A method for detecting Salmonella, its application and primers

Abstract

This invention discloses a method for detecting Salmonella, its applications, and primers. The method includes: identifying AT-rich sequences in the Salmonella genome as target sequences; automatically and with high throughput designing specific primers for the target sequences; designing a specific Tm value calculation formula to calculate the denaturation and annealing temperatures, and setting the nucleic acid amplification reaction conditions accordingly; and performing the nucleic acid amplification reaction under conditions of local template denaturation. This invention targets AT-rich sequences, which traditional nucleic acid amplification methods typically avoid, greatly expanding the candidate target region. Through specific screening based on massive genomic data and precise calculation of Tm values ​​for AT-rich regions, local denaturation of double-stranded DNA is achieved, significantly 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 while significantly improving reaction performance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for detecting Salmonella and an application and primers thereof. The method is a method for performing nucleic acid amplification specifically for the genome sequence of Salmonella. 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 composition of organisms in nature varies greatly. For example, the AT base content of the Plasmodium genome is about 82%. Zhou Huiqi et al. studied 2,670 strains of bacteria and archaea and found that the GC content of 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 technology ignores a large number of potential target sequences, which increases the difficulty of successfully detecting the target object; from the perspective of genetic manipulation, this selection preference makes it difficult for a considerable number of target sequences to be successfully amplified, making it difficult to carry out further molecular manipulation. If appropriate primers can be designed for the GC base content and AT base content imbalance domains avoided by traditional nucleic acid amplification methods to avoid the formation of secondary structures, and appropriate reaction conditions can be set to smoothly carry out nucleic acid amplification experiments, the success rate of target object detection will be significantly increased, and more sequences can be amplified, thereby facilitating the implementation of downstream molecular manipulation.

[0005] The key step in carrying out nucleic acid amplification reactions for regions with uneven GC and AT base contents is primer design. In view of the above analysis, species-specific genes (or sequences) publicly released in the field usually come from regions with GC contents of 40% to 60% or even 45% to 55%, and therefore cannot be applied to primer design for regions with uneven GC and AT base contents. In addition, the base composition diversity of the continuously existing GC-rich or AT-rich regions in such sequences is weaker than that of other regions, which brings additional difficulties to the design of specific primers. Therefore, it is first necessary to propose a set of high-throughput, automated, and efficient primer design algorithm processes for sequences with uneven GC and AT base contents.

[0006] In addition to the selection of the amplification region and the design of primers, the calculation of the Tm value of the amplification region and primers is also an important factor affecting whether the amplification process can be successfully completed. The Tm value of the amplification region and primers is usually calculated according to the nearest neighbor two-state model, but the specific calculation formulas used by different researchers and primer production companies are different. For example, the primer Tm calculation formula recommended in the fourth edition of the Molecular Cloning Experiment Guide is Tm = 4×(G, C number) + 2×(A, T number), and the primer Tm calculation formula recommended by TaKaRa is Tm = 4×(G, C number) + 2×(A, T number) + 32-2×(total number of bases); the primer Tm calculation formula of Sangon Biotechnology is Tm(0.05M Na + ) = 59.94 + 1 × (GC percentage) - (675 / primer sequence length). For regions with uneven GC and AT base content, how to design a model to calculate the primer Tm value becomes an important factor in ensuring the success of the experiment.

[0007] In summary, the field urgently needs to develop a nucleic acid amplification technology that targets areas that are difficult to reach with traditional nucleic acid amplification technology, 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, so as to expand the application scope of nucleic acid amplification technology, improve the specificity of amplification, and meet the needs 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 Salmonella. The method uses AT-rich nucleic acid sequences as amplification targets, and performs target sequence identification, primer design, denaturation temperature and annealing temperature calculation, and nucleic acid amplification of template partial melting. First, an automated primer design process is adopted to make full use of the rich genome sequence information in public data resources to design specific primer pairs for AT-rich target sequences with high throughput. 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 based on the theoretical amplification product sequence and the sequence length, base composition and other factors of the primer; considering that the denaturation temperature of AT-rich nucleic acid sequences is significantly lower than that of sequences with a GC content of 40% to 60% and high GC, the calculated theoretical amplification product sequence Tm value is used as the minimum denaturation temperature. At this temperature, the AT-rich region of the double-stranded DNA can be partially melted without adding any chemical denaturants, thereby initiating the nucleic acid amplification reaction.

[0009] On the one hand, the present invention introduces massive genomic data from public data resources in the primer design link to conduct sequence-specific screening; on the other hand, in the denaturation temperature setting link, unlike the traditional nucleic acid amplification method that opens all double-stranded structures at a denaturation temperature of 93 to 95°C, only AT-rich sequences are opened by lowering the denaturation temperature, thereby greatly reducing the possibility of non-specific amplification in non-targeted areas at the source. These two design strategies significantly reduce the possibility of common non-specific amplification 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 the method is implemented using C language and Perl language programming, and has the characteristics of high throughput and automation. The entire amplification method has strong specificity and high success rate, which effectively expands the application scope of traditional nucleic acid amplification technology and significantly improves the reaction performance.

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

[0011] The specific steps include:

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

[0013] (2) Automated and high-throughput design of primers that are both universal and specific for the target sequence;

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

[0015] (4) A nucleic acid amplification reaction is performed under conditions in which the template is partially melted to obtain an amplified product.

[0016] In the present invention, the method for detecting Salmonella comprises the following specific steps:

[0017] (1) Screening target sequences rich in AT bases in the Salmonella genome sequence;

[0018] (2) designing primers that are both universal and specific for the target sequence;

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

[0020] (4) A nucleic acid amplification reaction is performed under conditions in which the template is partially melted to obtain an amplified product.

[0021] In the present invention, the primer pair is:

[0022] Primer pair A:

[0023] Sal-F1: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.1)

[0024] Sal-R1: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO. 2).

[0025] 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 from the first base, with a step length of 5 to 100 bp. The AT base content is calculated for the sequence contained in the window position each time, and the region with an AT base content greater than 60% is retained as the target sequence. Preferably, the region with an AT base content of 60-80% is retained as the target sequence.

[0026] In step (1) of the present invention, for the Salmonella genome sequence, a window with a width of 1000 bp is slid from the first base, with a step length of 5 to 100 bp. The AT base content is calculated for the sequence contained in the window position each time, and the region with an AT base content greater than 60% is retained as the target sequence. Preferably, the region with an AT base content of 60-80% is retained as the target sequence.

[0027] In step (2) of the present invention, the primer design method comprises: (2.1) designing a single primer for a target sequence to obtain a candidate primer; (2.2) determining the physical and chemical properties of the candidate primer to screen for 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; and (2.5) outputting primer pairs that meet the conditions to obtain the specific primers.

[0028] In the present invention, any programming language capable of realizing high-throughput primer design can be used to implement it, such as C, Perl and other programming languages ​​with strong operability and high speed.

[0029] In step (2.1), candidate primers are designed for the target sequence, and the candidate primers must meet the following conditions: a) the primer sequence length is between 20bp and 36bp; b) the AT base content is between 55% and 80%, and c) the number of consecutive GCs is ≤5; at the same time, the position information, positive and negative strand information of the candidate primer matching the target sequence are recorded.

[0030] Preferably, the AT base content is 60% to 75%.

[0031] The "number of consecutive GCs" refers to the number of consecutive base Gs or consecutive base Cs in the primer sequence, such as the number of consecutive GCs in the primer sequence AAGGGGGTTCCAGGCATTA (SEQ ID NO. 15) is 5 / 2 / 2.

[0032] In step (2.2), the physicochemical properties of the single primers that meet the above conditions (2.1) are determined, including but not limited to physicochemical properties such as 3' end stability, 5' end stability and / or secondary structure stability, and the single primers that meet the requirements are retained.

[0033] The “single primer that meets the requirements” refers to a single primer that has 3' end stability, 5' end stability and secondary structure stability.

[0034] In step (2.3), the single primers screened in the above step (2.2) are combined into primer pairs according to their position information and positive and negative strand information on the target sequence; the length of the theoretical amplification product sequence of the primer pair should be between 200bp and 600bp. The Tm value of the single primer is calculated according to the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length); the primer pairs are screened under the conditions that the Tm difference of the primer pair is ≤3°C and the primers cannot interact with each other to obtain candidate primer pairs. Among them, the GC percentage refers to the percentage of the number of bases G and C in the primer to the total number of bases in the primer; the primer sequence length refers to the number of bases in the primer.

[0035] In step (2.4), the universality and specificity of each primer in the candidate primer pair obtained in step (3) are determined. The universality determination refers to checking whether the primer pair strictly matches all target sequences (such as the genomes of multiple strains of Salmonella);

[0036] Specificity determination refers to checking whether a single primer cannot specifically match non-target sequences other than all target sequences, wherein 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.

[0037] Candidate primers that pass the universality and specificity test can proceed to the next step.

[0038] In step (2.5), the primer pair that meets the conditions is output, that is, the primer pair for the nucleic acid amplification reaction is obtained.

[0039] In step (3) of the present invention, the reaction denaturation temperature is calculated by the formula of 0.357×(GC percentage)×100+70.582-(990 / theoretical amplification product sequence length of the primer pair), denoted as Tma; wherein the GC percentage refers to the percentage of the number of bases G and C in the theoretical amplification product sequence of the primer pair to the total number of bases in the theoretical amplification product sequence of the primer pair.

[0040] 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), which is recorded as Tmb. That is, the Tm value of a single primer is calculated according to the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), and the average value of the Tm value is the reaction annealing temperature, which is recorded as Tmb; wherein the GC percentage refers to the percentage of the number of bases G and C in the primer to the total number of bases in the primer.

[0041] In step (3) of the present invention, the nucleic acid amplification reaction conditions include: reacting at a denaturation temperature for 5 seconds, reacting at an annealing temperature for 5 seconds, and reacting at an extension temperature for 20 seconds, and the above process is repeated 30-40 times. The denaturation temperature can be adjusted between Tma±5°C as needed; the annealing temperature can be adjusted between Tmb±2°C as needed; and the extension temperature is 72°C.

[0042] The present invention also provides application of the method in nucleic acid amplification of a target nucleic acid sequence rich in AT region.

[0043] The present invention also provides a primer pair obtained by the design method as described above.

[0044] In the present invention, the primer pair is:

[0045] Primer pair A:

[0046] Sal-F1: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.1)

[0047] Sal-R1: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.2)

[0048] The present invention also provides the use of the above method or the above primer pair in amplification and / or detection of Salmonella-related genes or regions.

[0049] The present invention also provides the use of the primer pair as described above in preparing a product for amplifying and / or detecting a gene or region related to Salmonella.

[0050] The present invention also provides the use of the above method in the simultaneous detection of any two or more combinations of Salmonella, Staphylococcus aureus, Cronobacter sakazakii, and Yersinia enteritidis.

[0051] Specifically, the present invention also provides the use of the above method in the simultaneous detection of Salmonella and any one or more combinations of Staphylococcus aureus, Cronobacter sakazakii, and Yersinia enteritidis.

[0052] The present invention provides a reagent for diagnosing Salmonella and application thereof in detecting Salmonella. The diagnostic reagent comprises the primer pair as described above.

[0053] The present invention also provides a diagnostic reagent and its use in detecting Salmonella, wherein the diagnostic reagent comprises the primer pair as described above.

[0054] The present invention also provides a system for detecting Salmonella, the system comprising:

[0055] A screening module, used for screening target sequences rich in AT bases in the genome sequence of Salmonella;

[0056] A primer design module, which is used to design primers that are both universal and specific for the target sequence;

[0057] A calculation module is used to calculate the reaction denaturation temperature and annealing temperature based on the percentage of GC, the primer sequence length, and the theoretical amplification product sequence length of the primer pair, and to set the nucleic acid amplification reaction conditions;

[0058] The amplification module is used to perform a nucleic acid amplification reaction under the condition of partial melting of the template to obtain an amplification product.

[0059] The beneficial effect of the present invention is that the present invention targets sequences rich in AT bases that are usually avoided by traditional nucleic acid amplification methods, greatly expanding the candidate target region; through specific screening based on massive genomic data, and accurate calculation of the Tm value of the AT-rich region, double-stranded DNA is locally unwound, greatly reducing the possibility of nonspecific amplification that is prevalent in nucleic acid amplification technology. This method effectively expands the application scope of traditional nucleic acid amplification technology, and at the same time significantly improves the reaction performance in terms of primer design flux, reaction specificity, etc., which can meet the needs of nucleic acid detection, molecular genetics research, and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The figure is an amplification curve of the real-time fluorescence quantitative PCR at a suitable temperature for the nucleic acid amplification reaction of the Salmonella primer pair Sal-F1 / R1 of the present invention.

[0061] Figure 2 It is the melting curve of the real-time fluorescence quantitative PCR at a suitable temperature for the nucleic acid amplification reaction of Salmonella based on the primer pair Sal-F1 / R1 of the present invention.

[0062] Figure 3 The figure is an agarose gel electrophoresis at a suitable temperature for the nucleic acid amplification reaction of Salmonella based on the primer pair Sal-F1 / R1 of the present invention.

[0063] Figure 4 The figure is the sensitivity of the Salmonella primer pair Sal-F1 / R1 of the present invention for real-time fluorescence quantitative PCR using four Salmonella species as templates and denatured at 80°C.

[0064] Figure 5The figure is based on the sensitivity of the Salmonella primer pair Sal-F1 / R1 of the present invention to real-time fluorescence quantitative PCR using four Salmonella species as templates denatured at 81°C.

[0065] Figure 6-1 The sensitivity of the Salmonella primer pair Sal-F1 / R1 of the present invention for PCR with four Salmonella species as templates and denatured at 80° C. detected by the fluorescent dye method.

[0066] Figure 6-2 The sensitivity of the Salmonella primer pair Sal-F1 / R1 of the present invention for PCR with four Salmonella species as templates and denatured at 81° C. detected by the fluorescent dye method.

[0067] Figure 7-1 The sensitivity of the Salmonella primer pair Sal-F1 / R1 of the present invention for PCR with four Salmonella species as templates and denatured at 80° C. detected by agarose gel electrophoresis.

[0068] Figure 7-2 The sensitivity of the Salmonella primer pair Sal-F1 / R1 of the present invention for PCR with four Salmonella species as templates and denatured at 81° C. detected by agarose gel electrophoresis.

[0069] Figure 8 It is the fluorescent dye staining result of nucleic acid amplification specific detection based on the nucleic acid amplification reaction of Salmonella primer pair Sal-F1 / R1 of the present invention at a denaturation temperature of 81°C.

[0070] Fig. 9 It is the dye color development result of nucleic acid amplification specific detection of Cronobacter sakazakii at different denaturation temperatures based on the present invention.

[0071] Fig.10 It is the electrophoresis result of the amplification product based on the nucleic acid amplification specific detection of Cronobacter sakazakii at 94°C denaturation temperature of the present invention.

[0072] Fig.11 It is the electrophoresis result of the amplification product based on the nucleic acid amplification specific detection of Cronobacter sakazakii at 82°C denaturation temperature of the present invention.

[0073] Fig.12The fluorescent dye staining results of the nucleic acid amplification reaction of four bacteria, namely, Salmonella enteritidis subspecies, Staphylococcus aureus subspecies, Yersinia enterocolitica, and Cronobacter sakazakii, are shown in the present invention.

[0074] Fig.13 The agarose gel electrophoresis results of the nucleic acid amplification reaction of four bacteria, namely, Salmonella enteritidis subspecies, Staphylococcus aureus subspecies, Yersinia enterocolitica, and Cronobacter sakazakii, were obtained in the present invention.

[0075] Fig.14 The results of SYBRGreen I dye color development for specific detection of four bacteria, Salmonella enterica, Staphylococcus aureus, Yersinia enterocolitica and Cronobacter sakazakii, at a denaturation temperature of 81°C according to the present invention are shown.

[0076] Fig.15 The results of agarose gel electrophoresis of nucleic acid amplification reaction of two bacteria, Salmonella enteritidis subspecies and Staphylococcus aureus subspecies, are shown in the present invention.

[0077] Fig.16 The results are agarose gel electrophoresis results of nucleic acid amplification reaction of two bacteria, Salmonella enteritidis subspecies and Yersinia enterocolitica, according to the present invention.

[0078] Fig.17 The results are agarose gel electrophoresis results of nucleic acid amplification reaction of three bacteria, namely, Salmonella enteritidis subspecies, Staphylococcus aureus subspecies and Yersinia enteritidis, according to the present invention. DETAILED DESCRIPTION

[0079] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0080] The present invention also provides a system for detecting Salmonella, the system comprising:

[0081] A screening module, used for screening target sequences rich in AT bases in the genome sequence of Salmonella;

[0082] A primer design module, which is used to design primers that are both universal and specific for the target sequence;

[0083] A calculation module is used to calculate the reaction denaturation temperature and annealing temperature based on the percentage of GC, the primer sequence length, and the theoretical amplification product sequence length of the primer pair, and to set the nucleic acid amplification reaction conditions;

[0084] The amplification module is used to perform a nucleic acid amplification reaction under the condition of partial melting of the template to obtain an amplification product.

[0085] Example 1 Detection of Salmonella

[0086] The present invention screens AT-rich sequences for the Salmonella genome and designs specific primers, sets reaction conditions for nucleic acid amplification according to the calculated primer Tm and target sequence Tm, and determines whether the target sequence exists in the sample to be tested by judging whether the reaction result is positive, thereby determining whether Salmonella exists in the sample to be tested. The specific steps are as follows:

[0087] (1) Screening of target sequences rich in AT bases:

[0088] The data of bacteria, archaea and viruses with complete genome sequences downloaded from NCBI's FTP on August 5, 2019, totaling 2896 whole genome sequences. Set A, which contains all Salmonella genome sequences; set B, which contains all non-Salmonella genome sequences. The Salmonella genome sequence is used as the reference genome, and a window with a sliding width of 1000bp is slid from the first base of the genome with a step length of 50bp; the sequence AT base content is calculated once before each sliding, and the region with a sequence AT base content greater than 60% is retained as the candidate target sequence. The above process is implemented using a perl script.

[0089] (2) Design of specific primers:

[0090] According to the characteristics of PCR primers and the requirements of the present invention, the AT base content of the primers is set to 55% to 75%, the 3' end stability ΔG <4, the 5' end stability ΔG <3 and the primer sequence length (20 to 36 bp) and other characteristic parameters, and at the same time, the conditions that a single primer cannot produce a hairpin structure and cannot interact with itself are set, and the candidate target sequence in step (1) is used as the candidate sequence for the design of a single primer, and a single primer that meets the above set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), and the position of each primer on the target sequence, the positive and negative chain information (i.e., whether it is derived from the positive chain or the negative chain) and the length of the primer are recorded.

[0091] Primers were paired according to the position information of a single primer, and primer pairs that simultaneously met the two conditions of primer pair Tm difference <3°C and primer pair amplification region between 200 and 600 bp were retained as candidate primer pairs.

[0092] Using the alignment software Bowtie, each primer in the candidate primer pair designed in the previous step is aligned with the target genome sequence in set A and the non-target genome in set B. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in set A, the parameter setting "-an 0" is used, that is, the single primer is required to completely match the target sequence; in order to ensure the specificity of the primer, when the single primer is aligned with the non-target sequence in set B, the parameter setting "-an 3" is used, that is, the single primer is allowed to have no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, where the number of primer pairs can be preset, and in this embodiment, the number of primer pairs is preset to 20. The above high-throughput, automated primer design process is implemented using C and Perl scripts.

[0093] After the program was run, 20 PCR primer pairs were designed in the AT-rich region, and one primer set was randomly selected for validity verification. The sequence of the primer pair was:

[0094] Sal-F1: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.1)

[0095] Sal-R1: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.2)

[0096] The AT base contents of the primers are 60% and 55% respectively, and the AT percentage of the theoretical amplification product sequence of the primer pair is 74.43%.

[0097] Calculation of PCR reaction denaturation temperature and annealing temperature:

[0098] Using the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), it can be calculated that the Tm of primer Sal-F = 62.18°C, the Tm of Sal-R = 64.51°C, and the average Tm value of this primer pair is 63.35°C; using the formula 0.357×(GC percentage)×100+70.582-(990 / amplification product sequence length), the denaturation temperature of the amplification region is calculated to be 76.90°C.

[0099] (4) Nucleic acid amplification reaction and result detection:

[0100] The configuration of the nucleic acid amplification reaction system is shown in Table 1. Based on the calculation results, the 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 objects at the applicable denaturation temperature was tested; and the detection limit at the applicable denaturation temperature was tested. Denaturation for 5 seconds, annealing at 60°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 specific detection objects is shown in Table 2.

[0101] After the reaction is completed using an ordinary gradient PCR instrument, the amplification result can be determined in two ways. One is to add SYBRGreen I dye at 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 determine whether the amplification result is positive based on the electrophoresis bands, that is, whether the target sequence exists in the sample to be tested.

[0102] Table 1. Salmonella nucleic acid amplification reaction system

[0103] system Volume (μl) Final concentration <![CDATA[R300 MIX(TaKaRaTaq TM HS Perfect Mix)]]> 12.5 / Sal-F / Sal-R (10 μM) 1.25+1.25 0.5μM 100% DMSO 0.5 2％ DNA template 0 / 1 / ddH2O Up to 25 /

[0104] Table 2. List of specific detection targets for Salmonella nucleic acid amplification reaction

[0105]

[0106]

[0107] Among them, 1 to 9 are Staphylococcus aureus, Staphylococcus aureus subspecies, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria innocua, and Listeria ivanovii, and 14 to 30 are Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, and diarrheagenic Escherichia coli. Escherichia coli, enterotoxigenic Escherichia coli, enterotoxigenic Escherichia coli, hemorrhagic Escherichia coli, Cronobacter sakazakii, Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio flexneri, Vibrio cholerae and Shigella sonnei, N: negative control; 10-13 are Salmonella enteritidis subspecies, Salmonella enteritidis, Salmonella typhimurium and Salmonella paratyphi B, respectively.

[0108] Figures 1 to 3The amplification curve, melting curve and agarose gel electrophoresis result of the real-time fluorescence quantitative PCR of the nucleic acid amplification reaction for Salmonella at a suitable temperature of the present invention are shown, wherein "80°C" and "81°C" refer to the amplification results of the positive template at the denaturation temperature, "NTC" or "N" refers to the corresponding negative template amplification result, and "M" refers to Marker DL2000. Figure 1 The amplification of positive and negative templates of Salmonella Enteritidis subspecies, Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Paratyphi B was as expected. Figure 2 The melting curves of S. enteritidis subspecies, S. enteritidis, S. typhimurium and S. paratyphi B all showed a single peak without non-specific reaction. Figure 3 The left and right sides of "M" are the results of amplification electrophoresis at 81°C and 82°C denaturation respectively. If there is a single band at 352bp after electrophoresis of the amplified product, it is positive; if there is no band after electrophoresis of the amplified product, it is negative.

[0109] Figure 4 Figures 7 to 8 show three detection limit detection methods for the nucleic acid amplification reaction of Salmonella at denaturation temperatures of 80°C and 81°C. Figure 4 / 5 and Table 3, 10 ng to 10 fg were diluted 10 times; in the range of 10 ng to 100 pg, the amplification curve differed by more than 3 circles for every 10-fold difference, and the linearity was slightly worse below 10 pg. Therefore, above 10 pg, a standard curve was made and quantitative determination could be performed; Figure 6 shows the sensitivity after adding SYBRGreen I dye at a final concentration of 25x, 10 ng to 10 fg were diluted 10 times, and the staining results were judged as positive in the range of 10 ng to 100 pg ( Figure 6-1 and Figure 6-2 Figure 7 shows the sensitivity of the Salmonella primer pair Sal-F1 / R1 of the present invention to PCR with four Salmonella species as templates and denatured at 80°C and 81°C detected by the fluorescent dye method. Figure 7 shows the experimental results of agarose gel electrophoresis of the amplified product. There is a single band at 352 bp, which is judged as positive ( Figure 7-1 and Figure 7-2 They are respectively the sensitivity of the Salmonella primer pair Sal-F1 / R1 of the present invention for PCR with four Salmonella species as templates denatured at 80° C. and 81° C. detected by agarose gel electrophoresis).

[0110] Table 3 Figure 4 / 5Summary of sensitivity of real-time fluorescence quantitative PCR

[0111]

[0112]

[0113] Figure 8 The SYBRGreen I dye color development result of the nucleic acid amplification reaction of the present invention for specific detection of Salmonella at a denaturation temperature of 81° C. If the amplification product is bright green, it is positive; if the amplification product is orange, it is negative. Figure 8 In the test, the negative control (N) was orange, indicating a negative result, which was in line with expectations; the positive control (P) was green, indicating a positive result, which was in line with expectations. Enteritidis Salmonella subspecies 10 to 13, Enteritidis Salmonella, Salmonella Typhimurium and Salmonella Paratyphi B were bright green, indicating positive results, which were in line with expectations. At the same time, the detection results of other bacterial genomic DNA templates were all negative, as shown in tubes 1 to 9 and 14 to 30. All results were in line with expectations, indicating that no nonspecific amplification occurred at a denaturation temperature of 81°C, or even if a trace amount of nonspecific amplification occurred, it was not enough to affect the determination of the dye method results. Example 2 Detection of Cronobacter sakazakii

[0114] The genome for which the primers are designed in the present invention is changed to the genome of Cronobacter sakazakii, AT-rich sequences are screened and specific primers are designed, reaction conditions are set according to the calculated primer Tm and target sequence Tm 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 then whether Cronobacter sakazakii exists in the sample to be tested. The specific steps are as follows:

[0115] (1) Screening of target sequences rich in AT bases:

[0116] The data of bacteria, archaea and viruses with complete genome sequences downloaded from NCBI's FTP on August 5, 2019, totaling 2896 whole genome sequences. Set A, which contains all Cronobacter sakazakii genome sequences; 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 1000bp from the first base of the genome, and the step length is 50bp; calculate the sequence AT base content once before each slide, and retain the region with a sequence AT base content greater than 60% as the candidate target sequence. The above process is implemented using perl scripts.

[0117] (2) Design of specific primers:

[0118] According to the characteristics of PCR primers and the requirements of the present invention, the AT base content of the primers is set to 55% to 75%, the 3' end stability ΔG <4, the 5' end stability ΔG <3 and the primer sequence length (20 to 36 bp) and other characteristic parameters, and at the same time, the conditions that a single primer cannot produce a hairpin structure and cannot interact with itself are set, and the candidate target sequence in step (1) is used as the candidate sequence for the design of a single primer, and a single primer that meets the above set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), and the position of each primer on the target sequence, the positive and negative chain information (i.e., whether it is derived from the positive chain or the negative chain) and the length of the primer are recorded.

[0119] Primers were paired according to the position information of a single primer, and primer pairs that simultaneously met the two conditions of primer pair Tm difference <3°C and primer pair amplification region between 200 and 600 bp were retained as candidate primer pairs.

[0120] Using the alignment software Bowtie, each primer in the candidate primer pair designed in the previous step is aligned with the target genome sequence in set A and the non-target genome in set B. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in set A, the parameter setting "-an 0" is used, that is, the single primer is required to completely match the target sequence; in order to ensure the specificity of the primer, when the single primer is aligned with the non-target sequence in set B, the parameter setting "-an 3" is used, that is, the single primer is allowed to have no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, where the number of primer pairs can be preset, and in this embodiment, the number of primer pairs is preset to 20. The above high-throughput, automated primer design process is implemented using C and Perl scripts.

[0121] After the program was run, 20 PCR primer pairs were designed in the AT-rich region, and one primer set was randomly selected for validity verification. The sequence of the primer pair was:

[0122] Cro-F: 5'-CGCCATAACTGCATAATCAT-3' (SEQ ID NO.3)

[0123] Cro-R: 5'-ATAACGAGTTACCGTGCAGA-3' (SEQ ID NO.4)

[0124] The AT base contents of the primers are 60% and 55% respectively, and the AT percentage content of the theoretical amplification product sequence of the primer pair is 69%.

[0125] (3) Calculation of PCR reaction denaturation temperature and annealing temperature:

[0126] Using the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), it can be calculated that the Tm of primer Cro-F = 62.2°C, the Tm of Cro-R = 64.5°C, and the average Tm value of this primer pair is 63.4°C; using the formula 0.357×(GC percentage)×100+70.582-(990 / amplification product sequence length), the denaturation temperature of the amplification region is calculated to be 78.3°C.

[0127] (4) Nucleic acid amplification reaction and result detection:

[0128] The configuration of the nucleic acid amplification reaction system is shown in Table 4. According to the calculation results, the experiment was set up with 3 groups, respectively testing the specificity of primers for different detection objects at 94℃ / 90℃ / 82℃ denaturation temperatures. The nucleic acid amplification reaction conditions were 94℃ / 90℃ / 82℃ denaturation for 5 seconds, 63℃ annealing for 5 seconds, and 72℃ extension for 20 seconds. The above process was repeated 35 times. The list of specific detection objects is shown in Table 5.

[0129] After the reaction is completed, the amplification results are determined in two ways: one is to add SYBRGreen I dye at 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 determine whether the amplification result is positive based on the electrophoresis band, that is, whether the target sequence exists in the sample to be tested.

[0130] Table 4. Cronobacter sakazakii nucleic acid amplification reaction system

[0131] system Volume (μl) Final concentration <![CDATA[R300 MIX(TaKaRaTaq TM HS Perfect Mix)]]> 12.5 / Cro-F / Cro-R (50μM) 0.25+0.25 0.5μM 100% DMSO 0.5 2％ DNA template (10ng) 0 / 1 / ddH2O Up to 25 /

[0132] Table 5. List of specific detection targets for Cronobacter sakazakii nucleic acid amplification reaction

[0133] 1. Staphylococcus aureus 21600 16. Shigella flexneri 1.1868 2. Staphylococcus aureus subspecies 1.2465 17. Escherichia coli 10738 3. Staphylococcus epidermidis 1.4260 18. Pathogenic Escherichia coli 10372 4. Rhodococcus equi 1.4262 19. Diarrhea-causing 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. Hemorrhagic Escherichia coli 21530 8. Listeria innocua 10417 23. Bacillus sakazakii 21560 9. Listeria monocytogenes 21663 24. Yersinia enterocolitica 21669 10. Salmonella Enteritidis subspecies 1.1859 25. Yersinia pseudotuberculosis 53504 11. Salmonella Enteritidis 21482 26. Vibrio vulnificus 21615 12. Salmonella Typhimurium 10420 27. Vibrio parahaemolyticus 1.1997 13. Salmonella paratyphi B 10437 28. Vibrio freundii 1.1613 14. Shigella dysenteriae 1.1869 29. Vibrio cholerae 1.8676 15. Shigella boydii 1.10618 30. Shigella flexneri

[0134] The experimental results are shown in Fig. 9 , 10, 11, where 1-22 are Staphylococcus aureus, Staphylococcus aureus aureus subspecies, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Salmonella enteritidis subspecies, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (including meat toxin-producing Clostridium type A gene), pathogenic Escherichia coli, diarrheagenic Escherichia coli, enterotoxigenic Escherichia coli, enterotoxigenic Escherichia coli, hemorrhagic Escherichia coli, 24-30 are Yersinia enterocolitica, Yersinia pseudotuberculosis, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio flexneri, Vibrio cholerae and Shigella flexneri, N: negative control, P: positive control (plasmid containing the target sequence); 23 is Cronobacter sakazakii.

[0135] Fig. 9 The SYBRGreen I dye color development results of the nucleic acid amplification reaction of Cronobacter sakazakii at different denaturation temperatures of the present invention are shown. If the amplification product is bright green, it is positive; if the amplification product is orange, it is negative. Fig. 9 In the experiment, when the denaturation temperature was 94℃ / 90℃, the negative control (N) showed orange, which was a negative result, as expected; the positive control (P) and No. 23 Cronobacter sakazakii showed bright green, which was a positive result, as expected. This shows that the entire reaction system can work normally. However, a large number of positive results appeared in the amplification products of other bacterial genomic DNA templates, such as No. 3, 4, 10-18, 20-22, 24, 26, 27, 29, and 30 tubes under the denaturation temperature of 94℃, and No. 3, 7, 10-18, 20-22, 24, 26, 27, 29, and 30 tubes under the denaturation temperature of 90℃, which was inconsistent with expectations, indicating that nonspecific amplification is easy to occur at the denaturation temperature of 94℃ / 90℃, resulting in false positive results. When the denaturation temperature was 82℃, the negative control (N) showed orange, which was a negative result, as expected; the positive control (P) and No. 23 Cronobacter sakazakii showed bright green, which was a positive result, as expected. This indicates that the entire reaction system can work normally. At the same time, the test results of other bacterial genomic DNA templates were all negative, as shown in tubes 1 to 22 and 24 to 30. All results were in line with expectations, indicating that no nonspecific amplification occurred at the denaturation temperature of 82°C, or even if a trace amount of nonspecific amplification occurred, it was not enough to affect the determination of the dye method results.

[0136] Fig.10The electrophoresis results of the amplification products of the present invention for specific detection of Cronobacter sakazakii nucleic acid amplification reaction at a denaturation temperature of 94°C are shown. If there is a single band at 291 bp after electrophoresis of the amplification product, it is positive; if there is no band after electrophoresis of the amplification product, it is negative; if one or more bands appear outside 291 bp after electrophoresis of the amplification reaction product, it is a false positive caused by non-specific amplification. Fig.10 In the experiment, the negative control (N) had no band, which was in line with expectations; the positive control (P) and Cronobacter sakazakii 23 had a clear band at 291bp, which was in line with expectations. This showed that the entire reaction system could work normally. However, the amplification products of other bacterial genomic DNA templates showed multiple bands in the range of 500bp to 2000bp, which was inconsistent with expectations, indicating that the reaction system produced a large amount of nonspecific amplification at the 94℃ denaturation temperature. This result was consistent with Fig. 9 The dye color development results are consistent.

[0137] Fig.11 The electrophoresis results of the amplification products of the present invention for specific detection of Cronobacter sakazakii nucleic acid amplification reaction at a denaturation temperature of 82°C are shown. If there is a single band at 291 bp after electrophoresis of the amplification product, it is positive; if there is no band after electrophoresis of the amplification reaction product, it is negative; if one or more bands appear outside 291 bp after electrophoresis of the amplification reaction product, it is a false positive caused by non-specific amplification. Fig.11 In the experiment, the negative control (N) had no band, which was in line with expectations; the positive control (P) and Cronobacter sakazakii No. 23 had a clear band at 291bp, which was in line with expectations. This shows that the entire reaction system can work normally. At the same time, most of the amplification reaction products of other bacterial genomic DNA templates had no bands and showed negative results, as shown in lanes 2-6, 10, 12, 13, 15-22, and 26-30; a few template amplification reaction products showed very weak bands after electrophoresis, which were easy to distinguish from positive results, as shown in tubes 1, 7, 8, 9, 11, 14, 24, and 25, indicating that no or a trace amount of nonspecific amplification that was easy to distinguish in the electrophoresis results was produced at the denaturation temperature of 82°C.

[0138] comprehensive Figure 9-11 As can be seen from Table 5, the nucleic acid amplification method of the present invention has good strain specificity for Cronobacter sakazakii, that is, under the reaction system and reaction conditions proposed by the present invention, only Cronobacter sakazakii presents a positive result, while non-Cronobacter sakazakii presents a negative result. It is worth noting that when the denaturation temperature (90°C or 94°C) of the traditional PCR method is used for the reaction, although the electrophoresis results ( Fig.10) shows that the target detection object (Cronobacter sakazakii, lane 23) has a single clear band, while non-target detection objects generally show a large amount of non-specific amplification, such as Staphylococcus epidermidis in lane 3; if the dye colorimetric method is used to determine the test results, such as Fig. 9 As shown in the figure, under the denaturation conditions of 90°C and 94°C, a large number of false positive results are presented, making it impossible to distinguish between the detected object and the non-detected object. Under the same reaction system, when the denaturation temperature calculated by the present invention is 82°C, the electrophoresis results ( Fig.11 ) shows that the target detection object (Cronobacter sakazakii, lane 23) has a single clear band, while non-target detection objects rarely have non-specific amplification. Even if there is, the non-specific amplification band is very weak and can be easily distinguished from the positive result. If the dye colorimetric method is used to determine the test results, such as Fig. 9 As shown, under the denaturation condition of 82°C, only the target detection object (Cronobacter sakazakii, tube 23) showed a positive result, and the non-target detection objects all showed negative results, showing good discrimination.

[0139] Example 3 Simultaneous detection of Salmonella, Staphylococcus aureus, Yersinia enteritidis and Cronobacter sakazakii

[0140] The genomes targeted by the primer design in the present invention are changed to the genomes of Salmonella, Staphylococcus aureus, Yersinia enterica and Cronobacter sakazakii, AT-rich sequences are screened and specific primers are designed, primer combinations are screened according to the calculated primer Tm and target sequence Tm, 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 then it is determined whether one or more of Salmonella, Staphylococcus aureus, Yersinia enterica and Cronobacter sakazakii exists in the sample to be tested. The specific steps are as follows:

[0141] (1) Screening of target sequences rich in AT bases:

[0142] The data of bacteria, archaea and viruses with complete genome sequences downloaded from NCBI's FTP on August 5, 2019, totaling 2896 whole genome sequences. Set A, which contains all Salmonella genome sequences; set B, which contains all non-Salmonella genome sequences. The Salmonella genome sequence is used as the reference genome, and a window with a sliding width of 1000bp is slid from the first base of the genome with a step length of 50bp; the sequence AT base content is calculated once before each sliding, and the region with a sequence AT base content greater than 60% is retained as the candidate target sequence. The above process is implemented using a perl script.

[0143] (2) Design of specific primers:

[0144] According to the characteristics of PCR primers and the requirements of the present invention, the AT base content of the primers is set to 55% to 75%, the 3' end stability ΔG <4, the 5' end stability ΔG <3 and the primer sequence length (20 to 36 bp) and other characteristic parameters, and at the same time, the conditions that a single primer cannot produce a hairpin structure and cannot interact with itself are set, and the candidate target sequence in step (1) is used as the candidate sequence for the design of a single primer, and a single primer that meets the above set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), and the position of each primer on the target sequence, the positive and negative chain information (i.e., whether it is derived from the positive chain or the negative chain) and the length of the primer are recorded.

[0145] Primers were paired according to the position information of a single primer, and primer pairs that simultaneously met the two conditions of primer pair Tm difference <3°C and primer pair amplification region between 200 and 600 bp were retained as candidate primer pairs.

[0146] Using the alignment software Bowtie, each primer in the candidate primer pair designed in the previous step is aligned with the target genome sequence in set A and the non-target genome in set B. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in set A, the parameter setting "-an 0" is used, that is, the single primer is required to completely match the target sequence; in order to ensure the specificity of the primer, when the single primer is aligned with the non-target sequence in set B, the parameter setting "-an 3" is used, that is, the single primer is allowed to have no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, wherein the number of primer pairs can be preset, and in this embodiment, the number of primer pairs is preset to 100. The above high-throughput, automated primer design process is implemented using C and Perl scripts. After the program is run, 100 Salmonella amplification primer pairs are designed in the AT-enriched area.

[0147] By the same method, 100 primer pairs for amplification of Staphylococcus aureus, Yersinia enteritidis, and Cronobacter sakazakii were designed. The Tm of each primer was calculated using the formula 0.466×(GC percentage)×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 amplification region of each primer pair was calculated using the formula 0.357×(GC percentage)×100+70.582-(990 / amplification product sequence length).

[0148] The primer combinations are screened based on the conditions that the length of the amplified sequences of different bacteria differ by at least 50 bp, the reaction denaturation temperature differs by at most 2°C, and the reaction annealing temperature differs 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 is preset to 10. A primer set is randomly selected for validity verification. The sequence of the primer combination is:

[0149] Primer pair A:

[0150] Sal-F1: 5'-TCAGACATCCGTTCAGAAAAT-3' (SEQ ID NO.1)

[0151] Sal-R1: 5'-GTTCAACTGTCGACAAGATTAA-3' (SEQ ID NO.2)

[0152] Primer pair B:

[0153] Sta-F: 5'-GTAGGTATGGTAAATAGTTACAC-3' (SEQ ID NO.5)

[0154] Sta-R:5'-CACTAATGCCAAATTTACTTAAAATCG-3'(SEQ ID NO.6)

[0155] Primer pair C:

[0156] Cro-F: 5'-CGCCATAACTGCATAATCAT-3' (SEQ ID NO.3)

[0157] Cro-R:5'-ATAACGAGTTACCGTGCAGA-3'(SEQ ID NO.4)

[0158] Primer pair D:

[0159] Yer-F: 5'-TGTGCGGTGGATGTAAAATAATTC-3' (SEQ ID NO.7)

[0160] Yer-R:5'-GCTTTGAAACTCAAGGACTG-3'(SEQ ID NO.8)

[0161] 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 amplification product sequence fragment length of the primer pair is 355bp, the AT percentage content is 71%, and the theoretical denaturation temperature is 78.14°C.

[0162] 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 amplification product sequence fragment length of the primer pair is 465bp, the AT percentage is 73.76%, and the theoretical denaturation temperature is 77.82°C.

[0163] 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 amplification product sequence fragment length of the primer pair is 291bp, the AT percentage is 68.73%, and the theoretical denaturation temperature is 78.34°C.

[0164] 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 amplification product sequence fragment length of the primer pair is 235bp, the AT percentage is 68.09%, and the theoretical denaturation temperature is 77.76°C.

[0165] (4) Nucleic acid amplification reaction and result detection:

[0166] The configuration of the nucleic acid amplification reaction system is shown in Table 6. According to the calculation results, 81°C denaturation for 5 seconds, 62°C annealing for 5 seconds, and 72°C extension for 20 seconds are used. It is recommended to repeat the above process 35 times. The list of detection objects is shown in Table 7.

[0167] After the reaction is completed using an ordinary gradient PCR instrument, the amplification result can be determined in two ways. One is to add SYBRGreen I dye at 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 determine whether the amplification result is positive based on the electrophoresis bands, that is, whether the target sequence exists in the sample to be tested.

[0168] Table 6. Four bacterial nucleic acid amplification reaction systems

[0169]

[0170]

[0171] Table 7. List of specific detection targets for four bacterial nucleic acid amplification reactions

[0172] 1. Staphylococcus aureus 21600 16. Shigella flexneri 1.1868 2. Staphylococcus aureus subspecies 1.2465 17. Escherichia coli 10738 3. Staphylococcus epidermidis 1.4260 18. Pathogenic Escherichia coli 10372 4. Rhodococcus equi 1.4262 19. Diarrhea-causing 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. Hemorrhagic Escherichia coli 21530 8. Listeria innocua 10417 23. Cronobacter sakazakii 21560 9. Listeria monocytogenes 21663 24. Yersinia enterocolitica 21669 10. Salmonella Enteritidis subspecies 1.1859 25. Yersinia pseudotuberculosis 53504 11. Salmonella Enteritidis 21482 26. Vibrio vulnificus 21615 12. Salmonella Typhimurium 10420 27. Vibrio parahaemolyticus 1.1997 13. Salmonella paratyphi B 10437 28. Vibrio freundii 1.1613 14. Shigella dysenteriae 1.1869 29. Vibrio cholerae 1.8676 15. Shigella boydii 1.10618 30. Shigella sonnei

[0173] Fig.12 and 13The fluorescent dye staining results and agarose gel electrophoresis results of the nucleic acid amplification reaction of four bacteria, namely, Salmonella enteritidis subspecies, Staphylococcus aureus subspecies, Yersinia enterocolitica, and Cronobacter sakazakii, are shown in the present invention. "N" is the corresponding negative template amplification result, "M1" refers to Marker DL2000, "M2" refers to Marker B (100-600bp), and "mixed" refers to the mixed template of the four bacterial genomic DNAs. Fig.12 The amplification and color development results of each single bacteria, mixed template and negative template were in line with expectations. Fig.13 The amplified fragments of each single bacteria were consistent with the theoretical calculated values ​​and the amplified fragments of each bacteria in the mixed template species could be separated from each other.

[0174] Fig.14 The SYBRGreen I dye color development results of the nucleic acid amplification reaction of the present invention for specific detection of four bacteria, Salmonella, Staphylococcus aureus, Yersinia enterocolitica, and Cronobacter sakazakii at a denaturation temperature of 81°C are shown. If the amplified product is bright green, it is positive; if the amplified product is orange, it is negative. Fig.14 Among them, Staphylococcus aureus, Staphylococcus aureus subspecies 1-2, Salmonella enteritidis subspecies, Salmonella enteritidis, Salmonella typhimurium and Salmonella paratyphi B, Cronobacter sakazakii, and Yersinia enterococci, were bright green, which was a positive result, as expected; at the same time, the test results of other bacterial genomic DNA templates were all negative, as shown in tubes 3-9, 14-22, and 25-30. All results were in line with expectations, indicating that no nonspecific amplification was produced at the denaturation temperature of 81°C, or even if a trace amount of nonspecific amplification was produced, it was not enough to affect the determination of the dye method results.

[0175] Example 4 Simultaneous detection of Salmonella and Staphylococcus aureus

[0176] The present invention screens AT-rich sequences for the genomes of Salmonella and Staphylococcus aureus and designs specific primers, screens primer combinations based on the calculated primer Tm and target sequence Tm, sets reaction conditions for nucleic acid amplification, and determines whether the target sequence exists in the sample to be tested by judging whether the reaction result is positive, thereby determining whether one or more of Salmonella and Staphylococcus aureus exists in the sample to be tested. The specific steps are as follows:

[0177] (1) Screening of target sequences rich in AT bases:

[0178] The data of bacteria, archaea and viruses with complete genome sequences downloaded from NCBI's FTP on August 5, 2019, totaling 2896 whole genome sequences. Set A, which contains all Salmonella genome sequences; set B, which contains all non-Salmonella genome sequences. The Salmonella genome sequence is used as the reference genome, and a window with a sliding width of 1000bp is slid from the first base of the genome with a step length of 50bp; the sequence AT base content is calculated once before each sliding, and the region with a sequence AT base content greater than 60% is retained as the candidate target sequence. The above process is implemented using a perl script.

[0179] (2) Design of specific primers:

[0180] According to the characteristics of PCR primers and the requirements of the present invention, the AT base content of the primers is set to 55% to 75%, the 3' end stability ΔG <4, the 5' end stability ΔG <3 and the primer sequence length (20 to 36 bp) and other characteristic parameters, and at the same time, the conditions that a single primer cannot produce a hairpin structure and cannot interact with itself are set, and the candidate target sequence in step (1) is used as the candidate sequence for the design of a single primer, and a single primer that meets the above set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), and the position of each primer on the target sequence, the positive and negative chain information (i.e., whether it is derived from the positive chain or the negative chain) and the length of the primer are recorded.

[0181] Primers were paired according to the position information of a single primer, and primer pairs that simultaneously met the two conditions of primer pair Tm difference <3°C and primer pair amplification region between 200 and 600 bp were retained as candidate primer pairs.

[0182] Using the alignment software Bowtie, each primer in the candidate primer pair designed in the previous step is aligned with the target genome sequence in set A and the non-target genome in set B. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in set A, the parameter setting "-an 0" is used, that is, the single primer is required to completely match the target sequence; in order to ensure the specificity of the primer, when the single primer is aligned with the non-target sequence in set B, the parameter setting "-an 3" is used, that is, the single primer is allowed to have no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, wherein the number of primer pairs can be preset, and in this embodiment, the number of primer pairs is preset to 100. The above high-throughput, automated primer design process is implemented using C and Perl scripts. After the program is run, 100 Salmonella amplification primer pairs are designed in the AT-enriched area.

[0183] 100 primer pairs for Staphylococcus aureus amplification were designed by the same method. The Tm of each primer was calculated using the formula 0.466×(GC percentage)×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 amplification region of each primer pair was calculated using the formula 0.357×(GC percentage)×100+70.582-(990 / amplification product sequence length).

[0184] The primer combinations are screened based on the conditions that the length of the amplified sequences of different bacteria differ by at least 50 bp, the reaction denaturation temperature differs by at most 2°C, and the reaction annealing temperature differs 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 is preset to 10. A primer set is randomly selected for validity verification. The sequence of the primer combination is:

[0185] Primer pair A:

[0186] Sal-F1: 5'-TCAGACATCCGTTCAGAAAAT-3' (SEQ ID NO.1)

[0187] Sal-R1: 5'-GTTCAACTGTCGACAAGATTAA-3' (SEQ ID NO.2)

[0188] Primer pair E:

[0189] Sta-F2: 5'-CCTTTCATCTAAAAACCTCCA-3' (SEQ ID NO.9)

[0190] Sta-R2:5'-GAAATGGATGTTTTAAAAGAAGG-3'(SEQ ID NO.10)

[0191] 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 amplification product sequence fragment length of the primer pair is 355bp, the AT percentage content is 71%, and the theoretical denaturation temperature is 78.14°C.

[0192] 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 amplification product sequence fragment length of the primer pair is 595bp, the AT percentage is 76.30%, and the theoretical denaturation temperature is 77.38°C.

[0193] (4) Nucleic acid amplification reaction and result detection:

[0194] The configuration of the nucleic acid amplification reaction system is shown in Table 8. According to the calculation results, 81°C denaturation for 5 seconds, 62°C annealing for 5 seconds, and 72°C extension for 20 seconds are used. It is recommended to repeat the above process 35 times. The list of detection objects is shown in Table 7.

[0195] After the reaction is completed using an ordinary gradient PCR instrument, the amplified product is subjected to agarose gel electrophoresis to determine whether the amplification result is positive based on the electrophoresis bands, that is, whether the target sequence exists in the sample to be tested.

[0196] Table 8. Two bacterial nucleic acid amplification reaction systems

[0197]

[0198]

[0199] Fig.15 The agarose gel electrophoresis results of the nucleic acid amplification reaction of two bacteria, Salmonella enteritidis subspecies and Staphylococcus aureus subspecies, are shown in the present invention. "N" refers to the negative template amplification result corresponding to it, "M" refers to MarkerDL2000, "Salmonella" refers to Salmonella enteritidis subspecies, "Staphylococcus aureus" refers to Staphylococcus aureus subspecies, and "mixed" refers to the mixed template of the genomic DNA of the two species of bacteria. Fig.15 The amplified fragments of each single bacteria were consistent with the theoretical calculated values ​​and the amplified fragments of each bacteria in the mixed template species could be separated from each other.

[0200] Example 5 Simultaneous detection of Salmonella and Yersinia enteritidis

[0201] The present invention screens AT-rich sequences for the genomes of Salmonella and Yersinia enterica and designs specific primers, screens primer combinations according to the calculated primer Tm and target sequence Tm, sets reaction conditions for nucleic acid amplification, and determines whether the target sequence exists in the sample to be tested by judging whether the reaction result is positive, thereby determining whether one or more of Salmonella and Yersinia enterica exists in the sample to be tested. The specific steps are as follows:

[0202] (1) Screening of target sequences rich in AT bases:

[0203] The data of bacteria, archaea and viruses with complete genome sequences downloaded from NCBI's FTP on August 5, 2019, totaling 2896 whole genome sequences. Set A, which contains all Salmonella genome sequences; set B, which contains all non-Salmonella genome sequences. The Salmonella genome sequence is used as the reference genome, and a window with a sliding width of 1000bp is slid from the first base of the genome with a step length of 50bp; the sequence AT base content is calculated once before each sliding, and the region with a sequence AT base content greater than 60% is retained as the candidate target sequence. The above process is implemented using a perl script.

[0204] (2) Design of specific primers:

[0205] According to the characteristics of PCR primers and the requirements of the present invention, the AT base content of the primers is set to 55% to 75%, the 3' end stability ΔG <4, the 5' end stability ΔG <3 and the primer sequence length (20 to 36 bp) and other characteristic parameters, and at the same time, the conditions that a single primer cannot produce a hairpin structure and cannot interact with itself are set, and the candidate target sequence in step (1) is used as the candidate sequence for the design of a single primer, and a single primer that meets the above set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), and the position of each primer on the target sequence, the positive and negative chain information (i.e., whether it is derived from the positive chain or the negative chain) and the length of the primer are recorded.

[0206] Primers were paired according to the position information of a single primer, and primer pairs that simultaneously met the two conditions of primer pair Tm difference <3°C and primer pair amplification region between 200 and 600 bp were retained as candidate primer pairs.

[0207] Using the alignment software Bowtie, each primer in the candidate primer pair designed in the previous step is aligned with the target genome sequence in set A and the non-target genome in set B. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in set A, the parameter setting "-an 0" is used, that is, the single primer is required to completely match the target sequence; in order to ensure the specificity of the primer, when the single primer is aligned with the non-target sequence in set B, the parameter setting "-an 3" is used, that is, the single primer is allowed to have no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, wherein the number of primer pairs can be preset, and in this embodiment, the number of primer pairs is preset to 100. The above high-throughput, automated primer design process is implemented using C and Perl scripts. After the program is run, 100 Salmonella amplification primer pairs are designed in the AT-enriched area.

[0208] 100 primer pairs for Yersinia enteritidis amplification were designed by the same method. The Tm of each primer was calculated using the formula 0.466×(GC percentage)×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 amplification region of each primer pair was calculated using the formula 0.357×(GC percentage)×100+70.582-(990 / amplification product sequence length).

[0209] The primer combinations are screened based on the conditions that the length of the amplified sequences of different bacteria differ by at least 50 bp, the reaction denaturation temperature differs by at most 2°C, and the reaction annealing temperature differs 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 is preset to 10. A primer set is randomly selected for validity verification. The sequence of the primer combination is:

[0210] Primer pair F:

[0211] Sal-F2: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.11)

[0212] Sal-R2: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.12)

[0213] Primer pair G:

[0214] Yer-F2: 5'-ATGGAAAATAACATAATTTCTATTACCGG-3' (SEQ ID NO.13)

[0215] Yer-R2:5'-TCTCTGCGAATAACCTTGTG-3'(SEQ ID NO.14)

[0216] 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 amplification product sequence fragment length of the primer pair is 352bp, the AT percentage is 74.43%, and the theoretical denaturation temperature is 76.90°C.

[0217] 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 amplification product sequence fragment length of the primer pair is 242bp, the AT percentage is 72.02%, and the theoretical denaturation temperature is 76.48°C.

[0218] (4) Nucleic acid amplification reaction and result detection:

[0219] The configuration of the nucleic acid amplification reaction system is shown in Table 9. According to the calculation results, 81°C denaturation for 5 seconds, 62°C annealing for 5 seconds, and 72°C extension for 20 seconds are used. It is recommended to repeat the above process 35 times. The list of detection objects is shown in Table 7.

[0220] After the reaction is completed using an ordinary gradient PCR instrument, the amplified product is subjected to agarose gel electrophoresis to determine whether the amplification result is positive based on the electrophoresis bands, that is, whether the target sequence exists in the sample to be tested.

[0221] Table 9. Two bacterial nucleic acid amplification reaction systems

[0222] system Volume (μl) Final concentration Taq R300mix 15 1x SAL-F2 / R2 (50 μM) 0.25+0.25 0.5μM YER-F2 / R2 (50 μM) 0.25+0.25 0.5μM 100% DMSO 0.5 2％ Template (10ng) 0 / 1 / ddH2O Upto25 /

[0223] Fig.16 The agarose gel electrophoresis results of the nucleic acid amplification reaction of two bacteria, Salmonella enteritidis subspecies and Yersinia enterococci, are shown in the present invention. "N" refers to the corresponding negative template amplification result, "M" refers to MarkerDL2000, "Salmon" refers to Salmonella enteritidis subspecies, "Yersinia enterococci" refers to Yersinia enterococci subspecies, and "mixed" refers to the mixed template of the genomic DNA of the two species of bacteria. Fig.16 The amplified fragments of each single bacteria were consistent with the theoretical calculated values ​​and the amplified fragments of each bacteria in the mixed template species could be separated from each other.

[0224] Example 6 Simultaneous detection of Salmonella, Staphylococcus aureus and Yersinia enteritidis

[0225] The present invention screens AT-rich sequences for the genomes of Salmonella, Staphylococcus aureus and Yersinia enterica, designs specific primers, screens primer combinations according to the calculated primer Tm and target sequence Tm, sets reaction conditions for nucleic acid amplification, and determines whether the target sequence exists in the sample to be tested by judging whether the reaction result is positive, thereby determining whether one or more of Salmonella, Staphylococcus aureus and Yersinia enterica exists in the sample to be tested. The specific steps are as follows:

[0226] (1) Screening of target sequences rich in AT bases:

[0227] The data of bacteria, archaea and viruses with complete genome sequences downloaded from NCBI's FTP on August 5, 2019, totaling 2896 whole genome sequences. Set A, which contains all Salmonella genome sequences; set B, which contains all non-Salmonella genome sequences. The Salmonella genome sequence is used as the reference genome, and a window with a sliding width of 1000bp is slid from the first base of the genome with a step length of 50bp; the sequence AT base content is calculated once before each sliding, and the region with a sequence AT base content greater than 60% is retained as the candidate target sequence. The above process is implemented using a perl script.

[0228] (2) Design of specific primers:

[0229] According to the characteristics of PCR primers and the requirements of the present invention, the AT base content of the primers is set to 55% to 75%, the 3' end stability ΔG <4, the 5' end stability ΔG <3 and the primer sequence length (20 to 36 bp) and other characteristic parameters, and at the same time, the conditions that a single primer cannot produce a hairpin structure and cannot interact with itself are set, and the candidate target sequence in step (1) is used as the candidate sequence for the design of a single primer, and a single primer that meets the above set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466×(GC percentage)×100+66.04-(450 / primer sequence length), and the position of each primer on the target sequence, the positive and negative chain information (i.e., whether it is derived from the positive chain or the negative chain) and the length of the primer are recorded.

[0230] Primers were paired according to the position information of a single primer, and primer pairs that simultaneously met the two conditions of primer pair Tm difference <3°C and primer pair amplification region between 200 and 600 bp were retained as candidate primer pairs.

[0231] Using the alignment software Bowtie, each primer in the candidate primer pair designed in the previous step is aligned with the target genome sequence in set A and the non-target genome in set B. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in set A, the parameter setting "-an 0" is used, that is, the single primer is required to completely match the target sequence; in order to ensure the specificity of the primer, when the single primer is aligned with the non-target sequence in set B, the parameter setting "-an 3" is used, that is, the single primer is allowed to have no more than 3 mismatches with the non-target sequence. The system outputs primer pairs that meet the conditions, wherein the number of primer pairs can be preset, and in this embodiment, the number of primer pairs is preset to 100. The above high-throughput, automated primer design process is implemented using C and Perl scripts. After the program is run, 100 Salmonella amplification primer pairs are designed in the AT-enriched area.

[0232] 100 primer pairs for Yersinia enteritidis amplification were designed by the same method. The Tm of each primer was calculated using the formula 0.466×(GC percentage)×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 amplification region of each primer pair was calculated using the formula 0.357×(GC percentage)×100+70.582-(990 / amplification product sequence length).

[0233] The primer combinations are screened based on the conditions that the length of the amplified sequences of different bacteria differ by at least 50 bp, the reaction denaturation temperature differs by at most 2°C, and the reaction annealing temperature differs 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 is preset to 10. A primer set is randomly selected for validity verification. The sequence of the primer combination is:

[0234] Primer pair A:

[0235] Sal-F1: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.1)

[0236] Sal-R1: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.2)

[0237] Primer pair D:

[0238] Yer-F: 5'-ATGGAAAATAACATAATTTCTATTACCGG-3' (SEQ ID NO.7)

[0239] Yer-R:5'-TCTCTGCGAATAACCTTGTG-3'(SEQ ID NO.8)

[0240] Primer pair E:

[0241] Sta-F2: 5'-CCTTTCATCTAAAAACCTCCA-3' (SEQ ID NO.9)

[0242] Sta-R2:5'-GAAATGGATGTTTTAAAAGAAGG-3'(SEQ ID NO.10)

[0243] The AT base contents of the primer pair A are 60% and 55% respectively, and the theoretical average annealing temperature is 63°C; the theoretical amplification product sequence fragment length of the primer pair is 352bp, the AT percentage is 74.43%, and the theoretical denaturation temperature is 76.90°C.

[0244] The AT base contents of the primer pair D are 72.41% and 55% respectively, and the theoretical average annealing temperature is 63.5°C; the theoretical amplification product sequence fragment length of the primer pair is 242bp, the AT percentage is 72.02%, and the theoretical denaturation temperature is 76.48°C.

[0245] 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 amplification product sequence fragment length of the primer pair is 595bp, the AT percentage is 76.30%, and the theoretical denaturation temperature is 77.38°C.

[0246] (4) Nucleic acid amplification reaction and result detection:

[0247] The configuration of the nucleic acid amplification reaction system is shown in Table 10. According to the calculation results, 81°C denaturation for 5 seconds, 62°C annealing for 5 seconds, and 72°C extension for 20 seconds are used. It is recommended to repeat the above process 35 times. The list of detection objects is shown in Table 7.

[0248] After the reaction is completed using an ordinary gradient PCR instrument, the amplified product is subjected to agarose gel electrophoresis to determine whether the amplification result is positive based on the electrophoresis bands, that is, whether the target sequence exists in the sample to be tested.

[0249] Table 10. Three bacterial nucleic acid amplification reaction systems

[0250] system Volume (μl) Final concentration Taq R300mix 15 1x SAL-F / R (50μM) 0.25+0.25 0.5μM YER-F / R(50μM) 0.25+0.25 0.5μM STA-F2 / R2 (50 μM) 0.25+0.25 0.5μM 100% DMSO 0.5 2％ Template (10ng) 0 / 1 / ddH2O Upto25 /

[0251] Fig.17 The agarose gel electrophoresis results of the nucleic acid amplification reaction of three bacteria, namely, Salmonella enteritidis subspecies, Staphylococcus aureus subspecies and Yersinia enterococci, are shown. "N" refers to the corresponding negative template amplification result, "M" refers to Marker DL2000, "enteritis subspecies" refers to Salmonella enteritidis subspecies, "Yersinia enterococci" refers to Yersinia enterococci subspecies, "gold subspecies" refers to Staphylococcus aureus subspecies, and "mixed" refers to the mixed template of the three bacterial genomic DNAs. Fig.17 The amplified fragments of each single bacteria were consistent with the theoretical calculated values ​​and the amplified fragments of each bacteria in the mixed template species could be separated from each other.

[0252] References

[0253] Zhou Huiqi. (2014). Relationship between GC content of genome and base, codon and amino acid usage preference. (Mastermaster), University of Electronic Science and Technology of China, (67)

[0254] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims. SEQUENCE LISTING <110> Shanghai Bioinformatics Research Center, Shanghai Want Want Food Group Co., Ltd. <120> A method for detecting Salmonella, its application and primers <160> 15 <170> PatentIn version 3.3 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <400> 1 tcagacatcc gttcagaaaa t 21 <210> 2 <211> twenty two <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> twenty three <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> twenty three <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> twenty one <212> DNA <213> Artificial sequence <400> 9 cctttcatct aaaaacctcc a 21 <210> 10 <211> twenty three <212> DNA <213> Artificial sequence <400> 10 gaaatggatg ttttaaaaga agg 23 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <400> 11 tgggttgaaa tagcccatta 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <400> 12 gacgtgacac acttcgtttt 20 <210> 13 <211> 29 <212> DNA <213> Artificial sequence <400> 13 atggaaaata acataatttc tattaccgg 29 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <400> 14 tctctgcgaa taaccttgtg 20 <210> 15 <211> 19 <212> DNA <213> Artificial sequence <400> 15 aagggggttc caggcatta 19

Claims

1. A primer pair, characterized in that: The primer pairs are: Primer pair A: Sal-F1: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.1) Sal-R1: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO. 2).

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

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

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