Method for detecting multiple bacteria, system for detecting multiple bacteria and application thereof

Abstract

The present invention discloses a method, detection system, and application thereof for simultaneously detecting multiple bacteria. The method comprises screening target sequences rich in AT bases in different bacterial genome sequences; designing primers that are both universal and specific for a single bacterial genome sequence; calculating the reaction denaturation temperature and annealing temperature based on the GC percentage, primer sequence length, and theoretical amplification product sequence length of the primer pair; screening suitable single bacterial primer pairs based on the sequence amplification length, reaction denaturation temperature, and annealing temperature to form a primer set for detecting multiple bacteria; setting nucleic acid amplification reaction conditions, performing a nucleic acid amplification reaction under conditions that partially melt the template, and obtaining an amplified product. The present invention achieves partial melting of double-stranded DNA through specific screening based on massive genomic data and accurate calculation of the Tm value of AT-rich regions, greatly reducing the possibility of nonspecific amplification in nucleic acid amplification technology.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for detecting multiple bacteria, a system for detecting multiple bacteria and applications thereof. The method is a method for performing nucleic acid amplification specifically on genome sequences of multiple bacteria. Background Art

[0002] Nucleic acid amplification technology (NAAT) is a general term for a class of molecular biology techniques that achieve rapid and specific amplification of trace amounts of nucleic acid through reactions involving primers, DNA polymerase, and other reagents at specific temperatures. These techniques are widely used in disease diagnosis, pathogen detection, food safety testing, animal and plant quarantine, and various applications involving molecular cloning, such as sequencing, gene cloning, gene manipulation, allele analysis, and mutation detection. The polymerase chain reaction (PCR) is the earliest NAAT technique to be implemented. It mimics the process of DNA replication in vivo, using a pair of specific oligonucleotide primers that complement the DNA fragment to be amplified. After several cycles of denaturation, annealing, and extension, the DNA fragment is exponentially amplified. 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 shortcomings of early PCR technology, such as the need for continuous manual addition of polymerase due to the inactivation of DNA polymerase during thermal denaturation cycles. This greatly improved the efficiency of nucleic acid amplification and made the technology 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 well known, existing nucleic acid amplification techniques typically avoid GC- or AT-rich regions. This is because these regions are prone to forming hairpin loop secondary structures through self-complementary pairing, which hinders primer-template binding. Even if primers can barely bind to the template, they can easily interfere with DNA polymerase extension along the template, causing "stuttering" during amplification and disrupting DNA synthesis. Therefore, to improve reaction success rates, traditional nucleic acid amplification techniques have a preference for the base composition of the amplified region. Primer design often focuses on regions with a GC content of 40% to 60%, with an optimal range of 45% to 55%. This varies slightly between different amplification techniques. For example, PCR primer design recommends a G+C content of 40% to 60% (see Molecular Cloning Guide, Fourth Edition, Table 7-1, Primer Design), and LAMP primer design recommends a G+C content of 40% to 65% (see A Guide to LAMP Primer Design (PrimerExplorer V3)).

[0004] However, the base composition of nucleic acids varies greatly across organisms in nature. For example, the AT content of the Plasmodium genome is approximately 82%. Zhou Huiqi et al. studied 2,670 bacterial and archaeal strains and found that the GC content of their genomes varied from 14% to 75% (Zhou, 2014). From a detection perspective, the base composition bias of existing nucleic acid amplification technologies overlooks a large number of potential target sequences, increasing the difficulty of successfully detecting target sequences. From a genetic manipulation perspective, this bias makes it difficult to amplify many target sequences, hindering further molecular manipulation. If appropriate primers can be designed to target the imbalanced GC and AT base content regions that traditional nucleic acid amplification methods avoid, avoiding the formation of secondary structures, and if appropriate reaction conditions can be set to facilitate nucleic acid amplification experiments, the success rate of target detection will be significantly increased, allowing more sequences to be amplified and facilitating 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 continuous 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 Tm calculation formula for primers recommended in the fourth edition of the Molecular Cloning Experiment Guide is Tm = 4×(G, C number) + 2×(A, T number), the Tm calculation formula for primers recommended by TaKaRa is Tm = 4×(G, C number) + 2×(A, T number) + 32-2×(total number of bases); the Tm calculation formula for primers of Sangon Biotechnology is Tm(0.05MNa + ) = 59.94 + 1 × (GC percentage) - (675 / primer sequence length). For regions with uneven GC and AT base content, how to design a model for calculating primer Tm values ​​becomes an important factor in ensuring experimental success.

[0007] In summary, there is an urgent need in the field to develop a nucleic acid amplification technology that targets areas that are difficult to reach with traditional nucleic acid amplification technology. It 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 for detecting a variety of bacteria. This method targets AT-rich nucleic acid sequences and performs target sequence identification, primer design, denaturation and annealing temperature calculations, and nucleic acid amplification with partial template melting. First, an automated primer design process is employed to fully utilize the abundant genomic sequence information in public data resources to design specific primer pairs for AT-rich target sequences in a high-throughput manner. Second, a Tm value calculation formula that conforms to the characteristics of AT-rich sequences is fitted. The denaturation and annealing temperatures can be calculated based on the theoretical amplification product sequence and factors such as the 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% to 60% or high GC content, the calculated theoretical amplification product sequence Tm value is used as the minimum denaturation temperature. At this temperature, the AT-rich region of double-stranded DNA can be partially melted without the addition of 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 perform sequence specificity 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, it only opens AT-rich sequences 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 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 using C language and Perl language programming, and has the characteristics of high throughput and automation. The entire amplification method has strong specificity and a high success rate, which effectively expands the application range of traditional nucleic acid amplification technology, while significantly improving the reaction performance in terms of primer design throughput, reaction specificity, etc.

[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 screening a suitable primer combination; setting nucleic acid amplification reaction conditions, and performing the nucleic acid amplification reaction under conditions 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 using a specific formula and setting the nucleic acid amplification reaction conditions; that is, calculating the reaction denaturation temperature and annealing temperature based on the GC percentage, primer sequence length, and 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 where the template is partially melted to obtain an amplified product.

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

[0017] (1) Screening target sequences rich in AT bases in different bacterial genome sequences;

[0018] (2) Designing primers that are both universal and specific for a single bacterial genome 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;

[0020] (4) Screening suitable single bacterial primer pairs based on sequence amplification length, reaction denaturation temperature, and annealing temperature to form a primer set for detecting multiple bacteria;

[0021] (5) Setting nucleic acid amplification reaction conditions, performing nucleic acid amplification reaction under the condition of partial melting of the template, and obtaining an amplified product.

[0022] 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, with a step size of 5 to 100 bp. The AT base content is calculated for the sequence contained in each window position, 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.

[0023] In step (1) of the present invention, for a plurality of bacterial genome sequences, a window with a width of 1000 bp is slid starting from the first base, with a step length of 5 to 100 bp. The AT base content is calculated for the sequence contained in each window position, 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.

[0024] In step (2) of the present invention, the primer design method includes: (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 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 the primer pairs that meet the conditions to obtain the specific primers.

[0025] In the present invention, any programming language capable of achieving high-throughput primer design can be used, such as C, Perl and other programming languages ​​with strong operability and high speed.

[0026] In step (2.1), candidate primers are designed for the target sequence. The candidate primers must meet the following conditions: a) the primer sequence length is between 20 bp and 36 bp; 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 and positive and negative strand information of the candidate primer matching the target sequence are recorded.

[0027] Preferably, the AT base content is 60% to 75%.

[0028] The “number of consecutive GC residues” refers to the number of consecutive G bases or consecutive C bases in the primer sequence. For example, the number of consecutive GC residues in the primer sequence AAGGGGGTTCCAGGCATTA (SEQ ID NO. 15) is 5 / 2 / 2.

[0029] In step (2.2), the physicochemical properties of the single primers that meet the conditions in (2.1) above 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.

[0030] The “single primer that meets the requirements” refers to a single primer that has 3’ end stability, 5’ end stability and secondary structure stability.

[0031] In step (2.3), the single primers screened in step (2.2) above are combined into primer pairs based on their positional information on the target sequence and the positive and negative strand information. The theoretical amplification product sequence length of the primer pair should be between 200 bp and 600 bp. 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 that 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.

[0032] 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 Salmonella strains);

[0033] Specificity determination refers to checking whether a single primer cannot specifically match non-target sequences other than all target sequences. The non-target sequences refer to nucleic acid sequences other than the nucleic acid sequence to be amplified. The specific match refers to a match with no more than 2 mismatches.

[0034] Candidate primers that pass the universality and specificity test can proceed to the next step.

[0035] 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.

[0036] In step (3) of the present invention, the reaction denaturation temperature is calculated as 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.

[0037] In step (3) of the present invention, the reaction annealing temperature is the average of the Tm values ​​of the two primers in step (2.3) above, denoted 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 of the Tm values ​​is the reaction annealing temperature, denoted 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.

[0038] In step (4) of the present invention, the screening conditions of the primer combination include that the lengths 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.

[0039] In step (5) 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 repeating the above process 30-40 times. The denaturation temperature can be adjusted within ±5°C of the average denaturation temperature of the amplified fragments of all primer pairs as needed; the annealing temperature can be adjusted within ±2°C of the average annealing temperature of all primer pairs as needed; and the extension temperature is 72°C.

[0040] The present invention also provides application of the method in nucleic acid amplification of a target nucleic acid sequence rich in AT region.

[0041] The present invention also provides a primer pair obtained by the above-mentioned design method.

[0042] In the present invention, the primer pair is:

[0043] Primer pair A:

[0044] Sal-F1: 5'-TCAGACATCCGTTCAGAAAAT-3' (SEQ ID NO.1)

[0045] Sal-R1: 5'-GTTCAACTGTCGACAAGATTAA-3' (SEQ ID NO.2)

[0046] Primer pair B:

[0047] Sta-F1: 5’-GTAGGTATGGTAAATAGTTACAC-3’ (SEQ ID NO.3)

[0048] Sta-R1: 5’-CACTAATGCCAAATTTACTTAAAATCG-3’ (SEQ ID NO.4)

[0049] Primer pair C:

[0050] Cro-F1: 5’-CGCCATAACTGCATAATCAT-3’ (SEQ ID NO.5)

[0051] Cro-R1: 5’-ATAACGAGTTACCGTGCAGA-3’ (SEQ ID NO.6)

[0052] Primer pair D:

[0053] Yer-F1: 5’-TGTGCGGTGGATGTAAATAATTC-3’ (SEQ ID NO.7)

[0054] Yer-R1: 5’-GCTTTGAAACTCAAGGACTG-3’ (SEQ ID NO.8)

[0055] Primer pair E:

[0056] Sta-F2: 5’-CCTTTCATCTAAAAACCTCCA-3’ (SEQ ID NO.9)

[0057] Sta-R2: 5’-GAAATGGATGTTTTAAAAGAAGG-3’ (SEQ ID NO.10)

[0058] Primer pair F:

[0059] [[ID=3??]]Sal-F2: 5’-TGGGTTGAAATAGCCCATTA-3’ (SEQ ID NO.11)

[0060] Sal-R2: 5’-GACGTGACACACTTCGTTTT-3’ (SEQ ID NO.12)

[0061] Primer pair G:

[0062] Yer-F2: 5’-ATGGAAAATAACATAATTTCTATTACCGG-3’ (SEQ ID NO.13)

[0063] Note: There seems to be a typo in the original text where "Sal-F2" has an extra "?" in the tag ID "3??". It should be "36" as per the sequence. The translation is done as accurately as possible with the given text.Yer-R2:5'-TCTCTGCGAATAACCTTGTG-3'(SEQ ID NO.14)

[0064] The present invention also provides the use of the above method or primer pair in the amplification and / or detection of various bacteria-related genes or regions.

[0065] The present invention also provides the use of the above method or primer pair in preparing products for amplifying and / or detecting genes or regions related to multiple bacterial genomes.

[0066] The present invention provides a reagent for diagnosing multiple bacteria and its application in detecting multiple bacteria. The diagnostic reagent comprises the primer pair described above.

[0067] The present invention also provides a diagnostic reagent and its use in detecting multiple bacteria. The diagnostic reagent comprises the primer pair described above.

[0068] The multiple bacteria include Cronobacter sakazakii, Salmonella, Staphylococcus aureus, Yersinia enteritidis, etc.

[0069] The present invention also provides a system for simultaneously detecting multiple bacteria, which includes a screening module for screening target sequences rich in AT bases in different bacterial genome sequences; a primer design module for designing primers that are both universal and specific for a single bacterial genome sequence; a calculation module for calculating the reaction denaturation temperature and annealing temperature based on the GC percentage, primer sequence length, and theoretical amplification product sequence length of the primer pair; a composition module for screening suitable single bacterial primer pairs and composing a primer set for detecting multiple bacteria based on the sequence amplification length, reaction denaturation temperature, and annealing temperature; and an amplification module for performing a nucleic acid amplification reaction under conditions of partial template melting to obtain an amplified product.

[0070] The present invention also provides a device for simultaneously detecting multiple bacteria, the device 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 1-4 of the above-mentioned method are implemented.

[0071] The beneficial effects of the present invention lie in that it targets AT-rich sequences, which are typically avoided by traditional nucleic acid amplification methods, greatly expanding the candidate target regions. By performing specific screening based on massive genomic data and accurately calculating the Tm values ​​for AT-rich regions, it locally melts double-stranded DNA, significantly reducing the potential for nonspecific amplification, which is common in nucleic acid amplification techniques. This method effectively expands the application scope of traditional nucleic acid amplification techniques while significantly improving reaction performance in terms of primer design throughput and reaction specificity, meeting the needs of various applications, including nucleic acid detection and molecular genetics research. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 These are the 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, according to the present invention.

[0073] Figure 2 The results are agarose gel electrophoresis of nucleic acid amplification reaction of four bacteria, namely, Salmonella enteritidis subspecies, Staphylococcus aureus subspecies, Yersinia enterocolitica, and Cronobacter sakazakii.

[0074] Figure 3 The results of SYBRGreen I dye color development for specific detection of four bacteria, namely, Salmonella enteritidis subspecies, Staphylococcus aureus subspecies, Yersinia enterocolitica, and Cronobacter sakazakii, at a denaturation temperature of 81°C, were obtained by the present invention.

[0075] Figure 4 The dye color development results are based on the nucleic acid amplification specific detection of Cronobacter sakazakii at different denaturation temperatures of the present invention.

[0076] Figure 5 This is the electrophoresis result of the amplified product based on the nucleic acid amplification specific detection of Cronobacter sakazakii at a denaturing temperature of 94°C according to the present invention.

[0077] Figure 6 This is the electrophoresis result of the amplified product based on the nucleic acid amplification specific detection of Cronobacter sakazakii at a denaturation temperature of 82°C according to the present invention.

[0078] Figure 7 The results are agarose gel electrophoresis results of nucleic acid amplification reaction for two bacteria, Salmonella enteritidis subspecies and Staphylococcus aureus subspecies, according to the present invention.

[0079] Figure 8The 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.

[0080] Figure 9 The results are agarose gel electrophoresis results of nucleic acid amplification reaction of three bacteria, namely, Salmonella enteritidis subspecies, Staphylococcus aureus subspecies and Yersinia enterocolitica, according to the present invention. DETAILED DESCRIPTION

[0081] The present invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0082] The present invention also provides a system for simultaneously detecting multiple bacteria, which includes a screening module for screening target sequences rich in AT bases in different bacterial genome sequences; a primer design module for designing primers that are both universal and specific for a single bacterial genome sequence; a calculation module for calculating the reaction denaturation temperature and annealing temperature based on the GC percentage, primer sequence length, and theoretical amplification product sequence length of the primer pair; a composition module for screening suitable single bacterial primer pairs and composing a primer set for detecting multiple bacteria based on the sequence amplification length, reaction denaturation temperature, and annealing temperature; and an amplification module for performing a nucleic acid amplification reaction under conditions of partial template melting to obtain an amplified product.

[0083] Example 1 Simultaneous detection of Salmonella, Staphylococcus aureus, Yersinia enteritidis and Cronobacter sakazakii

[0084] The present invention screens AT-rich sequences for the genomes of Salmonella, Staphylococcus aureus, Yersinia enteritidis, and Cronobacter sakazakii and designs specific primers. Primer combinations are selected based on the calculated primer Tm and target sequence Tm. Reaction conditions are set for nucleic acid amplification. By determining whether the reaction result is positive, it is determined whether the target sequence is present in the test sample, and further whether one or more of Salmonella, Staphylococcus aureus, Yersinia enteritidis, and Cronobacter sakazakii are present in the test sample. The specific steps are as follows:

[0085] (1) Screening of target sequences rich in AT bases:

[0086] We used data from bacteria, archaea, and viruses with complete genome sequences downloaded from the NCBI FTP server on August 5, 2019, totaling 2,896 whole-genome sequences. We set Set A to contain all Salmonella genome sequences, and Set B to contain all non-Salmonella genome sequences. Using the Salmonella genome sequence as the reference genome, we slid a 1,000-bp window starting from the first base of the genome, with a step size of 50 bp. We calculated the AT content of the sequence before each slid, retaining regions with an AT content greater than 60% as candidate target sequences. This process was implemented using a Perl script.

[0087] (2) Design of specific primers:

[0088] Based on the characteristics of the PCR primers and the requirements of the present invention, the primers are set to have an AT base content of 55% to 75%, a 3' end stability ΔG < 4, a 5' end stability ΔG < 3, and a primer sequence length (20 to 36 bp). At the same time, conditions are set such that a single primer cannot produce a hairpin structure and cannot interact with itself. The candidate target sequence in step (1) is used as the candidate sequence for single primer design, and a single primer that meets the above set conditions is calculated. The Tm value of the single primer is calculated using the formula 0.466 × (GC percentage) × 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 derived from the positive strand or the negative strand), and the length of the primer are recorded.

[0089] Primer pairing was performed based on the position information of the individual primers, 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.

[0090] 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 versatility of the primers, when the single primer is aligned with the target sequence in set A, the parameter setting "-a -n 0" is used, that is, the single primer is required to completely match the target sequence; in order to ensure the specificity of the primers, when the single primer is aligned with the non-target sequence in set B, the parameter setting "-a -n 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 pre-set. In this embodiment, the number of primer pairs is pre-set 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-rich region.

[0091] Using the same method, 100 primer pairs were designed for Staphylococcus aureus, Yersinia enterocolitica, and Cronobacter sakazakii. The Tm of each primer was calculated using the formula 0.466 × (GC content percentage) × 100 + 66.04 - (450 / primer sequence length), and the average Tm value for the primer pair was used as the reaction annealing temperature. The denaturation temperature of the amplified region for each primer pair was calculated using the formula 0.357 × (GC content percentage) × 100 + 70.582 - (990 / amplified product sequence length).

[0092] Primer combinations were screened based on the conditions that the length of the amplified sequences of different bacteria differed by at least 50 bp, the reaction denaturation temperature differed by at most 2°C, and the reaction annealing temperature differed by at most 2°C. The system output primer combinations that met the conditions. The number of primer combinations can be preset. In this embodiment, the number of primer combinations was preset to 10. A primer set was randomly selected for validity verification. The sequence of the primer combination was:

[0093] Primer pair A:

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

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

[0096] Primer pair B:

[0097] Sta-F1: 5'-GTAGGTATGGTAAATAGTTACAC-3' (SEQ ID NO.3)

[0098] Sta-R1: 5'-CACTAATGCCAAATTTACTTAAAATCG-3' (SEQ ID NO. 4) Primer pair C:

[0099] Cro-F1: 5'-CGCCATAACTGCATAATCAT-3' (SEQ ID NO.5)

[0100] Cro-R1:5'-ATAACGAGTTACCGTGCAGA-3'(SEQ ID NO.6)

[0101] Primer pair D:

[0102] Yer-F1: 5'-TGTGCGGTGGATGTAAATAATTC-3' (SEQ ID NO.7)

[0103] Yer-R1:5'-GCTTTGAAACTCAAGGACTG-3'(SEQ ID NO.8)

[0104] The AT base contents of 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.

[0105] 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 content is 73.76%, and the theoretical denaturation temperature is 77.82°C.

[0106] 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 content is 68.73%, and the theoretical denaturation temperature is 78.34°C.

[0107] 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 content is 68.09%, and the theoretical denaturation temperature is 77.76°C.

[0108] (4) Nucleic acid amplification reaction and result detection:

[0109] The nucleic acid amplification reaction system configuration is shown in Table 1. Based on calculations, it is recommended to use 81°C denaturation for 5 seconds, 62°C annealing for 5 seconds, and 72°C extension for 20 seconds, repeating this process 35 times. See Table 2 for a list of test subjects.

[0110] After the reaction is completed using a conventional gradient PCR instrument, the amplification results can be determined in two ways. One is to add SYBR Green 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 test sample; the other is to perform agarose gel electrophoresis on the amplified product and determine whether the amplification result is positive by the electrophoresis band, that is, whether the target sequence exists in the test sample.

[0111] Table 1. Four bacterial nucleic acid amplification reaction systems

[0112]

[0113]

[0114] Table 2. List of specific detection targets for four bacterial nucleic acid amplification reactions

[0115] 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 15. Shigella boydii 1.10618 30. Shigella sonnei

[0116] Figure 1 and 2 The fluorescent dye staining and agarose gel electrophoresis results of the nucleic acid amplification reaction for four bacteria, Salmonella enteritidis subsp. enteritidis, Staphylococcus aureus subsp. aureus, Yersinia enterocolitica, and Cronobacter sakazakii, are shown. "N" represents the corresponding negative template amplification result, "M1" refers to Marker DL2000, "M2" refers to Marker B (100-600bp), and "Mixed" refers to a mixed template of the four bacterial genomic DNAs. Figure 1 The amplification and color development results of each single bacteria, mixed template and negative template were in line with expectations. Figure 2 The amplified fragments of each single bacterium were consistent with the theoretical calculated values ​​and the amplified fragments of each bacterium in the mixed template species could be separated from each other.

[0117] Figure 3 The SYBR Green I dye color development results for the nucleic acid amplification reaction of the present invention for specific detection of Staphylococcus aureus, Salmonella, Cronobacter sakazakii, and Yersinia enterocolitica 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. Figure 3 Among the samples, Staphylococcus aureus and Staphylococcus aureus subspecies 1-2, Salmonella Enteritidis subspecies, Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Paratyphi B, Cronobacter sakazakii, and Yersinia enterocolitica, respectively, showed a bright green color, indicating positive results, as expected. Meanwhile, the genomic DNA templates for the other bacteria tested were all negative, as shown in tubes 3-9, 14-22, and 25-30. All results were consistent with expectations, indicating that nonspecific amplification did not occur at the 81°C denaturation temperature, or that even if it did occur, it was not sufficient to affect the results of the dye-based assay.

[0118] Example 2 Detection of Cronobacter sakazakii

[0119] The present invention screens AT-rich sequences for the Cronobacter sakazakii genome and designs specific primers. The 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 is present in the test sample, and further whether Cronobacter sakazakii is present in the test sample. The specific steps are as follows:

[0120] (1) Screening of target sequences rich in AT bases:

[0121] We used data from bacteria, archaea, and viruses with complete genome sequences downloaded from the NCBI FTP server on August 5, 2019, totaling 2,896 whole-genome sequences. We set Set A to contain all Cronobacter sakazakii genome sequences, and Set B to contain all non-Cronobacter sakazakii genome sequences. Using the Cronobacter sakazakii genome sequence with GI number 156932229 as the reference genome, we slid a 1,000-bp window starting from the first base of the genome with a step size of 50 bp. We calculated the AT content of the sequence before each slid, retaining regions with an AT content greater than 60% as candidate target sequences. This process was implemented using a Perl script.

[0122] (2) Design of specific primers:

[0123] Based on the characteristics of the PCR primers and the requirements of the present invention, the primers are set to have an AT base content of 55% to 75%, a 3' end stability ΔG < 4, a 5' end stability ΔG < 3, and a primer sequence length (20 to 36 bp). At the same time, conditions are set such that a single primer cannot produce a hairpin structure and cannot interact with itself. The candidate target sequence in step (1) is used as the candidate sequence for single primer design, and a single primer that meets the above set conditions is calculated. The Tm value of the single primer is calculated using the formula 0.466 × (GC percentage) × 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 derived from the positive strand or the negative strand), and the length of the primer are recorded.

[0124] Primer pairing was performed based on the position information of the individual primers, 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.

[0125] 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 versatility 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 pre-set. In this embodiment, the number of primer pairs is pre-set to 20. The above high-throughput, automated primer design process is implemented using C and Perl scripts.

[0126] 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 sequences of the primer pairs were:

[0127] Cro-F1: 5'-CGCCATAACTGCATAATCAT-3' (SEQ ID NO: 5)

[0128] Cro-R1: 5'-ATAACGAGTTACCGTGCAGA-3' (SEQ ID NO: 6)

[0129] 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 69%.

[0130] (3) Calculation of PCR reaction denaturation temperature and annealing temperature:

[0131] 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 is 62.2°C, the Tm of Cro-R is 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.

[0132] (4) Nucleic acid amplification reaction and result detection:

[0133] The nucleic acid amplification reaction system configuration is shown in Table 3 below. Based on the calculation results, three sets of experiments were conducted to test the specificity of primers for different detection targets at denaturation temperatures of 94°C, 90°C, and 82°C, respectively. The nucleic acid amplification reaction conditions were denaturation at 94°C, 90°C, and 82°C for 5 seconds, annealing at 63°C for 5 seconds, and extension at 72°C for 20 seconds. This process was repeated 35 times. A list of specific detection targets is shown in Table 4.

[0134] After the reaction is completed, the amplification results are determined in two ways: one is to add SYBR Green I dye at a final concentration of 25x and determine whether the amplification result is positive by color, that is, whether the target sequence is present in the test sample; the other is to perform agarose gel electrophoresis on the amplified product and determine whether the amplification result is positive by the electrophoresis band, that is, whether the target sequence is present in the test sample.

[0135] Table 3. Cronobacter sakazakii nucleic acid amplification reaction system

[0136]

[0137]

[0138] Table 4. List of specific detection targets for Cronobacter sakazakii nucleic acid amplification reaction

[0139] 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 flexneri

[0140] The experimental results are shown in Figure 4 、 5 , 6, 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 (containing botulinum toxin 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.

[0141] Figure 4 The results of the SYBR Green I dye development for the specific detection of Cronobacter sakazakii nucleic acid amplification reaction at different denaturation temperatures are shown. If the amplified product is bright green, it is positive; if the amplified product is orange, it is negative. Figure 4In the experiment, at a denaturation temperature of 94°C / 90°C, the negative control (N) turned orange, indicating a negative result, as expected; the positive control (P) and Cronobacter sakazakii No. 23 turned bright green, indicating a positive result, as expected. This indicates that the entire reaction system is functioning properly. However, a large number of positive results were observed for amplification products of other bacterial genomic DNA templates, such as tubes 3, 4, 10-18, 20-22, 24, 26, 27, 29, and 30 at a denaturation temperature of 94°C, and tubes 3, 7, 10-18, 20-22, 24, 26, 27, 29, and 30 at a denaturation temperature of 90°C. This is inconsistent with expectations, suggesting that nonspecific amplification is prone to occur at a denaturation temperature of 94°C / 90°C, leading to false-positive results. At a denaturation temperature of 82°C, the negative control (N) turned orange, indicating a negative result, as expected; the positive control (P) and Cronobacter sakazakii No. 23 turned bright green, indicating a positive result, as expected. This indicates that the entire reaction system is functioning properly. Meanwhile, the test results for other bacterial genomic DNA templates were all negative, as shown in tubes 1-22 and 24-30. All results were consistent with expectations, indicating that nonspecific amplification did not occur at the 82°C denaturation temperature, or that even if minor nonspecific amplification occurred, it was insufficient to affect the results of the dye-based assay.

[0142] Figure 5 The electrophoresis results of the amplified products of the nucleic acid amplification reaction for specific detection of Cronobacter sakazakii at a denaturation temperature of 94°C are shown. A positive result is determined if a single band is present at the 291 bp position after electrophoresis; a negative result is determined if no band is present after electrophoresis; and a false positive result is caused by nonspecific amplification if one or more bands are present outside the 291 bp position after electrophoresis. Figure 5 In the negative control (N), there was no band, which was in line with expectations. The positive control (P) and Cronobacter sakazakii 23 both had a clear band at 291bp, which was in line with expectations. This showed that the entire reaction system could work properly. 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, suggesting that the reaction system produced a large amount of nonspecific amplification at the denaturation temperature of 94℃. This result is consistent with the results of the experiment. Figure 4 The dye color development results were consistent.

[0143] Figure 6The electrophoresis results of the amplification products of the nucleic acid amplification reaction for specific detection of Cronobacter sakazakii at a denaturation temperature of 82°C are shown. A positive result is determined if a single band is present at the 291 bp position after electrophoresis; a negative result is determined if no band is present after electrophoresis; and a false positive result is determined if one or more bands are present outside the 291 bp position after electrophoresis. Figure 6 The negative control (N) showed no bands, as expected; the positive control (P) and Cronobacter sakazakii strain 23 both showed a clear band at 291 bp, as expected. This indicates that the entire reaction system is functioning properly. Meanwhile, the majority of amplification products from the genomic DNA templates of other bacteria showed no bands, indicating negative results, as shown in lanes 2-6, 10, 12, 13, 15-22, and 26-30. A few template amplification products showed very faint bands after electrophoresis, easily distinguishable from positive results, as shown in tubes 1, 7, 8, 9, 11, 14, 24, and 25. This suggests that the 82°C denaturation temperature either did not produce any nonspecific amplification or produced a trace amount of nonspecific amplification that was easily distinguishable in the electrophoresis results.

[0144] comprehensive Figures 4-6 As can be seen from Table 4, 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 of the traditional PCR method (90°C or 94°C) is used for the reaction, although the electrophoresis results ( Figure 2 ) 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 Figure 4 As shown in the figure, under the denaturation conditions of 90℃ and 94℃, a large number of false positive results are presented, making it impossible to distinguish between the detection object and the non-detection object. Under the same reaction system, when the denaturation temperature of 82℃ calculated by the present invention is used for the reaction, the electrophoresis results ( Figure 6 ) 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 Figure 4 As shown in the figure, 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.

[0145] Example 3 Simultaneous detection of Salmonella and Staphylococcus aureus

[0146] The present invention screens for AT-rich sequences in the genomes of Salmonella and Staphylococcus aureus and designs specific primers. Primer combinations are selected based on the calculated primer Tm and target sequence Tm. Reaction conditions are set for nucleic acid amplification. By determining whether the reaction result is positive, the presence of the target sequence and, therefore, the presence of one or more of Salmonella and Staphylococcus aureus in the sample is determined. The specific steps are as follows:

[0147] (1) Screening of target sequences rich in AT bases:

[0148] We used data from bacteria, archaea, and viruses with complete genome sequences downloaded from the NCBI FTP server on August 5, 2019, totaling 2,896 whole-genome sequences. We set Set A to contain all Salmonella genome sequences, and Set B to contain all non-Salmonella genome sequences. Using the Salmonella genome sequence as the reference genome, we slid a 1,000-bp window starting from the first base of the genome, with a step size of 50 bp. We calculated the AT content of the sequence before each slid, retaining regions with an AT content greater than 60% as candidate target sequences. This process was implemented using a Perl script.

[0149] (2) Design of specific primers:

[0150] Based on the characteristics of the PCR primers and the requirements of the present invention, the primers are set to have an AT base content of 55% to 75%, a 3' end stability ΔG < 4, a 5' end stability ΔG < 3, and a primer sequence length (20 to 36 bp). At the same time, conditions are set such that a single primer cannot produce a hairpin structure and cannot interact with itself. The candidate target sequence in step (1) is used as the candidate sequence for single primer design, and a single primer that meets the above set conditions is calculated. The Tm value of the single primer is calculated using the formula 0.466 × (GC percentage) × 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 derived from the positive strand or the negative strand), and the length of the primer are recorded.

[0151] Primer pairing was performed based on the position information of the individual primers, 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.

[0152] 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 versatility 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 primers, 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 pre-set. In this embodiment, the number of primer pairs is pre-set 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-rich region.

[0153] Using the same method, 100 primer pairs were designed for Staphylococcus aureus amplification. The Tm of each primer was calculated using the formula 0.466 × (GC content percentage) × 100 + 66.04 - (450 / primer sequence length), and the average Tm value for the primer pair was calculated as the reaction annealing temperature. The denaturation temperature of the amplified region for each primer pair was calculated using the formula 0.357 × (GC content percentage) × 100 + 70.582 - (990 / amplified product sequence length).

[0154] Primer combinations were screened based on the conditions that the length of the amplified sequences of different bacteria differed by at least 50 bp, the reaction denaturation temperature differed by at most 2°C, and the reaction annealing temperature differed by at most 2°C. The system output primer combinations that met the conditions. The number of primer combinations can be preset. In this embodiment, the number of primer combinations was preset to 10. A primer set was randomly selected for validity verification. The sequence of the primer combination was:

[0155] Primer pair A:

[0156] Sal-F1: 5'-TCAGACATCCGTTCAGAAAAT-3' (SEQ ID NO.1)

[0157] Sal-R1: 5'-GTTCAACTGTCGACAAGATTAA-3' (SEQ ID NO.2)

[0158] Primer pair E:

[0159] Sta-F2: 5'-CCTTTCATCTAAAAACCTCCA-3' (SEQ ID NO.9)

[0160] Sta-R2:5'-GAAATGGATGTTTTAAAAGAAGG-3'(SEQ ID NO.10)

[0161] The AT base contents of 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 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 content is 76.30%, and the theoretical denaturation temperature is 77.38°C.

[0163] (4) Nucleic acid amplification reaction and result detection:

[0164] The nucleic acid amplification reaction system configuration is shown in Table 5. Based on calculations, it is recommended to use 81°C denaturation for 5 seconds, 62°C annealing for 5 seconds, and 72°C extension for 20 seconds, repeating this process 35 times. See Table 2 for a list of test subjects.

[0165] After the reaction is completed using a common 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 is present in the sample to be tested.

[0166] Table 5. Two bacterial nucleic acid amplification reaction systems

[0167] system Volume (μl) Final concentration TaqR300mix 15 1x SAL-F1 / R1 (50 μM) 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 /

[0168] Figure 7 The agarose gel electrophoresis results of the nucleic acid amplification reaction for two bacteria, Salmonella enterica subsp. enteritidis and Staphylococcus aureus subsp. aureus, are shown. "N" represents the corresponding negative template amplification result, "M" refers to MarkerDL2000, "Salmon" refers to Salmonella enterica subsp. enteritidis, "Staphylococcus aureus" refers to Staphylococcus aureus subsp. aureus, and "Mixed" refers to a mixed template of genomic DNA from both bacterial species. Figure 7 The amplified fragments of each single bacterium were consistent with the theoretical calculated values ​​and the amplified fragments of each bacterium in the mixed template species could be separated from each other.

[0169] Example 4 Simultaneous Detection of Salmonella and Yersinia Enterococci

[0170] The present invention screens AT-rich sequences for the genomes of Salmonella and Yersinia enterocolitica and designs specific primers. Primer combinations are selected based on the calculated primer Tm and target sequence Tm. Reaction conditions are set for nucleic acid amplification. By determining whether the reaction result is positive, it is determined whether the target sequence is present in the test sample, and further whether one or more of Salmonella and Yersinia enterocolitica are present in the test sample. The specific steps are as follows:

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

[0172] We used data from bacteria, archaea, and viruses with complete genome sequences downloaded from the NCBI FTP server on August 5, 2019, totaling 2,896 whole-genome sequences. We set Set A to contain all Salmonella genome sequences, and Set B to contain all non-Salmonella genome sequences. Using the Salmonella genome sequence as the reference genome, we slid a 1,000-bp window starting from the first base of the genome, with a step size of 50 bp. We calculated the AT content of the sequence before each slid, retaining regions with an AT content greater than 60% as candidate target sequences. This process was implemented using a Perl script.

[0173] (2) Design of specific primers:

[0174] Based on the characteristics of the PCR primers and the requirements of the present invention, the primers are set to have an AT base content of 55% to 75%, a 3' end stability ΔG < 4, a 5' end stability ΔG < 3, and a primer sequence length (20 to 36 bp). At the same time, conditions are set such that a single primer cannot produce a hairpin structure and cannot interact with itself. The candidate target sequence in step (1) is used as the candidate sequence for single primer design, and a single primer that meets the above set conditions is calculated. The Tm value of the single primer is calculated using the formula 0.466 × (GC percentage) × 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 derived from the positive strand or the negative strand), and the length of the primer are recorded.

[0175] Primer pairing was performed based on the position information of the individual primers, 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.

[0176] 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 versatility 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 primers, 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 pre-set. In this embodiment, the number of primer pairs is pre-set 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-rich region.

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

[0178] Primer combinations were screened based on the conditions that the length of the amplified sequences of different bacteria differed by at least 50 bp, the reaction denaturation temperature differed by at most 2°C, and the reaction annealing temperature differed by at most 2°C. The system output primer combinations that met the conditions. The number of primer combinations can be preset. In this embodiment, the number of primer combinations was preset to 10. A primer set was randomly selected for validity verification. The sequence of the primer combination was:

[0179] Primer pair F:

[0180] Sal-F2: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.11)

[0181] Sal-R2: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.12)

[0182] Primer pair G:

[0183] Yer-F2: 5'-ATGGAAAATAACATAATTTCTATTACCGG-3' (SEQ ID NO.13)

[0184] Yer-R2:5'-TCTCTGCGAATAACCTTGTG-3'(SEQ ID NO.14)

[0185] 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 content is 74.43%, and the theoretical denaturation temperature is 76.90°C.

[0186] 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 content is 72.02%, and the theoretical denaturation temperature is 76.48°C.

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

[0188] The nucleic acid amplification reaction system configuration is shown in Table 6. Based on calculations, it is recommended to use 81°C denaturation for 5 seconds, 62°C annealing for 5 seconds, and 72°C extension for 20 seconds, repeating this process 35 times. See Table 2 for a list of test subjects.

[0189] After the reaction is completed using a common 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 is present in the sample to be tested.

[0190] Table 6. Two bacterial nucleic acid amplification reaction systems

[0191] system Volume (μl) Final concentration TaqR300mix 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 /

[0192] Figure 8 The agarose gel electrophoresis results of the nucleic acid amplification reaction for Salmonella enteritidis subsp. enteritidis and Yersinia enterococci are shown. "N" represents the corresponding negative template amplification result, "M" refers to MarkerDL2000, "Salmon" refers to Salmonella enteritidis subsp. enteritidis, "Yersinia enterococci" refers to Yersinia enterococci subsp. enterococci, and "Mixed" refers to a mixed template of genomic DNA from both bacterial species. Figure 8 The amplified fragments of each single bacterium were consistent with the theoretical calculated values ​​and the amplified fragments of each bacterium in the mixed template species could be separated from each other.

[0193] Example 5 Simultaneous detection of Salmonella, Staphylococcus aureus and Yersinia enteritidis

[0194] The present invention screens AT-rich sequences for the genomes of Salmonella, Staphylococcus aureus, and Yersinia enterocolitica and designs specific primers. Primer combinations are selected based on the calculated primer Tm and target sequence Tm. Reaction conditions are set for nucleic acid amplification. By determining whether the reaction result is positive, it is determined whether the target sequence is present in the test sample, and further whether one or more of Salmonella, Staphylococcus aureus, and Yersinia enterocolitica are present in the test sample. The specific steps are as follows:

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

[0196] We used data from bacteria, archaea, and viruses with complete genome sequences downloaded from the NCBI FTP server on August 5, 2019, totaling 2,896 whole-genome sequences. We set Set A to contain all Salmonella genome sequences, and Set B to contain all non-Salmonella genome sequences. Using the Salmonella genome sequence as the reference genome, we slid a 1,000-bp window starting from the first base of the genome, with a step size of 50 bp. We calculated the AT content of the sequence before each slid, retaining regions with an AT content greater than 60% as candidate target sequences. This process was implemented using a Perl script.

[0197] (2) Design of specific primers:

[0198] Based on the characteristics of the PCR primers and the requirements of the present invention, the primers are set to have an AT base content of 55% to 75%, a 3' end stability ΔG < 4, a 5' end stability ΔG < 3, and a primer sequence length (20 to 36 bp). At the same time, conditions are set such that a single primer cannot produce a hairpin structure and cannot interact with itself. The candidate target sequence in step (1) is used as the candidate sequence for single primer design, and a single primer that meets the above set conditions is calculated. The Tm value of the single primer is calculated using the formula 0.466 × (GC percentage) × 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 derived from the positive strand or the negative strand), and the length of the primer are recorded.

[0199] Primer pairing was performed based on the position information of the individual primers, 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.

[0200] 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 versatility 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 primers, 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 pre-set. In this embodiment, the number of primer pairs is pre-set 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-rich region.

[0201] Using the same method, 100 primer pairs were designed for Yersinia enterocolitica amplification. The Tm of each primer was calculated using the formula 0.466 × (GC content percentage) × 100 + 66.04 - (450 / primer sequence length), and the average Tm value for the primer pair was calculated as the reaction annealing temperature. The denaturation temperature of the amplified region for each primer pair was calculated using the formula 0.357 × (GC content percentage) × 100 + 70.582 - (990 / amplified product sequence length).

[0202] Primer combinations were screened based on the conditions that the length of the amplified sequences of different bacteria differed by at least 50 bp, the reaction denaturation temperature differed by at most 2°C, and the reaction annealing temperature differed by at most 2°C. The system output primer combinations that met the conditions. The number of primer combinations can be preset. In this embodiment, the number of primer combinations was preset to 10. A primer set was randomly selected for validity verification. The sequence of the primer combination was:

[0203] Primer pair A:

[0204] Sal-F1: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.1)

[0205] Sal-R1: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.2)

[0206] Primer pair B:

[0207] Yer-F1: 5'-ATGGAAAATAACATAATTTCTATTACCGG-3' (SEQ ID NO.7)

[0208] Yer-R1:5'-TCTCTGCGAATAACCTTGTG-3'(SEQ ID NO.8)

[0209] Primer pair E:

[0210] Sta-F2: 5'-CCTTTCATCTAAAAACCTCCA-3' (SEQ ID NO.9)

[0211] Sta-R2:5'-GAAATGGATGTTTTAAAAGAAGG-3'(SEQ ID NO.10)

[0212] The AT base contents of 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 content is 74.43%, and the theoretical denaturation temperature is 76.90°C.

[0213] The AT base contents of the primer pair B 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 content is 72.02%, and the theoretical denaturation temperature is 76.48°C.

[0214] 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 content is 76.30%, and the theoretical denaturation temperature is 77.38°C.

[0215] (4) Nucleic acid amplification reaction and result detection:

[0216] The nucleic acid amplification reaction system configuration is shown in Table 7. Based on calculations, it is recommended to use 81°C denaturation for 5 seconds, 62°C annealing for 5 seconds, and 72°C extension for 20 seconds, repeating this process 35 times. See Table 2 for a list of test subjects.

[0217] After the reaction is completed using a common 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 is present in the sample to be tested.

[0218] Table 7. Three bacterial nucleic acid amplification reaction systems

[0219] system Volume (μl) Final concentration TaqR300mix 15 1x SAL-F1 / R1 (50 μM) 0.25+0.25 0.5μM YER-F1 / R1 (50 μM) 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 /

[0220] Figure 9The agarose gel electrophoresis results of the nucleic acid amplification reaction for three bacteria, Salmonella enteritidis subsp. enteritidis, Staphylococcus aureus subsp. enteritidis, and Yersinia enterococci, are shown. "N" represents the corresponding negative template amplification result, "M" refers to Marker DL2000, "Enteritis" refers to Salmonella enteritidis subsp. enteritidis, "Yersinia enterococci" refers to Yersinia enterococci subsp. enteritidis, "Aureus" refers to Staphylococcus aureus subsp. enterococci, and "Mixed" refers to a mixed template of the three bacterial genomic DNAs. Figure 9 The amplified fragments of each single bacterium were consistent with the theoretical calculated values ​​and the amplified fragments of each bacterium in the mixed template species could be separated from each other.

[0221] The present invention also provides a device for simultaneously detecting multiple bacteria, 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, the steps of the method described in Examples 1-4 of the present invention are implemented.

[0222] References

[0223] 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)

[0224] 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 appended claims. SEQUENCE LISTING <110> Shanghai Bioinformatics Research Center, Shanghai Want Want Food Group Co., Ltd. <120> Method for detecting multiple bacteria, system for detecting multiple bacteria and application thereof <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> twenty three <212> DNA <213> Artificial sequence <400> 3 gtaggtatgg taaatagtta cac 23 <210> 4 <211> 27 <212> DNA <213> Artificial sequence <400> 4 cactaatgcc aaatttactt aaaatcg 27 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <400> 5 cgccataact gcataatcat 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <400> 6 ataacgagtt accgtgcaga 20 <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'-TCAGACATCCGTTCAGAAAAT-3' (SEQ ID NO.1) Sal-R1: 5'-GTTCAACTGTCGACAAGATTAA-3' (SEQ ID NO.2) Primer pair B: Sta-F1: 5'-GTAGGTATGGTAAATAGTTACAC-3' (SEQ ID NO.3) Sta-R1: 5'-CACTAATGCCAAATTTACTTAAAATCG-3' (SEQ ID NO. 4) Primer pair C: Cro-F1: 5'-CGCCATAACTGCATAATCAT-3' (SEQ ID NO.5) Cro-R1:5'-ATAACGAGTTACCGTGCAGA-3'(SEQ ID NO.6) Primer pair D: Yer-F1: 5'-TGTGCGGTGGATGTAAATAATTC-3' (SEQ ID NO.7) Yer-R1:5'-GCTTTGAAACTCAAGGACTG-3'(SEQ ID NO.8) Primer pair E: Sta-F2: 5'-CCTTTCATCTAAAAACCTCCA-3' (SEQ ID NO.9) Sta-R2: 5'-GAAATGGATGTTTTAAAAGAAGG-3' (SEQ ID NO.10) Primer pair F: Sal-F2: 5'-TGGGTTGAAATAGCCCATTA-3' (SEQ ID NO.11) Sal-R2: 5'-GACGTGACACACTTCGTTTT-3' (SEQ ID NO.12) Primer pair G: Yer-F2: 5'-ATGGAAAATAACATAATTTCTATTACCGG-3' (SEQ ID NO. 13) Yer-R2: 5'-TCTCTGCGAATAACCTTGTG-3' (SEQ ID NO. 14).

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 a variety of bacterial genomes; characterized in that: The multiple bacteria are Cronobacter sakazakii, Salmonella, Staphylococcus aureus, and Yersinia enterocolitica.

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