A method for detecting Shigella and its application

Abstract

The present invention discloses a method for detecting Shigella and its application. The method includes: identifying a sequence rich in AT bases in the Shigella genomic sequence as a target sequence; automatically and high-throughput designing specific primers for the target sequence; designing a specific Tm value calculation formula to calculate the reaction denaturation and annealing temperatures, and setting nucleic acid amplification reaction conditions accordingly; and performing nucleic acid amplification reaction under the condition of local strand separation of the template. The present invention targets sequences rich in AT bases that are usually avoided in traditional nucleic acid amplification methods, greatly broadening the candidate target regions; through specific screening based on a large amount of genomic data and precise calculation of the Tm value for AT-rich regions, local strand separation of double-stranded DNA is achieved, greatly reducing the possibility of non-specific amplification in nucleic acid amplification technology. This method effectively expands the application scope of traditional nucleic acid amplification technology and significantly improves the reaction performance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for detecting Shigella and an application thereof. The method is a method for performing nucleic acid amplification specifically on the genome sequence of Shigella. Background Art

[0002] Nucleic acid amplification technology (NAAT) is a general term for a class of molecular biological techniques. This type of technology achieves rapid and specific amplification of trace nucleic acids through the reaction of primers, DNA polymerase and other reagents at a specific temperature. It can be widely used in disease diagnosis, pathogen detection, food safety detection, animal and plant quarantine, and various applications involving molecular cloning, such as sequencing, gene cloning, gene manipulation, allele analysis, mutation detection, etc. Among them, polymerase chain reaction (PCR) is the earliest nucleic acid amplification technology to be implemented. It imitates the process of DNA replication in the body, through a pair of specific oligonucleotide primers complementary to the DNA fragment to be amplified, and then through several cycles of denaturation, annealing, and extension, it achieves exponential amplification of DNA fragments. PCR technology was invented by Mullis of Cetus in 1985. Subsequently, Saiki et al. introduced thermostable DNA polymerase into the PCR reaction system, which solved the shortcoming of early PCR technology that DNA polymerase was passivated during thermal denaturation cycles, resulting in the need for continuous manual addition of polymerase. This greatly improved the efficiency of nucleic acid amplification and enabled the technology to be automated. Since then, nucleic acid amplification technology represented by PCR has been widely promoted and applied, and has also greatly promoted the development of molecular biology.

[0003] As is known to all, existing nucleic acid amplification techniques usually avoid GC or AT-rich regions because these regions are prone to form hairpin loop secondary structures through self-complementary pairing, thereby hindering the binding of primers to templates; even if the primers and templates can barely bind, they are likely to affect the extension of the DNA polymerase along the template chain, causing the DNA polymerase to "stuck" when amplifying along the template and interfering with DNA synthesis. Therefore, in order to improve the success rate of the reaction, traditional nucleic acid amplification techniques have a selection preference for the base composition of the amplification region, and their primer design is often concentrated in the region with a GC content of 40% to 60%, with 45% to 55% being the most suitable. Different amplification techniques are slightly different, such as the recommended G+C content for PCR primer design is 40% to 60% (see Molecular Cloning Experiment Guide 4th Edition, Table 7-1 Primer Design), and the recommended G+C content for LAMP primer design is 40% to 65% (see A Guide to LAMP primer designing (PrimerExplorer V3)).

[0004] However, the nucleic acid base compositions of organisms in nature vary greatly. For example, the AT base content in the genome of Plasmodium is approximately 82%. Zhou Huiqi et al. studied 2,670 strains of bacteria and archaea and found that the GC content in their genomes ranged from 14% to 75% (Zhou Huiqi, 2014). From the perspective of detection, the base composition selection preference of existing nucleic acid amplification technologies ignores a large number of potential target sequences, increasing the difficulty of successfully detecting the target object; from the perspective of gene manipulation, this selection preference makes it difficult to amplify a considerable number of target sequences, thus making it difficult to carry out further molecular operations. If appropriate primers can be designed for the unbalanced regions of GC base content and AT base content that are avoided by traditional nucleic acid amplification methods, to avoid the formation of secondary structures, and at the same time set appropriate reaction conditions to successfully carry out nucleic acid amplification experiments, it will significantly increase the success rate of detecting the target object, and at the same time enable more sequences to be amplified and thus facilitate the implementation of downstream molecular operations.

[0005] For nucleic acid amplification reactions carried out for the unbalanced regions of GC base content and AT base content, the key step is the primer design. In view of the above analysis, the species-specific genes (or sequences) publicly released in the field usually come from regions with a GC content of 40% - 60% or even 45% - 55%, so they cannot be applied to the primer design for the unbalanced regions of GC and AT base content. In addition, the continuous GC-rich or AT-rich regions in such sequences have relatively weak base composition diversity compared to other regions, which brings additional difficulties to the design of specific primers. Therefore, it is first necessary to propose a set of primer design algorithm processes that can be high-throughput, automated, and efficient for unbalanced sequences of GC and AT base content.

[0006] In addition to the selection of the amplification region and primer design, the calculation of the Tm values of the amplification region and primers is also an important influencing factor for the successful completion of the amplification process. The Tm values of the amplification region and primers are usually calculated according to the nearest neighbor two-state model, but the specific calculation formulas used by different researchers and primer production companies vary. For example, the primer Tm calculation formula recommended in the fourth edition of the Molecular Cloning Experiment Guide is Tm = 4×(number of G, C) + 2×(number of A, T), and the primer Tm calculation formula recommended by TaKaRa is Tm = 4×(number of G, C) + 2×(number of A, T) + 32 - 2×(total number of bases); the primer Tm calculation formula of Sangon Biotech is Tm(0.05M Na + ) = 59.94 + 1×(percentage of GC) - (675 / primer sequence length). For the unbalanced regions of GC and AT base content, how to design a model for calculating the Tm values of primers has become an important factor to ensure the success of the experiment.

[0007] In summary, there is an urgent need in the field to develop a nucleic acid amplification technology for regions that are difficult to cover by traditional nucleic acid amplification technologies, which can design specific primers in a high-throughput and automated manner, calculate their Tm values, and set appropriate reaction conditions such as denaturation temperature and annealing temperature, expand the application scope of nucleic acid amplification technology, improve the specificity of amplification, and meet the requirements of nucleic acid detection, molecular genetics research and other aspects. Summary of the Invention

[0008] The present invention proposes an innovative nucleic acid amplification method and applies it to the detection of Shigella. This method uses an AT-rich nucleic acid sequence as the amplification target and performs target sequence recognition, primer design, denaturation temperature and annealing temperature calculation, nucleic acid amplification with local unwinding of the template, etc. First, an automated primer design process is adopted, making full use of the rich genomic sequence information in public data resources to design specific primer pairs for AT-rich target sequences in a high-throughput manner. Secondly, a Tm value calculation formula that conforms to the characteristics of AT-rich sequences is fitted, and the denaturation temperature and annealing temperature can be calculated according to factors such as the theoretical amplification product sequence, primer sequence length, and base composition; considering that the denaturation temperature of AT-rich nucleic acid sequences is significantly lower than that of sequences with a GC content of 40%-60% and high-GC sequences, the Tm value of the calculated theoretical amplification product sequence is used as the lowest denaturation temperature. At this temperature, without adding any chemical denaturant, local unwinding of the AT-rich region of double-stranded DNA can be carried out, thereby initiating the nucleic acid amplification reaction.

[0009] On the one hand, the present invention introduces a large amount of genomic data in public data resources in the primer design step to perform sequence specificity screening; on the other hand, different from the traditional nucleic acid amplification method that opens all double-stranded structures at a denaturation temperature of 93-95°C, the denaturation temperature is set by only opening the AT-rich sequences, thereby greatly reducing the possibility of non-specific amplification in non-target regions at the source. These two design strategies significantly reduce the possibility of non-specific amplification commonly seen in nucleic acid amplification reactions, and on this basis, a nucleic acid amplification method for AT-rich sequences is formed. The primer design step of this method is implemented by programming in C language and Perl language, with the characteristics of high-throughput and automation. The entire amplification method has strong specificity and high success rate, effectively expanding the application scope of traditional nucleic acid amplification technology and significantly improving the reaction performance.

[0010] In the present invention, the nucleic acid amplification method includes identifying an AT-rich sequence in the nucleic acid sequence to be amplified as the target sequence; automatically and high-throughput designing specific primers for the target sequence; designing a specific Tm value calculation formula to calculate the reaction denaturation and annealing temperatures, and setting nucleic acid amplification reaction conditions accordingly; and performing nucleic acid amplification reaction under the condition of local unwinding of the template.

[0011] Specifically, it includes the following steps:

[0012] (1) Screen the target sequence rich in AT bases in the nucleic acid sequence to be amplified;

[0013] (2) Automatically and high-throughput design primers with both universality and specificity for the target sequence;

[0014] (3) Calculate the reaction denaturation temperature and annealing temperature through a specific formula, and set the nucleic acid amplification reaction conditions; that is, calculate the reaction denaturation temperature and annealing temperature based on the GC percentage content, primer sequence length, and theoretical amplification product sequence length of the primer pair, and set the nucleic acid amplification reaction conditions;

[0015] (4) Conduct a nucleic acid amplification reaction under the condition of local template denaturation to obtain an amplification product.

[0016] In the present invention, the method for detecting Shigella genus specifically comprises the following steps:

[0017] (1) Screen the target sequence rich in AT bases in the genomic sequence of Shigella genus;

[0018] (2) Design primers with both universality and specificity for the target sequence;

[0019] (3) Calculate the reaction denaturation temperature and annealing temperature based on the GC percentage content, primer sequence length, and theoretical amplification product sequence length of the primer pair, and set the nucleic acid amplification reaction conditions;

[0020] (4) Conduct a nucleic acid amplification reaction under the condition of local template denaturation to obtain an amplification product.

[0021] In step (1) of the present invention, for the nucleic acid sequence to be amplified, a window with a width of 1000 bp is slid starting from the first base, and the step size is 5 - 100 bp. Calculate the AT base content for the sequence contained in each window position, and retain the region where the AT base content of the sequence is greater than 60% as the target sequence. Preferably, retain the region where the AT base content of the sequence is 60 - 80% as the target sequence.

[0022] In step (1) of the present invention, for the genomic sequence of Shigella genus, a window with a width of 1000 bp is slid starting from the first base, and the step size is 5 - 100 bp. Calculate the AT base content for the sequence contained in each window position, and retain the region where the AT base content of the sequence is greater than 60% as the target sequence. Preferably, retain the region where the AT base content of the sequence is 60 - 80% as the target sequence.

[0023] In step (2) of the present invention, the method for designing the primer includes: (2.1) designing a single primer for a target sequence to obtain candidate primers; (2.2) determining the physicochemical properties of the candidate primers, and screening out single primers that meet the conditions; (2.3) combining the single primers screened in step (2.2) into primer pairs; (2.4) determining the universality and specificity of the primer pairs; (2.5) outputting the primer pairs that meet the conditions to obtain the specific primers.

[0024] In the present invention, any programming language capable of implementing high-throughput primer design can be used, such as programming languages like C and Perl with strong operability and relatively fast speed.

[0025] In step (2.1), when designing candidate primers for the target sequence, the candidate primers need to meet the following conditions: a) the primer sequence length is between 20bp and 36bp; b) the AT base content is between 55% and 80%; c) the number of consecutive GC bases ≤ 5; at the same time, record the position information and positive / negative strand information of the candidate primers matching to the target sequence.

[0026] Preferably, the AT base content is 60% - 75%.

[0027] The "number of consecutive GC bases" refers to the number of consecutive bases G or consecutive bases C in the primer sequence. For example, in the primer sequence AAGGGGGTTCCAGGCATTA (SEQ ID NO: 17), the number of consecutive GC bases is 5 / 2 / 2.

[0028] In step (2.2), determine the physicochemical properties of the single primers that meet the above conditions in (2.1), including but not limited to physicochemical properties such as 3'-end stability, 5'-end stability, and / or secondary structure stability, and retain the single primers that meet the requirements.

[0029] The "single primer that meets the requirements" refers to a single primer with 3'-end stability, 5'-end stability, and secondary structure stability.

[0030] In step (2.3), for each single primer screened in the above step (2.2), according to its position information and positive / negative strand information on the target sequence, the single primers are combined into primer pairs; the length of the theoretical amplification product sequence of the primer pairs should be between 200 bp and 600 bp. Calculate the Tm value of each single primer according to the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length); with the condition that the Tm difference of the primer pairs ≤ 3°C and no interaction occurs between the primers, screen the primer pairs to obtain candidate primer pairs. Among them, the percentage of GC content refers to the percentage of the number of bases G and C in the primer accounting for the total number of bases in the primer; the primer sequence length refers to the number of bases in the primer.

[0031] In step (2.4), perform universality and specificity determination on each primer in the candidate primer pairs obtained in step (3). Among them, universality determination refers to checking whether the primer pairs strictly match all target sequences (such as the genomes of multiple strains of Shigella).

[0032] Specificity determination refers to checking whether each single primer cannot specifically match non-target sequences other than all target sequences, where the non-target sequences refer to other nucleic acid sequences other than the nucleic acid sequence to be amplified, and the specific match refers to a match with no more than 2 mismatches.

[0033] The candidate primers that pass the universality and specificity determination can enter the next step.

[0034] In step (2.5), output the primer pairs that meet the conditions, that is, obtain the primer pairs for the nucleic acid amplification reaction.

[0035] In step (3) of the present invention, the calculation formula for the reaction denaturation temperature is 0.357×(percentage of GC content)×100 + 70.582 - (990 / the length of the theoretical amplification product sequence of the primer pair), denoted as Tma; among them, the percentage of GC content refers to the percentage of the number of bases G and C in the theoretical amplification product sequence of the primer pair accounting for the total number of bases in the theoretical amplification product sequence of the primer pair.

[0036] In step (3) of the present invention, the reaction annealing temperature is the average value of the Tm values of the two primers in the above step (2.3), denoted as Tmb. That is, calculate the Tm value of each single primer according to the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length), and take the average value of the Tm values as the reaction annealing temperature, denoted as Tmb; among them, the percentage of GC content refers to the percentage of the number of bases G and C in the primer accounting for the total number of bases in the primer.

[0037] In step (3) of the present invention, the nucleic acid amplification reaction conditions include: reacting for 5 seconds at the denaturation temperature, reacting for 5 seconds at the annealing temperature, and reacting for 20 seconds at the extension temperature, and the above process is repeated 30 - 40 times. Among them, the denaturation temperature can be adjusted between Tma ± 5°C as needed; the annealing temperature can be adjusted between Tmb ± 2°C as needed; the extension temperature is 72°C.

[0038] The present invention also provides the application of the above method in nucleic acid amplification of a target nucleic acid sequence rich in AT regions.

[0039] The present invention also provides a primer pair obtained by the above design method.

[0040] In the present invention, the primer pair is:

[0041] Primer pair H:

[0042] Shi-F: 5’-CGGCAGAACGTTCAAATATGA-3’ (SEQ ID NO: 9)

[0043] Shi-R: 5’-CGCAGAAGGATATCCTGATA-3’ (SEQ ID NO: 10).

[0044] The present invention also provides the application of the above method in simultaneously detecting any two or more combinations of Staphylococcus aureus, Cronobacter sakazakii, Salmonella, and Yersinia enterocolitica.

[0045] The present invention provides a reagent for diagnosing Shigella and its application in detecting Shigella, and the diagnostic reagent includes the primer pair as described above.

[0046] The present invention also provides a diagnostic reagent and its application in detecting Shigella, and the diagnostic reagent includes the primer pair as described above.

[0047] The present invention also provides a system for detecting Shigella, and the system includes:

[0048] A screening module for screening a target sequence rich in AT bases in the Shigella genomic sequence;

[0049] A primer design module for designing primers with both universality and specificity for the target sequence;

[0050] A calculation module for calculating the reaction denaturation temperature and annealing temperature based on the GC percentage content, primer sequence length, and theoretical amplification product sequence length of the primer pair, and setting the nucleic acid amplification reaction conditions;

[0051] An amplification module for performing a nucleic acid amplification reaction under conditions of local denaturation of a template to obtain an amplification product.

[0052] The present invention also provides a device for detecting Shigella, comprising: a memory, a processor, and an amplification module; a computer program is stored on the memory, and when the computer program is executed by the processor, steps 1-3 of the above-described method are implemented; the amplification module is used for performing a nucleic acid amplification reaction under conditions of local denaturation of a template to obtain an amplification product.

[0053] The beneficial effects of the present invention are as follows: The present invention targets sequences rich in AT bases that are usually avoided in traditional nucleic acid amplification methods, greatly expanding the candidate target regions; through specific screening based on a large amount of genomic data and precise calculation of the Tm value for regions rich in AT, local denaturation of double-stranded DNA is achieved, greatly reducing the possibility of non-specific amplification commonly present in nucleic acid amplification techniques. This method effectively expands the application scope of traditional nucleic acid amplification techniques, and at the same time significantly improves the reaction performance in terms of primer design throughput, reaction specificity, etc., and can meet the requirements of nucleic acid detection, molecular genetics research, and other aspects. Description of the Drawings

[0054] Figure 1 It is the amplification curve of real-time fluorescence quantitative PCR of the nucleic acid amplification reaction of primer pair H for Shigella Castellani based on the present invention at a suitable temperature.

[0055] Figure 2 It is the melting curve of real-time fluorescence quantitative PCR of the nucleic acid amplification reaction of primer pair H for Shigella Castellani based on the present invention at a suitable temperature.

[0056] Figure 3 It is the agarose gel electrophoresis of the nucleic acid amplification reaction of primer pair H for Shigella Castellani based on the present invention at a suitable temperature.

[0057] Figure 4 shows the sensitivity of real-time fluorescence quantitative PCR of the nucleic acid amplification reaction of primer pair H for Shigella Castellani based on the present invention using Shigella dysenteriae as a template at 81°C ( Figure 4A ) and 82°C ( Figure 4B ).

[0058] Figure 5 shows the nucleic acid amplification reaction of primer pair H for Shigella Castellani based on the present invention using Shigella flexneri as a template at 81°C ( Figure 5A ) and 82°C ( Figure 5B) Sensitivity of modified real-time fluorescence quantitative PCR.

[0059] Figure 6 shows the sensitivity of real-time fluorescence quantitative PCR for nucleic acid amplification reaction of primer pair H of Shigella Castellani based on the present invention with Shigella sonnei as the template at 81 °C ( Figure 6A ) and 82 °C ( Figure 6B ) for denaturation.

[0060] Figure 7 shows the sensitivity staining results of nucleic acid amplification reaction of primer pair H of Shigella Castellani based on the present invention at 81 °C ( Figure 7A ) and 82 °C ( Figure 7B ) for denaturation.

[0061] Figure 8 shows the sensitivity gel electrophoresis results of nucleic acid amplification reaction of primer pair H of Shigella Castellani based on the present invention at 81 °C ( Figure 8A ) and 82 °C ( Figure 8B ) for denaturation.

[0062] Figure 9 The staining result is for the detection of nucleic acid amplification specificity of primer pair H of Shigella Castellani based on the present invention at a denaturation temperature of 82 °C.

[0063] Figure 10 The dye color development result is for the detection of nucleic acid amplification specificity of Cronobacter sakazakii based on the present invention at different denaturation temperatures.

[0064] Figure 11 The electrophoresis result of the amplification product is for the detection of nucleic acid amplification specificity of Cronobacter sakazakii based on the present invention at a denaturation temperature of 94 °C.

[0065] Figure 12 The electrophoresis result of the amplification product is for the detection of nucleic acid amplification specificity of Cronobacter sakazakii based on the present invention at a denaturation temperature of 82 °C.

[0066] Figure 13 The fluorescence dye staining result is for the nucleic acid amplification reaction of four bacteria, namely Salmonella enterica subsp., Staphylococcus aureus subsp., Yersinia enterocolitica, and Cronobacter sakazakii, according to the present invention.

[0067] Figure 14This is the agarose gel electrophoresis result of the nucleic acid amplification reaction for four bacteria: Salmonella enterica subsp. enterica, Staphylococcus aureus subsp. aureus, Yersinia enterocolitica, and Cronobacter sakazakii, according to the present invention.

[0068] Figure 15 This is the SYBR Green I dye color development result of the specific detection of the nucleic acid amplification reaction for four bacteria: Salmonella enterica subsp. enterica, Staphylococcus aureus subsp. aureus, Yersinia enterocolitica, and Cronobacter sakazakii, according to the present invention, at a denaturation temperature of 81°C.

[0069] Figure 16 This is the agarose gel electrophoresis result of the nucleic acid amplification reaction for two bacteria: Salmonella enterica subsp. enterica and Staphylococcus aureus subsp. aureus, according to the present invention.

[0070] Figure 17 This is the agarose gel electrophoresis result of the nucleic acid amplification reaction for two bacteria: Salmonella enterica subsp. enterica and Yersinia enterocolitica, according to the present invention.

[0071] Figure 18 This is the agarose gel electrophoresis result of the nucleic acid amplification reaction for three bacteria: Salmonella enterica subsp. enterica, Staphylococcus aureus subsp. aureus, and Yersinia enterocolitica, according to the present invention. Detailed implementation manners

[0072] In combination with the following specific embodiments and the accompanying drawings, the present invention will be further described in detail. The processes, conditions, experimental methods, etc. for implementing the present invention are all common knowledge and well-known common sense in the art, except for the specifically mentioned content below. The present invention has no particularly restricted content.

[0073] The present invention also provides a system for detecting Shigella, the system comprising:

[0074] A screening module for screening a target sequence rich in AT bases in the Shigella genomic sequence;

[0075] A primer design module for designing primers with both universality and specificity for the target sequence;

[0076] A calculation module for calculating the reaction denaturation temperature and annealing temperature based on the GC percentage content, primer sequence length, and theoretical amplified product sequence length of the primer pair, and setting the nucleic acid amplification reaction conditions;

[0077] An amplification module for performing a nucleic acid amplification reaction under the condition of local denaturation of the template to obtain an amplified product.

[0078] Example 1 Detection of Shigella Castellani

[0079] The present invention screens for AT-rich sequences in the genome of Shigella Castellani and designs specific primers. Reaction conditions are set according to the calculated primer Tm and target sequence Tm for nucleic acid amplification. By determining whether the reaction result is positive, it is determined whether the target sequence exists in the sample to be tested, and further whether Shigella Castellani exists in the sample to be tested. The specific steps are as follows:

[0080] (1) Screening of target sequences rich in AT bases:

[0081] Using the data of bacteria, archaea, and viruses with complete genome sequences downloaded from the NCBI FTP on August 5, 2019, a total of 2,896 whole genome sequences. Set set A, which contains all Shigella Castellani genome sequences; set set B, which contains all non-Shigella Castellani genome sequences. Using the Shigella Castellani genome sequence as the reference genome, a window with a width of 1,000 bp is slid starting from the first base of the genome, with a step size of 50 bp; the AT base content of the sequence is calculated before each slide, and the region with an AT base content greater than 60% is retained as the candidate target sequence. The above process is implemented using a perl script.

[0082] (2) Design of specific primers:

[0083] According to the characteristics of PCR primers and the requirements of the present invention, set the characteristics parameters of the primers such as the AT base content of the primers being 55% - 75%, the 3'-end stability ΔG < 4, the 5'-end stability ΔG < 3, and the primer sequence length (20 - 36 bp). At the same time, set conditions such as that a single primer cannot form a hairpin structure and cannot interact with itself. Using the candidate target sequences in step (1) as the candidate sequences for single primer design, single primers that meet the above set conditions are calculated. Calculate the Tm value of a single primer through the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length), and record information such as the position of each primer on the target sequence, the positive and negative strand information (i.e., whether it is from the positive strand or the negative strand), and the length of the primer.

[0084] According to the position information of the single primers, primer pairs are paired, and the primer pairs that simultaneously meet the two conditions of the Tm difference between the primer pairs < 3°C and the amplification region of the primer pairs being 200 - 600 bp are retained as candidate primer pairs.

[0085] Using the alignment software Bowtie, each primer in the candidate primer pairs designed in the previous step was separately aligned with the target genomic sequences in set A and the non-target genomes in set B. To ensure the universality of the primers, when aligning a single primer with the target sequence in set A, the parameter settings "-a -n 0" were used, which requires the single primer to be completely matched with the target sequence; to ensure the specificity of the primers, when aligning a single primer with the non-target sequence in set B, the parameter settings "-a -n 3" were used, which allows the single primer to have no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, and the number of primer pairs can be preset. In this embodiment, the number of primer pairs was preset to 20. The above high-throughput and automated primer design process was implemented using C and Perl scripts.

[0086] After the program ran, 20 PCR reaction primer pairs were designed in the AT-rich region, and a primer group was randomly selected for validity verification. The sequences of the primer pairs are as follows:

[0087] Shi-F: 5'-CGGCAGAACGTTCAAATATGA-3' (SEQ ID NO.9)

[0088] Shi-R: 5'-CGCAGAAGGATATCCTGATA-3' (SEQ ID NO.10)

[0089] The AT base contents of the primers are 57.14% and 55% respectively, and the percentage content of AT in the theoretical amplification product sequence of the primer pair is 68.66%.

[0090] Calculation of the denaturation temperature and annealing temperature of the PCR reaction:

[0091] Using the formula 0.466×(percentage content of GC)×100 + 66.04 - (450 / primer sequence length), it can be calculated that the Tm of primer Shi-F is 64.58°C, and the Tm of Shi-R is 64.51°C. The average Tm value of this primer pair is 64.55°C; using the formula 0.357×(percentage content of GC)×100 + 70.582 - (990 / amplification product sequence length), the denaturation temperature of the amplification region is calculated to be 77.2°C.

[0092] (4) Nucleic acid amplification reaction and result detection:

[0093] The nucleic acid amplification reaction system is configured as shown in Table 1 below. According to the calculation results, a real-time fluorescence quantitative PCR instrument was used to determine the applicable denaturation temperature and melting curve; the specificity of the primers for different detection targets was tested at the applicable denaturation temperature; the detection limit at the applicable denaturation temperature was tested. Denaturation for 5 seconds, annealing at 63.5 °C for 5 seconds, and extension at 72 °C for 20 seconds. It is recommended that the above process be repeated 35 times. The list of specific detection targets is shown in Table 2.

[0094] After the reaction using an ordinary gradient PCR instrument is completed, the amplification results can be judged in two ways. One is to add SYBR Green I dye with a final concentration of 25x and judge whether the amplification result is positive by color, that is, whether the target sequence exists in the sample to be tested; the other is to perform agarose gel electrophoresis on the amplification product and judge whether the amplification result is positive according to the electrophoresis band, that is, whether the target sequence exists in the sample to be tested.

[0095] Table 1. Nucleic Acid Amplification Reaction System for Shigella Castellani

[0096] System Volume (μl) Final concentration <![CDATA[R300MIX(TaKaRaTaq TM HS Perfect Mix)]]> 12.5 / Shi-F / Shi-R (10 μM) 1.25+1.25 0.5 μM 100% DMSO 0.5 2％ DNA template 0 / 1 / ddH2O Up to 25 /

[0097] Table 2. List of Specific Detection Targets for Nucleic Acid Amplification Reaction of Shigella Castellani

[0098] 1. Staphylococcus aureus 21600 16. Shigella flexneri 1.1868 2. Staphylococcus aureus subsp. 1.2465 17. Escherichia coli 10738 3. Staphylococcus epidermidis 1.4260 18. Pathogenic Escherichia coli 10372 4. Rhodococcus equi 1.4262 19. Diarrheagenic Escherichia coli 10411 5. Bacillus cereus 1.3760 20. Enterotoxigenic Escherichia coli 10415 6. Bacillus mycoides 21473 21. Enterotoxigenic Escherichia coli 10665 7. Listeria monocytogenes 21635 22. Enterohemorrhagic Escherichia coli 21530 8. Listeria innocua 10417 23. Cronobacter sakazakii 21560 9. Listeria ivanovii 21663 24. Yersinia enterocolitica 21669 10. Salmonella enterica subsp. 1.1859 25. Yersinia pseudotuberculosis 53504 11. Salmonella enterica 21482 26. Vibrio vulnificus 21615 12. Salmonella typhimurium 10420 27. Vibrio parahaemolyticus 1.1997 13. Salmonella paratyphi B 10437 28. Vibrio furnissii 1.1613 14. Shigella dysenteriae 1.1869 29. Vibrio cholerae 1.8676 15. Enterobacter cloacae 10015 30. Shigella sonnei

[0099] The experimental results are shown in Figures 1-9 , where 1-13 are Staphylococcus aureus, Staphylococcus aureus subspecies, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Salmonella enterica subspecies, Salmonella enterica, Salmonella typhimurium, Salmonella paratyphi B, 15 is Enterobacter cloacae, 17-29 are Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, diarrheagenic Escherichia coli, enterotoxigenic Escherichia coli, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli, Cronobacter sakazakii, Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio fischeri, Vibrio cholerae; N: negative control; 14 and 16 are Shigella dysenteriae and Shigella flexneri respectively, and 30 is Shigella sonnei.

[0100] Figures 1-3The amplification curve, melting curve and agarose gel electrophoresis results of the real-time fluorescence quantitative PCR of the nucleic acid amplification reaction of the present invention against Shigella Castellani at a suitable temperature are shown. Among them, "81°C" and "82°C" refer to the amplification results of the positive template at this denaturation temperature, "NTC" or "N" is the amplification result of the corresponding negative template, and "M" refers to Marker DL2000. Figure 1 The amplification of the positive and negative templates of Shigella dysenteriae, Shigella flexneri and Shigella sonnei met the expectations. Figure 2 The melting curves of Shigella dysenteriae, Shigella flexneri and Shigella sonnei were all single peaks, without non-specific reactions. Figure 3 On both sides of "M" are the amplification electrophoresis identification results at denaturation temperatures of 80°C and 81°C respectively. If there is a single band at 217bp after electrophoresis of the amplification product, it is positive; if there is no band after electrophoresis of the amplification product, it is negative.

[0101] Figures 4-6, 7 and 8 show three detection limit detection methods of the nucleic acid amplification reaction of the present invention against Shigella Castellani at denaturation temperatures of 81°C and 82°C. In Figures 4-6 and Table 3, 10 ng to 10 fg were diluted 10-fold. In the range of 10 ng to 10 pg, for every 10-fold difference, the amplification curve differed by more than 3 cycles, and the linearity was worse below 10 pg. Therefore, above 10 pg, a standard curve can be made for quantitative determination. Figure 7 shows the sensitivity after adding SYBRGreen I dye at a final concentration of 25x. 10 ng to 10 fg were diluted 10-fold, and the staining results were judged as positive in the range of 10 ng to 100 pg. Figure 8 shows the agarose gel electrophoresis of the amplification product. There was a single band at 217bp in the range of 10 ng to 100 pg, which was judged as positive.

[0102] Table 3 Summary of the sensitivity of real-time fluorescence quantitative PCR in Figures 4-6

[0103]

[0104] Figure 9 The SYBRGreen I dye color development results of the specificity detection of the nucleic acid amplification reaction of the present invention against Shigella Castellani at a denaturation temperature of 82°C are shown. If the amplification product is bright green, it is positive; if the amplification product is orange, it is negative. Figure 9Among them, the negative control (N) showed orange, indicating a negative result, which was in line with expectations; 13 / 15 were Shigella dysenteriae and Shigella flexneri respectively, and 30 was Shigella sonnei, showing bright green, indicating a positive result, which was in line with expectations. At the same time, the detection results of other bacterial genomic DNA templates were all negative, as shown in tubes No. 1-13, 17-29. All results were in line with expectations, suggesting that no non-specific amplification occurred at a denaturation temperature of 82°C, or even if a small amount of non-specific amplification occurred, it was not sufficient to affect the result determination by the dye method.

[0105] This example perfectly verified that the method proposed in this invention can significantly reduce non-specific amplification by locally denaturing the template sequence by reducing the denaturation temperature.

[0106] Example 2 Detection of Cronobacter sakazakii

[0107] Change the Shigella genomic sequence targeted by the primer design of the present invention to the Cronobacter sakazakii genomic sequence, screen for AT-rich sequences and design specific primers, set the reaction conditions for nucleic acid amplification according to the calculated primer Tm and target sequence Tm, and determine whether the target sequence exists in the test sample by judging whether the reaction result is positive, and then determine whether Cronobacter sakazakii exists in the test sample. The specific steps are as follows:

[0108] (1) Screening of target sequences rich in AT bases:

[0109] Use the data of bacteria, archaea and viruses with complete genome sequences downloaded from the FTP of NCBI on August 5, 2019, a total of 2,896 whole genome sequences. Set set A, which contains all Cronobacter sakazakii genome sequences; set set B, which contains all non-Cronobacter sakazakii genome sequences. Take the Cronobacter sakazakii genome sequence with GI number 156932229 as the reference genome, slide a window with a width of 1000 bp from the first base of the genome, and the step size is 50 bp; calculate the AT base content of the sequence once before each slide, and retain the region with an AT base content greater than 60% of the sequence as the candidate target sequence. The above process is implemented using a perl script.

[0110] (2) Design of specific primers:

[0111] According to the characteristics of PCR primers and the requirements of the present invention, characteristic parameters such as the AT base content of the primers being 55% - 75%, the 3'-end stability ΔG < 4, the 5'-end stability ΔG < 3, and the primer sequence length (20 - 36 bp) are set. At the same time, conditions such as a single primer not generating a hairpin structure and not interacting with itself are set. Using the candidate target sequence in step (1) as the candidate sequence for single primer design, single primers that meet the above set conditions are calculated. The Tm value of a single primer is calculated by the formula 0.466×(percentage content of GC)×100 + 66.04 - (450 / primer sequence length), and information such as the position of each primer on the target sequence, the positive and negative strand information (i.e., whether it is from the positive strand or the negative strand), and the length of the primer is recorded.

[0112] According to the position information of single primers, primer pairs are paired, and primer pairs that simultaneously meet the two conditions of the Tm difference between primer pairs < 3°C and the amplification region of primer pairs being 200 - 600 bp are retained as candidate primer pairs.

[0113] Using the alignment software Bowtie, each primer in the candidate primer pairs designed in the previous step is respectively aligned with the target genomic sequence in set A and the non-target genome in set B. To ensure the universality of the primer, when this single primer is aligned with the target sequence in set A, the parameter setting "-a -n 0" is used, that is, it is required that this single primer be completely matched with the target sequence; to ensure the specificity of the primer, when this single primer is aligned with the non-target sequence in set B, the parameter setting "-a -n 3" is used, that is, it is allowed that this single primer has no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, and the number of primer pairs can be preset. In this embodiment, the number of primer pairs is preset to 20. The above high-throughput and automated primer design process is implemented using C and Perl scripts.

[0114] After the program runs, 20 PCR reaction primer pairs are designed in the AT-rich region, and a primer group is randomly selected for validity verification. The sequences of the primer pairs are:

[0115] Cro-F: 5'-CGCCATAACTGCATAATCAT-3' (SEQ ID NO: 3)

[0116] Cro-R: 5'-ATAACGAGTTACCGTGCAGA-3' (SEQ ID NO: 4)

[0117] The AT base contents of the primers are 60% and 55% respectively, and the percentage content of AT in the theoretical amplification product sequence of the primer pair is 69%.

[0118] (3) Calculation of the denaturation temperature and annealing temperature of the PCR reaction:

[0119] Using the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length), it can be calculated that the Tm of primer Cro-F is 62.2°C and the Tm of Cro-R is 64.5°C. The average Tm value of this primer pair is 63.4°C; using the formula 0.357×(percentage of GC content)×100 + 70.582 - (990 / amplified product sequence length), the denaturation temperature of the amplification region is 78.3°C.

[0120] (4) Nucleic acid amplification reaction and result detection:

[0121] The nucleic acid amplification reaction system is configured as shown in Table 4 below. According to the calculation results, 3 groups were set up in the experiment to test the specificity of the primer for different detection objects at denaturation temperatures of 94°C / 90°C / 82°C. The nucleic acid amplification reaction conditions were denaturation at 94°C / 90°C / 82°C for 5 seconds, annealing at 63°C for 5 seconds, and extension at 72°C for 20 seconds. The above process was repeated 35 times. The list of specific detection objects is shown in Table 5.

[0122] After the reaction is completed, the amplification results are determined in two ways. One is to add SYBR Green I dye with a final concentration of 25x and determine whether the amplification result is positive by color, that is, whether the target sequence exists in the sample to be tested; the other is to perform agarose gel electrophoresis on the amplified product and judge whether the amplification result is positive according to the electrophoresis band, that is, whether the target sequence exists in the sample to be tested.

[0123] Table 4. Nucleic acid amplification reaction system for Cronobacter sakazakii

[0124] System Volume (μl) Final concentration <![CDATA[R300MIX(TaKaRaTaq TM HS Perfect Mix)]]> 12.5 / Cro-F / Cro-R (50 μM) 0.25+0.25 0.5 μM 100% DMSO 0.5 2％ DNA template (10 ng) 0 / 1 / ddH2O Up to 25 /

[0125] Table 5. List of specific detection objects for nucleic acid amplification reaction of Cronobacter sakazakii

[0126]

[0127]

[0128] The experimental results are shown in Figures 10-12, where 1 - 22 are respectively Staphylococcus aureus, Staphylococcus aureus subsp. aureus, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Salmonella enterica subsp. enterica, Salmonella enterica, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, diarrheagenic Escherichia coli, enterotoxigenic Escherichia coli, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli, 24 - 30 are respectively Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio furnissii, Vibrio cholerae and Shigella flexneri, N: negative control, P: positive control (plasmid containing the target sequence); 23 is Cronobacter sakazakii.

[0129] Figure 10 The SYBR Green I dye color development results of the specific detection of the nucleic acid amplification reaction of the present invention against Cronobacter sakazakii at different denaturation temperatures are shown. If the amplification product shows bright green, it is positive; if the amplification product shows orange, it is negative. Figure 10 Among them, when the denaturation temperature is 94°C / 90°C, the negative control (N) shows orange, which is a negative result and meets the expectation; the positive control (P) and Cronobacter sakazakii No. 23 show bright green, which is a positive result and meets the expectation. This indicates that the entire reaction system can work normally. However, a large number of positive results appear in the amplification products of other bacterial genomic DNA templates. For example, in tubes No. 3, 4, 10 - 18, 20 - 22, 24, 26, 27, 29, 30 under the condition of a denaturation temperature of 94°C, and tubes No. 3, 7, 10 - 18, 20 - 22, 24, 26, 27, 29, 30 under the condition of a denaturation temperature of 90°C, which do not match the expectation, suggesting that non-specific amplification is likely to occur at the denaturation temperature of 94°C / 90°C, resulting in false positive results. When the denaturation temperature is 82°C, the negative control (N) shows orange, which is a negative result and meets the expectation; the positive control (P) and Cronobacter sakazakii No. 23 show bright green, which is a positive result and meets the expectation. This indicates that the entire reaction system can work normally. At the same time, the detection results of other bacterial genomic DNA templates are all negative, as shown in tubes No. 1 - 22, 24 - 30. All results meet the expectation, suggesting that no non-specific amplification occurs at the denaturation temperature of 82°C or even if a small amount of non-specific amplification occurs, it is not sufficient to affect the result determination by the dye method.

[0130] Figure 11The electrophoresis results of the amplification products for the specific detection of the nucleic acid amplification reaction of the present invention against Cronobacter sakazakii at a denaturation temperature of 94°C are shown. If there is a single band at 291 bp after electrophoresis of the amplification products, it is positive; if there is no band after electrophoresis of the amplification products, it is negative; if there is one or more bands outside 291 bp after electrophoresis of the amplification reaction products, it is a false positive caused by non-specific amplification. Figure 11 In Figure 11 , there is no band in the negative control (N), which is as expected; there is a clear band at 291 bp in both the positive control (P) and Cronobacter sakazakii No. 23, which is as expected. This indicates that the entire reaction system can work properly. However, multiple bands appear in the range of 500 bp to 2000 bp in the amplification products of other bacterial genomic DNA templates, which is inconsistent with the expectation, suggesting that a large amount of non-specific amplification occurs in the reaction system at a denaturation temperature of 94°C. This result is consistent with Figure 10 the dye color development results in Figure 10 .

[0131] Figure 12 The electrophoresis results of the amplification products for the specific detection of the nucleic acid amplification reaction of the present invention against Cronobacter sakazakii at a denaturation temperature of 82°C are shown. If there is a single band at 291 bp after electrophoresis of the amplification products, it is positive; if there is no band after electrophoresis of the amplification reaction products, it is negative; if there is one or more bands outside 291 bp after electrophoresis of the amplification reaction products, it is a false positive caused by non-specific amplification. Figure 12 In Figure 12 , there is no band in the negative control (N), which is as expected; there is a clear band at 291 bp in both the positive control (P) and Cronobacter sakazakii No. 23, which is as expected. This indicates that the entire reaction system can work properly. At the same time, most of the amplification reaction products of other bacterial genomic DNA templates have no bands and show negative results, as shown in lanes 2 - 6, 10, 12, 13, 15 - 22, 26 - 30; a very weak band appears after electrophoresis in a few template amplification reaction products, which is easy to distinguish from the positive result, as shown in tubes 1, 7, 8, 9, 11, 14, 24, 25, suggesting that no or a trace amount of non-specific amplification that is easy to distinguish in the electrophoresis result is generated at a denaturation temperature of 82°C.

[0132] Based on Figures 10-12 and Table 5, it can be seen that the application of the nucleic acid amplification method of the present invention against Cronobacter sakazakii has good strain specificity, that is, under the reaction system and reaction conditions proposed by the present invention, only Cronobacter sakazakii shows a positive result, while non-Cronobacter sakazakii shows a negative result. It should be noted that when using the denaturation temperature (90°C or 94°C) of the traditional PCR method for the reaction, although the electrophoresis results ( Figure 11)The target detection object (Cronobacter sakazakii, lane 23) shows a single clear band, while a large number of non-specific amplifications are generally present in non-target detection objects, such as Staphylococcus epidermidis in lane 3; if the dye colorimetric method is used to determine the detection result, as Figure 10 shown, under the denaturation conditions of 90 °C and 94 °C, a large number of false positive results appear, making it impossible to distinguish between the detection object and the non-detection object. Under the same reaction system, when the reaction is carried out at the denaturation temperature of 82 °C calculated and generated by the present invention, the electrophoresis result ( Figure 12 ) shows that the target detection object (Cronobacter sakazakii, lane 23) has a single clear band, while there is little non-specific amplification in non-target detection objects. Even if there is, the non-specific amplification band is very weak and is extremely easy to distinguish from the positive result; if the dye colorimetric method is used to determine the detection result, as Figure 10 shown, under the denaturation condition of 82 °C, only the target detection object (Cronobacter sakazakii, tube 23) shows a positive result, and all non-target detection objects show negative results, showing good discrimination.

[0133] Example 3 Simultaneously Detecting Salmonella, Staphylococcus aureus, Yersinia enterocolitica and Cronobacter sakazakii

[0134] Changing the Shigella genome targeted by the present invention to the genomes of Salmonella, Staphylococcus aureus, Yersinia enterocolitica and Cronobacter sakazakii, screening for AT-rich sequences and designing specific primers, screening primer combinations according to the calculated primer Tm and target sequence Tm, setting reaction conditions for nucleic acid amplification, and determining whether there is a target sequence in the test sample by judging whether the reaction result is positive, and then determining whether there is one or more of Salmonella, Staphylococcus aureus, Yersinia enterocolitica and Cronobacter sakazakii in the test sample. The specific steps are as follows:

[0135] (1) Screening of target sequences rich in AT bases:

[0136] Using the data of bacteria, archaea and viruses with complete genome sequences downloaded from the FTP of NCBI on August 5, 2019, a total of 2,896 whole genome sequences. Set set A, which contains all Salmonella genome sequences; set set B, which contains all non-Salmonella genome sequences. Using the Salmonella genome sequence as the reference genome, a window with a width of 1000 bp is slid starting from the first base of the genome, with a step size of 50 bp; calculate the AT base content of the sequence once before each slide, and retain the region with an AT base content greater than 60% of the sequence as the candidate target sequence. The above process is implemented using a perl script.

[0137] (2) Design of specific primers:

[0138] According to the characteristics of PCR primers and the requirements of the present invention, characteristic parameters such as the AT base content of the primers being 55% - 75%, the 3'-end stability ΔG < 4, the 5'-end stability ΔG < 3, and the primer sequence length (20 - 36 bp) are set. At the same time, conditions such as a single primer not generating a hairpin structure and not interacting with itself are set. Using the candidate target sequence in step (1) as the candidate sequence for single primer design, single primers that meet the above set conditions are calculated. Calculate the Tm value of each single primer through the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length), and record information such as the position of each primer on the target sequence, the positive / negative strand information (i.e., whether it is from the positive strand or the negative strand), and the length of the primer.

[0139] According to the position information of single primers, primer pairs are paired, and primer pairs that simultaneously meet the two conditions of the Tm difference of the primer pair < 3°C and the amplification region of the primer pair being 200 - 600 bp are retained as candidate primer pairs.

[0140] Using the alignment software Bowtie, each primer in the candidate primer pairs designed in the previous step is respectively aligned with the target genomic sequence in set A and the non-target genome in set B. To ensure the universality of the primer, when this single primer is aligned with the target sequence in set A, the parameter setting "-a -n 0" is used, that is, it is required that this single primer be completely matched with the target sequence; to ensure the specificity of the primer, when this single primer is aligned with the non-target sequence in set B, the parameter setting "-a -n 3" is used, that is, it is allowed that this single primer has no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, and the number of primer pairs can be preset. In this example, the number of primer pairs is preset to 100. The above high-throughput and automated primer design process is implemented using C and Perl scripts. After the program runs, 100 Salmonella amplification primer pairs are designed in the AT-rich region.

[0141] By the same method, 100 amplification primer pairs for Staphylococcus aureus, Yersinia enterocolitica, and Cronobacter sakazakii are designed respectively. Calculate the Tm of each primer using the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length) and calculate the average Tm value of the primer pair as the reaction annealing temperature; calculate the denaturation temperature of the amplification region of each primer pair using the formula 0.357×(percentage of GC content)×100 + 70.582 - (990 / amplification product sequence length).

[0142] Screen primer combinations on the condition that the amplified sequence lengths of different bacteria differ by at least 50 bp, the reaction denaturation temperatures differ by at most 2 °C, and the reaction annealing temperatures differ by at most 2 °C. The system outputs primer combinations that meet the conditions, and the number can be preset. In this embodiment, the number of primer combinations is preset to 10. Randomly select a primer group for validity verification. The sequences of the primer combinations are as follows:

[0143] Primer pair A:

[0144] Sal-F: 5’-TCAGACATCCGTTCAGAAAAT-3’ (SEQ ID NO.1)

[0145] Sal-R: 5’-GTTCAACTGTCGACAAGATTAA-3’ (SEQ ID NO.2)

[0146] Primer pair B:

[0147] Sta-F: 5’-GTAGGTATGGTAAATAGTTACAC-3’ (SEQ ID NO.5)

[0148] Sta-R: 5’-CACTAATGCCAAATTTACTTAAAATCG-3’ (SEQ ID NO.6)

[0149] Primer pair C:

[0150] Cro-F: 5’-CGCCATAACTGCATAATCAT-3’ (SEQ ID NO.3)

[0151] Cro-R: 5’-ATAACGAGTTACCGTGCAGA-3’ (SEQ ID NO.4)

[0152] Primer pair D:

[0153] Yer-F: 5’-TGTGCGGTGGATGTAAATAATTC-3’ (SEQ ID NO.7)

[0154] Yer-R: 5’-GCTTTGAAACTCAAGGACTG-3’ (SEQ ID NO.8)

[0155] The AT base contents of the primer pair A are 60% and 64% respectively, and the theoretical average annealing temperature is 62 °C; the theoretical amplified product sequence fragment length of the primer pair is 355 bp, the percentage content of AT is 71%, and the theoretical denaturation temperature is 78.14 °C.

[0156] The AT base contents of the primer pair B are 65.22% and 70.37% respectively, and the theoretical average annealing temperature is 62.5 °C; the theoretical amplified product sequence fragment length of the primer pair is 465 bp, the percentage content of AT is 73.76%, and the theoretical denaturation temperature is 77.82 °C.

[0157] The AT base contents of the primer pair C are 60% and 55% respectively, and the theoretical average annealing temperature is 63 °C; the theoretical amplified product sequence fragment length of the primer pair is 291 bp, the percentage content of AT is 68.73%, and the theoretical denaturation temperature is 78.34 °C.

[0158] The AT base contents of the primer pair D are 60.87% and 55% respectively, and the theoretical average annealing temperature is 64 °C; the theoretical amplified product sequence fragment length of the primer pair is 235 bp, the percentage content of AT is 68.09%, and the theoretical denaturation temperature is 77.76 °C.

[0159] (4) Nucleic acid amplification reaction and result detection:

[0160] The nucleic acid amplification reaction system is configured as shown in Table 6 below. According to the calculation results, denaturation is carried out at 81 °C for 5 seconds, annealing is carried out at 62 °C for 5 seconds, and extension is carried out at 72 °C for 20 seconds. It is recommended to repeat the above process 35 times. The list of detection objects is shown in Table 7.

[0161] After the reaction is completed using an ordinary gradient PCR instrument, the amplification results can be determined in two ways. One is to add SYBR Green I dye with a final concentration of 25x, and determine whether the amplification result is positive by color, that is, whether the target sequence exists in the sample to be tested; the other is to perform agarose gel electrophoresis on the amplified product, and judge whether the amplification result is positive according to the electrophoresis band, that is, whether the target sequence exists in the sample to be tested.

[0162] Table 6. Nucleic acid amplification reaction system for four kinds of bacteria

[0163] System Volume (μl) Final concentration Taq RR001 (5 U / μl) 0.4 2U dNTP (2.5 mM each) 3 0.3 mM each 10x buffer (20 mM Mg2+) 2.5 1x buffer SAL-F / R (50 μM) 0.2+0.2 0.4 μM STA-F / R (50 μM) 0.2+0.2 0.4 μM YER-F / R (50 μM) 0.3+0.3 0.6 μM CRO-F / R (50 μM) 0.2+0.2 0.4 μM 100% DMSO 0.5 2％ MgSO4 (100 mM) 0.5 4 (mM) Template (10 ng) 0 / 1 / ddH2O Upto25 /

[0164] Table 7. List of specific detection objects for nucleic acid amplification reaction of four kinds of bacteria

[0165]

[0166]

[0167] Figure 13 and 14The fluorescence dye staining results and agarose gel electrophoresis results of the nucleic acid amplification reactions of the present invention for four bacteria, namely Salmonella enterica subsp., Staphylococcus aureus subsp., Yersinia enterocolitica, and Cronobacter sakazakii, are shown. Among them, "N" is the amplification result of its corresponding negative template, "M1" refers to Marker DL2000, "M2" refers to Marker B (100 - 600bp), and "mixed" refers to the mixed template of the genomic DNAs of the four bacteria. Figure 13 The amplification and color development results of each single bacterium, mixed template, and negative template meet the expectations. Figure 14 The amplified fragments of each single bacterium are in line with the theoretical calculated values, and the amplified fragments of each bacterium in the mixed template can be separated from each other.

[0168] Figure 15 The SYBR Green I dye color development results of the specificity detection of the nucleic acid amplification reactions of the present invention for four bacteria, namely Salmonella, Staphylococcus aureus, Yersinia enterocolitica, and Cronobacter sakazakii, at a denaturation temperature of 81°C are shown. If the amplified product shows bright green, it is positive; if the amplified product shows orange, it is negative. Figure 15 Among them, Staphylococcus aureus and Staphylococcus aureus subsp. of No. 1 - 2, Salmonella enterica subspecies, Salmonella enterica, Salmonella typhimurium, and Salmonella paratyphi B of No. 10 - 13, Cronobacter sakazakii of No. 23, and Yersinia enterocolitica of No. 24 show bright green, which is a positive result and meets the expectations; at the same time, the detection results of other bacterial genomic DNA templates are all negative, as shown in tubes No. 3 - 9, 14 - 22, and 25 - 30. All results meet the expectations, indicating that no non-specific amplification occurs at a denaturation temperature of 81°C, or even if a small amount of non-specific amplification occurs, it is not sufficient to affect the result determination by the dye method.

[0169] Example 4 Simultaneously Detecting Salmonella and Staphylococcus aureus

[0170] The present invention screens for AT-rich sequences in the genomes of Salmonella and Staphylococcus aureus and designs specific primers. According to the calculated primer Tm and target sequence Tm, primer combinations are screened, reaction conditions are set for nucleic acid amplification, and by judging whether the reaction result is positive, it is determined whether the target sequence exists in the sample to be tested, and further whether one or more of Salmonella and Staphylococcus aureus exist in the sample to be tested. The specific steps are as follows:

[0171] (1) Screening of target sequences rich in AT bases:

[0172] Using the data of bacteria, archaea and viruses with complete genome sequences downloaded from the NCBI FTP on August 5, 2019, a total of 2,896 whole genome sequences were used. Set set A, which contains all Salmonella genome sequences; set set B, which contains all non-Salmonella genome sequences. The Salmonella genome sequence was used as the reference genome, and a window with a width of 1,000 bp was slid starting from the first base of the genome, with a step size of 50 bp; the AT base content of the sequence was calculated once before each slide, and the region with an AT base content greater than 60% was retained as the candidate target sequence. The above process was implemented using a perl script.

[0173] (2) Design of specific primers:

[0174] According to the characteristics of PCR primers and the requirements of the present invention, the characteristics parameters of the primers were set, such as the AT base content of the primers being 55% - 75%, the 3'-end stability ΔG < 4, the 5'-end stability ΔG < 3, and the primer sequence length (20 - 36 bp). At the same time, conditions such as a single primer not being able to produce a hairpin structure and not interacting with itself were set. The candidate target sequence in step (1) was used as the candidate sequence for the design of a single primer, and single primers that met the above set conditions were calculated. The Tm value of a single primer was calculated by the formula 0.466×(percentage content of GC)×100 + 66.04 - (450 / primer sequence length), and information such as the position of each primer on the target sequence, the positive and negative strand information (i.e., whether it is from the positive strand or the negative strand), and the length of the primer was recorded.

[0175] According to the position information of the single primers, primer pairs were paired, and primer pairs that simultaneously met the two conditions of the Tm difference between primer pairs < 3°C and the amplification region of primer pairs being 200 - 600 bp were retained as candidate primer pairs.

[0176] Using the alignment software Bowtie, each primer in the candidate primer pairs designed in the previous step was separately aligned with the target genome sequence in set A and the non-target genome in set B. To ensure the universality of the primers, when this single primer was aligned with the target sequence in set A, the parameter setting "-a -n 0" was used, that is, it was required that this single primer be completely matched with the target sequence; to ensure the specificity of the primers, when this single primer was aligned with the non-target sequence in set B, the parameter setting "-a -n 3" was used, that is, it was allowed that this single primer had no more than 3 mismatches with the non-target sequence. The system output primer pairs that met the conditions, and the number of primer pairs could be preset. In this example, the number of primer pairs was preset to 100. The above high-throughput and automated primer design process was implemented using C and Perl scripts. After the program ran, 100 Salmonella amplification primer pairs were designed in the AT-rich region.

[0177] 100 primer pairs for Staphylococcus aureus amplification were designed respectively by the same method. The Tm of each primer was calculated using the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length), and the average Tm value of the primer pair was calculated as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357×(percentage of GC content)×100 + 70.582 - (990 / amplified product sequence length).

[0178] The primer combinations were screened on the condition that the amplified sequence lengths of different bacteria differed by at least 50 bp, the reaction denaturation temperatures differed by at most 2 °C, and the reaction annealing temperatures differed by at most 2 °C. The system output the primer combinations that met the conditions, and the number could be preset. In this example, the number of primer combinations was preset to 10. A primer group was randomly selected for validity verification. The sequences of the primer combinations are as follows:

[0179] Primer pair A:

[0180] Sal-F: 5’-TCAGACATCCGTTCAGAAAAT-3’(SEQ ID NO.1)

[0181] Sal-R: 5’-GTTCAACTGTCGACAAGATTAA-3’(SEQ ID NO.2)

[0182] Primer pair E:

[0183] Sta-F1: 5’-CCTTTCATCTAAAAACCTCCA-3’(SEQ ID NO.11)

[0184] Sta-R1: 5’-GAAATGGATGTTTTAAAAGAAGG-3’(SEQ ID NO.12)

[0185] The AT base contents of the primer pair A were 60% and 64% respectively, and the theoretical average annealing temperature was 62 °C; the theoretical amplified product sequence fragment length of the primer pair was 355 bp, the percentage of AT was 71%, and the theoretical denaturation temperature was 78.14 °C.

[0186] The AT base contents of the primer pair E were 61.90% and 69.57% respectively, and the theoretical average annealing temperature was 61 °C; the theoretical amplified product sequence fragment length of the primer pair was 595 bp, the percentage of AT was 76.30%, and the theoretical denaturation temperature was 77.38 °C.

[0187] (4) Nucleic acid amplification reaction and result detection:

[0188] The nucleic acid amplification reaction system is configured as shown in Table 8 below. According to the calculation results, denaturation at 81°C for 5 seconds, annealing at 62°C for 5 seconds, and extension at 72°C for 20 seconds are recommended, and the above process is repeated 35 times. The list of test objects is shown in Table 7.

[0189] After the reaction is completed using a common gradient PCR instrument, agarose gel electrophoresis is performed on the amplification products, and the amplification result is judged to be positive according to the electrophoresis bands, that is, whether the target sequence exists in the test sample.

[0190] Table 8. Nucleic acid amplification reaction system for two bacteria

[0191] System Volume (μl) Final concentration Taq R300mix 15 1x SAL-F / R (50 μM) 0.25+0.25 0.5 μM STA-F1 / R1 (50 μM) 0.25+0.25 0.5 μM 100% DMSO 0.5 2％ Template (10 ng) 0 / 1 / ddH2O Upto25 /

[0192] Figure 16 The agarose gel electrophoresis results of the nucleic acid amplification reactions of two bacteria, Salmonella enterica subsp. enterica and Staphylococcus aureus subsp. aureus, of the present invention are shown. Among them, "N" is the amplification result of its corresponding negative template, "M" refers to MarkerDL2000, "Salmonella" refers to Salmonella enterica subsp. enterica, "Staphylococcus aureus" refers to Staphylococcus aureus subsp. aureus, and "mixed" refers to the mixed template of the genomic DNAs of the two bacteria. Figure 16 The amplified fragments of each single bacterium all conform to the theoretical calculated values, and the amplified fragments of each bacterium in the mixed template can be separated from each other.

[0193] Example 5 Simultaneously Detecting Salmonella and Yersinia enterocolitica

[0194] The present invention screens for AT-rich sequences in the genomes of Salmonella and Yersinia enterocolitica, designs specific primers, screens primer combinations according to the calculated primer Tm and target sequence Tm, sets reaction conditions for nucleic acid amplification, determines whether the target sequence exists in the test sample by judging whether the reaction result is positive, and further determines whether one or more of Salmonella and Yersinia enterocolitica exist in the test sample. The specific steps are as follows:

[0195] (1) Screening of target sequences rich in AT bases:

[0196] Using the data of bacteria, archaea, and viruses with complete genome sequences downloaded from the FTP of NCBI on August 5, 2019, a total of 2896 whole genome sequences. Set set A, which contains all Salmonella genome sequences; set set B, which contains all non-Salmonella genome sequences. The Salmonella genome sequence is used as the reference genome, and a window with a width of 1000 bp is slid starting from the first base of the genome, with a step size of 50 bp; the AT base content of the sequence is calculated once before each sliding, and the region with an AT base content greater than 60% of the sequence is retained as the candidate target sequence. The above process is implemented using a perl script.

[0197] (2) Design of specific primers:

[0198] According to the characteristics of PCR primers and the requirements of the present invention, characteristic parameters such as the AT base content of the primers being 55% - 75%, the 3'-end stability ΔG < 4, the 5'-end stability ΔG < 3, and the primer sequence length (20 - 36 bp) are set. At the same time, conditions such as a single primer not generating a hairpin structure and not interacting with itself are set. Using the candidate target sequence in step (1) as the candidate sequence for single primer design, single primers meeting the above set conditions are calculated. Calculate the Tm value of each single primer using the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length), and record information such as the position of each primer on the target sequence, positive and negative strand information (i.e., whether it is from the positive strand or the negative strand), and the length of the primer.

[0199] According to the position information of the single primers, primer pairs are paired, and the primer pairs that simultaneously meet the two conditions of the Tm difference between primer pairs < 3°C and the amplification region of the primer pairs being 200 - 600 bp are retained as candidate primer pairs.

[0200] Using the alignment software Bowtie, each primer in the candidate primer pairs designed in the previous step is respectively aligned with the target genomic sequence in set A and the non-target genome in set B. To ensure the universality of the primers, when this single primer is aligned with the target sequence in set A, the parameter setting "-a -n 0" is used, that is, it is required that this single primer be completely matched with the target sequence; to ensure the specificity of the primers, when this single primer is aligned with the non-target sequence in set B, the parameter setting "-a -n 3" is used, that is, it is allowed that this single primer has no more than 3 mismatches with the non-target sequence. The system outputs the primer pairs that meet the conditions, and the number of primer pairs can be preset. In this embodiment, the number of primer pairs is preset to 100. The above high-throughput and automated primer design process is implemented using C and Perl scripts. After the program runs, 100 pairs of Salmonella amplification primers are designed in the AT-rich region.

[0201] By the same method, 100 pairs of Yersinia enterocolitica amplification primers are designed respectively. Calculate the Tm of each primer using the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length) and calculate the average Tm value of this primer pair as the reaction annealing temperature; calculate the denaturation temperature of the amplification region of each primer pair using the formula 0.357×(percentage of GC content)×100 + 70.582 - (990 / amplification product sequence length).

[0202] The primer combinations were screened on the conditions that the amplified sequence lengths of different bacteria differed by at least 50 bp, the reaction denaturation temperatures differed by at most 2 °C, and the reaction annealing temperatures differed by at most 2 °C. The system outputs the primer combinations that meet the conditions, and the number can be preset. In this embodiment, the number of primer combinations was preset to 10. A primer group was randomly selected for validity verification. The sequences of the primer combinations are as follows:

[0203] Primer pair F:

[0204] Sal-F2: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.13)

[0205] Sal-R2: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.14)

[0206] Primer pair G:

[0207] Yer-F1: 5'-ATGGAAAATAACATAATTTCTATTACCGG-3' (SEQ ID NO.15)

[0208] Yer-R1: 5'-TCTCTGCGAATAACCTTGTG-3' (SEQ ID NO.16)

[0209] The AT base contents of the primer pair F were 60% and 55% respectively, and the theoretical average annealing temperature was 63 °C; the theoretical amplified product sequence fragment length of the primer pair was 352 bp, the percentage content of AT was 74.43%, and the theoretical denaturation temperature was 76.90 °C.

[0210] The AT base contents of the primer pair G were 72.41% and 55% respectively, and the theoretical average annealing temperature was 63.5 °C; the theoretical amplified product sequence fragment length of the primer pair was 242 bp, the percentage content of AT was 72.02%, and the theoretical denaturation temperature was 76.48 °C.

[0211] (4) Nucleic acid amplification reaction and result detection:

[0212] The nucleic acid amplification reaction system was configured as shown in Table 9 below. According to the calculation results, denaturation at 81 °C for 5 s, annealing at 62 °C for 5 s, and extension at 72 °C for 20 s were recommended, and the above process was repeated 35 times. The list of detection objects is shown in Table 7.

[0213] After the reaction was completed using a conventional gradient PCR instrument, agarose gel electrophoresis was performed on the amplified product, and the amplification result was judged as positive based on the electrophoresis band, that is, whether the target sequence was present in the sample to be tested.

[0214] Table 9. Nucleic acid amplification reaction system for two bacteria

[0215] System Volume (μl) Final concentration Taq R300mix 15 1x SAL-F2 / R2 (50 μM) 0.25+0.25 0.5 μM YER-F1 / R1 (50 μM) 0.25+0.25 0.5 μM 100% DMSO 0.5 2％ Template (10 ng) 0 / 1 / ddH2O Upto25 /

[0216] Figure 17 The agarose gel electrophoresis results of the nucleic acid amplification reactions of the present invention for two bacteria, Salmonella enterica subsp. and Yersinia enterocolitica, are shown. Among them, "N" is the amplification result of its corresponding negative template, "M" refers to Marker DL2000, "Salmon" refers to Salmonella enterica subsp., "Yersinia" refers to Yersinia enterocolitica subsp., and "Mix" refers to the mixed template of the genomic DNAs of the two bacteria. Figure 17 The amplified fragments of each single bacterium all conform to the theoretical calculated values, and the amplified fragments of each bacterium in the mixed template can be separated from each other.

[0217] Example 6 Simultaneously Detecting Salmonella, Staphylococcus aureus and Yersinia enterocolitica

[0218] The present invention screens for AT-rich sequences in the genomes of Salmonella, Staphylococcus aureus and Yersinia enterocolitica and designs specific primers. According to the calculated primer Tm and the target sequence Tm, primer combinations are screened, reaction conditions are set for nucleic acid amplification, and by judging whether the reaction result is positive, it is determined whether the target sequence exists in the sample to be tested, and further whether one or more of Salmonella, Staphylococcus aureus and Yersinia enterocolitica exist in the sample to be tested. The specific steps are as follows:

[0219] (1) Screening of target sequences rich in AT bases:

[0220] Using the data of bacteria, archaea and viruses with complete genome sequences downloaded from the FTP of NCBI on August 5, 2019, a total of 2,896 whole genome sequences. Set set A, which contains all Salmonella genome sequences; set set B, which contains all non-Salmonella genome sequences. The Salmonella genome sequence is used as the reference genome, and a window with a width of 1000 bp is slid starting from the first base of the genome, with a step size of 50 bp; the AT base content of the sequence is calculated once before each sliding, and the region with an AT base content greater than 60% of the sequence is retained as the candidate target sequence. The above process is implemented using a perl script.

[0221] (2) Design of specific primers:

[0222] According to the characteristics of PCR primers and the requirements of the present invention, characteristic parameters such as the AT base content of the primers are set to 55% - 75%, the 3'-end stability ΔG < 4, the 5'-end stability ΔG < 3, and the primer sequence length (20 - 36 bp). At the same time, conditions such as a single primer not generating a hairpin structure and not interacting with itself are set. Using the candidate target sequence in step (1) as the candidate sequence for single primer design, single primers that meet the above set conditions are calculated. Calculate the Tm value of a single primer through the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length), and record information such as the position of each primer on the target sequence, the positive and negative strand information (i.e., whether it is from the positive strand or the negative strand), and the length of the primer.

[0223] According to the position information of single primers, primer pairs are paired, and primer pairs that simultaneously meet the two conditions of the Tm difference of the primer pair < 3°C and the amplification region of the primer pair being 200 - 600 bp are retained as candidate primer pairs.

[0224] Using the alignment software Bowtie, each primer in the candidate primer pairs designed in the previous step is respectively aligned with the target genomic sequence in set A and the non-target genome in set B. To ensure the universality of the primer, when this single primer is aligned with the target sequence in set A, the parameter setting "-a -n 0" is used, that is, it is required that this single primer be completely matched with the target sequence; to ensure the specificity of the primer, when this single primer is aligned with the non-target sequence in set B, the parameter setting "-a -n 3" is used, that is, it is allowed that this single primer has no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, and the number of primer pairs can be preset. In this embodiment, the number of primer pairs is preset to 100. The above high-throughput and automated primer design process is implemented using C and Perl scripts. After the program runs, 100 Salmonella amplification primer pairs are designed in the AT-rich region.

[0225] By the same method, 100 Yersinia enterocolitica amplification primer pairs are designed respectively. Calculate the Tm of each primer using the formula 0.466×(percentage of GC content)×100 + 66.04 - (450 / primer sequence length) and calculate the average Tm value of the primer pair as the reaction annealing temperature; calculate the denaturation temperature of the amplification region of each primer pair using the formula 0.357×(percentage of GC content)×100 + 70.582 - (990 / amplification product sequence length).

[0226] Screen primer combinations on the condition that the amplified sequence lengths of different bacteria differ by at least 50 bp, the reaction denaturation temperatures differ by at most 2 °C, and the reaction annealing temperatures differ by at most 2 °C. The system outputs primer combinations that meet the conditions, and the quantity can be preset. In this embodiment, the quantity of primer combinations is preset to 10. Randomly select a primer group for validity verification. The sequences of the primer combinations are as follows:

[0227] Primer pair F:

[0228] Sal-F2: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.13)

[0229] Sal-R2: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.14)

[0230] Primer pair G:

[0231] Yer-F1: 5'-ATGGAAAATAACATAATTTCTATTACCGG-3' (SEQ ID NO.3)

[0232] Yer-R1: 5'-TCTCTGCGAATAACCTTGTG-3' (SEQ ID NO.4)

[0233] Primer pair E:

[0234] Sta-F1: 5'-CCTTTCATCTAAAAACCTCCA-3' (SEQ ID NO.11)

[0235] Sta-R1: 5'-GAAATGGATGTTTTAAAAGAAGG-3' (SEQ ID NO.12)

[0236] The AT base contents of the primer pair F are 60% and 55% respectively, and the theoretical average annealing temperature is 63 °C; the theoretical amplified product sequence fragment length of the primer pair is 352 bp, the percentage content of AT is 74.43%, and the theoretical denaturation temperature is 76.90 °C.

[0237] The AT base contents of the primer pair G are 72.41% and 55% respectively, and the theoretical average annealing temperature is 63.5 °C; the theoretical amplified product sequence fragment length of the primer pair is 242 bp, the percentage content of AT is 72.02%, and the theoretical denaturation temperature is 76.48 °C.

[0238] The AT base contents of the primer pair E are 61.90% and 69.57% respectively, and the theoretical average annealing temperature is 61°C; the theoretical amplified product sequence fragment length of the primer pair is 595 bp, the percentage content of AT is 76.30%, and the theoretical denaturation temperature is 77.38°C.

[0239] (4) Nucleic acid amplification reaction and result detection:

[0240] The nucleic acid amplification reaction system is configured as shown in Table 10 below. According to the calculation results, denaturation is carried out at 81°C for 5 seconds, annealing at 62°C for 5 seconds, and extension at 72°C for 20 seconds. It is recommended to repeat the above process 35 times. The list of detection objects is shown in Table 7.

[0241] After the reaction is completed using an ordinary gradient PCR instrument, agarose gel electrophoresis is performed on the amplified product, and the amplification result is judged as positive according to the electrophoresis band, that is, whether the target sequence exists in the sample to be tested.

[0242] Table 10. Nucleic acid amplification reaction system for three kinds of bacteria

[0243] System Volume (μl) Final concentration Taq R300mix 15 1x SAL-F2 / R2 (50 μM) 0.25+0.25 0.5 μM YER-F1 / R1 (50 μM) 0.25+0.25 0.5 μM STA-F1 / R1 (50 μM) 0.25+0.25 0.5 μM 100% DMSO 0.5 2％ Template (10 ng) 0 / 1 / ddH2O Upto25 /

[0244] Figure 18 The agarose gel electrophoresis results of the nucleic acid amplification reactions of three kinds of bacteria, namely Salmonella enteritidis subspecies, Staphylococcus aureus subspecies, and Yersinia enterocolitica, according to the present invention are shown. Among them, "N" is the amplification result of its corresponding negative template, "M" refers to Marker DL2000, "Entero-sub" refers to Salmonella enteritidis subspecies, "Yer-sub" refers to Yersinia enterocolitica subspecies, "Sta-sub" refers to Staphylococcus aureus subspecies, and "Mix" refers to the mixed template of the genomic DNAs of the three kinds of bacteria. Figure 18 The amplified fragments of each single bacterium all conform to the theoretical calculated values, and the amplified fragments of each bacterium in the mixed template can be separated from each other.

[0245] The present invention also provides a device for detecting Shigella, including: a memory, a processor, and an amplification module; a computer program is stored on the memory, and when the computer program is executed by the processor, the steps of the above-mentioned method are implemented; the amplification module is used to carry out nucleic acid amplification reaction under the condition of local strand separation of the template to obtain an amplified product.

[0246] References

[0247] Zhou, H. Q. (2014). Relationship between genomic GC content and base, codon, and amino acid usage preferences. (mastermaster), University of Electronic Science and Technology of China, (67)

[0248] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, all changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims. SEQUENCE LISTING <110> Shanghai Institute of Biotechnology, Shanghai Want Want Food Group Co., Ltd. <120> Method for Detecting Shigella and Its Application <160> 17 <170> PatentIn version 3.3 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <400> 1 tcagacatcc gttcagaaaa t 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <400> 2 gttcaactgt cgacaagatt aa 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 cgccataact gcataatcat 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 ataacgagtt accgtgcaga 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <400> 5 gtaggtatgg taaatagtta cac 23 <210> 6 <211> 27 <212> DNA <213> Artificial sequence <400> 6 cactaatgcc aaatttactt aaaatcg 27 <210> 7 <211> 23 <212> DNA <213> Artificial sequence <400> 7 tgtgcggtgg atgtaaataa ttc 23 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <400> 8 gctttgaaac tcaaggactg 20 <210> 9 <211> 21 <212> DNA <213> Artificial sequence <400> 9 cggcagaacg ttcaaatatg a 21 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 cgcagaagga tatcctgata 20 <210> 11 <211> 21 <212> DNA <213> Artificial sequence <400> 11 cctttcatct aaaaacctcc a 21 <210> 12 <211> 23 <212> DNA <213> Artificial sequence <400> 12 gaaatggatg ttttaaaaga agg 23 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <400> 13 tgggttgaaa tagcccatta 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <400> 14 gacgtgacac acttcgtttt 20 <210> 15 <211> 29 <212> DNA <213> Artificial sequence <400> 15 atggaaaata acataatttc tattaccgg 29 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <400> 16 tctctgcgaa taaccttgtg 20 <210> 17 <211> 19 <212> DNA <213> Artificial sequence <400> 17 aagggggttc caggcatta 19

Claims

1. A primer pair, characterized in that, The primer pair is as follows: Primer pair H: Shi-F: 5’-CGGCAGAACGTTCAAATATGA-3’(SEQ ID NO: 9) Shi-R: 5’-CGCAGAAGGATATCCTGATA-3’(SEQ ID NO: 10).

2. A diagnostic reagent, characterized in that, The diagnostic reagent comprises the primer pair as described in claim 1.

3. Use of the primer pair according to claim 1 in the preparation of a product for amplifying and / or detecting genes or regions related to the genome of Shigella.

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