. stewartii and obtaining the sequence of active target genes

[0033] Download the genomic information of Pantoea stewartii subsp. stewarti (PSS), a bacterium causing bacterial wilt in maize, and its four related species, Pantoea stewartii subsp. indologenes (Psi), Pantoea agglomerans, Pantoea allii - 7501 - 4, and Pantoea ananatis, from the NCBI database. Perform orthologous clustering analysis on the whole - genome protein sequences of the PSS strain and the four related strains using OrthoVenn 2 software (). The results show that a total of 4510 gene clusters are obtained, including 1742 orthologous gene clusters (containing at least two strains) and 2768 single - copy gene clusters. Figure 1 It can be seen that there are 120 gene clusters unique to the PSS strain, with a total of 673 genes on the genome, among which 103 are genes of known gene families, including 94 genes related to biological processes, 8 genes related to molecular functions, and 1 gene related to cellular components. Further screening based on the GO functional annotation results yields a total of 25 genes related to stress resistance, transcriptional, or metabolic activities, mainly including RNA polymerase sigma factor (RpoE), acetoacetyl - CoA synthase, and transcriptional regulator, etc. (see Table 1).

[0034] II. Screen the active target genes of Pantoea stewartii subsp. stewarti and obtain candidate active detection targets

[0035] 1 Primer design

[0036] Design specific primers using Oligo 7.0 software based on the sequences of the 25 screened active target genes. The primers and probes are synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown in Table 1.

[0037] Table 1 Sequence information of active target genes

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] 2 Primer Screening

[0045] Transfer the PSS colonies cultured at a constant temperature of 28 °C for 3 - 5 days into NA liquid medium (nutrient agar medium) and culture for 12 - 24 h. Take the bacterial solution with a concentration of about 10 -6 CFU / mL to extract RNA as a template for real-time fluorescence RT-PCR amplification to evaluate the amplification effect of PSS primers. The results of real-time fluorescence RT-PCR amplification showed that among the 39 pairs of primers designed above, a total of 15 pairs of primers could specifically amplify PSS, and the amplification curve was a standard "S" curve, and the melting curve showed only a single peak. The remaining 14 pairs of primers could not amplify or had non-specific amplification for the PSS amplification results (see Figure 2 ). Therefore, 15 pairs of PSS-specific primers selected were used for subsequent verification of target genes for activity detection.

[0046] 3 Evaluation of the Stability of Target Genes after Heat Treatment and Death

[0047] After the bacterial suspension was dry heat-treated at 70 °C for 1 hour, it was streaked on NA plates and cultured at a constant temperature of 28 °C for 3 - 5 days. No colony growth was found, indicating that the pathogen had been killed and inactivated after heat treatment. To evaluate the stability of 15 pairs of candidate target genes and 16S rRNA, 23S rRNA genes, RT-PCR detection was performed on the inactivated bacterial suspension, as shown in Table 2 and Figure 3 .

[0048] Table 2 Results of the Evaluation of the Stability of Target Genes after Heat Treatment and Death

[0049]

[0050]

[0051] Table 2 and Figure 3 The results showed that 4 mRNA genes (atpB, rpoE, MET, and IDH1) were rapidly degraded 12 - 72 hours after the pathogen lost its activity and could be used as candidate targets for activity detection.

[0052] III. Establishment of TaqMan Fluorescent Quantitative RT-PCR Detection Method

[0053] 1 Establishment of a TaqMan Fluorescent Quantitative RT-PCR Method for Detecting Xanthomonas stewartii subsp. stewartii

[0054] Design specific probes that match the primers of the 4 target genes screened above to establish a TaqMan fluorescent quantitative RT-PCR detection method.

[0055] Reaction instrument: Thermo QuantStudio 6.0 fluorescence quantitative PCR instrument (Thermo Fisher Scientific).

[0056] The reaction system is as follows: 12.5 μL of One Step PrimeScript III RT-qPCR Mix (TaKaRa), 0.6 μL each of 10 μmol / L forward and reverse primers, 0.4 μL of 10 μmol / L probe, 2.0 μL of RNA template, and made up to 25 μL with RNase Free ddH2O.

[0057] The reaction procedure is as follows: Reverse transcription at 42 °C for 5 min; pre-denaturation at 95 °C for 10 s; denaturation at 95 °C for 5 s, annealing at 60 °C for 60 s (collect fluorescence signal), for 40 cycles.

[0058] The detection efficiency of the 4 sets of primers and probes corresponding to the above 4 target genes for PSS was evaluated by TaqMan fluorescence quantitative RT-PCR. The fluorescence signals generated by the primers and probes in groups S1, S2, and S3 appeared the fastest, and reached the plateau at about 32 cycles; the fluorescence signal of the primers and probes in group S4 appeared after 26 cycles and reached the plateau at about 40 cycles. However, the fluorescence signal of group S4 at the plateau was much lower than that of the other 3 groups, indicating that the amplification efficiency of the primers and probes in group S4 was lower than that of the other 3 groups (see Figure 4 ). According to the fluorescence absorption values of the fluorescence signals generated by the primers and probes in groups S1, S2, and S3, the primers and probes of group S1 (i.e., using atpB as the target) were selected for subsequent experiments.

[0059] 3 Specificity analysis

[0060] The primers and probes of group S1 were evaluated for specificity. The real-time fluorescence RT-PCR reaction established with the primers and probes of group S1 was used for specific analysis of Pantoea stewartii subsp. stewartii (PSS) and its closely related species.

[0061] Table 3 Results of specificity analysis of primers and probes in group S1

[0062]

[0063]

[0064] The results in Table 3 show that the primers and probes in group S1 can specifically amplify PSS, have no amplification signal for its closely related species, and have good specificity for PSS.

[0065] 4 Sensitivity analysis

[0066] The sensitivity of primer and probe set S1 was evaluated. RNA extracted from Pantoea stewartii subsp. stewartii (PSS), which was serially diluted 10-fold at a concentration of 10 8 CFU / mL, was used as a template for real-time fluorescence RT-PCR reaction.

[0067] The results Figure 5 showed that the detection sensitivity of the real-time fluorescence RT-PCR established with primer and probe set S1 was 10 3 CFU / mL.

[0068] 5 Detection and analysis of actual samples

[0069] A total of 15 samples, including 5 samples of seeds with PSS bacteria, 5 samples of inactivated seeds with bacteria, and 5 samples of healthy seeds, were detected by RT-qPCR for PSS activity using the TaqMan real-time fluorescence RT-PCR method established in this study, as shown in Table 4.

[0070] Table 4 Detection results of the real-time fluorescence RT-PCR method for actual samples

[0071]

[0072]

[0073] The results in Table 4 showed that among the 15 tested samples, the Ct values of RT-qPCR detection were between 25.0 and 30.0, which were consistent with the qPCR detection results and the plate isolation results; 5 samples of seeds with bacteria were detected as positive, while the detection results of the 5 inactivated seeds and the healthy seeds were both negative, with the Ct values of RT-qPCR detection greater than 38.0 or no amplification signal, while the Ct values of qPCR detection were between 26.0 and 31.0, indicating that false positives would occur in qPCR detection using DNA as the detection template for inactivated seeds with bacteria, while the RT-qPCR detection method established in this study could effectively distinguish dead bacteria from live bacteria. The above results indicated that this method was applicable to the detection of dead and live bacteria of PSS in seed samples.

[0074] In summary, through genomic alignment analysis of Pantoea stewartii and its closely related species, the present invention can screen out suitable mRNA target genes, and the established live bacteria RT-qPCR detection system for Pantoea stewartii can solve the problem of inability to distinguish dead bacteria from live bacteria in molecular detection, providing technical support for the quarantine and prevention and control of Pantoea stewartii.

[0075] The above-described embodiments merely represent the preferred embodiments of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A method for mining targets for detecting the activity of corn bacterial wilt pathogen based on comparative genomics analysis, characterized in that: The steps include: S1. Compare and analyze the genomes of corn bacterial wilt PSS and its similar species to obtain the active target gene sequence; S2. Design specific primers based on the active target gene sequence and perform real-time fluorescence RT-PCR specific amplification to obtain candidate target genes; S3. Evaluate the stability of candidate target genes after heat treatment and obtain activity detection targets.

2. The method for mining maize bacterial wilt pathogen activity detection targets based on comparative genomics analysis according to claim 1, characterized in that: The comparison analysis method specifically comprises performing orthologous clustering analysis on the whole genome protein sequences of the PSS strain and its closely related strains, and then further screening according to the GO functional annotation results to obtain the active target gene sequences.

3. The method for mining maize bacterial wilt pathogen activity detection targets based on comparative genomics analysis according to claim 1, characterized in that: The activity detection targets are atpB, rpoE, MET and IDH1.

4. A primer for TaqMan fluorescent quantitative RT-PCR detection of live bacteria of corn bacterial wilt, characterized in that: The primers are any one of the primer pair atpB-1 for detecting the target atpB according to claim 3, the primer pair rpoE-2 for detecting the target rpoE according to claim 3, the primer pair MET-3 for detecting the target MET according to claim 3, and the primer pair IDH1-4 for detecting the target IDH1 according to claim 3; The primer pair atpB-1 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2; The primer pair rpoE-2 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 4 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 5; The primer pair MET-3 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8; The primer pair IDH1-4 includes an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 10 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO:

11.

5. A probe for TaqMan fluorescent quantitative RT-PCR detection of live bacteria of corn bacterial wilt, characterized in that: The nucleotide sequence of the probe used in combination with the primer pair atpB-1 described in claim 4 is shown in EQ ID NO: 3; the nucleotide sequence of the probe used in combination with the primer pair rpoE-2 described in claim 4 is shown in EQ ID NO: 6; the nucleotide sequence of the probe used in combination with the primer pair MET-3 described in claim 4 is shown in EQ ID NO: 9; the nucleotide sequence of the probe used in combination with the primer pair IDH1-4 described in claim 4 is shown in EQ ID NO:

12.

6. A kit for detecting live bacteria of corn bacterial wilt by TaqMan fluorescent quantitative RT-PCR, characterized in that: The method comprises at least the primers for detecting live bacteria of corn bacterial wilt pathogen TaqMan fluorescent quantitative RT-PCR as described in claim 4 and the probe for detecting live bacteria of corn bacterial wilt pathogen TaqMan fluorescent quantitative RT-PCR as described in claim 5.

7. A method for detecting live bacteria of corn bacterial wilt pathogen using TaqMan fluorescent quantitative RT-PCR using the primer, probe or kit according to any one of claims 4 to 6, characterized in that: The reaction system of the fluorescent quantitative RT-PCR detection consists of the following components: One Step PrimeScript III RT-qPCR Mix 12.5 μL, 10 μmol / L upstream primer 0.6 μL, 10 μmol / L downstream primer 0.6 μL, 10 μmol / L probe 0.4 μL, RNA template 2.0 μL, RNase Free ddH2O supplemented to 25 μL.

8. The method for detecting live bacteria of corn bacterial wilt pathogen TaqMan fluorescent quantitative RT-PCR according to claim 7, characterized in that: The reaction procedure of the fluorescent quantitative RT-PCR detection is: reverse transcription at 42°C for 5 minutes; pre-denaturation at 95°C for 10 seconds; denaturation at 95°C for 5 seconds, annealing at 60°C for 60 seconds, and 40 cycles.

9. Use of the primers, probes, kit, reaction system and / or reaction procedure of the method for detecting live bacteria of corn bacterial wilt pathogenic bacteria TaqMan fluorescent quantitative RT-PCR according to claim 8 in detecting live bacteria of corn bacterial wilt pathogenic bacteria.

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