TW201416453A - Nucleic acid molecule, diagnostic kit, biochip and method for the detection of Salmonella strains - Google Patents

Abstract

Disclosed herein are primer pairs and/or probes for use in the detection of Salmonella enterica. Also disclosed herein are diagnostic kits, biochips and methods for the detection of Salmonella enterica which presents in a sample using said primer pairs and/or probes.

Description

Nucleic acid molecule, test kit, biochip, and method for detecting a Salmonella strain

The present invention relates to primer pairs and/or probes for detecting Salmonella enterica . The invention also relates to a diagnostic kit, biochip, and method for detecting Salmonella enterica present in a sample using the primer pairs and/or probes.

Salmonella strains are a group of gram-negative facultative anaerobic bacteria that are ubiquitous in humans and animals or in food and the environment. Salmonella mainly contains two species, Salmonella enterica and Salmonella bongori , in which Salmonella enterica can be divided into six subspecies, and Bangor Salmonella can be divided into one subspecies. These Salmonella subspecies can be distinguished into different serogroups according to the difference in the performance of somatic antigens (ie, O antigens), and each serogroup can be based on The difference in expression of the flagellar antigen [ie, H antigen] is further differentiated into different serotypes.

Most of the Salmonella strains that infect humans and animals belong to Salmonella enterica , and the more common ones are Salmonella enterica serovars: Salmonella enteric serovar serotypes S. enterica subsp. enterica ser. Typhimurium) [also referred to as Salmonella Typhimurium], Salmonella enterica subsp. entericidal serotype ( S. enterica subsp. enterica ser. Enteritidis) [also Abbreviated as Salmonella Enteritidis, S. enterica subsp. enterica ser. Hadar (also referred to as Salmonella Hadar), intestine Salmonella typhimurium subtype of infantile serotype ( S. enterica subsp. enterica ser. Infantis) [also referred to as Salmonella Infantis] and Salmonella enterica subsp. ( S. enterica subsp. enterica ser. Virchow) [also referred to as Salmonella Virchow].

Salmonella enterica is a rod-shaped enterobacteria that when humans ingest food contaminated with such bacteria [eg raw meat, fresh produce, processed meat) (processed meat), fruit juice, and dairy product, can cause typhoid fever, paratyphoid fever, and food-borne illness [such as salmonellosis). ]Wait. Patients with Salmonella disease usually develop acute enterocolitis with sudden onset of headache, abdominal pain, diarrhea, nausea, and vomiting. Such as clinical symptoms, severe cases may even have septicemia or death.

Since Salmonella enterica has seriously threatened human health and caused losses in the livestock and food industries, how to detect these intestinal Salmonella early and quickly becomes a concern of researchers in the technical field. Focus with research.

In order to overcome the traditional strain culture and bacteriological characterization [eg, gram staining, API identification system, serotyping, physiological or biochemical characterization, antibiotic tolerance test, etc.] in the operation process On the more complicated and time-consuming drawbacks, many molecular biology methods have been applied to detect various strains of Salmonella. These biological methods include: polymerase chain reaction (PCR), pulse-field gel electrophoresis (PFGE), multiplex polymerase chain reaction, random amplification Random Amplified Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), Immunoassay method, DNA probe method, and biochip ( Biochip) and so on.

In recent years, the development of biochip technology has been gradually taken into consideration and has been widely used in the detection of various pathogens, the classification of cancer, the development of new drugs, and the sequencing and identification of genes. Advantages of biochips include: small size, high confidence and accuracy of analytical results, fast analysis, and versatile experimental data with fewer samples and reagents. Biochips can be classified into the following four categories according to their functionality: (1) gene chip, also known as microarray chip; (2) microfluidic chip; (3) Protein chip; and (4) Lab-on-a-chip.

In general, the height of the genomic DNA sequence of the bacteria will be included. Highly conserved and highly variable DNA sequences that are available for species-specific, subspecies-specific or serovar-specific (serovar-specific) Detection or identification. Studies have shown that the O antigen, H antigen of the Salmonella enterica strain and the biosynthesis gene of the capsular antigen [Vi antigen] can be used as intestinal sand gates. The serotype-specifically detected target gene of the Bacillus strain.

For example, in Yang Hong et al . (2008), BMC Microbiology , 8: 178-185, Yang Hong et al . target the O antigen synthesis gene in Salmonella serogroups [including the wba operon ( wba operon). )] and the nucleotide sequence of the central variable region of the H1 and H2 flagellin genes to design a specific primer pair and use it for allelotyping multiplex PCR). The experimental results confirmed via, by detecting antigen and O alleles composition H antigen (gene allele combination) can effectively identify Salmonella enteritidis, Salmonella Hadar, Salmonella Heidelberg (S .Heidelberg) and Salmonella typhimurium.

In Mohd Elbagir Elhassan Nori and Kwai Lin Thong (2010), African Journal of Microbiology Research , 4: 871-876, Mohd Elbagir Elhassan Nori and Kwai Lin Thong mainly discuss the continuous multi-polymerization of a target O, H and Vi antigens. The combination of a sequential multiplex polymerase chain reaction is useful in distinguishing Salmonella strains of different serotypes. The results of the study show that Salmonella enterica strains such as Salmonella typhimurium, Salmonella Paratyphi, Salmonella enteritidis, and Salmonella in Munich can be effectively used by a combination of continuous multiplex polymerase chain reaction against these antigens. Serotyping was performed by Salmonella Muenchen and Salmonella Hadar.

In addition, in Masato Akiba et al . (2011), Journal of Microbiological Methods , 85:9-15, Masato Akiba et al . by serotypes of seven Salmonella typhimurium [including Salmonella typhimurium, Salmonella choleraesuis ( Salmonella Choleraesuis), Salmonella infantile, Salmonella Hadar, Salmonella enteritidis, Salmonella Dublin, and Salmonella Gallinarum for genomic sequence comparison, and screening Serovar-specific genomic regions (SSGRs) specific for these Salmonella serotypes, respectively, and then serotypes were designed for the nucleotide sequences of these SSGRs - Specific primer pairs (3 sets of serotype-specific primer pairs were designed for each strain). The experimental results show that the serotype-specific primer pair designed according to the above can quickly and specifically identify the Salmonella gut serotype to be detected by multiplex polymerase chain reaction.

Although the above literature has been reported, applicants are actively working to find nucleic acid molecules that can be used to detect Salmonella enterica. In the present invention, the applicant screens for highly mutated DNA sequences among genomic DNAs of three Salmonella serotypes of Salmonella, including Salmonella typhimurium, Salmonella vilsoni, and Salmonella Hadar. Three kinds of Salmonella serotypes have high specificity and sensitivity of nucleic acid molecules for For rapid detection or identification.

Summary of invention

Thus, in a first aspect, the present invention provides a primer reagent for detecting Salmonella enterica comprising at least one of: (a) a first set of primer pairs for detecting Salmonella Hadrilla , comprising a forward sequence of a nucleotide sequence as shown in sequence identification number: 4, and a reverse primer having a nucleotide sequence as shown in sequence identification number: 5; (b) A second set of primer pairs for detecting Salmonella infestans, comprising a nucleotide sequence as shown in sequence identification number: 6, and a nucleoside having a sequence number: 7 a reverse primer of the acid sequence; (c) a third set of primer pairs for detecting Salmonella infestans, comprising a nucleotide sequence as shown in sequence identification number: 8, and a primer Like the reverse primer of the nucleotide sequence shown in sequence identification number: 7; (d) a fourth set of primer pairs for detecting Salmonella vilson, which contains a sequence identification number: 9 The nucleotide sequence shown before the primer, and a reverse primer having a nucleotide sequence as shown in Sequence Identification Number: 7; and (e) a Group 5 primer pair for detecting Salmonella vilsoni, comprising a sequence identification number: The nucleotide sequence shown in 10 is preceded by a primer, and one has a sequence identification number: 7 The reverse primer of the indicated nucleotide sequence.

In a second aspect, the present invention provides a probe reagent for detecting Salmonella enterica comprising at least one of: (a) a probe for detecting Salmonella Haddar having one a nucleotide sequence selected from the group consisting of: sequence identification number: 4, sequence identification number: 5, and sequence identification number: 11; (b) a probe for detecting Salmonella infantile, having a probe selected from Such as a sequence identification number: 6, a sequence identification number: 8 and a nucleotide sequence shown in sequence identification number: 12; and (c) a probe for detecting Salmonella vilsoni, which has a selected from Sequence identification number: 9, sequence identification number: 10, and nucleotide sequence number shown in sequence identification number: 13.

The primer pairs and/or probes described above have a high degree of specificity and sensitivity for the target Salmonella typhimurium to be detected, and thus can be used to detect Salmonella enterica in a sample. . Accordingly, in a third aspect, the present invention provides a method for detecting the presence or absence of a Salmonella enterica in a sample, comprising: subjecting the sample to DNA amplification using at least one set of primer pairs as described above And detecting a DNA fragment amplified by using the at least one set of primer pairs, wherein the presence of the DNA fragment indicates the presence of Salmonella enterica corresponding to the at least one set of primer pairs.

In a fourth aspect, the invention also provides a method for detecting the presence or absence of a Salmonella enterica in a sample, comprising: The sample is subjected to a hybridization reaction using at least one probe as described above; and detecting whether there is a hybrid formed by performing hybridization reaction using the at least one probe, wherein the presence of the hybrid indicates a correspondence The presence of Salmonella enterica in the at least one probe.

In a fifth aspect, the invention provides a test kit for detecting Salmonella enterica comprising an primer reagent as described above and/or a probe reagent as described above.

In a sixth aspect, the invention provides a biochip for detecting Salmonella enterica comprising a primer reagent as described above and/or a probe reagent as described above.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the pre-existing publication forms a common general knowledge in the art. Part of it.

For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to" and the term "comprises" has a corresponding meaning.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person familiar with the art will recognize many of those described in this article. Methods and materials similar or equivalent that can be used in the practice of the invention. Of course, the invention is in no way limited by the methods and materials described. For clarity, the following definitions are used herein.

As used herein, the terms "nucleic acid", "nucleic acid sequence" or "nucleotide sequence" and the like mean a deoxyribonucleotide sequence or a ribonucleotide sequence in the form of a single strand or a double strand, and includes There are known naturally occurring nucleotides or aritificial chemical mimics. As used herein, the term "nucleic acid" is used interchangeably with "gene," "DNA," "cDNA," "mRNA," "oligonucleotide," and "polynucleotide."

As used herein, the terms "nucleic acid fragment" and "DNA fragment" are used interchangeably and mean a DNA polymer in the form of a separate segment. Or as a component of a larger DNA construct, which may be derived from isolated DNA or chemically prepared by methods well known in the art. Or enzyme synthesis.

Unless otherwise indicated, a nucleic acid sequence encompasses complementary sequences, as well as their conservative analogs, associated naturally occurring structural variants, and in addition to the specific sequences disclosed herein. / or synthetic non-naturally occurring analogs.

As used herein, terms such as "hybridization" and "annealing" are used interchangeably and mean two single nucleotide sequences by virtue of the Watson-Crick rule ( Watson-Crick rules) And by their hydrogen bonding of purine and/or pyrimidine bases, they are specifically bound to each other in sequence, thereby forming a hybrid. The hybrid is a duplex nucleic acid complex having a stable nucleic acid structure comprising RNA: RNA, RNA: DNA or DNA: DNA double helix molecules.

Conventionally, in the detection of Salmonella enterica which causes food poisoning, most methods such as strain culture and bacteriological characterization are used, but these detection methods have disadvantages such as complicated operation, time consuming and inaccurate. Therefore, how to quickly and accurately detect Salmonella enterica in the biological specimen or food has become the goal of the food industry and the medical community. Molecular biology methods [such as polymerase chain reaction (PCR) and biochip technology] have the advantages of high speed, high reliability and accuracy of analysis results, so the applicant is committed to developing rapid detection methods in this area. .

In general, high-conserved and highly variable DNA sequences are included in the genomic DNA sequence of Salmonella enterica, which can be used for species-specific, subspecies-specific or serotype-specific detection or identification. . Therefore, the applicants attempted to design primer pairs and/or probes for the high specificity and sensitivity of these Salmonella serotypes from the genomic DNA sequences of Salmonella Harder, Salmonella typhimurium and Salmonella vilii. Needle for quick detection or identification.

In the present invention, Applicants are based on the Hadar-specific genomic region of the complete genome of Salmonella Harder (downloaded from ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/HADAR.dbs) Hadar-specific genomic region, HSR) and the complete base of infantile Salmonella Designed separately for the infant-specific genomic region (ISR) in the group (downloaded from ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/SIN.dbs) Group 1 has serotype-specific primer pair HADAF/HADAR for Salmonella Haddar and two groups of serotype-specific primer pairs for INFA1F/InViR and INFA2F/InViR for infant Salmonella.

On the other hand, the genome of Salmonella vilson has not yet been completely sequenced, and according to previous studies by the applicant, the nucleotide residues in the complete genome of Salmonella typhimurium are located at positions 3710138 to 3713529. It has a high degree of sequence similarity to those of Salmonella. Therefore, the applicant designed a set of Univer-F primers (SEQ ID NO: 1) and Univer-R primer (SEQ ID NO: 2) for the nucleotide sequence of the complete genome of the above-described Salmonella typhimurium, and then used The genomic DNA of Salmonella vilsona CA08.158 was used as a template for polymerase chain reaction (PCR). Thereafter, analysis was carried out by agarose gel electrophoresis, and it was found that Salmonella vilshii obtained two PCR products of about 3000 bp and 500 bp, respectively.

Next, the PCR product of Salmonella vilsonii having a size of about 500 bp showed by sequencing analysis that it contained a DNA fragment of 341 bp in size and having the nucleotide sequence as shown in SEQ ID NO: 3. Then, using the BLAST software (http://blast.ncbi.nlm.nih.gov/), the nucleotide sequence of the sequenced DNA fragment (SEQ ID NO: 3) is taken with the above-described Salmonella typhimurium Nucleotide sequence in the complete genome Line alignment analysis. The obtained alignment result shows that the DNA fragment of Salmonella wilsonii can substantially correspond to the nucleotide residue position 3713150 to 3713480 in the complete genome of the infant type Salmonella, wherein the DNA fragment and the nucleus The nucleotide sequence at position 3,731,251 to 3,713,480 of the nucleotide residue has a high degree of similarity, but has a higher variability with the nucleotide sequence at position 3713150 to 3713250 of the nucleotide residue (see Fig. 1). Therefore, Applicants designed two sets of primer pairs VIRC1F/InViR and VIRC2F/InViR which are serotype-specific for Salmonella vilson according to the DNA fragment. The nucleotide sequences of the five sets of serotype-specific primer pairs designed for the above three Salmonella serotypes were summarized in Table 2.

In addition to designing a primer pair for detection of Salmonella enterica, the applicant is also based on the Hadar-specific genomic region (HSR) in the complete genome of Salmonella Harder and the complete genome of the infant Salmonella. The serotype-specific probes HADAP and INFA1P for Salmonella hadar and Salmonella typhimurium were designed separately for the nucleotide sequence shown in the infant-specific genomic region (ISR). In addition, the complement of the VIRC1F primer designed according to the above was used as the serotype-specific probe VIRC1P against Salmonella vilsh. The nucleotide sequences of the three serotype-specific probes designed for the above three Salmonella serotypes were summarized in Table 5.

In order to evaluate the specificity and sensitivity of the primer pair and/or probe according to the present invention for Salmonella enterica, the applicant uses the genomic DNA of the bacterial strain obtained from the chicken sample as a template, and respectively uses the present invention. 5 sets of serotype-specific primers for PCR reactions with HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR, and VIRC2F/InViR, or using one of HADAF/HADAR, INFA1F/InViR, and VIRC1F/InViR The constructed primers are combined to perform multiplex PCR. The PCR amplification product obtained by PCR or multiplex PCR was analyzed by agarose gel electrophoresis and found that the primer pair according to the present invention has high specificity and sensitivity for the target Salmonella enterica to be detected.

Further, the applicant further takes a hybridization reaction based on the PCR amplification product obtained above and a biochip spotted with the probe according to the present invention. The map after hybridization shows that the probe according to the invention is also highly specific and sensitive to the target Salmonella enterica to be detected.

Based on the above, the primer pair and the probe according to the present invention are expected to be available for rapid detection of Salmonella enterica present in a sample. Accordingly, the present invention provides a primer reagent for detecting Salmonella enterica comprising at least one of: (a) a first set of primer pairs for detecting Salmonella Haddar, comprising one having one For example, the nucleotide sequence shown in sequence identification number: 4 is forward-introduced, and a reverse primer having a nucleotide sequence as shown in sequence identification number: 5; (b) one for detecting Salmonella infantile a second set of primer pairs comprising a nucleotide sequence as shown in sequence identification number: 6 and a primer having a sequence number: 7 a reverse primer of a nucleotide sequence; (c) a third set of primer pairs for detecting Salmonella infestans, comprising a nucleotide sequence forward primer as shown in SEQ ID NO: 8 a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 7; (d) a fourth set of primer pairs for detecting Salmonella vilson, comprising a sequence identification number: a nucleotide sequence shown before 9 to the primer, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 7; and (e) a 5th for detecting Salmonella vilsoni A primer set comprising a forward sequence of a nucleotide sequence as shown in sequence identification number: 10, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 7.

The invention also provides a probe reagent for detecting Salmonella enterica, comprising at least one of: (a) a probe for detecting Salmonella Haddar having a sequence selected from the group consisting of Identification number: 4, sequence identification number: 5 and nucleotide sequence number shown in sequence identification number: 11; (b) a probe for detecting Salmonella infantile, having a sequence selected from, for example, a sequence identification number: 6. Sequence identification number: 8 and a nucleotide sequence of sequence identification number: 12; and (c) a probe for detecting Salmonella vilsoni having a sequence selected from, for example, a sequence identification number: 9 , sequence identification number: 10 and the nucleotide sequence shown in sequence identification number: 13.

The primers and/or probes in accordance with the present invention may be further attached or incorporated to a detectable label using standard techniques well known to those of ordinary skill in the art to permit detection or quantification of the And other intestinal Salmonella. Detectable labels suitable for use in the present invention include, but are not limited to, hapten labels, for example, biotin and digoxigenin (Dig); chemiluminescent labels; Fluorescent labels, for example, fluorescein, rhodamine, FAM, TET, HEX, JOE, TAMA, NTB, TAMRA, ROX, VIC, NED, Yakima Yellow , BHQI, BHQ2, BHQ3, Iowa Black FQ, Iowa Black RQ, DABCYL, DABSYL, ElleQuencher, Eclipse Dark Quencher, Methyl Red, Texas Red, malachite green, Disperse Blue 3 (DisperseBlue3), Bodipy 493/503, cyanin (Cy) dye [such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7], AlexaFluor dye [such as AlexaFluor 488, 532, 546 , 555, 568, 594, 610, 647, and 680], PromoFluor dyes [such as PromoFluor 488, 555, 590, 633, 647, and 680] and LC-Red dyes [such as LC-Red 610, 640, 670, and 705] Enzyme label; radioactive label (eg, ³² P); particle label (particle) Labels), for example, gold colloids and quantum dots; and colorimetric labels such as dyes and colored glass or plastic beads.

According to the invention, either the 5' end and/or the 3' end of the probes may be added or At least one nucleotide residue is deleted such that it has a length that falls within the range of about 15 to 30 nucleotide residues. Furthermore, at least one thymidine may be added to the 5' end and/or the 3' end of the probes such that they may have better immobilization when spotted on the surface of the biochip. Immobilization, and enhance the detection signals they obtain when performing hybridization reactions. These are techniques that are familiar and familiar to those skilled in the art. Preferably, the number of thymidines added at the 5' and/or 3' ends of the probes falls within the range of about 10 to 35.

The present invention also provides a method for detecting the presence or absence of a Salmonella enterica in a sample, comprising: subjecting the sample to a DNA amplification reaction using at least one set of primer pairs as described above; and detecting whether there is a loan A DNA fragment amplified by using the at least one set of primer pairs, wherein the presence of the DNA fragment indicates the presence of Salmonella enterica corresponding to the at least one set of primer pairs.

According to the present invention, total genomic DNA is extracted from the sample as a template before the DNA amplification reaction is carried out.

According to the present invention, the DNA amplification reaction can be carried out by using at least one of the following methods: polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-) PCR), Real time quantitative PCR, nested PCR, hot-start PCR, in-situ PCR, degenerate oligonucleosides Acid primer PCR (Denogene oligonucleotide primer PCR, DOP PCR), micro-PCR, multiplex polymerase chain reaction, restriction fragment length polymorphism nucleic acid sequence-based amplification (NASBA) ), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), and rolling circle amplification (RCA). The setting of operating conditions for these methodologies falls within the professionalism and routine skills of those skilled in the art.

According to the present invention, the DNA amplification reaction can be carried out on a biochip selected from the group consisting of microfluidic wafers (for example, sample pretreatment wafers, reactive wafers, and analytical wafers) and Wafer Lab.

According to the present invention, the detection of the DNA fragment can be carried out by a hybridization reaction using a probe, wherein: (a) the DNA amplification reaction is carried out using the first set of primer pairs against Salmonella The probe has a nucleotide sequence selected from the group consisting of: sequence identification number: 4, sequence identification number: 5, and sequence identification number: 11; (b) when the second is used for infant type Salmonella When the primer pair is used for the DNA amplification reaction, the probe has a nucleotide selected from the group consisting of: sequence identification number: 6, sequence identification number: 7, sequence identification number: 8, and sequence identification number: 12. sequence; (c) when the DNA amplification reaction is carried out using the third set of primer pairs for Salmonella typhimurium, the probe has a core selected from the group consisting of sequence identification number: 7 and sequence identification number: a nucleotide sequence; (d) when the DNA amplification reaction is carried out using the fourth primer pair against Salmonella vilsoni, the probe has a sequence selected from, for example, a sequence identification number: 7. Sequence identification number: 9. Sequence identification number: 10 and a nucleotide sequence of sequence identification number: 13; and (e) when the DNA amplification reaction is carried out using the group 5 primer pair against Salmonella vilsonii, The probe has a nucleotide sequence selected from the group consisting of, for example, sequence identification number: 7 and sequence identification number: 10.

According to the present invention, the hybridization reaction can be carried out on a biochip selected from the group consisting of a gene wafer, a microfluidic wafer (for example, a sample pretreatment wafer, a reactive wafer, and an analytical wafer). And the wafer lab. In a preferred embodiment of the invention, the hybridization reaction is carried out on a gene wafer.

According to the invention, the sample may be selected from the group consisting of a food sample, a biological sample, and an environmental sample.

Preferably, the sample is a food sample including, but not limited to, fluid milk products such as milk and goat milk; beverages; meat products such as chicken, beef, lamb, and pork; Sea foods, such as fish and shellfish; animal feeds; water; dairy products such as yogurt, ice cream, and cheese Cheeses); vegetables; fruits; and canned foods. In a preferred embodiment of the invention, the food sample is chicken.

As used herein, the term "biological sample" means a sample derived from an organism (such as a human or an animal) or components of the organism (such as cells and tissues). The biological sample includes, but is not limited to, blood, plasma, serum, urine, saliva, cerebrospinal fluid, interstitial fluid, Peritoneal fluid, muscle, and feces.

As used herein, the term "environmental sample" means a sample derived from inanimate objects or reservoirs in an indoor or outdoor environment. The environmental samples include, but are not limited to, soil samples, dust samples, air samples, bulk samples [such as building materials, furniture, and landfill contents] and Other reservoir samples [such as animal refuse, harvested grains, and foodstuffs].

The probe according to the present invention can be directly subjected to hybridization reaction with the above-mentioned food sample, biological sample or environmental sample. Accordingly, the present invention provides a method for detecting the presence or absence of a Salmonella enterica in a sample, comprising: The sample is subjected to a hybridization reaction using at least one probe as described above; and detecting whether there is a hybrid formed by performing hybridization reaction using the at least one probe, wherein the presence of the hybrid indicates a correspondence The presence of Salmonella enterica in the at least one probe.

In a preferred embodiment of the present invention, the at least one probe has a nucleotide sequence selected from the group consisting of sequence identification number: 11, sequence identification number: 12, and sequence identification number: 13.

It can be understood that the operating conditions of the hybridization reaction are further changed according to factors such as the apparatus used, the type of the sample and the manner of pretreatment, and the proportion of the reaction contents used, etc., in order to achieve The best detection results. The choice of these operating conditions is routinely determined by those skilled in the art.

According to the present invention, the hybridization reaction can be carried out on a biochip selected from the group consisting of a gene wafer, a microfluidic wafer (for example, a sample pretreatment wafer, a reactive wafer, and an analytical wafer). And the wafer lab. In a preferred embodiment of the invention, the hybridization reaction is carried out on a gene wafer.

The primer pair and/or probe according to the present invention can be applied to prepare a test kit for detecting Salmonella enterica. In addition to the primer pairs and/or probes, the test kit can further comprise a nucleic acid dye for monitoring DNA amplification products. Preferably, the nucleic acid dye is selected from the group consisting of: ethidium bromide (EtBr), SYBR GREEN I, SYBR GREEN II, SYBR Orange, SYBR Gold, Propidium Iodide (PI), SYTOX Blue, and SYPRO Ruby.

Primer pairs and/or probes in accordance with the present invention are also contemplated for use in biochips to rapidly detect Salmonella enterica present in a sample.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example Experimental Materials:

1. The bacterial strains of Salmonella enteric serotype used in the following examples are isolated from the meat or feces of chickens in the BETAGRO chicken farm in Thailand, or Since the Centers for Disease Control (ROC (Taiwan)) in Taiwan, these strains include: (1) strains isolated from meat or feces of chickens: Salmonella tonatus ( S. Altona), Austria Barney Salmonella (S .Albany), Salmonella Agona (S .Agoma), Bota Salmonella (S .Berta), Bonn Salmonella (S .Bonn), cows Salmonella (S .Bovismorbificans) , Salmonella paratyphi A ( S. Bredeny), S. Bradenup, S. Brandenburg, S. Brandenburg, S. Cannstatt, Lexington Duisha door bacilli (S .Lexington), Gu Basha door bacillus (S .Cubana), Cairo Salmonella (S .Cairo), California Salmonella (S .California), red Manchester Salmonella (S .Chester), Chittagong Port Salmonella (S .Chittagong), Colorado Salmonella (S .Col Orado), S. Coleypark, S. Coleypark, S. Eppendorf, S. Essen, S. G., Salmonella Heidelberg ( S. heidelberg), Salmonella Indiana (S .Indiana), Salmonella Java (S .Java), Salmonella Kentucky (S .Kentucky), Salmonella non Craig (S .Krefeld), Levin ink tesha door subtilis (S .Limete ), Manhattan Salmonella (S .Manhattan), turkey Salmonella (S .Meleagridis), Miami Salmonella (S .Miami), Montevideo Jasa door bacilli (S .Montevideo), En Chang Gaza door bacilli (S. Nchanga), Salmonella Newton, S. Newington, S. Nigeria, S. Ohio, S. Oslo, S. Oslo, S. Oranienburg), S. Panama, S. Paratyphi A, S. Ponna, S. Sandiego bacilli (S .Senftenberg), Tennessee Salmonella (S .Tennessee), soup Vysha door bacilli (S .Thomasville) and Salmonella Thompson (S .Thompson); and (2) obtained from a strain of Taiwan CDC: Salmonella enteritidis CF09.001, CF09.002, CF09.003, CF09. 009, CF09.011, CF09.012, CF09.013, CF09.017, CF09.018 and CF09.023, Salmonella typhimurium CF09.015, CF09.016, CF09.019, CF09.020, CF09.021, CF09.037, CF09.039, SD09.016, SD09.018 and SD09.023, Salmonella Hadar. CA08.102, CA08.106, CA08.124, CA08.129, CA08.157, CA08.159, CA08. 160, CB09.011 and CF09.010, Salmonella typhimurium CC07.003, CC07.016, CC07.063, CC07.090, NQ08.053, SA08.050, SA08.059, SB08.005, SB08.038 and SD08.109, Salmonella salmonella CA08.075, CA08.116, CA08.135, CA08.158, CF08.016, CF08.030, CF08.039, CF08.041, CF08.050 and CF08.054, gona Salmonella SA09.039 and SQ09.012, Albany and Salmonella CF09.014 SD09.001, duck Salmonella (S .Anatum) CA08.145 and CA06.166, Augustus field salmonella Berger ( S .Augustenborg) CA06.114 and CG06.201, Salmonella rod Barrile (S .Bareilly) SA09.063 and SB09.004, Klebsiella Salmonella Blow (S .Blockley) NJ08.069 and WHO08.006, Bullinger have Lubai Salmonella SD08.079 and SD08.096, Cerro Salmonella ( S. Cerro) SB08.046 and SB08.071, Salmonella red serrata NA07.025 and NL07.041, Salmonella choleraesuis SB08.003 and SB08.008, S. Derby N (Serby) NJ09.006 With NJ09.015, S. Dublin, CA07.122 and CA07.126, S. Haifa, S.Haifa, NB06.053 and NB06.062, S. Havana, SB07.026 SD07.148, ho Datansha door subtilis (S .Houteu) CH08.005 and SA08.089, Indiana and Salmonella WHO08.007 CG04132, Salmonella Yisang Ji (S .Isangi) SQ07.020 and SQ07.044, open Du S.Kedougou NO08.042 and NQ08.034, S. Litchfield CC08.008 and CC08.067, and S. severidae O14 mutant ( S. Livingstone var . O14) NL08.069 and NL08.105, London Salmonella (S .London) CA07.069 and CA07.072, Ma Senya Salmonella (S .Massenya) NJ08.061 and NJ08.064, wood Bhandarkar salmonella ( S .Mbanda Ka) CB09.010 and NL09.004, Monteverre's Salmonella SA09.003 and SA09.024, Salmonella NL07.005 and CG05.044, S. Newport, CF09.005 and CF09.006 , Salmonella panicula CH07.059 and NG06.062, typhoid bacillus SB07.060 and SB07.068, type B paratyphoid Salmonella typhimurium Java variant strain ( S. Paratyphi B var . Java ) CF09.004 and SA09.012 , Salmonella Potsdam ( S. Potsdam) CF08.071 and CF08.092, S. Paulian S. Saintpaul SA09.014 and SA09.016, S. serrata ( S Schwarzengrund) CF08.009 and CF08.040, mountain Cardiff Fort Stockton Salmonella (S .Senftenberg) NC04.037 and SA04.051, hibiscus Salmonella (S .Seremban) SA08.144 and SA08.275, Singapore Salmonella (S .Singapore) SP08.013 and CC06.097, Shi Tanlai Salmonella (S .Stanley) CF09.026 with CF09.028, Shah Tamba hole door coli (S .Tambacounda) CA08.128 and CG06.008, Tennessee and Salmonella CA08134 CA06.018, Thompson salmonella SG05 .251 and CA06.191, Salmonella typhimurium ( S. Typhi) CH09.016, CH09.017, 04-901-356988, 04-901-764625, 04-901-925948, 04 -901-925983, 04-901-989321, 04-901-995173, 06-901-234525 and 06-901-234524, Uganda Salmonella ( S. Uganda) SA08.111 and SA08.185, Salmonella Victoria ( S .Victoria) SD08.011 and SD08.100, and S. Weltevreden SB09.001 and SQ09.016.

2. The bacterial strains of various non-intestinal Salmonella used in the following examples are biological resource conservation and research centers purchased from the Food Industry Research and Development Institute (FIRDI) in Taiwan, respectively. Biosource Collection and Research Center, BCRC) (300 Food Road, Hsinchu City, Taiwan), American Type Culture Collection (ATCC), and Culture Collection, Gothenburg University, Sweden (Culture Collection, University of Göteborg, CCUG), these strains include: (1) Staphylococcus spp.: Staphylococcus aureus BCRC 13824, 13825, 13830, 12654, 12656 and ATCC 700699, Staphylococcus aureus ( Staphylococcus xylosus ) BCRC 15252, Staphylococcus intermedius BCRC 15236 and 12157, Staphylococcus epidermidis BCRC 10785, Staphylococcus hyicus BCRC 12925, Staphylococcus haemolyticus BCRC 12923 Staphylococcus species BCRC 12660; (2) species of Listeria (Listeria spp.): Listeria monocytogenes bacteria (Listeria monocytogenes) BCRC 14845,14846,14847,14848,14930,15327,15330,15331,15332,15333 and 15334 and Listeria murrayi BCRC 15362, 15360, 14849 and 14843; (3) Pseudomonas spp.: Pseudomonas fluorescens BCRC 16016, 13902, 11028,13904 and 10304, Pseudomonas putida (P putida.) BCRC 10459, Pseudomonas aeruginosa (P aeruginosa.) BCRC 10944 and Pseudomonas mendocina (P mendocina.) BCRC 10458; (4) does not move Acinetobacter spp.: Acinetobacter species strains BCRC 15420 and 17454, A. calcoaceticus BCRC 11562, Acinetobacter baumannii ( A. baumannii ) BCRC 15556 and 15318, Acinetobacter baumannii (Acinetobacter baumannii ) A. ursingii ) BCRC 17329 and Acinetobacter johnsonii ( A. johnsonii ) BCRC 14853; (5) Eschericha coli BCRC 14824 and 14825, E. coli O157:H7 ( Eschrichia coli O157:H7) BCRC 13086, 13094, 13095 ATCC 35150 and 43890; (6) Streptococcus species (Streptococcus spp.): Streptococcus agalactiae (Streptococcus agalactiae) BCRC 10787, Streptococcus uberis (Streptococcus uberis) ATCC 700407 and S. bovis (Streptococcus bovis) CCUG 4214; ( 7) Cronobacter sakazakii BCRC 14122, 13988 and 14153; (8) Vibrio spp.: Vibrio parahaemolyticus BCRC 13023 and 13025 and Vibrio alginolyticus ( Vibrio) Alginolyticus ) BCRC 12829; (9) Clostridium spp.: Clostridium perfringens BCRC 10914, Clostridium acetobutylicum BCRC 10639, Clostridium difficile BCRC 10642 and Clostridium haemolyticum BCRC 10643; (10) Moraxella spp.: Moraxella catarrhalis BCRC 10629 and Moraxella Oslo Osloensis ) BCRC 10705; (11) Bacillus cereus BCRC 10250; (12) Corynebacterium renale BCRC 10657; (1) 3) Streptomyces filipinensis BCRC 11472; (14) Rahnella aquatilis BCRC 14814; and (15) Saccharomyces cerevisiae BCRC 20577.

3. Tryptic Soy Broth (TSB) and Plate Count Agar (PCA) used in the following examples were purchased from Difco (Difco Uboratories, Detroit, Michigan U.S.A.).

4. Biotin-labeled primer pairs and probes used in the following examples were synthesized by Biotech (Taipei, Taiwan).

General experimental method: 1. Extraction of genomic DNA:

The extraction of the genomic DNA of the bacterial strain was carried out using the Blood and Tissue Genomic DNA Miniprep System (VIOGENE). First, 1 mL of the bacterial culture or food sample was placed in a microcentrifuge tube, followed by centrifugation at 10,000 rpm for 5 minutes. After the supernatant was removed, the pellets were washed twice with 1 mL of sterile water. Thereafter, 200 μL of sterile water was added to fully suspend the cells, followed by the addition of 20 μL of lysozyme (2 mg/mL) and 20 μL of RNase A (2 mg/mL) and reacted at 37 ° C for 3 hours. Next, 200 μL of Ex buffer and 15 μL of proteinase K (10 mg/mL) were added and shake-mixed, whereby the resulting mixture was placed in a 60 ° C water bath for overnight reaction. Thereafter, the mixture was placed in a 70 ° C water bath for 30 minutes to deactivate the proteinase K, followed by the addition of 250 μL of absolute alcohol and a slight oscillating mixture for a few seconds. then, A B/T Genomic DNA Mini Column was placed in a collection tube, and all the mixture was transferred to the column, followed by centrifugation at 8,000 rpm. 2 minutes. Thereafter, the column was placed in a new collection tube, and the precipitate in the column was washed twice with 500 μL of WS buffer, followed by centrifugation at 8,000 rpm for 2 minutes. After the eluate was removed, the column was placed back into the collection tube and centrifuged at 12,000 rpm for 3 minutes. Finally, the column was placed in a new microcentrifuge tube, and then 100 μL of sterile water (preheated to 70 ° C) was added to the center of the column and allowed to stand for 5 minutes, then at 13,000 rpm. The centrifugation was carried out for 30 seconds to elute the genomic DNA of the bacterial strain. The genomic DNA thus obtained was stored at -20 ° C for use.

Example 1. Design of primer pairs for detecting Salmonella enterica

First, the applicant relied on Masato Akiba et al . (2011) (same above) from the Sanger Institute's FTP address ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/HADAR.dbs and ftp: //ftp.sanger.ac.uk/pub/pathogens/Salmonella/SIN.dbs download the complete genome sequence of Salmonella Harder and Salmonella typhimurium, then according to the complete genome of Salmonella The Hadar-specific genomic region (HSR) and the nucleus shown in the infant-specific genomic region (ISR) in the complete genome of the infant Salmonella The nucleotide sequence, and multiple sets of primer pairs were designed for Salmonella Hadda and Salmonella typhimurium, respectively.

In addition, the genome of Salmonella vilson has not yet been completely sequenced, and according to the applicant's previous research, the nucleotide residues in the complete genome of Salmonella typhimurium are located at 3710138 to 3713529. Salmonella sphaeroides has a high degree of sequence similarity. Therefore, the Applicant designed the nucleotide sequence shown below for the nucleotide residues 3710138 to 3710158 in the entire genome of the above-described Salmonella typhimurium and at the nucleotide residue positions 3713529 to 3713507. Primers are used for Univer-F primers and Univer-R primers.

Univer-F primer

5'-cctgatgaaaagcggacaaag-3' (sequence identification number: 1)

Univer-R primer

5'-gccttgataatgcctgtgcagaa-3' (sequence identification number: 2)

Thereafter, the Salmonella burgdorferi CA08.158 described in item 1 of the above "Experimental Materials" was inoculated into TSB broth medium and cultured overnight in a constant temperature shaking incubator (37 ° C, 150 rpm). . Next, 1 mL of the bacterial culture was taken and the genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Then, using the obtained genomic DNA of Salmonella vilsonella CA08.158 as a template, and using the primer designed above, the Univer-F primer and the Univer-R primer were used as shown in Table 1 below. Polymerase chain reaction (PCR) of the reaction conditions.

After the completion of the PCR, the obtained PCR reaction solution was analyzed by 2% agarose gel electrophoresis, and the experimental results showed that Salmonella vilshii had two sizes of about 3000 bp. And a 500 bp PCR product. Next, the PCR product of Salmonella vilsonii, which is about 500 bp in size, is purified and recovered from the agarose gel by using a Gel Extraction Kit (Viogene, USA), which is then entrusted. Hexin Biotech Co., Ltd. performed sequencing analysis, and the sequencing result showed that the purified PCR product contained a DNA fragment of 341 bp in size and having the nucleotide sequence as shown in SEQ ID NO: 3. Then, using the BLAST software (http://blast.ncbi.nlm.nih.gov/), the nucleotide sequence of the sequenced DNA fragment (SEQ ID NO: 3) is taken with the above-described Salmonella typhimurium The nucleotide sequence in the complete genome is subjected to an alignment analysis to obtain The results of the sequence alignment analysis are shown in Figure 1. As can be seen from Figure 1, the DNA fragment of Salmonella vilsoni substantially corresponds to the nucleotide residue position 3713150 to 3713480 in the complete genome of the infant type Salmonella, wherein the DNA fragment and the nucleotide residue The nucleotide sequence at base positions 3,732,251 to 3,713,480 has a high degree of similarity, but has a higher variability with the nucleotide sequence at positions 3713150 to 3713250 of the nucleotide residue. Therefore, the applicant designed a plurality of sets of primer pairs against Salmonella vilson according to the DNA fragment.

Thereafter, the above-mentioned sets of primers designed for the three types of Salmonella serotypes of the intestinal tract were taken from the bacterial gene database of the NCBI website using the Gene Blast software provided on the NCBI website (http://www.ncbi. All gene sequences in nlm.nih.gov/sutils/genom_table.cgi) were subjected to an alignment analysis to screen out a set of serovar-specfic primer pairs for Salmonella harvey. HADAF/HADAR, two groups of serotype-specific primer pairs for infantile Salmonella, INFA1F/InViR and INFA2F/InViR, and two groups of serotype-specific primer pairs for Salmonella serovar. VIRC1F/InViR and VIRC2F /InViR (see Table 2), and then the five sets of primer pairs were calibrated with biotin for subsequent experiments.

Example 2. Analysis of the specificity and sensitivity of the primers of the present invention in the detection of Salmonella enterica

In order to evaluate the specificity and sensitivity of the five sets of serotype-specific primer pairs designed for the three Salmonella serotypes of Salmonella in Example 1 above, the following experiment was conducted.

A. The specificity test of the pair of primers:

First, 259 bacterial strains described in Items 1 and 2 of the above "Experimental Materials" were inoculated separately into TSB broth medium, and cultured overnight in a constant temperature shaking incubator (37 ° C, 150 rpm). Next, 1 mL of the bacterial culture was taken and the genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Then, the obtained genomic DNA of each bacterial strain was used as a template, and the biotin-labeled primer pairs designed in the above Example 1 were respectively used for HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR and VIRC2F/InViR were used for polymerase chain reaction (PCR), and the reaction conditions of PCR are shown in Table 3 below.

After the completion of the PCR, it was confirmed by 2% agarose gel electrophoresis whether or not a PCR product of a predetermined size as shown in Table 2 was obtained.

B. Sensitivity test of the pair of primers:

To understand the sensitivity of the five groups of serotype-specific primers disclosed in the present invention for the detection of Salmonella enterica strains, Salmonella Havalella CA08.159, Salmonella serotype SB08.005 and Salmonella salmonella CA08. 158 was taken for the following experiment. First, these three strains were separately inoculated into TSB broth medium and cultured overnight in a constant temperature shaking incubator (37 ° C, 150 rpm). The resulting bacterial culture was adjusted to a concentration of 10 ⁹ CFU/mL (counted by plate count medium) in TSB broth medium, and used as a stock for 10-fold serial dilution ( 10-fold serial dilution), thereby obtaining diluted bacterial solutions having different concentrations (10 ² to 10 ⁹ CFU/mL). Thereafter, 1 mL of each of the diluted bacterial solutions containing different bacterial concentrations was taken, and genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in the "General Experimental Method" above.

Further, a part of the diluted bacterial solution containing the different bacterial concentrations obtained in the above manner was placed in a constant temperature shaking incubator (37 ° C, 150 rpm) for enrichment culturing for 10 hours. Thereafter, 1 mL of each of the diluted bacterial solutions containing different bacterial concentrations in the proliferation culture was taken, and genomic DNA was extracted according to the method described in the first item "Extraction of genomic DNA" in the "General Experimental Method" above.

The genomic DNA of each of the non-proliferating cultures and the cultured bacterial strains obtained above was used as a template, and PCR was carried out using five sets of serotype-specific primer pairs as shown in Table 2 above, respectively. The reaction conditions for PCR were as shown in Table 3 above.

After the completion of the PCR, it was confirmed by 2% agarose gel electrophoresis whether or not a PCR product of a predetermined size as shown in Table 2 was obtained.

result: A. The specificity test of the pair of primers:

As a result of agarose gel electrophoresis analysis, it was found that when PCR was carried out by using genomic DNA of each bacterial strain as a template and using biotin-labeled primers for HADAF/HADAR, only Salmonella Haddar had a size of about 427 bp. PCR amplification products.

In addition, when using the biotin-labeled primer pair for INFA1F/InViR and INFA2F/InViR for PCR, only the infant type Salmonella can obtain PCR amplification products of about 268 and 240 bp, respectively; The primed primer pair VIRC1F/InViR and When VIRC2F/InViR was used for PCR, only Salmonella vilsonii obtained PCR amplification products of approximately 273 and 234 bp, respectively.

The results of this experiment show that the five groups of serotype-specific primers according to the present invention have detection specificity for HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR and VIRC2F/InViR for the target strain to be tested.

B. Sensitivity test of the pair of primers:

Table 4 below shows the results of sensitivity tests of five sets of serotype-specific primer pairs according to the present invention. As can be seen from Table 4, biotin-labeled primers can detect HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR, and VIRC2F/InViR for unproliferated strains of S. cerevisiae strains up to 10 ³ CFU. /mL, and the detection sensitivity of the cultured strain of Salmonella enterica can reach 10 ⁰ CFU/mL. The results of this experiment show that the five groups of serotype-specific primers according to the present invention have high sensitivity to HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR, and VIRC2F/InViR, and can be used for detecting intestinal sand gates. Bacillus strain.

Example 3. Design of probes for detecting Salmonella enterica

Based on the complete genome of Salmonella Hadar (downloaded from ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/HADAR.dbs), the Hadar-specific genomic region (HSR) and infant Salmonella Intact-specific genomic region (ISR) of the complete genome (downloaded from ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/SIN.dbs) for Salmonella Haddar and infants Salmonella type to design a variety of probes.

These probes then use the Gene Blast software provided on the NCBI website to share all genes in the bacterial gene database (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) on the NCBI website. The sequences were subjected to alignment analysis, whereby the serotype-specific probes HADAP and INFA1P were screened for Salmonella Harder and Salmonella typhimurium, respectively.

In addition, the mutual interaction of the VIRC1F primers designed in the above embodiment 1 Complementary sequences were used as the serotype-specific probe VIRC1P against Salmonella vilsh.

For clarity, the primer pairs and probe related information (including: nucleotide sequence, size of the amplified PCR product, corresponding to the target gene or the location of the complete genomic DNA, etc.) according to the present invention have been integrated. In Table 5 below.

Example 4. Evaluation of the ability of a primer according to the present invention to detect Salmonella enterica in foods

In order to evaluate the detection specificity and sensitivity of the five groups of serotype-specific primers according to the present invention for Salmonella enterica strains present in foods, chicken samples containing various strains of Salmonella enterica were used for PCR.

A. Prepare chicken samples containing Salmonella Hadar, Salmonella typhimurium or Salmonella vilson:

First, Salmonella Habardella CA08.159, Salmonella serotype SB08.005 and Salmonella salmonella CA08.158 were inoculated into TSB broth medium and shaken in a constant temperature incubator (37 ° C, 150 rpm). The culture was carried out overnight. The formed bacterial culture was adjusted to a concentration of 10 ⁹ CFU/mL (counted by the plate count medium) in TSB broth medium, and 10 times serial dilution was performed as a stock solution, thereby Diluted bacterial solutions with different concentrations (10 ² ~ 10 ⁹ CFU/mL) were obtained. Then, take 100 μL of each diluted bacterial solution containing different bacterial concentrations, and then add 1 mL of chicken homogenate and 8.9 mL of TSB broth to mix and mix, thereby obtaining different bacterial concentrations (10 ⁰ ~ 10 ⁷ CFU/mL) chicken samples.

In addition, a part of the chicken meat samples containing different bacterial concentrations obtained in the above manner were subjected to proliferation culture in a constant temperature shaking incubator (37 ° C, 150 rpm) for 10 hours, thereby obtaining a culture containing proliferation. Chicken samples of different bacterial concentrations.

B. The specificity test of the pair of primers:

A chicken sample containing Salmonella hamodus, Salmonella typhimurium or Salmonella vilsoni, which has not been proliferated in the above item A (each sample contains a bacterial concentration of 10 ⁷ CFU/mL) Take 1 mL each, and extract the genomic DNA according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Then, PCR was performed using the obtained genomic DNA of each strain of Salmonella typhimurium as a template and using biotin-labeled primers for HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR, and VIRC2F/InViR, respectively. The reaction conditions for PCR were as shown in Table 3 above. After the completion of the PCR, each of the strains obtained 5 PCR amplification products, respectively, so that a total of 15 PCR amplification products were obtained, which were obtained by using primer pairs HADAF/HADAR, INFA1F/InViR, and VIRC1F/InViR. The PCR amplification products are collectively referred to as the first group PCR product.

The obtained 15 PCR products were each subjected to 2% agarose gel electrophoresis analysis to confirm whether a PCR product of a predetermined size as shown in Table 5 was obtained.

C. Sensitivity test of the pair of primers:

1 mL of chicken samples containing different concentrations (10 ⁰ ~ 10 ⁷ CFU/mL) of Salmonella Harder produced in the above-mentioned item A without proliferation and culture, and according to the above Extraction of genomic DNA by the method described in the first item "Extraction of genomic DNA" in the general experimental method. Next, the obtained genomic DNA of the non-proliferated culture and the cultured and cultured Salmonella harveyii was used as a template, and the biotin-labeled primer was used to carry out PCR on HADAF/HADAR, and the reaction conditions of the PCR were as shown in the above table. The one shown in 3. After completion of the PCR, a total of 16 PCR amplification products were obtained, which are collectively referred to as the second group PCR product.

Sensitivity tests for chicken samples containing Salmonella infantile or Salmonella vilsonii are generally carried out in the manner described above for chicken samples containing Salmonella Harder, except that infantile sand gates are used. When the genomic DNA of Bacillus is used as a template for PCR, the selected primer pair is INFA1F/InViR and INFA2F/InViR; and when PCR is carried out using genomic DNA of Salmonella breve, as the template, the selected primer pair is VIRC1F. /InViR and VIRC2F/InViR. After the completion of the PCR, both Salmonella infantile and Salmonella burgdorferi each obtained 32 PCR amplification products, wherein the PCR amplification products obtained by using the primers for INFA1F/InViR and VIRC1F/InViR are collectively referred to as Group 3 and 4 PCR products.

Finally, the 80 PCR products obtained above were each subjected to 2% agarose gel electrophoresis analysis to confirm whether a PCR product of a predetermined size as shown in Table 5 was obtained.

result: A. The specificity test of the pair of primers:

As a result of agarose gel electrophoresis analysis, it was found that the genomic DNA of Salmonella Harder, S. cerevisiae or Salmonella vilsoni obtained from chicken samples was used as a template and biotin-labeled primer pair HADAF was used. /HADAR for PCR, only Hadar Shamen The strain has a PCR amplification product of about 427 bp in size; when PCR is performed using the biotin-labeled primer pair INFA1F/InViR and INFA2F/InViR, only the infant type Salmonella has a size of about 268 and A 240 bp PCR amplification product; and when using biotin-labeled primer pairs for VIRC1F/InViR and VIRC2F/InViR for PCR, only Salmonella vilson has a PCR amplification of approximately 273 and 234 bp, respectively. Add product.

The results of this experiment show that the five groups of serotype-specific primers according to the present invention have specificity for HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR, and VIRC2F/InViR for the target strains present in foods.

B. Sensitivity test of the pair of primers:

Table 6 below shows the detection sensitivity of five sets of serotype-specific primer pairs according to the present invention for Salmonella enterica strains present in chicken samples. As can be seen from Table 6, the sensitivity of biotin-labeled primers to HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR, and VIRC2F/InViR for Salmonella enterica strains in non-proliferated chicken samples can be Achieving 10 ³ CFU/mL, and the detection sensitivity of the strain of Salmonella enterica in the cultured chicken samples can reach 10 ⁰ CFU/mL.

The results of this experiment show that the five groups of serotype-specific primers according to the present invention have high detection sensitivity for HADAF/HADAR, INFA1F/InViR, INFA2F/InViR, VIRC1F/InViR, and VIRC2F/InViR for the target strains present in foods. .

Example 5. Detection of Salmonella enterica in food by multiplex polymerase chain reaction (multiplex PCR)

In order to evaluate the detection effect of a primer combination consisting of three serotype-specific primers of the present invention on HADAF/HADAR, INFA1F/InViR, and VIRC1F/InViR on multiplex polymerase chain reaction, in the above example A of Example 4 The chicken samples containing the different strains of Salmonella typhimurium prepared in the non-proliferation culture were used for the following experiment.

experimental method:

First, a chicken sample containing Salmonella Harder, Salmonella typhimurium or Salmonella vilsoni, which was prepared in the above-mentioned item A of Example 4, was cultured in an uncultured manner (each sample contained one of 10 ⁷ CFU). Take 1 mL each of the bacterial concentration of /mL, and extract the genomic DNA according to the method described in the first item "Extraction of genomic DNA" in "General Experimental Methods" above. Next, the genomic DNA (concentration of 150 μg/μL) of the three Salmonella serotypes obtained from the chicken samples was mixed according to the combination and the ratio shown in Table 7 below, thereby obtaining the genome. DNA mixture A to D. Then, multiplex polymerase chain reaction was carried out using the obtained genomic DNA mixtures A to D as templates and using a combination of primers composed of HADAF/HADAR, INFA1F/InViR and VIRC1F/InViR, which were labeled with biotin. The reaction conditions for the multiplex polymerase chain reaction were as shown in Table 8 below.

After completion of multiplex PCR, the four PCR amplification products (which are collectively referred to as the fifth group PCR products) were separately subjected to 2% agarose gel electrophoresis analysis to confirm whether a PCR product of a predetermined size was available. .

result:

Figure 2 shows the PCR products obtained by multiplex PCR using genomic DNA mixtures A to D as templates and a primer combination of HADAF/HADAR, INFA1F/InViR and VIRC1F/InViR, respectively, using biotin-labeled primers. The result of agarose gel electrophoresis analysis, wherein the diameter M represents a DNA ladder marker, and the diameter 1 to the diameter 4 represent the genomic DNA mixtures A to D, respectively. As can be seen from Figure 2, the serotype-specific primer in the primer combination is for HADAF/HADAR, INFA1F/InViR and VIRC1F/InViR can simultaneously and specifically detect the target strains present in each genomic DNA mixture.

The results of this experiment confirmed that a primer combination consisting of three serotype-specific primer pairs of the present invention (i.e., HADAF/HADAR, INFA1F/InViR, and VIRC1F/InViR) can be used for multiplex PCR, thereby enabling simultaneous specificity. Salmonella Harder, infantile Salmonella, and Salmonella vilson, which are present in food, were detected.

Example 6. Evaluation of the ability of a probe according to the present invention to detect Salmonella enterica in food

In order to evaluate the detection specificity and sensitivity of the three probes designed in Example 3 above (as shown in Table 5) for Salmonella enterica in food, in Examples 4 and 5 above The obtained 1st to 5th PCR products were taken for the following experiment.

A. Preparation of biochips:

First, in order to allow the three probes according to the present invention to have better immobilization when spotted on the surface of the biochip, the 5' or 3' end of the three probes The oligo (dT) [oligo (dT)] was modified, respectively, and the 40-mer probes thus obtained were shown in Table 9 below.

Thereafter, the synthesized 40-member probes 1 to 3 were each dissolved in sterile water, whereby a probe stock solution 1 to 3 having a concentration of 10 to 20 μM was obtained. Next, 25 μL of each of the probe stock solutions 1 to 3 was taken and mixed with 25 μL of 2X Probe solution (Dr. Chip Biotech., Taiwan) to form wafer probe solutions 1 to 3. The formed wafer probe solutions 1 to 3 were added to a 96-well culture dish, and then 40-member probes were prepared by an Automatic Arrayer (Ezspot SR-A300, Shuai Ran Precision, Taiwan). 1 to 3 points were placed on a plastic membrane (DR. Chip, Dr. Chip Biotech., Taiwan), followed by UV crosslinker C1508 (UVItec, Cambridge, England) (254 Nm/0.6-1.2 joules) These 40-member probes were immobilized on a plastic film, thereby forming a biochip containing the three probes according to the present invention. In addition, the biotin-labeled random sequence fragment and the 2X probe solution were used as a positive control and a negative control, respectively. The DNA probe spot position of the biochip is shown in Figure 3, where A3 represents probe 2; B1 represents probe 3; B2 represents Probe 1; C1 indicates a positive control group; and C2 indicates a negative control group.

B. Specificity test of biochip:

First, 25 μL of the first group PCR product (total of 9 PCR amplification products) obtained in the "B, primer pair specificity test" of Example 4 above was placed in a 1.5 mL microcentrifuge tube. (microtube) and added 200 μL DR.Hyb ^TM buffer ^{(DR.Hyb TM buffer) (Dr.Chip Biotech} ., Taiwan). Subsequently, denaturation was carried out at 99 ° C for 10 minutes, followed by standing on ice for 5 minutes, thereby obtaining 9 mixed solutions containing single-stranded DNA. Thereafter, the biochip prepared in item A above is placed in a hybridization box (DR. Chip Biotech., Taiwan) and the nine mixed solutions obtained above are respectively added, and then the lid is placed and placed. a temperature is set at 50 deg.] C oven ^{(DR.Mini TM oven, Dr.Chip Biotech.} , Taiwan) for shaking the reaction for 1 hour. Thereafter, the solution in each hybridization cassette was removed and the biochips were washed once with 250 μL of Wash Buffer (Dr. Chip Biotech., Taiwan) at room temperature, followed by 250 μL. The wash buffer was washed once to remove non-specifically bound DNA-DNA hybrids (hybrids). Next, 200 μL of Blocking Reagent (Dr. Chip Biotech., Taiwan) was mixed with 0.2 μL of Streptomycin-AP (NEL751, Perkin Elmer Co., MA, USA). They were separately added to each hybridization box, and then placed in an oven set at 30 ° C for an oscillating reaction for 30 minutes. After removing the liquid from the hybridization cassettes, 500 μL of wash buffer was added and allowed to stand for 1 minute, followed by removal of the wash buffer. This cleaning-resting step is repeated 4 times. Thereafter, 196 μL of Detection Buffer (Dr. Chip Biotech., Taiwan) and 4 μL of BCIP/NBT substrate solution (NEL937, Perkin Elmer Co., MA, USA) Mix well and add separately to each hybridization cassette and then let stand for 15 to 30 minutes for chromogenic reaction. Thereafter, the cassette hybridisation oven ^{(DR.Mini TM Oven, Dr.Chip Biotech.} , Taiwan) in dried for 10 to 15 minutes and then taken out to such biochips and image analysis system (DR.AiM ^TM Reader, Dr. Chip Biotech., Taiwan) to scan.

In addition, in order to further evaluate whether the biochip can detect a plurality of strains of Salmonella enterica simultaneously present in a food, the applicant selects the 5th PCR product obtained in the above Example 5 (a total of 4 PCR amplifications) The product is subjected to a denaturation reaction in the manner described above, whereby the resulting mixed solution containing the single-stranded DNA is separately subjected to a hybridization reaction with the biochip, and the reaction conditions and operation steps relating to the hybridization reaction are carried out. It is as described above.

C. Sensitivity test of biochip:

The second to fourth group PCR products obtained in the "C, sensitivity test of the primer pair" of Example 4 above (there are 16 PCR amplification products in each group, for a total of 48 PCR amplification products) refer to the above The denaturation reaction is carried out in the manner described in item B, whereby the obtained mixed solution containing single-stranded DNA is separately subjected to hybridization reaction with the biochip, and the reaction conditions and operation steps relating to the hybridization reaction are as above. The one described in item B.

result: A, the specificity test of biochip:

To understand the specificity of detection of Salmonella enterica strains when the DNA probe of the present invention is applied to a biochip, to obtain Salmonella harveyi, Salmonella typhimurium or Salmonella vilsoni in chicken samples. The genomic DNA was used as a template and the PCR products obtained by PCR using HADAF/HADAR, INFA1F/InViR and VIRC1F/InViR, respectively, were extracted with a biotin-labeled primer, respectively, and one of the three serums containing the serum according to the present invention. The biochip of the type-specific probe performs a hybridization reaction. Figure 4 shows the map obtained after hybridization. As can be seen from Fig. 4A, the PCR product obtained by PCR using H. falciparum genomic DNA as a template and using biotin-labeled primers for HADAF/HADAR was taken. When a hybridization reaction was carried out with the biochip, a hybridization signal was observed at B2. As can be seen from Fig. 4B, when the PCR product obtained by PCR using the genomic DNA of Salmonella typhimurium as a template and using the biotin-labeled primer pair INFA1F/InViR is subjected to hybridization reaction with the biochip, A hybrid signal was observed at A3. As can be seen from Fig. 4C, when the PCR product obtained by PCR using the genomic DNA of Salmonella vilsonii as a template and using a biotin-labeled primer pair VIRC1F/InViR, respectively, is subjected to hybridization reaction with the biochip A hybrid signal was observed at B1.

Four mixtures containing different bacterial genomic DNAs (ie, genomic DNA mixtures A to D listed in Table 7 above) were used as templates and a biotin-labeled primer pair HADAF/HADAR, The four PCR products (i.e., the fifth group PCR products) obtained by performing multiplex PCR using INFA1F/InViR and VIRC1F/InViR primer combinations were respectively subjected to hybridization reaction with the biochip. Figure 5 shows a map obtained after hybridization, as seen from Figure 5A, when a multiplex PCR was performed using a mixture of genomic DNA of Salmonella Harder and Salmonella typhimurium (i.e., genomic DNA mixture A) as a template. When the resulting PCR product was subjected to a hybridization reaction with the biochip, a hybridization signal was observed at B2 and A3, respectively. As can be seen from Fig. 5B, when a PCR product obtained by performing multiplex PCR using a mixture of genomic DNA of Salmonella harvey and Salmonella vilsoni (i.e., genomic DNA mixture B) was taken and When the biochip was subjected to a hybridization reaction, a hybridization signal was observed at B2 and B1, respectively. As can be seen from Fig. 5C, the PCR product obtained after multiplex PCR using a mixture of genomic DNA of Salmonella typhimurium and Salmonella vilsoni (i.e., genomic DNA mixture C) was used as a template. When the biochip was subjected to a hybridization reaction, a hybridization signal was observed at A3 and B1, respectively. As can be seen from Fig. 5D, PCR was performed after multiplex PCR using a mixture of genomic DNA of Salmonella, Salmonella typhimurium and Salmonella vilsoni (i.e., genomic DNA mixture D) as a template. When the product was subjected to a hybridization reaction with the biochip, a hybridization signal was observed at B2, A3, and B1, respectively.

The above experimental results confirmed that the serotype-specific probes 1 to 3 according to the present invention can specifically detect Salmonella Harder's, Baby's Salmonella, and Salmonella vilson which are present in foods, respectively. therefore, The serotype-specific probes 1 to 3 according to the present invention can be applied to a gene chip for rapid detection of a strain of Salmonella enterica.

B. Sensitivity test of biochip:

Table 10 shows the results of sensitivity tests obtained using a biochip containing three serotype-specific probes according to the present invention to detect Salmonella enterica strains present in chicken samples. As can be seen from Table 10, the detection sensitivity of the biochip to the Salmonella enterica strain in the unfertilized chicken sample can reach 10 ² CFU/mL, and for the Salmonella enteric strain in the cultured chicken sample. The detection sensitivity can reach 10 ⁰ CFU/mL. The results of this experiment show that the serotype-specific probes 1 to 3 according to the present invention have high sensitivity to the target strain to be detected, and thus can be applied to a gene wafer for rapid detection of the presence in food. Trace intestinal Salmonella.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Figure 1 shows the results of an alignment analysis of the nucleotide sequences of Salmonella infantile and Salmonella vilson using BLAST software, wherein the two symbols indicated by the symbol "*" represent Salmonella enterica. Conserved nucleotide residues, while the numbers indicated on the gray background represent the positions of nucleotide residues in the complete genome corresponding to Salmonella infantile; Figure 2 shows the use of genomic DNA mixtures A through D as templates and separate use Agarose gel electrophoresis analysis of a PCR product obtained by multiplex PCR using a combination of primers composed of HADAF/HADAR, INFA1F/InViR, and VIRC1F/InViR, which are biotin-labeled primers, wherein the diameter M represents a DNA ladder Marked, and diatom 1 to diameter 4 represent genomic DNA mixtures A to D, respectively; Figure 3 shows DNA probe spotting positions of a biochip containing three probes according to the present invention, wherein A3 represents probe 2; B1 represents Probe 3; B2 represents probe 1; C1 represents a positive control group; and C2 represents a negative control group; and Figures 4A to 4C respectively show Salmonella Harder, Salmonella typhimurium or vitamins obtained from chicken samples Genomic DNA Shore Salmonella as a template, and were used by the biotin-calibrated primer pair HADAF / HADAR, INFA1F / InViR and VIRC1F / InViR performed after PCR The resulting PCR product was brought to a containing accordance with the present invention. Hybridization profiles observed for hybridization reactions of three serotype-specific probe biochips; and Figures 5A through 5D show genomic DNA mixtures A through D as templates, respectively, and using a biotin-labeled The PCR product obtained by performing multiplex PCR with the primer combination of HADAF/HADAR, INFA1F/InViR and VIRC1F/InViR was taken with a biochip containing three serotype-specific probes according to the present invention. The hybridization pattern observed when performing the hybridization reaction.

<110> Hongguang University of Science and Technology

<120> Nucleic acid molecules, test kits, biochips, and methods for detecting Salmonella strains

<130> Introduction and probe for detecting Salmonella enterica

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<170> PatentIn version 3.5

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<223> Univer-F primer for amplifying random DNA fragments of Salmonella vilsoni

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Claims (17)

An primer reagent for detecting Salmonella enterica comprising at least one of: (a) a first set of primer pairs for detecting Salmonella Haddar, comprising a sequence identification number: The nucleotide sequence shown in 4 is preceded by a primer, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 5; (b) a second primer for detecting Salmonella in infants. Pair, comprising a forward sequence of a nucleotide sequence as shown in sequence identification number: 6, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 7; (c) A third set of primer pairs for detecting Salmonella infestans, comprising a nucleotide sequence forward primer as shown in sequence identification number: 8 and a core having a sequence number: 7 a reverse primer of a nucleotide sequence; (d) a fourth set of primer pairs for detecting Salmonella vilson, comprising a nucleotide sequence forward primer as shown in sequence identification number: 9, and One has the same as the sequence identification number: 7 a reverse primer of the nucleotide sequence; and (e) a fifth set of primer pairs for detecting Salmonella vilson, comprising a nucleotide sequence as shown in sequence identification number: 10, And a reverse primer having a nucleotide sequence as shown in Sequence Identification Number: 7. A probe reagent for detecting Salmonella enterica, which contains the following At least one of the columns: (a) a probe for detecting Salmonella Haddar having a nucleoside selected from the group consisting of: Sequence ID: 4, Sequence ID: 5, and Sequence ID: An acid sequence; (b) a probe for detecting Salmonella infantile having a nucleotide sequence selected from the group consisting of: Sequence ID: 6, Sequence ID: 8 and Sequence ID: 12. And (c) a probe for detecting Salmonella vilsoni having a nucleotide sequence selected from the group consisting of: Sequence ID: 9, Sequence ID: 10, and Sequence ID: 13. A method for detecting the presence or absence of a Salmonella enterica in a sample, comprising: subjecting the sample to a DNA amplification reaction using at least one set of primer pairs, wherein the at least one set of primer pairs is selected from the group consisting of The resulting group: (a) a first set of primer pairs for detecting Salmonella Haddar, comprising a nucleotide sequence as shown in sequence identification number: 4, and a one having a a reverse primer of a nucleotide sequence as shown in Sequence Identification No.: 5; (b) a second set of primer pairs for detecting Salmonella infestans, comprising a sequence having a sequence identification number: The nucleotide sequence is preceded by a primer, and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 7; (c) a third set of primer pairs for detecting Salmonella infestans, the package thereof a forward primer having a nucleotide sequence as shown in sequence identification number: 8 and a reverse primer having a nucleotide sequence as shown in sequence identification number: 7; (d) one for detection A fourth set of primer pairs of Salmonella vilson, comprising a nucleotide sequence forward primer as shown in sequence identification number: 9, and a nucleotide having a sequence number: 7 a reverse primer of the sequence; and (e) a fifth set of primer pairs for detecting Salmonella vilson, comprising a nucleotide sequence as shown in sequence identification number: 10, and a primer a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 7; and detecting whether there is a DNA fragment amplified by using the at least one primer pair, wherein the presence of the DNA fragment indicates The presence of Salmonella enterica corresponding to the at least one set of primer pairs. The method of claim 3, wherein the sample is selected from the group consisting of a food sample, a biological sample, and an environmental sample. The method of claim 4, wherein the sample is a food sample selected from the group consisting of: fluid dairy, beverage, meat, seafood, animal feed, water, dairy, vegetable , fruit and canned food. The method of claim 3, wherein the total genomic DNA is extracted from the sample as a template before the DNA amplification reaction is carried out. The method of claim 3, wherein the DNA amplification reaction is carried out by using at least one of the following methods: polymerase chain reaction Should, reverse transcriptase polymerase chain reaction, real-time quantitative polymerase chain reaction, nested PCR, hot start PCR, in situ PCR, degenerate oligonucleotide primer PCR, microPCR, multiplex polymerase chain reaction, to limit The fragment length polymorphic nucleic acid sequence is mainly an amplification reaction, a transcription-regulated amplification reaction, a cyclic medium isothermal amplification reaction, and a rolling circle amplification reaction. The method of claim 3, wherein the detecting of the DNA fragment is carried out by a hybridization reaction using a probe. The method of claim 8, wherein: (a) when the DNA amplification reaction is carried out using the first set of primer pairs, the probe has a sequence selected from, for example, a sequence identification number: 4. : 5 and the nucleotide sequence shown in sequence identification number: 11; (b) when the DNA amplification reaction is carried out using the second set of primer pairs, the probe has a selected from, for example, a sequence identification number: 6 , sequence identification number: 7, sequence identification number: 8 and sequence identification number: nucleotide sequence shown in 12; (c) when the third group of primer pairs is used to perform the DNA amplification reaction, the probe has a nucleotide sequence selected from the group consisting of sequence identification number: 7 and sequence identification number: 8; (d) when the DNA amplification reaction is carried out using the group 4 primer pair, the probe has a selection From the nucleotide sequence of sequence identification number: 7, sequence identification number: 9, sequence identification number: 10, and sequence identification number: 13; and (e) when the group 5 primer pair is used to perform the DNA In the amplification reaction, the probe has a sequence selected from, for example, sequence identification number: 7 and sequence Identification number: The nucleotide sequence shown in 10. The method of claim 8, wherein the hybridization reaction is carried out on a biochip selected from the group consisting of: a gene wafer, a microfluidic wafer, and a wafer laboratory. A method for detecting the presence or absence of a Salmonella enterica in a sample, comprising: subjecting the sample to a hybridization reaction using at least one probe, wherein the at least one probe is selected from the group consisting of Group: (a) a probe for detecting Salmonella Haddar having a nucleotide sequence selected from the group consisting of: sequence identification number: 4, sequence identification number: 5, and sequence identification number: 11; b) a probe for detecting Salmonella infantile having a nucleotide sequence selected from the group consisting of: Sequence ID: 6, Sequence ID: 8 and Sequence ID: 12; and (c) a probe for detecting Salmonella vilsoni having a nucleotide sequence selected from the group consisting of: sequence identification number: 9, sequence identification number: 10, and sequence identification number: 13; and detecting whether there is a loan A hybrid formed by performing a hybridization reaction using the at least one probe, wherein the presence of the hybrid indicates the presence of a Salmonella enterica corresponding to the at least one probe. The method of claim 11, wherein the sample is selected from the group consisting of: a food sample, a biological sample, and an environmental sample. The method of claim 12, wherein the sample is a food sample selected from the group consisting of: fluid dairy, beverage, Meat, seafood, animal feed, water, dairy products, vegetables, fruits and canned food. The method of claim 11, wherein the hybridization reaction is carried out on a biochip selected from the group consisting of a gene wafer, a microfluidic wafer, and a wafer laboratory. The method of claim 11, wherein the at least one probe has a nucleotide sequence selected from the group consisting of: sequence identification number: 11, sequence identification number: 12, and sequence identification number: 13. A test kit for detecting Salmonella enterica, comprising a primer reagent as claimed in claim 1 and/or a probe reagent as in claim 2 of the patent application. A biochip for detecting Salmonella enterica comprising a primer reagent as claimed in claim 1 and/or a probe reagent as in claim 2 of the patent application.