Method for mining active detection target of bacterial wilt of corn based on comparative genomics analysis

Abstract

The present application relates to the technical field of microorganism detection, and discloses a method for mining active detection targets of corn bacterial wilt based on comparative genomics analysis, comprising the following steps: S1, performing genome alignment analysis on corn bacterial wilt PSS and its approximate species to obtain active target gene sequences; S2, designing specific primers according to the active target gene sequences, performing specific amplification of real-time fluorescence RT-PCR, and obtaining candidate target genes; S3, evaluating the stability of the candidate target genes after heat treatment death to obtain active detection targets. The live RT-qPCR detection system of corn bacterial wilt established according to the screened mRNA target genes can solve the problem that dead bacteria and live bacteria cannot be distinguished in molecular detection, and provides technical support for the quarantine and prevention and control of corn bacterial wilt.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a method for identifying activity detection targets of maize bacterial wilt pathogens based on comparative genomics analysis. Background Technology

[0002] Bacterial wilt of maize is one of the most devastating bacterial diseases, caused by the fungus *Pantoea stewartii* subsp. *stewartii* (Pss). It is a vascular disease, causing stunted or wilted plants and resulting in significant economic losses, particularly to sweet maize production. Bacterial wilt primarily spreads over long distances through infected seeds. For major maize-producing and consuming countries that import large quantities of maize, the introduction of this pathogen would inevitably lead to substantial economic losses. Therefore, establishing efficient and sensitive detection methods for *Pantoea stewartii* is crucial for early diagnosis and the development of effective control measures.

[0003] Currently, most methods for detecting bacterial wilt pathogens in maize, both domestically and internationally, employ molecular biology techniques such as conventional PCR, nested PCR, and real-time quantitative PCR. However, these methods are all based on genomic DNA as the target and cannot distinguish between live and dead bacteria of the bacterial wilt pathogen, which can easily lead to false positives. Therefore, there is an urgent need to establish a rapid method for detecting live bacteria of the bacterial wilt pathogen in maize samples.

[0004] In recent years, RT-PCR technology with RNA as the detection target has shown promising application prospects in the field of viable bacterial detection of medical diseases and food microorganisms, but no reports have been found on viable bacterial detection methods for plant pathogenic bacteria based on RNA as the target. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting the activity targets of maize bacterial wilt pathogens based on comparative genomics analysis. By comparing and analyzing the genomes of maize bacterial wilt and its similar species, differentially expressed genes specific to the species are screened, and their signaling pathways and functions are predicted and analyzed. Suitable mRNA target genes are screened, and the linear relationship between the loss of pathogen activity and the degradation time of mRNA after heat treatment is evaluated. A live bacteria RT-qPCR detection system for maize bacterial wilt is established to solve the problem of not being able to distinguish between dead and live bacteria in molecular detection, and to provide technical support for the quarantine and control of maize bacterial wilt.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a method for identifying activity detection targets of *Fusarium wilt* fungus in maize based on comparative genomics analysis, comprising the following steps:

[0008] S1. Genomic alignment analysis of maize bacterial wilt PSS and its similar species to obtain active target gene sequences;

[0009] S2. Based on the active target gene sequence, design specific primers and perform real-time fluorescent RT-PCR for specific amplification to obtain candidate target genes;

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

[0011] Preferably, the method of comparison analysis specifically involves performing orthologous clustering analysis on the whole genome protein sequences of PSS strain and its closely related strains, and then further screening based on GO functional annotation results to obtain active target gene sequences.

[0012] Preferably, the activity detection target is atpB, rpoE, MET, and IDH1.

[0013] The present invention also provides primers for TaqMan real-time quantitative RT-PCR detection of live bacteria of maize bacterial wilt fungus, wherein the primers are any one of the following primer pairs: atpB-1 for detecting the target atpB, rpoE-2 for detecting the target rpoE, MET-3 for detecting the target MET, and IDH1-4 for detecting the 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 for 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 real-time quantitative RT-PCR detection of live bacterium wilt of maize. The nucleotide sequence of the probe used with the above primer pair atpB-1 is shown as EQ ID NO: 3; the nucleotide sequence of the probe used with the above primer pair rpoE-2 is shown as EQ ID NO: 6; the nucleotide sequence of the probe used with the above primer pair MET-3 is shown as EQ ID NO: 9; and the nucleotide sequence of the probe used with the above primer pair IDH1-4 is shown as EQ ID NO: 12.

[0019] The present invention also provides a kit for TaqMan quantitative RT-PCR detection of live bacterium wilt of maize, comprising at least the primers and probes described above for TaqMan quantitative RT-PCR detection of live bacterium wilt of maize.

[0020] The present invention also provides a method for detecting live *Fusarium wiltii* bacteria of maize using the above-mentioned primers, probes, or kits via TaqMan real-time RT-PCR. The reaction system for the real-time RT-PCR detection consists of the following components: 12.5 μL of One Step PrimeScript III RT-qPCR Mix, 0.6 μL of 10 μmol / L upstream primer, 0.6 μL of 10 μmol / L downstream primer, 0.4 μL of 10 μmol / L probe, 2.0 μL of RNA template, and RNase-free ddH2O to a final volume of 25 μL.

[0021] Preferably, the reaction procedure for the real-time RT-PCR detection is as follows: reverse transcription at 42℃ for 5 min; pre-denaturation at 95℃ for 10 s; denaturation at 95℃ for 5 s; annealing at 60℃ for 60 s; 40 cycles.

[0022] This invention also provides the application of the primers, probes, kits, reaction systems, and / or reaction procedures of the above-described method for detecting live *Fusarium wiltii* bacteria in maize in the detection of live *Fusarium wiltii* bacteria.

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

[0024] This invention, through genome comparison analysis of maize bacterial wilt and its similar species, can screen out suitable mRNA target genes. Furthermore, the live bacteria RT-qPCR detection system for maize bacterial wilt established based on the screened mRNA target genes can solve the problem of not being able to distinguish between dead and live bacteria in molecular detection, providing technical support for the quarantine and control of maize bacterial wilt. Attached Figure Description

[0025] Figure 1 This is the result of comparative genomic PSS-specific gene analysis in this invention;

[0026] Figure 2 This is a graph showing the screening results of PSS-specific primers in this invention;

[0027] Figure 3 This is an electrophoresis diagram of the stability assessment of the target gene after lethal treatment in this invention;

[0028] Figure 4 The results of PSS amplification detected by the four sets of primers and probes in this invention are as follows (S1 is atpB; S2 is rpoE; S3 is MET; S4 is IDH1).

[0029] Figure 5 This invention analyzes the sensitivity of the S1 group primers and probes in the TaqMan real-time fluorescent RT-PCR method (1-7 represent concentrations of 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 bacterial wilt pathogens in maize). Detailed Implementation

[0030] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0031] Example:

[0032] I. Identifying key metabolically active genes of *Fusarium wilt*, the pathogen of maize bacterial wilt, and obtaining the sequences of active target genes.

[0033] The genomic information of *Pantoea stewartii* subsp. *stewarti* (PSS) and its four closely related species, *Pantoea stewartii* subsp. *indologenes* (Psi), *Pantoea agglomerans*, *Pantoea allii-7501-4*, and *Pantoea ananatis*, was downloaded from the NCBI database. Orthologous clustering analysis was performed on the whole genome protein sequences of the PSS strain and the four closely related strains using OrthoVenn 2 software. The results showed a total of 4510 gene clusters, including 1742 orthologous gene clusters (containing at least two strains) and 2768 single-copy gene clusters. Figure 1 It can be seen that strain PSS has 120 unique gene clusters, totaling 673 genes in the genome. Among them, 103 are 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 its GO functional annotation results revealed 25 genes related to stress resistance, transcription, or metabolic activity, mainly including RNA polymerase sigma factor (RpoE), acetoacetyl-CoA synthase, and transcriptional regulators (see Table 1).

[0034] II. Screening for active target genes of maize bacterial wilt pathogen to obtain candidate activity detection targets.

[0035] 1 Primer Design

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

[0037] Table 1. Active target gene sequence information

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] 2 Primer screening

[0045] Take PSS colonies that have been cultured at 28℃ for 3–5 days, transfer them to NA liquid medium (nutrient agar medium), and incubate for 12–24 hours. Take a sample with a concentration of approximately 10... -6 RNA extracted from CUF / mL bacterial culture was used as a template for real-time fluorescent RT-PCR amplification to evaluate the amplification effect of PSS primers. The real-time fluorescent RT-PCR results showed that of the 39 primer pairs designed above, 15 pairs could specifically amplify PSS, with amplification curves of a standard "S" shape and melting curves showing only a single peak. The remaining 14 primer pairs either failed to amplify PSS or showed non-specific amplification (see...). Figure 2 Therefore, 15 pairs of PSS-specific primers were selected for subsequent activity detection and target gene validation.

[0046] 3. Stability assessment of target genes after heat treatment-induced death

[0047] The bacterial suspensions were subjected to dry heat treatment at 70℃ for 1 hour, then streaked on NA plates and incubated at 28℃ for 3–5 days. No colony growth was observed, indicating that the pathogens were killed and inactivated after heat treatment. To assess the stability of 15 candidate target genes and 16S rRNA and 23S rRNA genes, RT-PCR was performed on the inactivated bacterial suspensions, as shown in Table 2. Figure 3 .

[0048] Table 2. Stability assessment results of target genes after heat treatment lethality.

[0049]

[0050]

[0051] Table 2 and Figure 3 The results showed that four 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 activity detection targets.

[0052] III. Establishment of TaqMan Real-Time RT-PCR Detection Method

[0053] 1. Establishment of a TaqMan real-time quantitative RT-PCR method for detecting bacterial wilt pathogens in maize

[0054] We designed specific probes that work with the primers of the four target genes screened above and established a TaqMan real-time RT-PCR detection method.

[0055] The instrument used for the reaction was a Thermo QuantStudio 6.0 real-time PCR instrument (Thermo Fisher Scientific).

[0056] The reaction system consisted of: 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 RNase-free ddH2O to a final volume of 25 μL.

[0057] The reaction procedure was as follows: reverse transcription at 42℃ for 5 min; pre-denaturation at 95℃ for 10 s; denaturation at 95℃ for 5 s; annealing at 60℃ for 60 s (collecting fluorescence signal); 40 cycles.

[0058] The detection efficiency of four sets of primers and probes for the four target genes was evaluated using TaqMan quantitative RT-PCR. Primers and probes in groups S1, S2, and S3 produced the fastest fluorescence signals, reaching a plateau around 32 cycles. Primers and probes in group S4 produced fluorescence signals after 26 cycles and reached a plateau around 40 cycles; however, the fluorescence signal in group S4 at the plateau was significantly lower than the other three groups, indicating that the amplification efficiency of primers and probes in group S4 was lower than the other three groups (see TaqMan quantitative RT-PCR). Figure 4 Based on the fluorescence absorption values ​​of the fluorescence signals generated by the primers and probes of groups S1, S2, and S3, primers and probes of group S1 (i.e., with atpB as the target) were selected for subsequent experiments.

[0059] 3. Specificity analysis

[0060] The specificity of primers and probes in group S1 was evaluated, and the real-time fluorescent RT-PCR reaction established using primers and probes in group S1 was used to perform specificity analysis on Pantoea stewartii subsp. stewwartii (PSS) and its similar species.

[0061] Table 3. Specificity analysis results 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, with no amplification signal for its similar species, and have good specificity for PSS.

[0065] 4. Sensitivity Analysis

[0066] Sensitivity evaluation of S1 group primers and probes was performed at 10 8 RNA was extracted from Pantoea stewartii subsp. stewartii (PSS) at CFU / mL serially diluted 10-fold to be used as a template for real-time fluorescent RT-PCR.

[0067] The result is Figure 5 It can be seen that the detection sensitivity of real-time fluorescent RT-PCR established using primers and probes in group S1 is 10. 3 CFU / mL.

[0068] 5. Actual Sample Testing and Analysis

[0069] Using the TaqMan real-time fluorescent RT-PCR method established in this study, PSS activity was detected in 15 samples, including 5 seeds with PPS bacteria, 5 samples of inactivated seeds with PPS bacteria, and 5 healthy seeds. The results are shown in Table 4.

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

[0071]

[0072]

[0073] Table 4 shows that among the 15 tested samples, the Ct values ​​for RT-qPCR were 25.0–30.0, consistent with the qPCR results and plate isolation results. Five samples of infected seeds tested positive for bacteria, while the five inactivated seeds and healthy seeds tested negative. The RT-qPCR Ct values ​​for these samples were greater than 38.0 or showed no amplification signal, while the qPCR Ct values ​​were 26.0–31.0. This indicates that qPCR testing using DNA as a template in inactivated infected seeds can produce false positives, while the RT-qPCR method established in this study can effectively distinguish between dead and live bacteria. These results demonstrate that this method is suitable for detecting dead and live PSS in seed samples.

[0074] In summary, this invention, through genome comparison analysis of maize bacterial wilt and its similar species, can screen out suitable mRNA target genes. Furthermore, the live bacteria RT-qPCR detection system for maize bacterial wilt established based on the screened mRNA target genes can solve the problem of not being able to distinguish between dead and live bacteria in molecular detection, providing technical support for the quarantine and control of maize bacterial wilt.

[0075] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Primers and probes for TaqMan real-time quantitative RT-PCR detection of live *Fusarium wilt* var. *maize*, characterized in that, The primers are any one of the following: primer pair atpB-1 for detecting target atpB, primer pair rpoE-2 for detecting target rpoE, and primer pair MET-3 for detecting target MET. 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; 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; The primer pair for 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; The nucleotide sequence of the probe used with primer pair atpB-1 is shown in EQ ID NO: 3; the nucleotide sequence of the probe used with primer pair rpoE-2 is shown in EQ ID NO: 6; and the nucleotide sequence of the probe used with primer pair MET-3 is shown in EQ ID NO:

9.

2. A kit for the detection of live bacteria of *Fusarium wilt* in maize using TaqMan real-time quantitative RT-PCR, characterized in that... It includes at least the primers and probes for TaqMan real-time quantitative RT-PCR detection of live bacterium wilt of maize as described in claim 1.

3. A method for detecting live *Fusarium wiltii* bacteria of maize using the primers and probes of claim 1 or the kit of claim 2, characterized in that... The reaction system for the quantitative RT-PCR detection consists of the following components: 12.5 μL of One Step PrimeScript III RT-qPCR Mix, 0.6 μL of 10 μmol / L upstream primer, 0.6 μL of 10 μmol / L downstream primer, 0.4 μL of 10 μmol / L probe, 2.0 μL of RNA template, and RNase-free ddH2O to a final volume of 25 μL.

4. The method for detecting live bacteria of *Fusarium wilt* in maize using TaqMan real-time quantitative RT-PCR according to claim 3, characterized in that... The reaction procedure for the quantitative RT-PCR detection was as follows: reverse transcription at 42℃ for 5 min; pre-denaturation at 95℃ for 10 s; denaturation at 95℃ for 5 s; annealing at 60℃ for 60 s; 40 cycles.

5. The application of the primers and probes of claim 1 or the kit of claim 2 in the detection of live bacteria of Fusarium wilt in maize.

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