KR100984785B1 - Primer set and probe for detecting Salmonella typhimurium - Google Patents

Abstract

The present invention is a Salmonella typhimurium (Salmonella typhimurium) primer set for detecting, Salmonella typhimurium detection kit comprising the primer set, Method for detecting Salmonella typhimurium in the sample using the primer set, Salmonella typhimurium The present invention relates to oligonucleotide probes for detecting groupings and to microarrays to which the probes are immobilized.

Using the primer sets and probes of the present invention, Salmonella typhimurium can be detected accurately, quickly and quantitatively.

Salmonella typhimurium, primer set, probe, kit, microarray

Description

Primer set and probe for detecting Salmonella typhimurium}

The present invention relates to primer sets and probes for Salmonella typhimurium detection.

The genus Salmonella is a Gram-negative bacterium and consists of two species, Salmonella enterica and Salmonella bongori (subspecies V). Salmonella enterica is divided into six subspecies: I, II, IIIa, IIIb, IV and VI. Salmonella is classified as 2500 serovars based on the Kauffmann-White table (Popoff et al. 2001. Res . Microbiol . 152: 907-909). Among the Salmonella subspecies I, Salmonella enterica serovar Typhimurium is the serotype most frequently isolated from the global food poisoning Salmonella infection. Therefore, the rapid detection and identification of these serums is necessary in the food industry.

Salmonella detection using the bacterial method takes about 3-4 days. In addition, this method is very labor intensive, expensive, complex and time consuming. Accurate and rapid identification of pathogen bacteria from food is important for the food industry and public health. PCR is recognized as a potentially effective method for microbiological diagnostics because of its simplicity, rapidity and accuracy (Kang et al. 2007. J. Microbiol . Biotechnol. 17: 516-519). Recently, a real-time PCR method has been reported to detect food poisoning with the advantages of Salmonella quantification, sensitivity and rapidity. Most of the previously published real-time PCR primer pairs and probes were based on various target genes and responded to Salmonella and Salmonella enterica serovars (Cheung et al. 2004. Lett . Appl . Microbiol . 39: 509-515). Moreover, real-time PCR methods used an enrichment step prior to genomic DNA extraction for Salmonella detection in food (Bhagwat, AA 2003. Food). Microbiol . 21: 73-78).

Salmonella serovars were genotyped by comparative analysis of the genome sequences of various salmonella serovars, and new identifications of major food poisoning Salmonella serovars were presented.

Korean Patent Registration No. 10-671501 discloses a food poisoning detection primer and a bacterial food poisoning detection method using the same, but different from the primer of the present invention.

The present invention has been made in accordance with the above requirements, and produced specific primers and probes based on the STM4497 gene of S. Typhimurium LT2 through gene comparison, and specifically detecting S. Typhimurium among various Salmonella serovars In addition, the present invention was completed by developing a quantitative detection method of S. Typhimurium without an enrichment step through real-time PCR.

In order to solve the above problems, the present invention provides a primer set capable of accurately and quickly detecting Salmonella typhimurium.

The present invention also provides a kit for Salmonella typhimurium detection comprising the primer set.

The present invention also provides a method for detecting Salmonella typhimurium in a subject sample using the primer set.

The present invention also provides an oligonucleotide probe for Salmonella typhimurium detection.

The present invention also provides a microarray to which the probe is immobilized.

Using the primer sets and probes of the present invention, Salmonella typhimurium can be detected accurately and quickly. In addition, direct quantification of S. Typhimurium was evaluated by DNA extracted from artificially inoculated chicken samples without enrichment step by real-time PCR. As a result, this real-time PCR method can be applied to the detection of food poisoning pathogenic bacteria inoculated into food.

In order to achieve the object of the present invention, the present invention provides a primer set capable of accurately, quickly and quantitatively detecting Salmonella typhimurium. The primer set is at least one oligonucleotide selected from the group consisting of fragments of at least 15 contiguous nucleotides in sequence SEQ ID NO: 1 and at least 15 contiguous nucleotide segments in sequence SEQ ID NO: 2 It may consist of a primer set comprising one or more oligonucleotides selected from the group consisting of.

The primer set is preferably one or more oligonucleotides selected from the group consisting of oligonucleotides consisting of segments of at least 16, at least 17 and at least 18 consecutive nucleotides in the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 2 It may be a primer set comprising one or more oligonucleotides selected from the group consisting of oligonucleotides consisting of at least 16, at least 17, at least 18, at least 19, at least 20 contiguous nucleotide segments within.

The primer set may most preferably consist of a primer set of SEQ ID NO: 1 and SEQ ID NO: 2.

In the present invention, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. The specific length and sequence of the primers will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

In the present invention, oligonucleotides used as primers may also comprise nucleotide analogues such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

In order to achieve another object of the present invention, the present invention

Primer sets of SEQ ID NOs: 1 and 2; And Salmonella typhimurium (Salmonella typhimurium) detection kit comprising a reagent for performing an amplification reaction.

In the kit, the reagents for carrying out the amplification reaction may include DNA polymerase, dNTPs, and buffers. In addition, the kit of the present invention may further include a user manual describing the conditions for performing the optimal reaction.

In order to achieve another object of the present invention, the present invention

Separating genomic DNA from the subject sample;

Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using primer sets of SEQ ID NOs: 1 and 2; And

It provides a method for detecting Salmonella typhimurium in a subject sample, comprising detecting the amplification product.

The method includes separating genomic DNA from a subject sample. The subject sample may be, for example, chicken, but is not limited to a sample suspected of being infected with Salmonella typhimurium. As a method of separating genomic DNA from the sample, a method known in the art may be used, for example, a wizard prep kit (Promega) may be used. Using the isolated genomic DNA as a template, amplification reactions can be amplified using a primer set of SEQ ID NOs: 1 and 2 according to an embodiment of the present invention as a primer. The target sequence is the STM4497 gene of Salmonella typhimurium LT2.

Methods for amplifying target nucleic acids include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification systems, There are any suitable methods for amplifying via strand displacement amplification or Qβ replication or for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a primer pair that specifically binds to the target nucleic acid using a polymerase. Such PCR methods are well known in the art and commercially available kits may be used.

The method of the present invention includes detecting the amplification product. Detection of the amplification product can be performed quantitatively through real-time PCR. In the method of the present invention, the amplified target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be, but is not limited to, a material that emits fluorescence, phosphorescence, or radioactivity. Preferably, the labeling substance is a fluorescent probe, SYBR Green I, Cy-5 or Cy-3. In the case of fluorescent probes, the probes are hybridized to the template before PCR, and the probe fluoresces as the probe is separated from the template as the PCR proceeds. Fluorescent reporter dye is labeled with 6-FAM (carboxyfluorescein) at the 5 'end and minor groove binder (MGB) at the end of the quencher 3', a non-fluorescent material. In the case of SYBR Green I, when the dye is mixed with the PCR reaction mixture and the PCR is performed, the SYBR Green I is fluoresced as the PCR is inserted into the PCR product. In the case of Cy-5 or Cy-3, when PCR is performed by labeling Cy-5 or Cy-3 at the 5'-end of the primer when amplifying the target sequence, the target sequence may be labeled with a detectable fluorescent labeling substance.

In addition, the label using the radioactive material may add radioactive isotopes such as ³² P or ³⁵ S to the PCR reaction solution when PCR is performed, and the amplification product may be incorporated into the amplification product while the amplification product is synthesized. One or more sets of oligonucleotide primers used to amplify the target sequence are as described above.

In order to achieve another object of the present invention, the present invention

Provided is an oligonucleotide probe for detecting Salmonella typhimurium, consisting of the nucleotide sequence of SEQ ID NO: 3.

As used herein, "probe" refers to a single stranded nucleic acid sequence that hybridizes with a complementary single stranded target sequence to form a double stranded molecule (hybrid).

As used herein, oligonucleotides used as probes may also include nucleotide analogs, such as phosphorothioate, alkylphosphothioate or peptide nucleic acid, or It may comprise an intercalating agent.

The present invention also provides a microarray having a substrate on which an oligonucleotide probe according to an embodiment of the present invention is immobilized.

In the microarray of the present invention, "microarray" refers to a microarray in which a group of oligonucleotides is immobilized at a high density on a substrate, and each of the oligonucleotide groups is immobilized in a predetermined region. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.

The term “substrate” refers to any substrate to which an oligonucleotide probe can be attached under conditions that retain hybridization properties and keep the background level of hybridization low. Typically, the substrate may be a microtiter plate, membrane (eg, nylon or nitrocellulose) or microspheres (beads) or chips. Prior to application or immobilization on the membrane, nucleic acid probes can be modified to promote immobilization or to improve hybridization efficiency. Such modifications may include homopolymer tailing, coupling with different reactive functional groups such as aliphatic groups, NH ₂ groups, SH groups and carboxyl groups, or coupling with biotin, hapten or protein.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example

Materials and methods

Microorganism Types and Growth Conditions

The pathogenic microorganisms known as major food poisoning bacteria and their related microorganisms were purchased from the American Type Culture Collection (ATCC) and used in the present invention, and the types thereof are shown in Table 1.

Table 1. List of strains used in real-time PCR experiments and results.

Species Strain result Salmonella serovar Typhimurium ATCC 19585 + Salmonella serovar Typhimurium ATCC 14028 + Salmonella serovar Typhimurium ATCC 13311 + Salmonella serovar Typhi ATCC 6539 - Salmonella serovar Typhi ATCC 33459 - Salmonella serovar Paratyphi C ATCC 13428 - Salmonella serovar Paratyphi B ATCC 10719 - Salmonella serovar Enteritidis ATCC 4931 - Salmonella bongori ATCC 43975 - Salmonella enterica salamae ATCC 15793 - Salmonella enterica arizonae ATCC 13314 - Salmonella enterica diarizonae ATCC 43973 - Salmonella enterica houtenae ATCC 43974 - Salmonella enterica indica ATCC 43976 - Salmonella serovar Choleraesuis ATCC 13312 - Salmonella serovar Gallinarum ATCC 9184 - Salmonella serovar pullorum ATCC 9120 - Escherichia coli O157: H7 ATCC 43894 - Esherichia coli ATCC 27325 - Yersinia enterocolitica ATCC 23715 - Bacillus cereus ATCC11778 - Bacillus subtilis ATCC 6633 - Enterococcus faecalis ATCC 19433 - Klebsiella pneumoniae subsp. Pneumoniae ATCC 8724 - Bacillus thuringiensis ATCC 10792 - Bacillus anthracis ATCC 14578 - Listeria monocytogenes ATCC 19118 - Listeria ivanovii ATCC 19119 - Listeria welshimeri ATCC 35897 - Listeria inocua ATCC 33090 - Listeria grayi ATCC 25401 - Listeria seeligeri ATCC 35967 - Staphylococcus aureus ATCC 6538 - Staphylococcus haemolyticus ATCC 29970 - Staphylococcus epidermidis ATCC 14990 - Vibrio parahaemolyticus ATCC 17802 - Vibrio cholerae ATCC 14547 - Vibrio vulnificus ATCC 33815 - Campylobacter jejuni ATCC 43429 - Clostridium botulinum ATCC 3502 - Clostridium perfringens ATCC 3624 - Citrobacter frenudii ATCC 8090 - Enterobacter aerogenes ATCC 13048 - Enterobacter cloacae ATCC 13047 - Enterobacter sakazakii ATCC 29544 - Enterobacter cloacae ATCC 13047 - Enterobacter sakazakii ATCC 29544 - Proteus vulgaris ATCC 29905 - Shigella flexneri ATCC 12022 - Shigella sonnei ATCC 25931 -

Standard culture conditions were used. Salmonella serovar Typhimurium ATCC19585 was incubated overnight at 37 ° C. in Luria-Bertani broth medium (Difco laboratories, Detroit, MI, U.S.A), which is a standard culture condition.

Preparation and Genome of Samples from Chickens DNA Extraction of

Packaged chicken breasts were purchased at a nearby mart and stored in sterile packs for analysis on the same day. The chicken was washed in running water and chopped into pieces. Thereafter, 225 ml of PBS (phosphate buffered saline) and 25 g of chicken meat were mixed in a sterile filter pack (BA6141 / STR filter bag, Seward Laboratories, West Sussex, UK). 1 ml of a 10 ⁸ CFU / ml bacterial culture solution with an optical density of 0.66 at 600 nm was placed in a 9 ml PBS solution, followed by serial dilution. Chicken samples and 1 ml of serially diluted bacterial cultures were contained in PBS solution and artificially inoculated to make bacterial-PBS solution. Six samples were prepared as ^4.5 × 10 ⁰ chicken-PBS samples at 4.5 × 10 ⁵ CFU / ml. 1 ml of PBS buffer was added as a standard control sample, and chicken and PBS solution were mixed. After inoculation the samples are immediately mixed for 30 seconds at 230 rpm using a stomacher 400 circulator, Seward Laboratories, West Sussex, UK. Harvest 1 ml of sample from 10,000 g for 10 minutes for real time PCR. Genomic DNA is extracted from the harvested samples for real-time PCR using the DNeasy Tissue Kit (QIAGEN, Hilden, Germany) according to the instructions in the manual. 60 μl of 3rd sterile water (GIBCO ™, Grand Island, NY, USA) is placed in a spin column for the separation of genomic DNA and then centrifuged for 10 minutes. Decimal cell suspensions diluted in decimal form for plate counting are determined in three replicates.

real time PCR Using Salmonella Typhimurium Genome DNA Standard curve

Standard genomic curves of real-time PCR were drawn using purified genomic DNA of S. Typhimurium ATCC19585. The genomic DNA of S. Typhimurium ATCC 19585, which was incubated overnight, was then extracted and quantified using Qubit ^TM Fluorometer and Qubit-it ^TM dsDNA HS assay Kit (Invitrogen, Carlsbad, CA, USA). Purified genomic DNA was quantified in ⁵ μl final concentrations 4X10 ⁵ , 4X10 ⁴ , 4X10 ³ , 4X10 ² , 4X10 ¹ , 4X10 ⁰ and 0 genome equivalent. DNA genome equivalent was calculated by the following equation: DNA genome equivalent = (A x 6.023 x 10 ²³ ) (660 x B) ^-1 , where A is the concentration of DNA and B is the length of the genomic DNA.

Salmonella Typhimurium For quantification of With primer Probe

Primers and probes used for quantification and detection of Salmonella Typhimurium are shown in Table 2.

Table 2. Primers and probes used for PCR.

Oligonucleotide Sequence (5'-3 ') Sal-F GCG CAC CTC AAC ATC TTT C (SEQ ID NO: 1) Sal-R CGG TCA AAT AAC CCA CGT TCA (SEQ ID NO: 2) Sal-probe FAM ^a ATC ATC GTC GAC ATG C MGB / NFQ (SEQ ID NO: 3)

^a FAM, 6-carboxyfluorescein (reporter dye).

PCR primers and fluorescent probes to be used for TaqMan analysis were prepared for the amplification of specific DNA fragments of S. Typhimurium LT2 using Primer Express 2.0 software (Applied Biosystems, Forest City, CA, U.S.A). Probes were purchased through Applied Biosystem (Applied Biosystems, Forest City, CA, U.S.A) and primers were purchased through bionics Corp (Seoul, Korea). The probe was labeled with 6-FAM (carboxyfluorescein) at the 5 'end of the fluorescent reporter dye, and MGB (minor groove binder) at the end of the quencher 3', a non-fluorescent material.

real time PCR  Condition

The amplification reaction is adjusted to 25 μl final volume and the sample is repeated three times each. 12.5 μl Mastermix (TaqMan® Universal PCR Master Mix, Applied Biosystems), 0.4 μM Primer, 0.2 μM Probe, 5 μl Sample DNA, and 5 μl Tertiary Distilled Water (GIBCO ™, Grand Island) , NY, USA). Thermal cycling is carried out using a two-step PCR protocol as follows: 50 cycles of 50 ° C. 2 min, 95 ° C. 10 min, and 95 ° C. 15 sec and 60 ° C. 1 min, and the fluorescence signal at each end of the extension phase. Measured in cycles.

Example  One: Salmonella Typhimurium for someone Primer pair Inclusivity  And  Exclusivity

In order to verify and evaluate the specificity of the primers and probes used, the primer pairs and probes shown in Table 2 were identified using various microbial strains and DNA of S. Typhimurium using real-time PCR. In addition, the prepared primers and probes used in the present invention amplify specific PCR products of S. Typhimurium, and are shown in Table 2.

Example  2: Salmonella Typhimurium Quantified genome DNA Real-time PCR  result

Amplification curves and standard curves of real-time PCR were obtained from genomic DNA serially diluted to 1/10 of S. Typhimurium ATCC 19585 and shown in FIGS. 1, 2 and Table 3. Rn is related to the concentration of the PCR product, and the larger the Rn, the higher the concentration of the PCR product. Five individual experiments were repeated three times and one amplification curve is shown in FIG. 1. According to the known size (4.99 Mb) of the complete S. Typhimurium ATCC 19585, one genome equivalent was calculated to be 5.46904 fg (femtogram). S. Typhimurium in one individual experiment repeated three times using real-time PCR Quantitative genomic DNA of ATCC 19585 ranges from 4.5 × 10 ⁵ to 0 genome equivalents. S. Typhimurium as shown in Figure 1 and Table 3 The DNA of 0 genome equivalent of ATCC 19585 was not amplified, and the amplification curves of 4.5X10 ⁵ to 4.5 genomic DNA were shown as shown.

Genomic DNA diluted to 1/10 showed almost identical threshold cycle (Ct) values in three replicates. Ct refers to the number of cycles in which a detectable fluorescence signal appears in a real-time PCR reaction. That is, the larger the initial DNA concentration, the greater the amplification at low Ct, and thus the fluorescence signal can be detected. The smaller the initial DNA concentration, the larger the Ct. And S. Typhimurium The mean values and standard deviations of the threshold cycles based on the DNA genome equivalent of ATCC 19585 are shown in Table 3.

Table 3. Salmonella Typhimurium ATCC 19585 quantification of genomic DNA genome equivalents Real-time PCR Threshold cycle (Ct) results

Genome equivalents ^a Ct value Average Standard Deviation One 2 3 4 5 400000 17.99 17.65 17.41 17.51 17.31 17.57 0.26 40000 21.55 21.12 20.99 21.08 20.79 21.11 0.28 4000 25.05 24.94 24.53 24.61 24.42 24.71 0.27 400 28.64 28.49 28.74 28.62 28.44 28.59 0.12 40 31.81 31.18 32.53 32.11 32.09 31.94 0.50 0 N. D. N. D. N. D N. D. N. D.

^a Genome equivalents of Salmonella Typhimurium ATCC 19585.

^b ND: Not detected.

The real-time PCR standard curve of quantified Salmonella genomic DNA was repeated three times, and the average result proved linearly with the log cell number in FIG. 2. The Ct value of the standard curve showed a high correlation coefficient of 0.9951 and the calculated slope was -3.4037.

Example  3: chicken Salmonella enteric serovar Typhimurium Artificially inoculate PCR And plate counting

Figure 3 shows the quantification of the amplification curve extracted genomic DNA by artificially inoculated chicken. Quantitative curves ranged from 4.5 × 10 ⁵ to 4.5 × 10 ⁰ CFU / ml of chicken-PBS solution. The Ct values of the six experiments ranged from 20.72 ± 0.23 to 36.25 ± 0.63, and each Ct value was averaged from three replicates. Table 4 shows the change in Ct value to genome equivalent from the standard amplification curve in FIG. 2.

Table 4. S. Typhimurium Real-time PCR results quantified by artificial inoculation of chicken with ATCC 19585.

Inoculated S. Typhimurium
(CFU / ml of chicken-PBS solution) TaqMan Real Time PCR Plate count 4.5 X 10 ⁵ (6.3 ± 0.9) X 10 ⁵ (4.3 ± 0.3) X 10 ⁵ 4.5 X 10 ⁴ (6.8 ± 0.6) X 10 ⁴ (3.3 ± 1.6) X 10 ⁴ 4.5 X 10 ³ (7.2 ± 0.9) X 10 ³ (4.0 ± 1.2) X 10 ³ 4.5 X 10 ² (8.1 ± 0.1) X 10 ² (5.5 ± 1.4) X 10 ² 4.5 X 10 ¹ (1.2 ± 0.6) X 10 ² (1.2 ± 1.0) X 10 ² 4.5 ^X 10 ⁰ 35 ± 15 15 ± 8 NI ^a ND ^b N.D.

^a NI: not inoculated, ^b ND: not detected.

Real-time quantitative PCR results of S. Typhimurium were higher than those inoculated with S. Typhimurium in the chicken artificially. S. Typhimurium artificially inoculated in chicken The amount was determined by the traditional plate count method (Table 4). Cell counts calculated from the plate counting method were similar to those of S. Typhimurium, which were artificially contaminated with real-time PCR in the sample.

1 shows S. Typhimurium ranging from 400,000 to 0 genome equivalents, using real time PCR Amplification plot of genomic DNA serially diluted 10-fold of ATCC 19585. Delta Rn is plotted on the Y-axis and the number of amplification cycles is plotted on the X-axis.

A, 400,000; B, 40,000; C, 4,000; D, 400; E, 40; F, 0; DNA genome equivalents of S. Typhimurium.

Figure 2 is a S. Typhimurium prepared using the average result of three repeated real-time PCR experiments Standard curve of genomic DNA serially diluted 10-fold by ATCC 19585. Threshold cycle (Ct) is plotted on the Y-axis, and log genome equivalent is plotted on the X-axis.

FIG. 3 is an amplification plot of genomic DNA extracted from chicken samples inoculated serially at 10-fold in the range of 450,000-0 CFU / ml of chicken-PBS solution using real-time PCR. A, 4.5 × 10 ⁵ ; B, 4.5 × 10 ⁴ ; C, 4.5 × 10 ³ ; D, 4.5 × 10 ² ; E, 4.5 × 10 ¹ ; F, 4.5 × 10 ⁰ ; G, 0; Number of S. Typhimurium Inoculated (CFU / ml of chicken-PBS solution).

<110> University-Industry Cooperation Group of Kyung Hee University <120> Primer set and probe for detecting Salmonella typhimurium <130> PN08100 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Sal-F primer <400> 1 gcgcacctca acatctttc 19 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Sal-R primer <400> 2 cggtcaaata acccacgttc a 21 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Sal-probe <400> 3 atcatcgtcg acatgc 16  

Claims (7)

A primer set for Salmonella typhimurium specific detection, consisting of a primer set of SEQ ID NOs: 1 and 2. delete Primer sets of SEQ ID NOs: 1 and 2; And Salmonella typhimurium specific detection kit comprising a reagent for performing an amplification reaction. Separating genomic DNA from the subject sample; Amplifying a target sequence by using the isolated genomic DNA as a template and performing an amplification reaction using primer sets of SEQ ID NOs: 1 and 2; And A method of specifically detecting Salmonella typhimurium in a subject sample, comprising detecting the amplification product. The method of claim 4, wherein the detection of the amplification product is performed quantitatively through real-time PCR. delete delete

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