CN115261494A - System and method for detecting yersinia enterocolitica and application of system and method - Google Patents

System and method for detecting yersinia enterocolitica and application of system and method Download PDF

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CN115261494A
CN115261494A CN202110486116.0A CN202110486116A CN115261494A CN 115261494 A CN115261494 A CN 115261494A CN 202110486116 A CN202110486116 A CN 202110486116A CN 115261494 A CN115261494 A CN 115261494A
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primer
sequence
nucleic acid
amplification
primer pair
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曹永梅
陈俊江
陈清艳
杨政华
吉玲
林彦君
李园园
刘伟
陆晓婷
李雪玲
陆长德
王涤松
李亦学
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SHANGHAI CENTER FOR BIOINFORMATION TECHNOLOGY
Shanghai Wangwang Food Group Co ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a system and a method for detecting yersinia enterocolitica and application thereof. The method comprises the following steps: identifying a sequence rich in AT base in a genome sequence of yersinia enterocolitica as a target sequence; designing specific primers aiming at a target sequence automatically in a high-throughput manner; designing a specific Tm value calculation formula to calculate the reaction denaturation and annealing temperatures, and setting the nucleic acid amplification reaction conditions according to the reaction denaturation and annealing temperatures; the nucleic acid amplification reaction is performed under conditions in which the template is locally melted. The target-oriented traditional nucleic acid amplification method generally needs to avoid sequences rich in AT bases, so that the candidate target area is greatly widened; by specific investigation based on massive genome data and accurate calculation aiming AT the Tm value of the AT-rich region, the local melting of double-stranded DNA is realized, and the possibility of non-specific amplification in the nucleic acid amplification technology is greatly reduced. The method effectively expands the application range of the traditional nucleic acid amplification technology and obviously improves the reaction performance.

Description

System and method for detecting yersinia enterocolitica and application of system and method
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a system and a method for detecting yersinia enterocolitica and application thereof.
Background
Nucleic Acid Amplification Technology (NAAT) is a general name of a class of molecular biology technology, which can realize rapid and specific amplification of trace nucleic acid by reacting primers, DNA polymerase and other reagents at a specific temperature, and can be widely applied to disease diagnosis, pathogenic microorganism detection, food safety detection, animal and plant quarantine and various applications related to molecular cloning, such as sequencing, gene cloning, gene operation, allele analysis, mutation detection and the like. The Polymerase Chain Reaction (PCR) is the earliest nucleic acid amplification technology, which simulates the process of DNA replication in vivo, and realizes exponential amplification of DNA fragments through the complementation of a pair of specific oligonucleotide primers and the DNA fragments to be amplified, and through several cycles of denaturation, annealing and extension. The PCR technology was invented in 1985 by Mullis of Cetus company, and then Saiki et al introduced thermostable DNA polymerase into the PCR reaction system, which solves the problem of continuous manual addition of polymerase caused by inactivation of DNA polymerase in the thermal denaturation cycle process in the early PCR technology, greatly improves the nucleic acid amplification efficiency and enables the technology to be automated, and since then, the nucleic acid amplification technology represented by PCR is widely popularized and applied, and the development of molecular biology is greatly promoted.
It is well known that existing nucleic acid amplification techniques generally avoid GC-or AT-rich regions because these regions tend to form hairpin secondary structures by self-complementary pairing, thereby hindering primer binding to the template; even if the primer is capable of binding to the template only marginally, it is susceptible to extension of the template strand by the DNA polymerase, resulting in "katton" of the DNA polymerase as it is amplified along the template and interfering with DNA synthesis. Therefore, in order to increase the success rate of the reaction, the conventional nucleic acid amplification technology has selective preference for the base composition of the amplification region, the primer design thereof is usually concentrated in the region with a GC content of 40% to 60%, 45% to 55% is the most suitable, and the different amplification technologies are slightly different, such as the primer design suggestion of PCR with a G + C content of 40% to 60% (see the fourth edition of the molecular cloning experimental guidance, table 7-1 primer design) and the LAMP primer design suggestion with a G + C content of 40% to 65% (see a Guide to LAMP primer design (PrimerExplorer V3)).
However, the nucleobase composition of organisms in nature is very different, for example, AT base content of plasmodium genome is about 82%, while the GC content of 2670 strains of bacteria and archaebacteria was studied by zhouchihen et al, and found to vary from 14% to 75% (zhouchihen, 2014). From the detection perspective, the base composition selection preference of the existing nucleic acid amplification technology ignores a large number of potential target sequences, and amplifies the difficulty of successfully detecting a target object; from a genetic manipulation perspective, this selective preference results in considerable target sequences being difficult to amplify smoothly, thereby making further molecular manipulations difficult. If proper primers can be designed aiming AT the unbalanced domains of GC base content and AT base content avoided by the traditional nucleic acid amplification method, the formation of a secondary structure is avoided, and meanwhile, proper reaction conditions are set, so that a nucleic acid amplification experiment is smoothly carried out, the success rate of detecting a target object is obviously increased, and more sequences can be amplified to conveniently implement downstream molecular operation.
The method is characterized in that a nucleic acid amplification reaction is carried out aiming AT a region with non-equilibrium GC base content and AT base content, and the key step is a primer design link. In view of the above analysis, the species-specific genes (or sequences) published in the field usually originate from a region with GC content of 40-60% or even 45-55%, and thus cannot be applied to primer design for regions with non-equilibrium GC and AT base contents. In addition, the continuous GC-rich or AT-rich region in such sequences has a weak diversity of base composition compared to other regions, which brings additional difficulties in designing specific primers. Therefore, a primer design algorithm process with high throughput, automation and high efficiency aiming AT the base content non-equilibrium sequences of GC and AT needs to be provided.
In addition to the selection of the amplification region and the design of the primers, the calculation of Tm values of the amplification region and the primers is also an important influence factor on the smooth completion of the amplification process. The Tm values of the amplified region and the primers are usually calculated according to the nearest neighbor binary model, but the specific calculation formulas used by different researchers and primer manufacturers are different, for example, the Tm calculation formula of the primers proposed in the fourth edition of the molecular cloning experimental instruction manual is Tm =4 (G, C) +2 (A, T), and the Tm calculation formula of the primers proposed by TaKaRa is Tm =4 (G, C) +2 (A, T) +32-2 (total base); the Tm formula of the primers of the organism is Tm (0.05M Na)+) =59.94+1 × (GC percentage) - (675/primer sequence length). For GC, AT base content non-uniformityIn terms of the equilibrium region, how to design a model for calculating the Tm value of a primer becomes an important factor for ensuring the success of an experiment.
In summary, there is a need in the art to develop a nucleic acid amplification technology for regions that are difficult to be involved in the conventional nucleic acid amplification technology, which can automatically design specific primers with high throughput, calculate Tm values thereof, set appropriate reaction conditions such as denaturation temperature and annealing temperature, expand the application range of the nucleic acid amplification technology, improve the amplification specificity, and satisfy the requirements of nucleic acid detection, molecular genetics research and other aspects.
Disclosure of Invention
The invention provides an innovative nucleic acid amplification method and is applied to detecting yersinia enterocolitica. The method takes an AT-rich nucleic acid sequence as an amplification target, and performs target sequence identification, primer design, denaturation temperature and annealing temperature calculation, template local melting nucleic acid amplification and the like. Firstly, an automatic primer design process is adopted, abundant genome sequence information in public data resources is fully utilized, and a specific primer pair aiming AT an AT-rich target sequence is designed in a high-throughput manner. Secondly, fitting a Tm value calculation formula conforming to the characteristics of the AT-rich sequence, and calculating the denaturation temperature and the annealing temperature according to the theoretical amplification product sequence, the sequence length of the primer, the base composition and other factors; considering that the denaturation temperature of the AT-rich nucleic acid sequence is significantly lower than that of the sequences with GC content of 40-60% and high GC content, the calculated Tm value of the theoretical amplification product sequence is used as the lowest denaturation temperature, and AT the lowest denaturation temperature, the AT-rich region of the double-stranded DNA can be locally melted without adding any chemical denaturant, so that the nucleic acid amplification reaction is started.
On one hand, massive genome data in public data resources are introduced in a primer design link to carry out sequence specificity investigation; on the other hand, in the denaturation temperature setting link, different from the traditional nucleic acid amplification method, all double-stranded structures are opened AT the denaturation temperature of 93-95 ℃, and only AT-rich sequences are opened by reducing the denaturation temperature, so that the possibility of non-specific amplification of non-target regions is greatly reduced AT the source. The design strategies of the two aspects remarkably reduce the possibility of non-specific amplification common in nucleic acid amplification reaction, and a nucleic acid amplification method aiming AT AT-rich sequences is formed on the basis of the non-specific amplification strategy. The primer design step of the method is realized by adopting C language and Perl language programming, the method has the characteristics of high throughput and automation, the whole amplification method has strong specificity and high success rate, the application range of the traditional nucleic acid amplification technology is effectively expanded, and the reaction performance is obviously improved.
The invention provides a system for detecting yersinia enterocolitica, which comprises the following components:
the screening module is used for screening a target sequence which is rich in AT bases in a genome sequence of yersinia enterocolitica;
a primer design module for designing a primer having both versatility and specificity for the target sequence;
the calculation module is used for calculating the inverse dependent temperature and the annealing temperature based on the percentage content of GC, the sequence length of the primer and the sequence length of a theoretical amplification product of the primer pair, and setting the reaction condition of nucleic acid amplification;
and the amplification module is used for carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
In the present invention, the nucleic acid amplification method comprises identifying, as a target sequence, an AT base-rich sequence in a nucleic acid sequence to be amplified; designing specific primers aiming at a target sequence automatically in a high-throughput manner; designing a specific Tm value calculation formula to calculate the reaction denaturation and annealing temperatures, and setting the nucleic acid amplification reaction conditions according to the reaction denaturation and annealing temperatures; the nucleic acid amplification reaction is performed under conditions where the template is locally melted.
The method specifically comprises the following steps:
(1) Screening a target sequence rich in AT basic groups in a nucleic acid sequence to be amplified;
(2) Designing primers with universality and specificity aiming at the target sequence automatically and in a high-throughput manner;
(3) Calculating the reaction denaturation temperature and the annealing temperature by a specific formula, and setting the reaction conditions of nucleic acid amplification; calculating the inverse strain temperature and the annealing temperature based on the percentage content of GC, the length of a primer sequence and the length of a theoretical amplification product sequence of a primer pair, and setting the reaction conditions of nucleic acid amplification;
(4) And carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
In the invention, the method for detecting yersinia enterocolitica comprises the following specific steps:
(1) Screening a target sequence rich in AT base in a genome sequence of yersinia enterocolitica;
(2) Designing a primer with both universality and specificity aiming at the target sequence;
(3) Calculating the inverse strain temperature and the annealing temperature based on the percentage content of GC, the length of the primer sequence and the length of the theoretical amplification product sequence of the primer pair, and setting the reaction conditions of nucleic acid amplification;
(4) And carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
In step (1) of the present invention, for the nucleic acid sequence to be amplified, a window having a width of 1000bp is slid from the first base, and the step length is 5 to 100bp. Calculating the AT base content of the sequence contained in the position of each window, and reserving the region with the AT base content of the sequence more than 60 percent as the target sequence. Preferably, the region with the base content of AT sequence of 60-80% is reserved as the target sequence.
In the step (1) of the present invention, the genomic sequence of Yersinia enterocolitica is slid over a window having a width of 1000bp from the first base, and the step length is 5 to 100bp. Calculating the AT base content of the sequence contained in the position of each window, and reserving the region with the AT base content of the sequence more than 60 percent as the target sequence. Preferably, the region with the base content of AT sequence of 60-80% is reserved as the target sequence.
In step (2) of the present invention, the method for designing the primer comprises: (2.1) designing a single primer aiming at a target sequence to obtain a candidate primer; (2.2) judging the physicochemical properties of the candidate primers, and screening single primers meeting the conditions; (2.3) combining the single primers obtained by screening in the step (2.2) into a primer pair; (2.4) judging the universality and the specificity of the primer pair; and (2.5) outputting a primer pair meeting the conditions to obtain the specific primer.
In the invention, any programming language capable of realizing high-flux primer design can be adopted, such as C, perl and other programming languages with strong operability and higher speed.
In the step (2.1), a candidate primer is designed aiming at a target sequence, and the candidate primer needs to satisfy the following conditions: a) The length of the primer sequence is between 20bp and 36 bp; b) The AT base content is 55-80%, c) the continuous GC number is less than or equal to 5; and simultaneously recording the position information and the positive and negative chain information of the candidate primers matched on the target sequence.
Wherein, the AT base content is preferably 60 to 75%.
Wherein the "consecutive GC number" refers to the number of consecutive bases G or consecutive bases C in the primer sequence, e.g., the number of consecutive GCs in the primer sequence AAGGGGGTTCCAGGCATTA (SEQ ID NO. 15) is 5/2/2.
In the step (2.2), the single primer meeting the condition (2.1) is subjected to physicochemical property judgment, including but not limited to physicochemical properties such as 3 'end stability, 5' end stability and/or secondary structure stability, and the single primer meeting the requirement is reserved.
Wherein, the 'single primer meeting the requirement' refers to a single primer with 3 'end stability, 5' end stability and secondary structure stability.
In the step (2.3), the single primers obtained by screening in the step (2.2) are combined into primer pairs according to the position information and the positive and negative chain information matched to the target sequence; the length of the theoretical amplification product sequence of the primer pair should be between 200bp and 600 bp. Calculating Tm value of single primer according to formula 0.466X (GC percentage content) 100+66.04- (450/primer sequence length); and screening the primer pairs under the conditions that the Tm difference value of the primer pairs is less than or equal to 3 ℃ and the primers cannot interact with each other to obtain candidate primer pairs. Wherein, the percentage content of GC refers to the percentage of the number of bases G and C in the primer to the total number of bases of the primer; the length of the primer sequence refers to the number of bases of the primer.
In the step (2.4), the universality and specificity of each primer in the candidate primer pair obtained in the step (3) are judged. Wherein the determination of commonality refers to checking whether the primer pairs exactly match all target sequences (e.g., multiple strain genomes of yersinia enterocolitica);
the specificity determination means to examine whether or not a single primer cannot be specifically matched with a non-target sequence other than all target sequences, i.e., nucleic acid sequences other than the nucleic acid sequence to be amplified, and the specific match means a match of not more than 2 mismatches.
Candidate primers judged by the versatility and specificity can be subjected to the next step.
And (2.5) outputting a primer pair meeting the conditions to obtain the primer pair for the nucleic acid amplification reaction.
In step (3) of the present invention, the reaction denaturation temperature is calculated as 0.357 x (GC percentage content) × 100+70.582- (990/theoretical amplification product sequence length of primer pair), and is denoted as Tma; wherein, the percentage content of GC 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.
In the step (3) of the present invention, the reaction annealing temperature is an average value of Tm values of the two primers in the step (2.3) and is represented as Tmb. Namely, calculating the Tm value of a single primer according to the formula 0.466X (GC percentage content) 100+66.04- (450/primer sequence length), and taking the average value of the Tm values to obtain the reaction annealing temperature which is marked as Tmb; wherein, the percentage content of GC refers to the percentage of the number of bases G and C in the primer to the total number of bases of the primer.
In the step (3) of the present invention, the nucleic acid amplification reaction conditions include: the reaction was carried out at the denaturation temperature for 5 seconds, at the annealing temperature for 5 seconds and at the elongation temperature for 20 seconds, and the above procedure was repeated 30 to 40 times. Wherein the denaturation temperature can be adjusted within the range of Tma +/-5 ℃ according to the requirement; the annealing temperature can be adjusted between Tmb plus or minus 2 ℃ according to the requirement; the extension temperature was 72 ℃.
The invention also provides the application of the method in nucleic acid amplification of target nucleic acid sequences rich in AT regions.
The invention also provides a primer pair obtained by the design method.
In the invention, the primer pair is as follows:
and (3) primer pair G:
Yer-F1：5’-ATGGAAAATAACATAATTTCTATTACCGG-3’(SEQ ID NO.1)
Yer-R1:5 'TCTCTGCGAATACCTTGTG-3' (SEQ ID NO. 2); and/or the presence of a gas in the atmosphere,
and (3) primer pair D:
Yer-F2：5’-TGTGCGGTGGATGTAAATAATTC-3’(SEQ ID NO.3)
Yer-R2：5’-GCTTTGAAACTCAAGGACTG-3’(SEQ ID NO.4)。
the invention also provides the application of the method or the primer in amplification and/or detection of relevant genes or regions of the yersinia enterocolitica.
The invention also provides application of the primer pair in preparation of products for amplifying and/or detecting the yersinia enterocolitica related genes or regions.
The invention also provides application of the method in simultaneous detection of any two or more of yersinia enterocolitica, staphylococcus aureus, cronobacter sakazakii and salmonella.
Specifically, the invention also provides the application of the method in simultaneously detecting the yersinia enterocolitica and any one or the combination of staphylococcus aureus, salmonella and cronobacter sakazakii.
The invention also provides a diagnostic reagent for diagnosing the yersinia enterocolitica, and application of the diagnostic reagent in detecting the yersinia enterocolitica, wherein the diagnostic reagent comprises the primer pair.
The invention also provides a diagnostic reagent and an application thereof in detecting yersinia enterocolitica, wherein the diagnostic reagent comprises the primer pair.
The invention has the advantages that the target traditional nucleic acid amplification method usually needs to avoid sequences rich in AT bases, thereby greatly expanding candidate target regions; by specific investigation based on massive genome data and accurate calculation of Tm value of AT-rich region, double-stranded DNA is locally unzipped, and the possibility of non-specific amplification commonly existing in nucleic acid amplification technology is greatly reduced. The method effectively expands the application range of the traditional nucleic acid amplification technology, obviously improves the reaction performance in the aspects of primer design flux, reaction specificity and the like, and can meet the requirements of nucleic acid detection, molecular genetics research and the like.
Drawings
FIG. 1 shows the results of dye development based on the detection of nucleic acid amplification specificity at different denaturation temperatures of Cronobacters sakazakii (Cronobacters sazakii) according to the present invention.
FIG. 2 is a graph showing the results of electrophoresis of amplification products for detection of nucleic acid amplification specificity at a denaturation temperature of 94 ℃ based on Cronobacter sakazakii (Cronobacter sazakii) according to the present invention.
FIG. 3 is an electrophoresis result of an amplification product based on the detection of nucleic acid amplification specificity at a denaturation temperature of 82 ℃ of Cronobacters sakazakii (Cronobacters sazakii) according to the present invention.
FIG. 4 is a graph showing the amplification curve of real-time fluorescent quantitative PCR based on the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentericolytica) primer pair G of the present invention at a suitable temperature.
FIG. 5 is a melting curve of real-time fluorescent quantitative PCR based on the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentocolitica) primer pair G of the present invention at a suitable temperature.
FIG. 6 is an agarose gel electrophoresis at a suitable temperature based on the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaenterocolitica) primer pair G of the present invention.
FIG. 7 shows the sensitivity of real-time fluorescent quantitative PCR based on the denaturation of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaenterocolitica) primer pair G at 80 ℃.
FIG. 8 shows the sensitivity of real-time fluorescent quantitative PCR based on the denaturation of the primer pair G of Yersinia enterocolitica (Yersiniaentericolytica) of the present invention at 81 ℃.
FIG. 9 shows the results of sensitive staining based on the denaturation at 80 ℃ and 81 ℃ of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaenterocolitica) primer pair G of the present invention.
FIG. 10 shows the results of sensitive staining based on the denaturation at 80 ℃ and 81 ℃ of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaenterocolitica) primer pair G of the present invention.
FIG. 11 shows the result of staining for specific detection of nucleic acid amplification in the nucleic acid amplification reaction based on the Yersinia enterocolitica (Yersiniaenterocolitica) primer set G of the present invention at a denaturation temperature of 81 ℃.
FIG. 12 is a graph showing the amplification curve of real-time fluorescent quantitative PCR based on the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentericolytica) primer pair D of the present invention at a suitable temperature.
FIG. 13 is a melting curve of real-time fluorescent quantitative PCR based on the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentocolitica) primer pair D of the present invention at a suitable temperature.
FIG. 14 is an agarose gel electrophoresis at a suitable temperature based on the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentocolitica) primer pair D of the present invention.
FIG. 15 shows the sensitivity of real-time fluorescent quantitative PCR based on the denaturation of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentericolytica) primer pair D of the present invention at 80 ℃.
FIG. 16 shows the sensitivity of real-time fluorescent quantitative PCR based on the denaturation at 81 ℃ of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaenterocolitica) primer pair D of the present invention.
FIG. 17 shows the results of sensitive staining based on the denaturation at 80 ℃ and 81 ℃ of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaenterocolitica) primer pair D of the present invention.
FIG. 18 shows the results of sensitive staining based on the denaturation at 80 ℃ and 81 ℃ of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaenterocolitica) primer pair D of the present invention.
FIG. 19 shows the staining results of nucleic acid amplification specific detection at a denaturation temperature of 81 ℃ in a nucleic acid amplification reaction based on the Yersinia enterocolitica (Yersiniaenterocolitica) primer set D of the present invention.
FIG. 20 shows the results of the fluorescent dye staining of the nucleic acid amplification reactions of four bacteria, salmonella enteritidis subspecies, staphylococcus aureus subspecies, yersinia enterocolitica, and Cronobacter sakazakii, according to the present invention.
FIG. 21 shows the result of agarose gel electrophoresis performed by the present invention on four kinds of bacteria, i.e., salmonella enteritidis subspecies, staphylococcus aureus subspecies, yersinia enterocolitica, and Cronobacter sakazakii, for nucleic acid amplification reactions.
FIG. 22 shows the results of the color development of the SYBRGreen I dye in the specific detection of the nucleic acid amplification reaction of the present invention against four bacteria, salmonella enteritidis subspecies, staphylococcus aureus subspecies, yersinia enterocolitica, and Cronobacter sakazakii, at a denaturation temperature of 81 ℃.
FIG. 23 shows the result of agarose gel electrophoresis performed by the present invention on the nucleic acid amplification reaction of Salmonella enteritidis subspecies and Staphylococcus aureus subspecies.
FIG. 24 shows the result of agarose gel electrophoresis of the nucleic acid amplification reaction of two bacteria, salmonella enteritidis subspecies and Yersinia enterocolitica according to the present invention.
FIG. 25 shows the result of agarose gel electrophoresis of nucleic acid amplification reactions of three bacteria, salmonella enteritidis subspecies, staphylococcus aureus subspecies and Yersinia enterocolitica, according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
The invention provides a system for detecting yersinia enterocolitica, which comprises the following components:
the screening module is used for screening a target sequence which is rich in AT bases in a genome sequence of yersinia enterocolitica;
a primer design module for designing a primer having both versatility and specificity for the target sequence;
the calculation module is used for calculating the inverse strain temperature and the annealing temperature based on the percentage content of GC, the length of the primer sequence and the length of the theoretical amplification product sequence of the primer pair, and setting the reaction conditions of nucleic acid amplification;
and the amplification module is used for carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
Example 1 detection of Yersinia enterocolitica (Yersiniaenterocolitica)
The invention screens AT-rich sequences and designs specific primers aiming AT the genome of Yersinia enterocolitica (Yersiniaenterocolitica), sets reaction conditions according to the calculated primer Tm and the target sequence Tm to carry out nucleic acid amplification, determines whether the target sequence exists in a sample to be detected by judging whether the reaction result is positive, and further determines whether the Yersinia enterocolitica exists in the sample to be detected. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 whole genome sequences were used for bacteria, archaea and virus data with complete genome sequences downloaded from the FTP at NCBI at 8/5 in 2019. Set a, which contains all enterocolitis yersinia genome sequences; set B was set up, which contained all non-enterocolitis yersinia genome sequences. Taking a genome sequence of yersinia enterocolitica as a reference genome, sliding a window with the width of 1000bp from the first base of the genome, and setting the step length to be 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the 3 'end stability delta G <4, the 5' end stability delta G <3, the primer sequence length (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure and cannot interact with the single primer are set, and the single primer meeting the set conditions is calculated by taking the candidate target sequence in the step (1) as a candidate sequence for designing the single primer. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the positive and negative strand information (i.e. whether it is from the positive strand or the negative strand), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, a parameter setting of "-a-n 3" was used when the single primer was aligned to the non-target sequences in pool B, i.e., the single primer was allowed no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying the condition, in which the number of primer pairs is preset, and in this embodiment, the number of primer pairs is preset to 20. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts.
After the program is operated, 20 PCR reaction primer pairs are designed in the AT enrichment region, and one primer group is randomly selected for validity verification. The sequence of the primer pair G is as follows:
Yer-F1：5’-ATGGAAAATAACATAATTTCTATTACCGG-3’(SEQ ID NO.1)
Yer-R1：5’-TCTCTGCGAATAACCTTGTG-3’(SEQ ID NO.2)
the AT base content of the primer is 72.41 percent and 55 percent respectively, and the percentage content of the theoretical amplification product sequence AT of the primer pair is 70.66 percent.
Calculation of denaturation temperature and annealing temperature for PCR reaction:
tm =63.8 ℃ for primer Yer-F1, tm =64.5 ℃ for Yer-R1, calculated using the formula 0.466X (GC percentage) X100 +66.04- (450/primer sequence length), the primer pair average Tm value is 64.2 ℃; the denaturation temperature of the amplified region was calculated to be 76.96 ℃ using the formula 0.357X (GC percentage) X100 +70.582- (990/amplification product sequence length).
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in table 1 below. Determining an applicable denaturation temperature and a dissolution curve by using a real-time fluorescence quantitative PCR instrument according to the calculation result; the specificity of the primers for different detection objects at the applicable denaturation temperature is tested; the limit of detection at the applicable denaturation temperature was tested. Denaturation 5 sec, annealing at 62.5 ℃ for 5 sec, and extension at 72 ℃ for 20 sec, 35 repetitions of the above procedure are recommended. The list of specific test subjects is shown in Table 2.
After the reaction is finished by using a common gradient PCR instrument, the amplification result can be judged by two modes, firstly, SYBRGreen I dye with the final concentration of 25x is added, and whether the amplification result is positive or not is judged by color, namely whether a target sequence exists in a sample to be detected or not is judged; and secondly, carrying out agarose gel electrophoresis on the amplification product, and judging whether the amplification result is positive or not according to the electrophoresis strip, namely whether the target sequence exists in the sample to be detected or not.
TABLE 1 nucleic acid amplification reaction System of Yersinia enterocolitica (Yersiniaentericolytica)
System of Volume (μ l) Final concentration
R300 MIX(TaKaRaTaqTMHS Perfect Mix) 12.5 /
Yer-F1/Yer-R1(10μM) 1.25+1.25 0.5μM
100％DMSO 0.5 2％
DNA template
0/1 /
ddH2O Up to 25 /
TABLE 2 list of specific detection targets for nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaenterocolitica)
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 engleri 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. Collateral injury of B typeSalmonella typhosa 10437 28. Vibrio frenuli 1.1613
14. Shigella dysenteriae 1.1869 29. Vibrio cholerae 1.8676
15. Shigella bodyii 1.10618 30. Shigella flexneri
The results are shown in FIGS. 4 to 11, wherein 1 to 23 are respectively Staphylococcus aureus, staphylococcus aureus subspecies aurantiae, staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inoke, listeria eheliae, salmonella enteritidis subspecies, salmonella enteritidis, salmonella typhimurium, salmonella paratyphi B, salmonella dysentery, shigella boydii, shigella flexneri, escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, escherichia coli diarrheal, escherichia coli toxin-producing Escherichia coli, escherichia coli toxigenic Escherichia coli, escherichia coli hemorrhagic, shinobilis sakazakii, and 25 to 30 are respectively Yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio cholerae, vibrio and Shigella forbergii, N: negative control, P: positive control (plasmid containing sequence of interest); and 24 is yersinia enterocolitica.
FIGS. 4/5/6 show the amplification curve, the dissolution curve and the agarose gel electrophoresis result of the real-time fluorescence quantitative PCR of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentericolytica) of the present invention at a suitable temperature, wherein "80 ℃", "81 ℃" indicates the amplification result of the positive template at the denaturation temperature, "NTC" or "N" is the corresponding amplification result of the negative template, and "M" indicates the Marker DL2000. FIG. 4 shows that the amplification of the positive and negative templates is expected. FIG. 5 shows a single peak of the dissolution curve without nonspecific reaction. FIG. 6 shows the results of the amplification electrophoresis with 80 ℃ and 81 ℃ denaturation on the left and right sides of M ", and is positive if a single band is present at 242bp after electrophoresis of the amplification product; if the amplified product has no band after electrophoresis, the amplified product is negative.
FIGS. 7/8/9/10 show 3 detection limit detection methods of nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentericolytica) at denaturation temperatures of 80 ℃ and 81 ℃ in accordance with the present invention. FIG. 7/8 and Table 3, 10-fold dilutions from 15ng to 15fg were made, with 10-fold differences in amplification curves, more than 3-fold difference, and less than 1.5pg linearity, in the 15ng to 15pg range. Therefore, a standard curve can be prepared at 15pg or more, and quantitative measurement can be carried out. FIG. 9 shows the sensitivity of SYBRGreen I dye added at a final concentration of 25X, with 10-fold dilutions between 15ng and 15fg, and positive staining results in the 15ng to 15pg range. FIG. 10 shows that the amplification product was subjected to agarose gel electrophoresis, and a single band at 242bp in the range of 15ng to 1.5pg was determined to be positive.
TABLE 3 FIG. 7/8 summary of real-time fluorescent quantitative PCR sensitivity
Primer numbering Temperature of denaturation 15fg 150fg 1.5pg 15pg 150pg 1.5ng 15ng
Yer-F1/R1 80℃ √ √ √ √
Yer-F1/R1 81℃ √ √ √ √ √
FIG. 11 shows the results of the color development of SYBRGreen I dye for the specific detection of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersinianterocolitica) at a denaturation temperature of 81 ℃ in accordance with the present invention. If the amplification product is bright green, the amplification product is positive; if the amplification product is orange, it is negative. In fig. 11, the negative control (N) appeared orange, a negative result, as expected; yersinia enterocolitica No. 24 shows bright green, is a positive result, accords with expectation, and simultaneously, the detection results of the genomic DNA templates of other bacteria are negative, as shown in the tubes 1 to 23 and 25 to 30. All results were as expected, suggesting that no or even a slight amount of non-specific amplification occurred at the denaturation temperature of 81 ℃ but not enough to affect the dye-based outcome determination.
Example 2 detection of Yersinia enterocolitica (Yersiniaenterocolitica)
The primer design process refers to the steps (1) and (2) in example 1, 20 PCR reaction primer pairs are designed in the AT enrichment region after the program is run, and one primer group is randomly selected for validity verification. The sequence of the primer pair D is as follows:
Yer-F2：5’-TGTGCGGTGGATGTAAATAATTC-3’(SEQ ID NO.3)
Yer-R2：5’-GCTTTGAAACTCAAGGACTG-3’(SEQ IDNO.4)
the AT base content of the primer is respectively 60.87 percent and 55 percent, and the percentage content of the theoretical amplification product sequence AT of the primer pair is 68.09 percent.
Calculation of denaturation temperature and annealing temperature of PCR reaction:
the Tm =64.7 ℃ for the primer Yer-F2, tm =64.5 ℃ for the Yer-R2, calculated using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), the average Tm value for this primer pair is 64.6 ℃; the denaturation temperature of the amplified region was 77.76 ℃ as calculated using the formula 0.357X (GC percentage) X100 +70.582- (990/amplification product sequence length).
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 4 below. Determining an applicable denaturation temperature and a dissolution curve by using a real-time fluorescence quantitative PCR instrument according to the calculation result; the specificity of the primers for different detection objects at the applicable denaturation temperature is tested; the limit of detection at the applicable denaturation temperature was tested. Denaturation 5 sec, annealing at 63.5 ℃ for 5 sec, and extension at 72 ℃ for 20 sec, and it is recommended to repeat the above process 35 times. The list of specific test subjects is shown in Table 5.
After the reaction is finished by using a common gradient PCR instrument, the amplification result can be judged by two modes, firstly, SYBRGreen I dye with the final concentration of 25x is added, and whether the amplification result is positive or not is judged by color, namely whether a target sequence exists in a sample to be detected or not is judged; and secondly, carrying out agarose gel electrophoresis on the amplification product, and judging whether the amplification result is positive or not according to the electrophoresis strip, namely whether the target sequence exists in the sample to be detected or not.
TABLE 4 nucleic acid amplification reaction System of Yersinia enterocolitica (Yersiniaentericolytica)
System of Volume (μ l) Final concentration
R300 MIX(TaKaRaTaqTMHS Perfect Mix) 12.5 /
Yer-F2/Yer-R2(10μM) 1.25+1.25 0.5μM
100％DMSO 0.5 2％
DNA template
0/1 /
ddH2O Up to 25 /
TABLE 5 list of specific test subjects for Yersinia enterocolitica (Yersiniaenterocolitica) nucleic acid amplification reaction
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 engleri 10417 23. Cronobacter sakazakii 21560
9. IshikiListeria 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. Paratyphoid B salmonella 10437 28. Vibrio frenuli 1.1613
14. Shigella dysenteriae 1.1869 29. Vibrio cholerae 1.8676
15. Shigella boydii 1.10618 30. Shigella flexneri
The results are shown in FIGS. 13 to 19, wherein 1 to 23 are respectively Staphylococcus aureus, staphylococcus aureus subspecies aurantiacus, staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inoke, listeria eheliae, salmonella enteritidis subspecies, salmonella enteritidis, salmonella typhimurium, salmonella paratyphi B, salmonella dysentery, shigella boydii, shigella flexneri, escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, escherichia coli diarrheal, escherichia coli toxin-producing Escherichia coli, escherichia coli toxigenic Escherichia coli, escherichia coli hemorrhagic Escherichia coli, cronobacter sakazakii, and 25 to 30 are respectively Yersia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio cholerae, vibrio and Shigella forbergii, N: negative control, P: positive control (plasmid containing sequence of interest); and 24 is yersinia enterocolitica.
FIGS. 12 to 14 show the amplification curve, the dissolution curve and the agarose gel electrophoresis result of the real-time fluorescence quantitative PCR of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentericolytica) of the present invention at a suitable temperature, wherein "81 ℃", "82 ℃" indicates the amplification result of the positive template at the denaturation temperature, "NTC" or "N" is the corresponding amplification result of the negative template, and "M" indicates Marker DL2000. FIG. 12 positive and negative template amplifications were expected. FIG. 13 shows a single peak of the dissolution curve without nonspecific reaction. FIG. 14 shows the results of the identification of the amplification electrophoresis with 81 ℃ and 82 ℃ denaturation on the left and right sides of M ", and if a single band is present at 235bp after the electrophoresis of the amplification product, the result is positive; if the amplified product has no band after electrophoresis, the amplified product is negative.
FIGS. 15 to 18 show 3 detection limit detection methods of the present invention for nucleic acid amplification reaction of Yersinia enterocolitica (Yersiniaentericolytica) at denaturation temperatures of 81 ℃ and 82 ℃. FIG. 15/16 and Table 6, 10-fold dilutions from 15ng to 15fg, with 10-fold difference per 15ng to 15pg, more than 3-fold difference in amplification curves, and less than 15pg of linearity. Therefore, a standard curve can be prepared at 15pg or more, and quantitative measurement can be carried out. FIG. 17 shows the sensitivity of SYBRGreen I dye added at a final concentration of 25 Xwith 10-fold dilutions from 15ng to 15fg, and positive results in the range of 15ng to 150 pg. FIG. 18 shows that the amplification product was subjected to agarose gel electrophoresis, and a single band at 235bp in the range of 15ng to 15pg was judged to be positive.
TABLE 6 FIG. 15/16 sensitivity summary of real-time fluorescent quantitative PCR
Primer numbering Temperature of denaturation 15fg 150fg 1.5pg 15pg 150pg 1.5ng 15ng
Yer-F2/R2 81℃ √ √ √ √
Yer-F2/R2 82℃ √ √ √ √
FIG. 19 shows the results of the color development of SYBRGreen I dye for the specific detection of the nucleic acid amplification reaction of Yersinia enterocolitica (Yersinianterocolitica) according to the present invention at a denaturation temperature of 82 ℃. If the amplification product is bright green, the amplification product is positive; if the amplification product is orange, it is negative. In fig. 19, the negative control (N) appeared orange, being a negative result, as expected; yersinia enterocolitica # 24 shows bright green, is a positive result, and accords with the expectation, and meanwhile, the detection results of the genomic DNA templates of other bacteria are negative, as shown in the tubes 1-23 and 25-30. All results were as expected, suggesting that at the denaturation temperature of 82 ℃ no or even a slight amount of non-specific amplification occurred but not enough to affect the dye-based outcome determination.
This example perfectly verifies that the non-specific amplification can be significantly reduced by local melting of the template sequence by lowering the denaturation temperature, as proposed by the present method.
Example 3 detection of Cronobacter sakazakii (Cronobacters sazakii)
The method comprises the steps of screening AT-rich sequences and designing specific primers for a genome of Cronobacter sakazakii (Cronobacter sakazakii), setting reaction conditions according to the calculated primer Tm and the target sequence Tm to carry out nucleic acid amplification, determining whether the target sequence exists in a sample to be detected by judging whether the reaction result is positive, and further determining whether the Cronobacter sakazakii exists in the sample to be detected. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 total genome sequences were used for the bacteria, archaea and virus data with complete genome sequences downloaded from FTP at NCBI, 8/5 in 2019. Set a, which comprises all cronobacter sakazakii genomic sequences; set B was set up to include all non-sakazakii cronobacter sakazakii genomic sequences. Taking a cronobacter sakazakii genome sequence with the GI number of 156932229 as a reference genome, sliding a window with the width of 1000bp from the first base of the genome, and setting the step length as 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the 3 'end stability delta G <4, the 5' end stability delta G <3, the primer sequence length (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure and cannot interact with the single primer are set, and the single primer meeting the set conditions is calculated by taking the candidate target sequence in the step (1) as a candidate sequence for designing the single primer. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, a parameter is set to be '-a-n 0', namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, a parameter setting of "-a-n 3" was used when the single primer was aligned to the non-target sequences in pool B, i.e., the single primer was allowed no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying the condition, in which the number of primer pairs is preset, and in this embodiment, the number of primer pairs is preset to 20. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts.
After the program is operated, 20 PCR reaction primer pairs are designed in the AT enrichment region, and one primer group is randomly selected for validity verification. The sequences of the primer pairs are as follows:
Cro-F：5’-CGCCATAACTGCATAATCAT-3’(SEQ ID NO：5)
Cro-R：5’-ATAACGAGTTACCGTGCAGA-3’(SEQ ID NO：6)
the content of AT base of the primer is 60% and 55%, and the percentage content of AT in the theoretical amplification product sequence of the primer pair is 69%.
(3) Calculation of denaturation temperature and annealing temperature of PCR reaction:
tm =62.2 ℃ for primer Cro-F, tm =64.5 ℃ for Cro-R, calculated using the formula 0.466X (GC percentage) X100 +66.04- (450/primer sequence length), the primer pair average Tm value is 63.4 ℃; the denaturation temperature of the amplified region was calculated to be 78.3 ℃ using the formula 0.357X (GC percentage) X100 +70.582- (990/amplification product sequence length).
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 7 below. According to the calculation result, 3 groups of experiments are set, the specificity of the primers for different detection objects at the denaturation temperature of 94 ℃/90 ℃/82 ℃ is respectively tested, the nucleic acid amplification reaction conditions are 94 ℃/90 ℃/82 ℃ denaturation for 5 seconds, 63 ℃ annealing for 5 seconds and 72 ℃ extension for 20 seconds, and the processes are repeated for 35 times. The list of specific test subjects is shown in Table 8.
After the reaction is finished, judging the amplification result by two modes, namely adding SYBRGreen I dye with the final concentration of 25x, judging whether the amplification result is positive or not by color, namely judging whether a target sequence exists in the sample to be detected or not; secondly, agarose gel electrophoresis is carried out on the amplification product, and whether the amplification result is positive or not is judged according to the electrophoresis strip, namely whether the target sequence exists in the sample to be detected or not is judged.
TABLE 7 nucleic acid amplification reaction System of Cronobacter sakazakii (Cronobacters sazakii)
System of Volume (μ l) Final concentration
R300 MIX(TaKaRaTaqTMHS Perfect Mix) 12.5 /
Cro-F/Cro-R(50μM) 0.25+0.25 0.5μM
100％DMSO 0.5 2％
DNA template (10 ng) 0/1 /
ddH2O Up to 25 /
TABLE 8 List of subjects for specific detection of Cronobacter sakazakii (Cronobacters sazakii) nucleic acid amplification reaction
Figure BDA0003050350070000171
Figure BDA0003050350070000181
The experimental results are shown in figures 1, 2 and 3, wherein 1-22 are staphylococcus aureus, staphylococcus aureus subspecies aureoflavus, staphylococcus epidermidis, rhodococcus equi, bacillus cereus, bacillus mycoides, listeria monocytogenes, listeria inoke, listeria ehelii, salmonella enteritidis subspecies, salmonella enteritidis, salmonella typhimurium, salmonella paratyphi b, shigella dysenteriae, shigella boydii, shigella flexneri, escherichia coli (containing clostridium botulinum type a gene), pathogenic escherichia coli, escherichia coli diarrheal, enterotoxigenic escherichia coli, enterohemorrhagic escherichia coli, and 24-30 are respectively yersinia enterocolitica, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio cholerae and shigella flexneri, N: negative control, P: positive control (plasmid containing the sequence of interest); 23 is cronobacter sakazakii.
FIG. 1 shows the results of the color development of the SYBRGreen I dye for the specific detection of the nucleic acid amplification reaction of Cronobacter sakazakii (Cronobacters sazakii) according to the invention at different denaturation temperatures. If the amplification product is bright green, the amplification product is positive; if the amplification product is orange, it is negative. In FIG. 1, the negative control (N) appeared orange when the denaturation temperature was 94 ℃/90 ℃ and was a negative result, as expected; the positive control (P) and cronobacter sakazakii No. 23 exhibited a bright green color, which was a positive result, and was expected. The whole reaction system can work normally. However, the amplification products of the genomic DNA templates of other bacteria showed a lot of positive results, as indicated by tubes 3, 4, 10-18, 20-22, 24, 26, 27, 29 and 30 at a denaturation temperature of 94 ℃ and tubes 3, 7, 10-18, 20-22, 24, 26, 27, 29 and 30 at a denaturation temperature of 90 ℃, which is contrary to the expectation, suggesting that non-specific amplification is likely to occur at a denaturation temperature of 94 ℃/90 ℃ and the result is false positive. When the denaturation temperature is 82 ℃, the negative control (N) is orange, and the negative result is a negative result, which is in line with expectation; the positive control (P) and cronobacter sakazakii No. 23 exhibited bright green color, which was a positive result, and was as expected. The whole reaction system can work normally. Meanwhile, the detection results of the genome DNA templates of other bacteria are negative, as shown in the No. 1-22 and No. 24-30 tubes. All results were as expected, suggesting that at the denaturation temperature of 82 ℃ no or even a slight amount of non-specific amplification occurred but not enough to affect the dye-based outcome determination.
FIG. 2 shows the results of electrophoresis of amplification products of the nucleic acid amplification reaction of the present invention against Cronobacter sakazakii (Cronobacters sazakii) with specific detection at a denaturation temperature of 94 ℃. If a single band exists at the 291bp position after the electrophoresis of the amplification product, the amplification product is positive; if the amplified product has no band after electrophoresis, the amplified product is negative; if one or more bands appear outside 291bp after electrophoresis of the amplification reaction product, the amplification reaction product is false positive caused by non-specific amplification. In FIG. 2, the negative control (N) had no bands, as expected; the positive control (P) and the Cronobacter sakazakii No. 23 both have a clear band at 291bp, which is expected. The whole reaction system can work normally. However, the amplified products of genomic DNA templates from other bacteria showed many bands in the range of 500bp to 2000bp, which is not in line with the expectation, suggesting that the reaction system produced a large amount of non-specific amplification at a denaturation temperature of 94 ℃ and the result was consistent with the dye color results in FIG. 1.
FIG. 3 shows the results of electrophoresis of amplification products of the nucleic acid amplification reaction of the present invention against Cronobacters sakazakii (Cronobacters sazakii) with specific detection at a denaturation temperature of 82 ℃. If a single band exists at the 291bp position after the electrophoresis of the amplification product, the amplification product is positive; if the amplification reaction product has no band after electrophoresis, the amplification reaction product is negative; if one or more bands appear outside 291bp after electrophoresis of the amplification reaction product, the amplification reaction product is false positive caused by non-specific amplification. In FIG. 3, the negative control (N) has no band, as expected; the positive control (P) and the Cronobacter sakazakii No. 23 both have a clear band at 291bp, which is expected. The whole reaction system can work normally. Meanwhile, most of the amplification reaction products of the genome DNA templates of other bacteria have no band and show negative results, such as lanes 2-6, 10, 12, 13, 15-22 and 26-30; the products of a few template amplification reactions showed very weak bands after electrophoresis, which were easily distinguished from the positive results, as shown in 1, 7, 8, 9, 11, 14, 24, 25 tubes, indicating that no or very little non-specific amplification was produced at the denaturation temperature of 82 ℃ which was easily distinguished in the electrophoresis results.
As can be seen from fig. 1 to 3 and table 8, the nucleic acid amplification method of the present invention has good strain specificity for the application of cronobacter sakazakii, i.e., only cronobacter sakazakii exhibits a positive result, but not cronobacter sakazakii exhibits a negative result, in the reaction system and the reaction conditions proposed in the present invention. It is to be noted that, when the reaction is carried out using the denaturation temperature (90 ℃ or 94 ℃) of the conventional PCR method, although the electrophoresis result (FIG. 2) shows that the target detection object (Cronobacter sakazakii, lane 23) has a single clear band, the non-target detection object generally exhibits a large amount of non-specific amplification, such as Staphylococcus epidermidis in lane 3; when the detection result is judged by the dye-color method, as shown in FIG. 1, a large number of false positive results are obtained under the denaturing conditions of 90 ℃ and 94 ℃, and thus the detection object and the non-detection object cannot be distinguished. In the same reaction system, when the reaction is carried out at the denaturation temperature of 82 ℃ calculated by the method, the electrophoresis result (figure 3) shows that a target detection object (cronobacter sakazakii, lane 23) has a single clear band, while non-target detection objects have less non-specific amplification, and even if the non-target detection objects have the single clear band, the non-specific amplification band is weak and is easily distinguished from a positive result; when the detection result was judged by the dye-color method, as shown in fig. 1, only the target assay-object (cronobacter sakazakii, tube No. 23) showed a positive result and all the non-target assay-objects showed a negative result under the denaturation condition at 82 ℃.
Example 4 Simultaneous detection of Salmonella, staphylococcus aureus, yersinia enterocolitica and Cronobacter sakazakii
The primer design in the invention aims AT the genome of yersinia enterocolitica, is respectively changed into the genomes of salmonella, staphylococcus aureus, yersinia enterocolitica and cronobacter sakazakii, AT-rich sequences are screened, specific primers are designed, a primer combination is screened according to the calculated primer Tm and target sequence Tm, reaction conditions are set for nucleic acid amplification, whether a target sequence exists in a sample to be detected is determined by judging whether the reaction result is positive, and then whether one or more of salmonella, staphylococcus aureus, yersinia enterocolitica and cronobacter sakazakii exists in the sample to be detected is determined. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 total genome sequences were used for the bacteria, archaea and virus data with complete genome sequences downloaded from FTP at NCBI, 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set up, which contained all non-salmonella genomic sequences. A salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the 3 'end stability delta G <4, the 5' end stability delta G <3, the primer sequence length (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure and cannot interact with the single primer are set, and the single primer meeting the set conditions is calculated by taking the candidate target sequence in the step (1) as a candidate sequence for designing the single primer. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, when the single primer is aligned to the non-target sequences in pool B, a parameter setting of "-a-n 3" is used, i.e., the single primer is allowed to have no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying a condition in which the number of primer pairs is preset, and in this embodiment, the number of primer pairs is preset to 100. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts. After the program is operated, 100 salmonella amplification primer pairs are designed in the AT enrichment region.
100 amplification primer pairs of staphylococcus aureus, yersinia enterocolitica and cronobacter sakazakii are respectively designed by the same method. Calculating Tm of each primer using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length) and calculating the average Tm value of the primer pair as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357X (percent GC). Times.100 +70.582- (990/amplification product sequence length).
Screening the primer combination by taking the conditions that the difference of the amplification sequence lengths of different bacteria is at least 50bp, the reaction denaturation temperature is at most 2 ℃ and the reaction annealing temperature is at most 2 ℃. The system outputs primer combinations satisfying the condition, and the number thereof may be set in advance, and in this embodiment, the number of primer combinations is set in advance to 10. And randomly selecting a primer group for validity verification. The sequences of the primer combination are as follows:
primer pair A:
Sal-F：5’-TCAGACATCCGTTCAGAAAAT-3’(SEQ ID NO.7)
Sal-R：5’-GTTCAACTGTCGACAAGATTAA-3’(SEQ ID NO.8)
and (3) primer pair B:
Sta-F：5’-GTAGGTATGGTAAATAGTTACAC-3’(SEQ ID NO.9)
Sta-R:5’-CACTAATGCCAAATTTACTTAAAATCG-3’(SEQ ID NO.10)
and (3) primer pair C:
Cro-F：5’-CGCCATAACTGCATAATCAT-3’(SEQ ID NO.5)
Cro-R:5’-ATAACGAGTTACCGTGCAGA-3’(SEQ ID NO.6)
and (3) primer pair D:
Yer-F2：5’-TGTGCGGTGGATGTAAATAATTC-3’(SEQ ID NO.3)
Yer-R2:5’-GCTTTGAAACTCAAGGACTG-3’(SEQ ID NO.4)
the AT base content of the primer pair A is 60 percent and 64 percent respectively, and the theoretical average annealing temperature is 62 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 355bp, the percentage content of AT is 71%, and the theoretical denaturation temperature is 78.14 ℃.
The AT base content of the primer pair B is 65.22 percent and 70.37 percent respectively, and the theoretical average annealing temperature is 62.5 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 465bp, the percentage content of AT is 73.76 percent, and the theoretical denaturation temperature is 77.82 ℃.
The AT base content of the primer pair C is 60 percent and 55 percent respectively, and the theoretical average annealing temperature is 63 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 291bp, the percentage content of AT is 68.73%, and the theoretical denaturation temperature is 78.34 ℃.
The AT base content of the primer pair D is 60.87 percent and 55 percent respectively, and the theoretical average annealing temperature is 64 ℃; the length of the theoretical amplification product sequence fragment of the primer pair is 235bp, the percentage content of AT is 68.09%, and the theoretical denaturation temperature is 77.76 ℃.
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 9 below. According to the calculation results, the above process is recommended to be repeated 35 times by using denaturation at 81 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds and elongation at 72 ℃ for 20 seconds. The list of test subjects is shown in table 10.
After the reaction is finished by using a common gradient PCR instrument, the amplification result can be judged by two modes, firstly, SYBRGreen I dye with the final concentration of 25x is added, and whether the amplification result is positive or not is judged by color, namely whether a target sequence exists in a sample to be detected or not is judged; and secondly, carrying out agarose gel electrophoresis on the amplification product, and judging whether the amplification result is positive or not according to the electrophoresis strip, namely whether the target sequence exists in the sample to be detected or not.
TABLE 9 four bacterial nucleic acid amplification reaction systems
Figure BDA0003050350070000221
Figure BDA0003050350070000231
TABLE 10 list of four bacteria nucleic acid amplification reaction-specific assay targets
1 Staphylococcus aureus 21600 16 Shigella flexneri 11868
2 Staphylococcus aureus subspecies 12465 17 Escherichia coli 10738
3 Staphylococcus epidermidis 14260 18 pathogenic escherichia coli 10372
4 Rhodococcus equi 14262 19 diarrhea causing escherichia coli 10411
5 Bacillus cereus 13760 20 enterotoxigenic Escherichia coli 10415
6 Bacillus mycoides 21473 21 enterotoxigenic Escherichia coli 10665
7 Listeria monocytogenes 21635 22 hemorrhagic escherichia coli 21530
Listeria monocytogenes 10417 23 cronobacter sakazakii 21560
9 listeria monocytogenes 21663 Yersinia enterocolitica 21669
10 Salmonella enteritidis subspecies 11859 25 Yersinia pseudotuberculosis 53504
11 Salmonella enteritidis 21482 26 Vibrio vulnificus 21615
12 Salmonella typhimurium 10420 27 Vibrio parahaemolyticus 11997
13 salmonella paratyphi type b 10437 28 Vibrio parahaemolyticus 11613
14. Shigella dysenteriae 1.1869 29. Vibrio cholerae 1.8676
15. Shigella bodyii 1.10618 30. Shigella sonnei
FIGS. 20 and 21 show the results of the fluorescent dye staining and the agarose gel electrophoresis of the nucleic acid amplification reaction of the present invention against four bacteria, salmonella enteritidis subspecies, staphylococcus aureus subspecies, yersinia enterocolitica, cronobacter sakazakii. Wherein, N is the amplification result of the corresponding negative template, M1 refers to Marker DL2000, M2 refers to Marker B (100-600 bp), and mixed refers to the mixed template of four bacterial genome DNAs. FIG. 20 shows that the results of single-strain, mixed-template and negative-template amplification and color development are expected. FIG. 21 shows that the individual amplified fragments of the individual bacteria match the theoretical values and that the amplified fragments of the bacteria of the mixed template species can be separated from each other.
FIG. 22 shows the SYBRGreen I dye color development results of specific detection of nucleic acid amplification reactions of the present invention against four bacteria, salmonella, staphylococcus aureus, yersinia enterocolitica, and Cronobacter sakazakii, at a denaturation temperature of 81 ℃. If the amplification product is bright green, the amplification product is positive; if the amplification product is orange, it is negative. In fig. 22, staphylococcus aureus No. 1-2, staphylococcus aureus subspecies, salmonella enteritidis subspecies No. 10-13, salmonella enteritidis, salmonella typhimurium and salmonella paratyphi b, salmonella sakazakii No. 23, and yersinia enterocolitica No. 24 exhibited bright green color, which was a positive result, and was expected; meanwhile, the detection results of the genomic DNA templates of other bacteria are negative, as shown in the tubes 3-9, 14-22 and 25-30. All results were as expected, suggesting that no or even a slight amount of non-specific amplification occurred at the denaturation temperature of 81 ℃ but not enough to affect the dye-based outcome determination.
Example 5 Simultaneous detection of Salmonella and Staphylococcus aureus
The method comprises the steps of screening sequences rich in AT aiming AT salmonella and staphylococcus aureus genomes, designing specific primers, screening primer combinations according to the calculated primer Tm and the target sequence Tm, setting reaction conditions for nucleic acid amplification, determining whether the target sequence exists in a sample to be detected by judging whether a reaction result is positive, and further determining whether one or more of salmonella and staphylococcus aureus exists in the sample to be detected. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 total genome sequences were used for the bacteria, archaea and virus data with complete genome sequences downloaded from FTP at NCBI, 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set up, which contained all non-salmonella genomic sequences. The salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the stability delta G AT the 3 'end of less than 4, the stability delta G AT the 5' end of less than 3, the length of the primer sequence (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure, cannot generate interaction per se and the like are set, the candidate target sequence in the step (1) is taken as the candidate sequence for designing the single primer, and the single primer meeting the set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, a parameter is set to be '-a-n 0', namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, a parameter setting of "-a-n 3" was used when the single primer was aligned to the non-target sequences in pool B, i.e., the single primer was allowed no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying a condition in which the number of primer pairs is preset, and in this embodiment, the number of primer pairs is preset to 100. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts. After the program is operated, 100 salmonella amplification primer pairs are designed in the AT enrichment region.
100 amplification primer pairs of staphylococcus aureus were designed by the same method. Calculating Tm of each primer using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length) and calculating the average Tm value of the primer pair as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357X (percent GC content). Times.100 +70.582- (990/amplification product sequence length).
The primer combination is screened under the conditions that the difference of the amplification sequence lengths of different bacteria is at least 50bp, the reaction denaturation temperature is at most 2 ℃, and the reaction annealing temperature is at most 2 ℃. The system outputs primer combinations satisfying the condition, and the number may be set in advance, and in this embodiment, the number of primer combinations is set in advance to 10. One primer set was randomly selected for validation. The sequences of the primer combination are as follows:
and (3) primer pair A:
Sal-F：5’-TCAGACATCCGTTCAGAAAAT-3’(SEQ ID NO.8)
Sal-R：5’-GTTCAACTGTCGACAAGATTAA-3’(SEQ ID NO.9)
and (3) primer pair E:
Sta-F1：5’-CCTTTCATCTAAAAACCTCCA-3’(SEQ ID NO.11)
Sta-R1:5’-GAAATGGATGTTTTAAAAGAAGG-3’(SEQ ID NO.12)
the AT base content of the primer pair A is 60 percent and 64 percent respectively, and the theoretical average annealing temperature is 62 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 355bp, the percentage content of AT is 71%, and the theoretical denaturation temperature is 78.14 ℃.
The AT base content of the primer pair E is 61.90 percent and 69.57 percent respectively, and the theoretical average annealing temperature is 61 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 595bp, the percentage content of AT is 76.30%, and the theoretical denaturation temperature is 77.38 ℃.
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 11 below. According to the calculation results, the above process is recommended to be repeated 35 times by using denaturation at 81 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds and elongation at 72 ℃ for 20 seconds. The list of test subjects is shown in table 10.
After the reaction is finished by using a common gradient PCR instrument, the amplification product is subjected to agarose gel electrophoresis, and whether the amplification result is positive or not is judged according to the electrophoresis band, namely whether the target sequence exists in the sample to be detected or not is judged.
TABLE 11 two bacterial nucleic acid amplification reaction systems
System of Volume (μ l) Final concentration
TaqR300mix
15 1x
SAL-F/R(50μM) 0.25+0.25 0.5μM
STA-F1/R1(50μM) 0.25+0.25 0.5μM
100％DMSO 0.5 2％
Stencil (10 ng) 0/1 /
ddH2O Upto25 /
FIG. 23 shows the results of agarose gel electrophoresis of the nucleic acid amplification reaction of the invention against two bacteria, salmonella enteritidis subspecies and Staphylococcus aureus subspecies. Wherein, N is the amplification result of the corresponding negative template, M is Marker DL2000, salmonella is Salmonella enteritidis subspecies, staphylococcus aureus subspecies, and mixed template of the two bacterial genome DNAs. FIG. 23 shows that each single amplified fragment of the bacteria meets the theoretical calculation and that each amplified fragment of the bacteria of the mixed template species can be separated from each other.
Example 6 Simultaneous detection of Salmonella and Yersinia enterocolitica
The method comprises the steps of screening AT-rich sequences and designing specific primers aiming AT salmonella and yersinia enterocolitica genomes, screening primer combinations according to calculated primer Tm and target sequence Tm, setting reaction conditions for nucleic acid amplification, determining whether a target sequence exists in a sample to be detected by judging whether a reaction result is positive, and further determining whether one or more of salmonella and yersinia enterocolitica exists in the sample to be detected. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 whole genome sequences were used for bacteria, archaea and virus data with complete genome sequences downloaded from the FTP at NCBI at 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set, which contained all non-salmonella genomic sequences. A salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the stability delta G AT the 3 'end of less than 4, the stability delta G AT the 5' end of less than 3, the length of the primer sequence (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure, cannot generate interaction per se and the like are set, the candidate target sequence in the step (1) is taken as the candidate sequence for designing the single primer, and the single primer meeting the set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the two conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is 200-600 bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, a parameter is set to be '-a-n 0', namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, when the single primer is aligned to the non-target sequences in pool B, a parameter setting of "-a-n 3" is used, i.e., the single primer is allowed to have no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying a condition in which the number of primer pairs is preset, and in this embodiment, the number of primer pairs is preset to 100. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts. After the program is operated, 100 salmonella amplification primer pairs are designed in the AT enrichment region.
100 Yersinia enterocolitica amplification primer pairs are designed by the same method. Calculating Tm of each primer using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length) and calculating the average Tm value of the primer pair as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357X (percent GC content). Times.100 +70.582- (990/amplification product sequence length).
Screening the primer combination by taking the conditions that the difference of the amplification sequence lengths of different bacteria is at least 50bp, the reaction denaturation temperature is at most 2 ℃ and the reaction annealing temperature is at most 2 ℃. The system outputs primer combinations satisfying the condition, and the number thereof may be set in advance, and in this embodiment, the number of primer combinations is set in advance to 10. And randomly selecting a primer group for validity verification. The sequences of the primer combination are as follows:
and (3) primer pair F:
Sal-F2：5’-TGGGTTGAAATAGCCCATTA-3’(SEQ ID NO.13)
Sal-R2：5’-GACGTGACACACTTCGTTTT-3’(SEQ ID NO.14)
a primer pair G:
Yer-F1：5’-ATGGAAAATAACATAATTTCTATTACCGG-3’(SEQ ID NO.1)
Yer-R1:5’-TCTCTGCGAATAACCTTGTG-3’(SEQ ID NO.2)
the AT base content of the primer pair F is 60 percent and 55 percent respectively, and the theoretical average annealing temperature is 63 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 352bp, the percentage content of AT is 74.43%, and the theoretical denaturation temperature is 76.90 ℃.
The AT base content of the primer pair G is 72.41 percent and 55 percent respectively, and the theoretical average annealing temperature is 63.5 ℃; the theoretical amplification product sequence fragment length of the primer pair is 242bp, the percentage content of AT is 72.02%, and the theoretical denaturation temperature is 76.48 ℃.
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 12 below. According to the calculation results, the above process is recommended to be repeated 35 times by using denaturation at 81 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds and elongation at 72 ℃ for 20 seconds. The list of test subjects is shown in table 10.
After the reaction is finished by using a common gradient PCR instrument, the amplification product is subjected to agarose gel electrophoresis, and whether the amplification result is positive or not is judged according to the electrophoresis band, namely whether the target sequence exists in the sample to be detected or not is judged.
TABLE 12 two bacterial nucleic acid amplification reaction systems
Figure BDA0003050350070000281
Figure BDA0003050350070000291
FIG. 24 shows the result of agarose gel electrophoresis of the nucleic acid amplification reaction of the present invention against two bacteria, salmonella enteritidis subspecies and Yersinia enterocolitica. Wherein "N" is the amplification result of the corresponding negative template, "M" refers to Marker DL2000, "Salmonella refers to Salmonella enteritidis subspecies," Yersi "refers to Yersinia enteronitis subspecies, and" mixed "refers to the mixed template of the genomic DNA of the two species of bacteria. FIG. 24 shows that each single-strain amplified fragment is in accordance with the theoretical calculation value and each strain amplified fragment of the mixed template can be separated from each other.
Example 7 Simultaneous detection of Salmonella, staphylococcus aureus and Yersinia enterocolitica
The method comprises the steps of screening AT-rich sequences and designing specific primers aiming AT salmonella, staphylococcus aureus and yersinia enterocolitica genomes, screening primer combinations according to calculated primer Tm and target sequence Tm, setting reaction conditions for nucleic acid amplification, determining whether a target sequence exists in a sample to be detected by judging whether a reaction result is positive, and further determining whether one or more of salmonella, staphylococcus aureus and yersinia enterocolitica exists in the sample to be detected. The method comprises the following specific steps:
(1) Screening of target sequences rich in AT bases:
2896 total genome sequences were used for the bacteria, archaea and virus data with complete genome sequences downloaded from FTP at NCBI, 8/5 in 2019. Set a, which contains all salmonella genomic sequences; set B was set up, which contained all non-salmonella genomic sequences. A salmonella genome sequence is used as a reference genome, a window with the width of 1000bp slides from the first base of the genome, and the step length is 50bp; calculating the base content of the sequence AT once before each sliding, and reserving a region with the base content of the sequence AT more than 60% as a candidate target sequence. The above process is implemented using perl scripts.
(2) Design of specific primers:
according to the characteristics of the PCR primer and the requirements of the invention, the characteristic parameters of the primer, such as the AT base content of 55-75%, the stability delta G AT the 3 'end of less than 4, the stability delta G AT the 5' end of less than 3, the length of the primer sequence (20-36 bp) and the like, are set, meanwhile, the conditions that a single primer cannot generate a hairpin structure, cannot generate interaction per se and the like are set, the candidate target sequence in the step (1) is taken as the candidate sequence for designing the single primer, and the single primer meeting the set conditions is calculated. The Tm value of a single primer is calculated by the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length), and the position of each primer on the target sequence, the plus and minus chain information (i.e., whether derived from the plus chain or the minus chain), and the length of the primer are recorded.
And (3) carrying out primer pairing according to the position information of the single primer, and reserving a primer pair which simultaneously meets the conditions that the Tm difference of the primer pair is less than 3 ℃ and the amplification area of the primer pair is between 200 and 600bp as a candidate primer pair.
And (3) using alignment software Bowtie to perform sequence alignment on each primer in the candidate primer pair designed in the last step with the target genome sequence in the set A and the non-target genome sequence in the set B respectively. In order to ensure the universality of the primers, when the single primer is aligned with the target sequence in the set A, the parameter setting of '-a-n 0' is used, namely the single primer is required to be completely matched with the target sequence; to ensure primer specificity, a parameter setting of "-a-n 3" was used when the single primer was aligned to the non-target sequences in pool B, i.e., the single primer was allowed no more than 3 mismatches to the non-target sequences. The system outputs primer pairs satisfying a condition in which the number of primer pairs is preset, and in this embodiment, the number of primer pairs is preset to 100. The high-throughput and automatic primer design process is realized by adopting C and Perl scripts. After the program is operated, 100 salmonella amplification primer pairs are designed in the AT enrichment region.
100 Yersinia enterocolitica amplification primer pairs are designed by the same method. Calculating Tm of each primer using the formula 0.466X (GC percentage content). Times.100 +66.04- (450/primer sequence length) and calculating the average Tm value of the primer pair as the reaction annealing temperature; the denaturation temperature of the amplified region of each primer pair was calculated using the formula 0.357X (percent GC). Times.100 +70.582- (990/amplification product sequence length).
The primer combination is screened under the conditions that the difference of the amplification sequence lengths of different bacteria is at least 50bp, the reaction denaturation temperature is at most 2 ℃, and the reaction annealing temperature is at most 2 ℃. The system outputs primer combinations satisfying the condition, and the number may be set in advance, and in this embodiment, the number of primer combinations is set in advance to 10. And randomly selecting a primer group for validity verification. The sequences of the primer combination are as follows:
and (3) primer pair A:
Sal-F：5’-TGGGTTGAAATAGCCCATTA-3’(SEQ ID NO.7)
Sal-R：5’-GACGTGACACACTTCGTTTT-3’(SEQ ID NO.8)
and (3) primer pair D:
Yer-F2：5’-ATGGAAAATAACATAATTTCTATTACCGG-3’(SEQ ID NO.3)
Yer-R2:5’-TCTCTGCGAATAACCTTGTG-3’(SEQ ID NO.4)
and (3) primer pair E:
Sta-F1：5’-CCTTTCATCTAAAAACCTCCA-3’(SEQ ID NO.11)
Sta-R1:5’-GAAATGGATGTTTTAAAAGAAGG-3’(SEQ ID NO.12)
the AT base content of the primer pair A is 60 percent and 55 percent respectively, and the theoretical average annealing temperature is 63 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 352bp, the percentage content of AT is 74.43 percent, and the theoretical denaturation temperature is 76.90 ℃.
The AT base content of the primer pair D is 72.41 percent and 55 percent respectively, and the theoretical average annealing temperature is 63.5 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 242bp, the percentage content of AT is 72.02%, and the theoretical denaturation temperature is 76.48 ℃.
The AT base content of the primer pair E is 61.90 percent and 69.57 percent respectively, and the theoretical average annealing temperature is 61 ℃; the length of a theoretical amplification product sequence fragment of the primer pair is 595bp, the percentage content of AT is 76.30%, and the theoretical denaturation temperature is 77.38 ℃.
(4) Nucleic acid amplification reaction and result detection:
the nucleic acid amplification reaction system configuration is shown in Table 13 below. According to the calculation results, the above process is recommended to be repeated 35 times by using denaturation at 81 ℃ for 5 seconds, annealing at 62 ℃ for 5 seconds and elongation at 72 ℃ for 20 seconds. The list of test subjects is shown in table 10.
After the reaction is finished by using a common gradient PCR instrument, the amplification product is subjected to agarose gel electrophoresis, and whether the amplification result is positive or not is judged according to an electrophoresis strip, namely whether a target sequence exists in a sample to be detected or not is judged.
TABLE 13 nucleic acid amplification reaction systems for three bacteria
Figure BDA0003050350070000311
Figure BDA0003050350070000321
FIG. 25 shows the results of agarose gel electrophoresis of nucleic acid amplification reactions of three bacteria of Salmonella enteritidis subspecies, staphylococcus aureus subspecies, and Yersinia enterocolitica according to the present invention. Wherein "N" is the amplification result of the corresponding negative template, "M" refers to Marker DL2000, "Enteria" refers to Salmonella enteritidis subspecies, "Small Ye" refers to Yersinia enteronitis subspecies, "Kinia" refers to Staphylococcus aureus subspecies, and "Mixed" refers to the mixed template of the genomic DNAs of the three bacteria. FIG. 25 shows that each single amplified fragment of the bacteria meets the theoretical calculation and that each amplified fragment of the bacteria of the mixed template species can be separated from each other.
Reference to the literature
Zhou Hui Qi (2014) relationship between GC content of genome and usage preference of base, codon and amino acid (master master), university of electronic technology (67)
The protection content of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.
SEQUENCE LISTING
<110> Shanghai Wangwang food group Co., ltd., shanghai Bioinformation technology research center
<120> system and method for detecting yersinia enterocolitica and application thereof
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 29
<212> DNA
<213> Artificial sequence
<400> 1
atggaaaata acataatttc tattaccgg 29
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
tctctgcgaa taaccttgtg 20
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
tgtgcggtgg atgtaaataa ttc 23
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
gctttgaaac tcaaggactg 20
<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> 21
<212> DNA
<213> Artificial sequence
<400> 7
tcagacatcc gttcagaaaa t 21
<210> 8
<211> 22
<212> DNA
<213> Artificial sequence
<400> 8
gttcaactgt cgacaagatt aa 22
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence
<400> 9
gtaggtatgg taaatagtta cac 23
<210> 10
<211> 27
<212> DNA
<213> Artificial sequence
<400> 10
cactaatgcc aaatttactt aaaatcg 27
<210> 11
<211> 21
<212> DNA
<213> Artificial sequence
<400> 11
cctttcatct aaaaacctcc a 21
<210> 12
<211> 23
<212> DNA
<213> Artificial sequence
<400> 12
gaaatggatg ttttaaaaga agg 23
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence
<400> 13
tgggttgaaa tagcccatta 20
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence
<400> 14
gacgtgacac acttcgtttt 20
<210> 15
<211> 19
<212> DNA
<213> Artificial sequence
<400> 15
aagggggttc caggcatta 19

Claims (13)

1. A system for detecting yersinia enterocolitica, comprising:
the screening module is used for screening a target sequence rich in AT bases in a genome sequence of yersinia enterocolitica; a primer design module for designing a primer having both versatility and specificity for the target sequence;
the calculation module is used for calculating the inverse dependent temperature and the annealing temperature based on the percentage content of GC, the sequence length of the primer and the sequence length of a theoretical amplification product of the primer pair, and setting the reaction condition of nucleic acid amplification;
and the amplification module is used for carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
2. A method of detecting yersinia enterocolitica comprising the steps of:
(1) Screening a target sequence rich in AT base in a genome sequence of yersinia enterocolitica;
(2) Designing a primer with both universality and specificity aiming at the target sequence;
(3) Calculating the inverse strain temperature and the annealing temperature based on the percentage content of GC, the sequence length of the primer and the sequence length of a theoretical amplification product of the primer pair, and setting the reaction conditions of nucleic acid amplification;
(4) And carrying out nucleic acid amplification reaction under the condition of local melting of the template to obtain an amplification product.
3. The method of claim 2, wherein in step (1), for the Yersinia enterocolitica genomic sequence, a window of 1000bp in width is slid starting from the first base, with a step size of 5-100 bp; calculating the AT base content of the sequence contained in the position of each window, and reserving the region with the AT base content of the sequence more than 60 percent as the target sequence.
4. The method of claim 2, wherein in step (2), the primer is designed by a method comprising: (2.1) designing a single primer aiming at a target sequence to obtain a candidate primer; (2.2) judging the physicochemical properties of the candidate primers, and screening single primers meeting the requirements; (2.3) combining the single primers obtained by screening in the step (2.2) into a primer pair; (2.4) judging the universality and the specificity of the primer pair; and (2.5) outputting a primer pair meeting the conditions to obtain the specific primer.
5. The method of claim 4, wherein in step (2.1), the candidate primers satisfy the following condition: a) The length of the primer sequence is between 20bp and 36 bp; b) The AT base content is 55-80%; c) The continuous GC number is less than or equal to 5; simultaneously recording position information and positive and negative chain information of the candidate primers matched on the target sequence; and/or, in the step (2.2), judging the physicochemical property of the single primer which meets the condition (2.1), wherein the physicochemical property comprises 3 'end stability, 5' end stability and/or secondary structure stability.
6. The method according to claim 4, wherein in step (2.3), the single primers obtained by screening in step (2.2) are combined into primer pairs according to the position information and the positive and negative chain information of the single primers matched with the target sequence; the length of the theoretical amplification product sequence of the primer pair is between 200bp and 600 bp; calculating Tm value of single primer according to formula 0.466X (GC percentage content) 100+66.04- (450/primer sequence length); screening the primer pairs under the conditions that the Tm difference value of the primer pairs is less than or equal to 3 ℃ and the primers cannot interact with each other to obtain candidate primer pairs;
wherein, the percentage content of GC is the percentage of the number of bases G and C in the primer to the total number of bases of the primer;
the length of the primer sequence refers to the number of bases of the primer.
7. The method of claim 4, wherein in step (2.4), the commonality determination is a check of whether the primer pairs exactly match all target sequences; the specificity determination refers to checking whether a single primer cannot be specifically matched with non-target sequences except for all target sequences, the non-target sequences refer to nucleic acid sequences except for the nucleic acid sequence to be amplified, and the specific matching refers to matching with no more than 2 mismatches.
8. The method of claim 2, wherein in step (3), the reaction denaturation temperature is calculated as 0.357 x (GC percentage content) x 100+70.582- (990/primer pair theoretical amplification product sequence length) and is recorded as Tma; wherein, the percentage content of GC 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; and/or the presence of a gas in the atmosphere,
calculating the Tm value of a single primer according to a formula of 0.466 multiplied by 100+66.04- (450/primer sequence length of GC percentage content), and taking the average value of the Tm value to obtain the reaction annealing temperature which is marked as Tmb; wherein, the percentage content of GC refers to the percentage of the number of bases G and C in the primer to the total number of bases of the primer.
9. The method of claim 8, wherein in step (3), the nucleic acid amplification reaction conditions comprise: reacting at the denaturation temperature for 5 seconds, at the annealing temperature for 5 seconds, and at the extension temperature for 20 seconds, wherein the above processes are repeated for 30-40 times; wherein the denaturation temperature can be adjusted within the range of Tma +/-5 ℃ according to requirements; the annealing temperature can be adjusted between Tmb +/-2 ℃ according to requirements; the extension temperature was 72 ℃.
10. Primer pairs obtainable by the method according to any one of claims 2 to 9.
11. The primer pair of claim 10, wherein the primer pair is:
a primer pair G:
Yer-F1：5’-ATGGAAAATAACATAATTTCTATTACCGG-3’(SEQ ID NO.1)
Yer-R1：5’-TCTCTGCGAATAACCTTGTG-3’(SEQ ID NO.2)；
and (3) primer pair D:
Yer-F2：5’-TGTGCGGTGGATGTAAATAATTC-3’(SEQ ID NO.3)
Yer-R2：5’-GCTTTGAAACTCAAGGACTG-3’(SEQ ID NO.4)。
12. a diagnostic reagent comprising the primer pair of claim 10 or 11.
13. Use of the method of any one of claims 2 to 9 for nucleic acid amplification of a target nucleic acid sequence enriched in AT region, use of the method of any one of claims 2 to 9 or the primer pair of claim 10 or 11 for amplification and/or detection of a yersinia enterocolitica-associated gene or region, use of the method of any one of claims 2 to 9 or the primer pair of any one of claims 10 or 11 for the manufacture of a product for amplification and/or detection of a yersinia enterocolitica-associated gene or region, use of the diagnostic reagent of claim 12 for detection of yersinia enterocolitica, use of the method of any one of claims 2 to 9 for simultaneous detection of any two or more of yersinia enterocolitica, staphylococcus aureus, crohn's sakazakii, salmonella.
CN202110486116.0A 2021-04-30 2021-04-30 System and method for detecting yersinia enterocolitica and application of system and method Pending CN115261494A (en)

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