CN110951898A - Specific novel molecular target of 4 species in Cronobacter and rapid detection method thereof - Google Patents

Abstract

The invention discloses a specific new molecular target of 4 species in Cronobacter and a rapid detection method thereof, and provides 19 new specific molecular detection targets for identifying 4 species in Cronobacter, namely Cronobacter sakazakii, Cronobacter malonate, Cronobacter zuchenii and Cronobacter dubliniensis, a corresponding PCR primer group and a PCR rapid detection method. The method can detect the Cronobacter species level without physiological and biochemical identification, and has the advantages of short detection time, low cost, simple operation and strong specificity; the probability of false positive or false negative is effectively reduced, the detection result is more accurate, the result judgment is simpler, the practicability is stronger, and the method has important significance for identifying the Cronobacter sakazakii.

Description

Specific novel molecular target of 4 species in Cronobacter and rapid detection method thereof

Technical Field

The invention belongs to the technical field of microbiological inspection, relates to a method for identifying Cronobacter sakazakii, and particularly relates to a new specific molecular target of 4 species in Cronobacter sakazakii and a rapid detection method thereof.

Background

Cronobacter spp is an important food-borne pathogenic bacterium, can cause diseases of people of all ages, has a great threat to premature infants, low-weight infants and immunocompromised newborns, can cause septicemia, meningitis or necrotizing enterocolitis in severe cases, can cause nervous system sequelae or rapid death, and has the mortality rate of 40-80%. Due to its high hazard, the international food microbial standards committee listed this strain as a microorganism that poses serious life risks to certain populations or chronic sequelae in 2002.

With the intensive research on Cronobacter and the evolving modern classification techniques, the nomenclature and class-typing of the bacterium has undergone a change from species to genus. Currently, the genus cronobacter contains 7 species: cronobacter sakazakii (Cronobacter sazakii), Cronobacter malonate (Cronobacter malonticus), Cronobacter zukii (Cronobacter turiensis), Cronobacter mokinsonii (Cronobacter mutyjensii), Cronobacter comforti (Cronobacter conditioner), Cronobacter ewingii (Cronobacter universalis), and Cronobacter dubliniensis (Cronobacter dubliniensis), wherein Cronobacter sakazakii is a model species of the genus. Studies have shown that there are differences between the toxicity of 7 species within the genus, not all species are associated with infection, only cronobacter sakazakii, cronobacter malonate and cronobacter zurich can cause neonatal infection. In the twelve-five period, the research team systematically completes the risk investigation of pathogenic microorganisms in 2717 foods in 28 major cities in China for the first time, 1092 Cronobacters are separated and stored, and the total pollution rate is as high as 17.96%, wherein the Cronobacter sakazakii accounts for 64.78%, the Cronobacter malonate accounts for 23.36%, the Cronobacter zukii Zurich accounts for 1.37%, the Cronobacter dublin accounts for 10.13%, and the Cronobacter mokinsonii accounts for 0.36%.

The detection method of Cronobacter sakazakii mainly comprises two main types, namely a traditional culture method and molecular biological detection. The traditional culture method can separate target bacteria, but has the defects of long time consumption (more than 5 d), complex operation (pre-enrichment, selective culture, chromogenic culture and biochemical identification), high cost, low accuracy and specificity and easy occurrence of false positive or false negative results. The molecular biology detection method based on PCR gradually becomes one of the most potential detection techniques to replace the traditional detection method due to the characteristics of rapidness, accuracy and simplicity.

At present, the main detection object of the PCR method reported at home and abroad is Cronobacter sakazakii, and the PCR method for detecting different species in the Cronobacter sakazakii is rare. Stoop et al established a PCR detection method capable of simultaneously detecting six species of Cronobacter sakazakii for the first time based on rpoB gene, but two pairs of primers are required for C.sakazakii detection, and two PCR reactions are performed. Carter et al established a PCR method for detecting different cronobacter species using the cgcA gene, but the primers used to detect c.sakazakii were relatively poor in specificity and non-specific fragments could be observed on an agarose gel. The PCR detection method established by Huang and the like based on the gyrB gene can effectively distinguish C.sakazakii from C.dublinensis, but cannot distinguish C.sakazakii from C.malonatus. Therefore, the method systematically excavates a specific new target on the Cronobacter species level, establishes an accurate and rapid PCR detection method, and has important significance for the detection of Klnobacter and pollution investigation research.

Disclosure of Invention

In view of the above problems, the present invention is to overcome the disadvantages of the prior art and to provide a novel specific molecular target and a corresponding detection method for identifying 4 species within the genus Cronobacter, namely Cronobacter sakazakii (Cronobacter sakazakii), Cronobacter malonate (Cronobacter malonticus), Cronobacter zukii (Cronobacter turiensis) and Cronobacter dubliniensis (Cronobacter dubliniensis).

In order to achieve the purpose, the invention adopts the technical scheme that: the invention claims a group of nucleotide sequences for identifying Cronobacter sakazakii, wherein the nucleotide sequences are shown in SEQ ID NO: 1-19, wherein the Cronobacter sakazakii, the Cronobacter malonate, the Cronobacter zuchenii or the Cronobacter dubliniensis.

Specific sequence fragments contained in 4 species of Cronobacter sakazakii (Cronobacter sakazakii, Cronobacter malonate, Cronobacter zuchenii, and Cronobacter dubliniensis) respectively are obtained by bioinformatics analysis; the fragment can be used as a specific new molecular target for identifying 4 species of Cronobacter sakazakii, and the nucleotide sequence is shown in SEQ ID NO. 1-19. Whether the object to be detected contains at least one of Cronobacter sakazakii, Cronobacter malonate, Cronobacter zukii or Cronobacter dubliniensis in the Cronobacter sakazakii can be obtained by detecting whether the object to be detected contains the corresponding sequence.

Further, the sequences SEQ ID NO.1 and SEQ ID NO.2 are used for identifying Cronobacter sakazakii; the sequence SEQ ID NO. 3-7 is used for identifying the malonates Cronobacter malonate; the sequence SEQ ID NO. 8-12 is used for identifying Cronobacter zurich; the sequence SEQ ID NO. 13-19 is used for identifying Cronobacter dublin.

Further, the invention also claims a primer group for identifying Cronobacter sakazakii, wherein the primer group is designed according to the nucleotide sequence shown in SEQ ID NO. 1-19.

The product corresponding to the primer group is all or part of the nucleotide sequence shown in SEQ ID NO. 1-19.

A nucleotide sequence is detected by referring to corresponding primer sets for PCR based on the corresponding nucleotide sequences, each primer set comprising a forward primer and a reverse primer. The amplification product of the primer group corresponds to all or part of the nucleotide sequence shown as SEQID NO. 1-19.

As a preferred embodiment of the invention, the nucleotide sequence of the primer group is shown as SEQ ID NO. 20-57 from 5 'to 3'; wherein:

SEQ ID NO.20 and SEQ ID NO.21 are primer sets corresponding to the sequence of SEQ ID NO. 1;

SEQ ID NO.22 and SEQ ID NO.23 are primer sets corresponding to the sequence of SEQ ID NO. 2;

SEQ ID NO.24 and SEQ ID NO.25 are primer sets corresponding to the sequence of SEQ ID NO. 3;

SEQ ID NO.26 and SEQ ID NO.27 are primer sets corresponding to the sequence of SEQ ID NO. 4;

SEQ ID NO.28 and SEQ ID NO.29 are primer sets corresponding to the sequence of SEQ ID NO. 5;

SEQ ID NO.30 and SEQ ID NO.31 are primer sets corresponding to the sequence of SEQ ID NO. 6;

SEQ ID NO.32 and SEQ ID NO.33 are primer sets corresponding to the sequence of SEQ ID NO. 7;

SEQ ID NO.34 and SEQ ID NO.35 are primer sets corresponding to the sequence of SEQ ID NO. 8;

SEQ ID NO.36 and SEQ ID NO.37 are primer sets corresponding to the sequence of SEQ ID NO. 9;

SEQ ID NO.38 and SEQ ID NO.39 are primer sets corresponding to the sequence of SEQ ID NO. 10;

SEQ ID NO.40 and SEQ ID NO.41 are primer sets corresponding to the sequence of SEQ ID NO. 11;

SEQ ID NO.42 and SEQ ID NO.43 are primer sets corresponding to the sequence of SEQ ID NO. 12;

SEQ ID NO.44 and SEQ ID NO.45 are primer sets corresponding to the sequence of SEQ ID NO. 13;

SEQ ID NO.46 and SEQ ID NO.47 are primer sets corresponding to the sequence of SEQ ID NO. 14;

SEQ ID NO.48 and SEQ ID NO.49 are primer sets corresponding to the sequence of SEQ ID NO. 15;

SEQ ID NO.50 and SEQ ID NO.51 are primer sets corresponding to the sequence of SEQ ID NO. 16;

SEQ ID NO.52 and SEQ ID NO.53 are primer sets corresponding to the sequence of SEQ ID NO. 17;

SEQ ID NO.54 and SEQ ID NO.55 are primer sets corresponding to the sequence of SEQ ID NO. 18;

SEQ ID NO.56 and SEQ ID NO.57 are primer sets corresponding to the sequence of SEQ ID NO. 19.

Further, the primer set can be used for identifying Cronobacter sakazakii, Cronobacter malonate, Cronobacter zuchenotii, or Cronobacter dubliniensis.

The present invention also provides a method for identifying Cronobacter sakazakii, comprising the steps of:

s1: carrying out PCR amplification on the DNA of a sample to be detected by using one of the primer groups;

s2: carrying out gel electrophoresis to detect the amplification product;

s3: observing whether the amplification product is in accordance with the expectation.

Generally, a PCR system contains only one set of primers; by setting a plurality of PCR systems and simultaneously amplifying the DNA of a single bacterium by using different primers, the detection efficiency is improved. The primer of the invention has good product specificity, and whether the Cronobacter sakazakii exists can be judged by observing whether the amplification product is at the expected position.

In a preferred embodiment of the present invention, the PCR amplification system in S1 includes 2 × PCR Mix, template DNA, a primer set, and sterilized double distilled water.

As a preferred embodiment of the present invention, the PCR amplification system is 2 XPCR Mix 12.5. mu.L, template DNA100ng, 10. mu.M primers are 1. mu.L each, and sterilized double distilled water is added to make up the volume to 25. mu.L.

In a preferred embodiment of the present invention, the PCR amplification procedure in S1 is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 50-62 ℃ for 30 s; extension at 72 ℃ for 30 s; 35 cycles of denaturation, annealing and extension are carried out; finally, extension is carried out for 10min at 72 ℃.

The invention discloses specific molecular targets for identifying 4 species (Cronobacter sakazakii, Cronobacter malonate, Cronobacter zuchenii and Cronobacter dubliniensis) in Cronobacter, and provides a related primer group and a corresponding PCR detection method. Compared with the prior art, the method can detect the Cronobacter species level without physiological and biochemical identification, and has the advantages of short detection time, low cost, simple operation and strong specificity; and the probability of false positive or false negative is reduced, the detection result is more accurate, the result judgment is simpler, and the practicability is stronger.

Drawings

FIG. 1 shows the results of the electrophoresis of the PCR detection using the primer set 1 in example 3.

FIG. 2 shows the results of the electrophoresis of the PCR detection using the primer set 2 in example 3.

FIG. 3 shows the results of the electrophoresis of the PCR detection using the primer set 3 in example 4.

FIG. 4 shows the results of the electrophoresis of the PCR detection using the primer set 4 in example 4.

FIG. 5 shows the results of the electrophoresis of the PCR detection using the primer set 5 in example 4.

FIG. 6 shows the results of the electrophoresis of the PCR detection using the primer set 6 in example 4.

FIG. 7 shows the results of the electrophoresis of the PCR detection using the primer set 7 in example 4.

FIG. 8 shows the results of the electrophoresis in the PCR detection using the primer set 8 in example 5.

FIG. 9 shows the results of the electrophoresis in the PCR detection using the primer set 9 in example 5.

FIG. 10 shows the results of the electrophoresis in the PCR detection using the primer set 10 in example 5.

FIG. 11 shows the results of the electrophoresis in the PCR detection using the primer set 11 in example 5.

FIG. 12 shows the results of the electrophoresis of the PCR detection using the primer set 12 in example 5.

FIG. 13 shows the results of the electrophoresis in the PCR detection using the primer set 13 in example 6.

FIG. 14 shows the results of the electrophoresis of the PCR detection using the primer set 14 in example 6.

FIG. 15 shows the results of the electrophoresis in the PCR detection using the primer set 15 in example 6.

FIG. 16 shows the results of the electrophoresis of the PCR detection using the primer set 16 in example 6.

FIG. 17 shows the results of the electrophoresis in the PCR detection using the primer set 17 in example 6.

FIG. 18 shows the results of the electrophoresis in the PCR detection using the primer set 18 in example 6.

FIG. 19 shows the results of the electrophoresis in the PCR detection using the primer set 19 in example 6.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

Example 1 excavation of novel molecular targets specific for different species within Cronobacter

Performing bioinformatics analysis according to a GenBank database and a Cronobacter sakazakii whole genome DNA sequence self-tested by the team; screening to obtain specific gene fragments of 4 species of Cronobacter sakazakii (Cronobacter sakazakii, Cronobacter malonate, Cronobacter zurich and Cronobacter dubliniensis), wherein the nucleotide sequences of the gene fragments are shown as SEQ ID NO. 1-19.

Wherein, the sequences SEQIDNO.1 and SEQIDNO.2 are used for identifying Cronobacter sakazakii; the sequence SEQID NO. 3-7 is used for identifying the malonates Cronobacter malonate; the sequence SEQ ID NO. 8-12 is used for identifying Cronobacter zurich; the sequence SEQ ID NO. 13-19 is used for identifying Cronobacter dublin.

Example 2 Rapid detection method of Cronobacter sakazakii

1) Primer design

Specific PCR amplification primer sets (including forward primers and reverse primers) were designed according to the sequences of SEQ ID Nos. 1-19 in example 1, and the sequences of the primer sets are shown in Table 1 below.

TABLE 1 specific PCR detection primer set

2) The method for identifying the Cronobacter comprises the following steps:

s1DNA template preparation: respectively culturing the strains to be detected in an LB liquid culture medium in an enrichment manner, and respectively extracting bacterial genome DNA of the strains to be detected by using a commercialized bacterial genome DNA extraction kit to serve as templates to be detected;

s2, PCR amplification: one of the primer groups 1-19 is used for carrying out PCR amplification on the DNA of a sample to be detected

① PCR detection System:

wherein:

when the template DNA is used for identifying the presence or absence of cronobacter sakazakii, the primer is a primer in the primer set 1 or 2;

when the template DNA is used for identifying whether the malonate Cronobacter malonate exists, the primer is any one of primer groups 3-7;

when the template DNA is used for identifying whether Cronobacter huicuensis exists, the primer is any one of the primer groups 8-12;

when the template DNA is used for identifying whether the Cronobacter dublin exists, the primer is any one of the primer groups 13-19;

② PCR amplification procedure:

wherein:

the primers in the primer groups 1-19 are used, and the annealing temperature is 50-62 ℃.

S3: taking the PCR amplification product to carry out gel electrophoresis;

s4: and observing whether a single amplification band exists at the position of each primer group corresponding to the size of the product. If present, indicating that the sample contains the corresponding Cronobacter sakazakii; if no corresponding single amplification band is present, the sample does not contain the corresponding Cronobacter sakazakii.

Example 3 evaluation of specificity of PCR detection method for Cronobacter sakazakii

PCR was carried out using 113 strains of Cronobacter sakazakii, 32 strains of other Cronobacters (including Cronobacter malonate, Cronobacter zuchenensis, etc.) and 18 strains of non-Cronobacter (including Escherichia coli, Shigella, etc.) according to the method of example 2. Wherein, the S1DNA template is prepared by respectively extracting the genome DNA of each bacterium; in the S2PCR amplification, the primers used are the primers in the primer set 1 or 2. A control group is set, and the template of the control group is an aqueous solution without genome.

The results of the PCR amplification product gel are shown in fig. 1 and 2 (fig. 1 shows the amplification product corresponding to the primer set 1, and fig. 2 shows the amplification product corresponding to the primer set 2), where (a) shows the detection result of 113 strains of cronobacter sakazakii in this example, (b) shows the detection result of 32 other cronobacters sakazakii in this example, and (c) shows the detection result of 18 strains of non-cronobacters sakazakii in this example. M is markerDL 2000 (2000 bp, 1000bp, 750bp, 500bp, 250bp and 100bp of 6 fragments from top to bottom in sequence), and C is a control group.

The strains used and the results of the tests are shown in table 2 below; in the table, "+" indicates positive and "-" indicates negative in the test result column.

TABLE 2 test results of evaluation of detection specificity of Cronobacter sakazakii of the present invention

Strain name	Type (B)	Strain numbering	Number of strains	Results
					C.sakazakii	Cronobacter sakazakii	ATCC29544		1	+
C.sakazakii	Cronobacter sakazakii	Food source isolate	112	+
					C.malonaticus	Other Cronobacter sakazakii	Food source isolate	10	-
C.dublinensis	Other CrohnNonobacterium sp	Food source isolate	10	-
					C.turicensis	Other Cronobacter sakazakii	Food source isolate	10	-
C.muytjensii	Other Cronobacter sakazakii	Food source isolate	2	-
					P.aeruginosa	non-Cronobacter sakazakii	ATCC 15442	1	-
P.vulgaris	non-Cronobacter sakazakii	CMCC(B)49027	1	-
					E.cloacae	non-Cronobacter sakazakii	CMCC(B)45301	1	-
E.aerogenes	non-Cronobacter sakazakii	ATCC13048	1	-
					S.marcescens	non-Cronobacter sakazakii	CMCC41002	1	-
P.mirabilis	non-Cronobacter sakazakii	CMCC49005	1	-
					S.flexneri	non-Cronobacter sakazakii	ATCC12022	1	-
A.nitrate	non-Cronobacter sakazakii	NICPBP25001	1	-
					A.baumannii	non-Cronobacter sakazakii	ATCC19606	1	-
S.aureus	non-Cronobacter sakazakii	ATCC25923	1	-
					L.monocytogenes	non-Cronobacter sakazakii	ATCC19115	1	-
Y.enterocolitica	non-Cronobacter sakazakii	CMCC52204	1	-
					E.coli	non-Cronobacter sakazakii	ATCC25922	1	-
S.enteritidis	non-Cronobacter sakazakii	CMCC50335	1	-
					S.sonnei	non-Cronobacter sakazakii	CMCC(B)51592-G2	1	-
B.subtilis	non-Cronobacter sakazakii	ATCC6633	1	-
					B.cereus	non-Cronobacter sakazakii	ATCC14579	1	-
B.thuringiensis	non-Cronobacter sakazakii	ATCC10792	1	-

From FIGS. 1, 2 and Table 2, it can be seen that only Cronobacter sakazakii showed specific amplification bands in all of the detection results of the 2 primer sets, and no specific bands were observed in any of the other Cronobacter sakazakii strains and the non-Cronobacter sakazakii strains, indicating that the method of the present invention has high specificity.

Example 4 evaluation of specificity of the method for PCR detection of Cronobacter malonate

PCR was carried out using 91 strains of Cronobacter malonate, 32 strains of other Cronobacters (including Cronobacter sakazakii, Cronobacter zurich, etc.) and 18 strains of non-Cronobacter (including Escherichia coli, Shigella, etc.) according to the method of example 2. Wherein, the S1DNA template is prepared by respectively extracting the genome DNA of each bacterium; and S2, during PCR amplification, the used primers are primers of any one of the primer groups 3-7. A control group is set, and the template of the control group is an aqueous solution without genome.

Gel results of PCR amplification products corresponding to the primer groups 3-7 are shown in FIGS. 3-7; wherein (d) is the detection result of 91 strains of Cronobacter malonate in the example, (e) is the detection result of 32 strains of other Cronobacters in the example, (f) is the detection result of 18 strains of non-Cronobacter in the example, markerDL 20000, and C is a control group.

The strains used and the results of the tests are shown in table 3 below; in the table, "+" indicates positive and "-" indicates negative in the test result column.

TABLE 3 evaluation test results of the detection specificity of Cronobacter malonate according to the present invention

As can be seen from FIGS. 3 to 7 and Table 3, the detection results of 5 primer sets showed only specific amplification bands of the malonat Cronobacter, and no specific bands of other Cronobacter and non-Cronobacter strains, indicating that the method of the present invention has high specificity.

Example 5 evaluation of specificity of PCR detection method for Cronobacter zurich

14 Cronobacter zuchenkianus strains, 32 other Cronobacters strains (including Cronobacter sakazakii, Cronobacter malonate, etc.) and 18 non-Cronobacter strains (including Escherichia coli, Shigella, etc.) were subjected to PCR detection in accordance with the method of example 2. Wherein, the S1DNA template is prepared by respectively extracting the genome DNA of each bacterium; and in S2PCR amplification, the used primers are primers of any one of the primer groups 8-12. A control group is set, and the template of the control group is an aqueous solution without genome.

Gel results of PCR amplification products corresponding to the primer groups 8-12 are shown in FIGS. 8-12; in this example, (g) shows the results of detection of 14 strains of Cronobacter zucheniformis in this example, (h) shows the results of detection of 32 other Cronobacters in this example, and (i) shows the results of detection of 18 strains of non-Cronobacters in this example. M is markerDL 2000, C is control group.

The strains used and the results of the tests are shown in table 4 below; in the table, "+" indicates positive and "-" indicates negative in the test result column.

TABLE 4 test results of the test for evaluating the detection specificity of Cronobacter zurich of the present invention

Strain name	Type (B)	Strain numbering	Number of strains	Results
					C.turicensis	Cronobacter zurich Li	Food source isolate	14	+
C.sakazakii	Other Cronobacter sakazakii	Food source isolate	10	-
					C.malonaticus	Other Cronobacter sakazakii	Food source isolate	10	-
C.dublinensis	Other Cronobacter sakazakii	Food source isolate	10	-
					C.muytjensii	Other Cronobacter sakazakii	Food source isolate	2	-
P.aeruginosa	non-Cronobacter sakazakii	ATCC 15442	1	-
					P.vulgaris	non-Cronobacter sakazakii	CMCC(B)49027	1	-
E.cloacae	non-Cronobacter sakazakii	CMCC(B)45301	1	-
					E.aerogenes	non-Cronobacter sakazakii	ATCC13048	1	-
S.marcescens	non-Cronobacter sakazakii	CMCC41002	1	-
					P.mirabilis	non-Cronobacter sakazakii	CMCC49005	1	-
S.flexneri	non-Cronobacter sakazakii	ATCC12022	1	-
					A.nitrate	non-Cronobacter sakazakii	NICPBP25001	1	-
A.baumannii	non-Cronobacter sakazakii	ATCC19606	1	-
					S.aureus	non-Cronobacter sakazakii	ATCC25923	1	-
L.monocytogenes	non-Cronobacter sakazakii	ATCC19115	1	-
					Y.enterocolitica	non-Cronobacter sakazakii	CMCC52204	1	-
E.coli	non-Cronobacter sakazakii	ATCC25922	1	-
					S.enteritidis	non-Cronobacter sakazakii	CMCC50335	1	-
S.sonnei	non-Cronobacter sakazakii	CMCC(B)51592-G2	1	-
					B.subtilis	non-Cronobacter sakazakii	ATCC6633	1	-
B.cereus	non-Cronobacter sakazakii	ATCC14579	1	-
					B.thuringiensis	non-Cronobacter sakazakii	ATCC10792	1	-

As can be seen from FIGS. 8 to 12 and Table 4, only Cronobacter zuchenensis showed specific amplification bands in all of the detection results of the 5 primer sets, and no specific bands were observed in all of the other Cronobacter sachenensis and non-Cronobacter sachenensis strains, indicating that the method of the present invention has high specificity.

Example 6 evaluation of specificity of PCR detection method for Cronobacter zurich

PCR was carried out using 82 Cronobacter dubliniensis, 32 other Cronobacter sakazakii (including Cronobacter sakazakii, Cronobacter malonate, etc.) and 18 non-Cronobacter sakazakii (including Escherichia coli, Shigella, etc.) according to the method of example 2. Wherein, the S1DNA template is prepared by respectively extracting the genome DNA of each bacterium; and in S2PCR amplification, the used primers are primers in any one of the primer groups 13-19. A control group is set, and the template of the control group is an aqueous solution without genome.

Gel results of PCR amplification products corresponding to the primer groups 13-19 are shown in FIGS. 13-19; wherein (j) is the detection result of 82 Cronobacter dubliniensis in this example, (k) is the detection result of 32 other Cronobacters in this example, and (l) is the detection result of 18 non-Cronobacter sakazakii in this example. M is marker DL 2000, C is control group.

The strains used and the results of the tests are shown in table 5 below; in the table, "+" indicates positive and "-" indicates negative in the test result column.

TABLE 5 test results of the test for evaluating the detection specificity of Cronobacter dublin of the present invention

As can be seen from FIGS. 13 to 19 and Table 5, the detection results of 5 primer sets showed specific amplification bands only for Cronobacter dubliniensis, and no specific bands were observed for other Cronobacter sakazakii and non-Cronobacter sakazakii strains, indicating that the method of the present invention has high specificity.

Example 7 detection of suspected Cronobacter sakazakii Strain

The suspected Cronobacter sakazakii strain isolated from 736 strains of food samples collected from supermarkets and commercial markets in Guangdong province was detected by PCR detection method in example 2, and the sample treatment and isolation of the suspected strain were performed according to the national standard method. Wherein, the S1DNA template is prepared by respectively extracting the genome DNA of each bacterium; and in S2PCR amplification, the used primers are primers of any one of the primer groups 1-19. The results are shown in Table 6.

TABLE 6 detection results of suspected Cronobacter sakazakii strains

As can be seen from Table 6, the numbers of strains detected by the method of the present invention for the target Cronobacter sakazakii were as follows: cronobacter sakazakii 482 strain, Cronobacter malonate 155 strain, Cronobacter zurich 11 strain, Cronobacter dublin 84 strain, Cronobacter mokinsonii, Cronobacter comforti, and Cronobacter ewingii were not detected; through 16s rDNA identification, the identification results of the cronobacter sakazakii, the cronobacter malonate, the cronobacter zurich and the cronobacter dublin are consistent, and other suspected strains are identified as other cronobacter species.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> institute of microorganisms of Guangdong province (center for analysis and detection of microorganisms of Guangdong province), Guangdong Huaqiao Biotech Co., Ltd

<120> specific novel molecular target of 4 species in Cronobacter and rapid detection method thereof

<130>2019

<160>57

<170>PatentIn version 3.3

<210>1

<211>513

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<213>Cronobacter sakazakii

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agcggcaacg aagtgtccgg aatgccgggc gtttacgaaa caggcgtgga aggcatcggc 300

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cgtaatacac cgctgggcta tgtctccagc gatggcaaca atacgttcga cttcaccgtg 420

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agcagctttg aggtcaacgc cacctgcaac gatccggtac aagtcggagt gaaagtctcc 720

agcgccaatg gctacgcatc caccgagcca tccgtgatta atctcacttc cgaacccggc 780

gtcgccagcg gcataggcgt tcagatgacg ctttttgggc aaaacatcga ttttgaccat 840

tacacctttg ttgcctcatt gaggccgaac tcaacgctca atatcccgtt tgctgttcag 900

tactatcaaa ctgccgacac cgtgacgccg ggcgttgcaa acgccgtcgc gaccattacc 960

gtgtcttacc ggtag 975

<210>3

<211>510

<212>DNA

<213>Cronobacter malonaticus

<400>3

atgaaacaaa tactgatact gatgatcctt accatcaccg ctgcgcaggc actggccgac 60

gatattcatc tgcaaattaa cggtgccatt atggcagcgt cctgtgaagt ggaaggtggc 120

agtaaaaaca aaacggtaga aatgggtgag gcgctggcca gcgacttttc acaaccccac 180

gactgggggc cgagagtgcc ttttgaactg agcgtgatca actgccctgc aacgatcaca 240

gccgttgagg ctgcctttag cggagtaaga gatccgcatg aaaataacgc ttatttaaat 300

aatggtacag gacgcgggct ggcgttacag attattgaaa gcaacagcgg aatgtatatg 360

ttgccagacg gtgaacccgc cactgcgccc gtcgacccca cgacgcacag cgccacctgg 420

gactttagcg cacggtacat ccgcaccacc gacgctttcg caccaggctc attcgatacg 480

gcggtccagg tgacgtttac ttaccggtga 510

<210>4

<211>471

<212>DNA

<213>Cronobacter malonaticus

<400>4

atgtccacct cggatatcat tgttgatggt gccattacct ccggagatgg cgcatgtacc 60

gtcttgatgg ataaagatgt ggttaacctg ttggccgaca aaactaccat tgtccctcag 120

agcaagagga aggccgatca ttttgacggt acgttttccg tttctatgac aggggatgag 180

atctgtatcc ggcgtattca ggaaggcaaa attgctgtcc gctttacagg gcctgccgat 240

gaggttgaag gcagcgtttt tgctaataca gcgaccggtg aaaatgcggc taaaggcgtt 300

ggggttggcc tgtttttaag tagccttgga cgaatcgcga ttaatcaagg ggctattcct 360

gttacaccta aacctttgag tctgagtgcg caagtggttg gccttaccgg tcaggaagtt 420

gtcgaaggta atgtccaggc gtccattaca gtagaagttg ttcgcctgta a 471

<210>5

<211>678

<212>DNA

<213>Cronobacter malonaticus

<400>5

atgaaacctg taattattgc cctgttaggc gtcctttctg tcggcgcagc tcaggcgagt 60

gtggtcgtag gcggcacccg cgtggtattt gatggtcagc aaaaagcagc ctcattatcg 120

gtgcagaata aagataaaac agcggatctt gtgcagtcat ggatttcgcc cgtcgacgcc 180

acctcaggcg caaaagacac catgattatc accccgccac tgtttcgtct ggatgctggt 240

gaaaaagcgt ctgtgcgtat tgtacgttcc ggtaagcctt tgcccgaaga tcgcgaatcg 300

atgttttggc tgaatgtgaa aggtattccg gcaatggaca acgcgccagc caaaaatgaa 360

ctgcaaatcg cgattaattc gcggataaaa ttaatttacc gcccggtaag cttgcatggc 420

agcgtacctg aatcggcaac ggataaactg agctggagca ccgaaggcaa tatgctgaaa 480

gtaaataatc cgacgccctt ttacatgaac ttctctacgg tcatggtgaa taacacgccg 540

gttaaagacg ttacatttgt ggcaccgttt tccagcacta cctttgcaat gcctaaagca 600

caaaatcatg ccgatgttaa atggcacata attaacgatt ttggcatggc tggcagcgaa 660

cacaacgcac atttctaa 678

<210>6

<211>351

<212>DNA

<213>Cronobacter malonaticus

<400>6

atgttaatga gtacgctaat taacgccgta ggccaggcgc tggcttcacc acagtttcaa 60

aaaagtgcct tgaaaatcgt cgcggacgag tttcttggtc tcctgagaca gggcctgaac 120

gatcgtgaaa cctttattaa caccagcgcg gagattgccg ctcaggcggt acttgatcgc 180

tcgcaggggt taattagtga agaggatatg gcagcgctgc tgaataaaca gaaaacgatc 240

gcgcagatcc atgccaacag cagtgagata gcgttacgca cgcgcattca atcgatcact 300

atgcgtcttc tcgatctggc agccagtgct atagcatgcg gactgaagta a 351

<210>7

<211>468

<212>DNA

<213>Cronobacter malonaticus

<400>7

atggtcgcga tatggatctc agcatatgtg ctgttaaacg cagggctcgt gattaaagat 60

attcgtgatg gtctgctacc tgaccgtctc acctgccctc tcctgtggat gggtctttct 120

taccatctca tatttgcgcc acagcatctg gcggatgccg tcgccggggc gttggtcgga 180

taccttctat tatgtttttt ctactgggct tatcgttttc tgcgtggtta tgaagggctg 240

ggatatggcg atgtgaaatt cttaggtgcc ctgggcgcct ggcacggttg gcagttactt 300

agtctgttgc taatcattgc atctgtaatg ggtcttatag ctgcgtttgt tgtctgtcag 360

ataaaaggca aagctccctt acgtaaaacc ccgctacctt ttgggccatg tctggcggca 420

gcggggctga tatgcagcgt ctttacattc caggtagcaa atctttga 468

<210>8

<211>885

<212>DNA

<213>Cronobacter turicensis

<400>8

attgtcagtg tgatttttct gacaacgttt tccgctaagg cgcaagacgt gcctcatgtg 60

aaatatcttt tctcgttcaa atccagtaaa agcacctgcc ttctgcgggc taatgatctg 120

cccgcgtttg acaataccac ggacgatgac ggcacgatcg ccgcaggctt taatatgacc 180

gcctttctgg aaaacggtaa taacgatatt gagttattaa tggggccgca ggatgtgaaa 240

caaccgcaga cgctgtgggc tgactccgca tgccaggtaa ccatctccga tgataccact 300

gacaacagcg ttaaactggc cgactacagc ctgacggtga atgataaaaa agagataagc 360

gcggtcaata cggttattta tgcgaccggt acgaaaacgt ttgaaggtta caccccatca 420

gatgatgatt acgggttata taatatgaag ggggttatta cacttgaccg tctgcctgag 480

tggtcatggg tgaatgcgac gccggtaacg gagaacgatc tgccgaaaat tcgtaaggct 540

tatgaagata tctggatgca gataaaacgc cgcgatgtgg aaggcctgaa acgcacagcg 600

aaaatttcga atgaagagat ggcccaggcg gaaggcgcca cgccagatat tattttcctt 660

tccactgatt ttccacaaca tgtgagtgac ccacaactga ctccggtgga cattgaatgg 720

caagactatc gcctgatgcg ttatcgcggt gggcgtcttt tccgactggc tgccggattc 780

ttccagaatt ctccgctgat gtttaaaaat agcgaagggg aagtggtctt cgtatacaac 840

ccttattttt ccattatcga tggtaatgtc gttttggttc gctaa 885

<210>9

<211>417

<212>DNA

<213>Cronobacter turicensis

<400>9

atgaacacca ggacactgct tgtaggtttt tttctgtttg ctggctcgct gaataacgcg 60

atagcagaca actgcagggc caatatttat ggcggagagg attgccgcta tgacaatgga 120

accacatcaa gcagcagagc caacattttc ggcgggcaga acaccaaata tagcgacggc 180

agaagctcaa gcagccgtcc caatattttc ggcggagagg atacccgaaa cagcgatggc 240

agctcttcaa gtagccgcgc caatatcttt gatggtcagg aaacgcagta cagcgacggc 300

cgctcttcca gcagccgcgc caatatcttc gacggcctgg acacgcaata cagcgacggc 360

cgctcttcca gcagccgtgc gaatatcttc gacagccagg acaccataaa taattaa 417

<210>10

<211>414

<212>DNA

<213>Cronobacter turicensis

<400>10

atgagcgccg ccctgacgct tcccgcgcgg ctcgatgcgc tgggcccgct cgccgacgcg 60

ctggcgcagt ttatggcgtc gctgccggtg gatgacgact ggcgtttcgc gttcgattta 120

gcggtgtgcg aagcggcggc aaacgtcatc cgtcatgcgc tggaagagac gccgacgcgt 180

gagtttaccg ttgagtttca gcacgacgac gcgagcgcaa gcgtgacgtt caccgatgac 240

ggtaacgcta ttcccgatga caaactggat gccgcgcgcg attgcgcgca ggacgacgac 300

acggcgctga tgagtgaagg cgggcgaggg ctgatgctga ttttcgcctg tgtcgatgaa 360

gtggagtatc acgcgggcgg ggttaaccgc ctgacgctga gcaagcggct ttaa 414

<210>11

<211>543

<212>DNA

<213>Cronobacter turicensis

<400>11

atggcgtcga cagaacaggg agccacaatg acctttcgta tcagaaagac cgtaaccgca 60

gatattggct gtcttcaagc cattgagcgt tcggcggaca gcgcatttga gacgatcccg 120

acgctcgcct gggtggcgag cgatggcgtc cagcctgagg atttacatca ccggctttgc 180

gagcagggat attctgcggt cgtggtgaat gaggatgaca ttcccgtcgg gtttatcaac 240

ggggaatata ccgccgacgc gctgcatatt ctgggcgtgg cggtgatgcg cgactgtcag 300

ggaatggggc ttgggaaaat gctgatggcc ggggccattc atcacgcacg ggaaaaacag 360

ctcgccgcgc tgacgctgac gacgttccga aacgtgccgt ggaatcagcc gttttatgcg 420

cgtctgggtt tccacgtgat ttcagacgca gagatgccgg aacgtctggc tcgcctgctt 480

gatgaagaag ccgcgcacgg ctttgcgcgc gccgggcgtt gcgccatggc gttacggctt 540

tag 543

<210>12

<211>336

<212>DNA

<213>Cronobacter turicensis

<400>12

atgcactggc gggtggttga ttctgtagta agcaccgaca ccaattctgt atttagcctg 60

atttcctcgc aacgcgcttt caaacttatc ctgtggtaca aagccacgtt ttatttatcg 120

ccaggcgata agctcactct taatggcgag gcgatcgtgg tgaatgatca ccctgttcag 180

ataaccctgt accggaccac gttttataat ccccgtttct ggcaaaccat cgttcacagc 240

aatacccact gcgcaggcaa ccatcagcga agcgtgagcc gatgtattta ccggcgtaaa 300

tgtaaactcc tctactgtcc gttccagcga cactga 336

<210>13

<211>789

<212>DNA

<213>Cronobacter dublinensis

<400>13

atgtccaccg gcgatttatc cagaatgggt gccgcagcag tggtgttgaa tacccgcgac 60

agccatcatc actatattga aaaatgccct gttggcgacg tggaatacca tttttatcat 120

gacgctgcgg cggcgcttaa tcaggcgggc attgcgacgc cagcgctgtt atccgcggat 180

gcgacccggc gcgcattgag actggagtat attcctcacc ccgttgacca ggctgctttc 240

gccagtgatg acgccctcgc catgctggcg cggctgcatc gttaccctgc ccattctgca 300

tggcgctacc acacgcacgc ctggtcagag gccgcgcttg aaaaatcgct gtcgctcctt 360

gcgttgccag caaacagtgc gcagcagcta cggcgctttc agcagcatag tgacgagttg 420

tttggctacc tgagtctggt gtccggtgac agcaatgccg gcaactgggg aaggcgtgaa 480

aacggtgatg tggtgctttt cgactgggag cggtttggaa aaggcagtcc tgctatcgat 540

ctggctccgc tgataaaggg aatggggacg aagcaagcaa tgctgagcct ggctgaacgc 600

tacggccaga tagcgcgtca tcacaatcca ccggcattag ccagagagat agccattgcc 660

aaagcctgga ttgtcaccga agtcatcgtg cttcttgatg agcggcaaaa ggcagagttt 720

ccgctgtatc gcgactggta cagagcacat cttcccgact ggttagcaac cagcatcacc 780

atgctgtga 789

<210>14

<211>468

<212>DNA

<213>Cronobacter dublinensis

<400>14

atggttacgg tatggatgtt tggatacgta ttgctaaacg cagggctcgt gctcaaggat 60

ctgcgccatg gtctgttacc cgacagtctg acctgccccc tactctggct gggcctgtcg 120

taccatctta tttcccggcc agacgcactc tccgatgcgg tagcaggcgc gcttctcggt 180

tatctctcat tggcgctgct ttactgggct taccgatggt tgcggggagt tgaagggctg 240

ggctatggcg atgtgaaata tgttgccgcg cttagcgcct ggcatggatg gcagatgctg 300

ggtctgttgc ttatgactgc cgctctactg ggccttgccg cgtcttttgt gatgtatcgg 360

aaaagagagc cggctctttt acgaaaaacc ccgctgcctt ttggtccatt tctggcagca 420

gcagggttgg tatgcagtat ctctaccttc cagatactga aggtatag 468

<210>15

<211>1086

<212>DNA

<213>Cronobacter dublinensis

<400>15

atgtccatac gcacactgat cctctctttt acgctacttc tgagcggcat aacccaggcc 60

gacaccgctt ttcgccagtt gcatctcgat gaagataagg ctcgcccgct cgatgtcgcc 120

gtctggtatc ccacggcgca aacgggcaaa actgaaacgg tgggcgacaa cccggttttt 180

gttggcacgc cagcattacg taacgcacag cccgctggtg gcgcgcatcc gttactactt 240

ctctcgcacg ggtatggcgg caactggcgc aacctcaact ggctggcgca acgcatggcg 300

gcgcaggggt atattgtcgc ggcacccgat catccgggaa caaccagccg caataaagcg 360

cagcaggatg cctggcagct ctggcagcgc ccgcgcgatc tgcgccgcgt gatgaatagg 420

cttatcgaca accccgccat tgctggcgat gtggataccg gtcgcattgc cgcgctcggc 480

cattctctgg gcggctggac ggtaatggag ctggcaggcg cgcgttttga tgccgggcac 540

tttcaacggg attgtaaaaa ccatgccgta ctgtcgagtt gtaagctcat ccctgcgctg 600

ggtatcgatc gcccggcggc atccggcccg ttaatggaaa gccagcgcga gccacgaatc 660

aacgcggtgg tctctctcga tttggggctg gcgcgcggtt ttacgcccgg gagcctcgct 720

cagctgaatg tgccggtgct tatcttgtcc gcgcaggccg acagcgacgc gctacccgcc 780

cggctggagt ctggctattt gcagcgttac attccggcgg cgaagcagcg ggcgcaaagc 840

gtcacgggcg cgacgcattt tagctttatg cagctctgca agcctggcgc gaaggcgctg 900

attgaggaac acgatccggg cgaaggcatt gtgtgtgacg atggcggctc gctaagccgg 960

gcagacattc atcagacgct tagcgacacg atcagcgctt ttctgcagca ggcgcttgat 1020

tatccgccgc tgcacgacga aacgccgttg ggctcatccc gctcaaccgc acaaactcac 1080

ggttga 1086

<210>16

<211>291

<212>DNA

<213>Cronobacter dublinensis

<400>16

atgaaggcac tatccatgag cccgattaga tctgaaactg caattccggt cgcgttgcct 60

gatgccgcgc tcagcgacta ttttctccgt gccggcgaaa gactggcgca ggaggccgcg 120

ctgctgggac ggatcgtcaa aagtatgtcg acggacgggg ctgtgctgac gcataaaacc 180

atcattcagc aactgattgt cgaactcgat ggcacgaaag atgtggtgac gggagaggtg 240

atccgcagga cgctggggat tgtggtcgat catacgctcg atgaccggta a 291

<210>17

<211>225

<212>DNA

<213>Cronobacter dublinensis

<400>17

atgggcattg ttaagccaga agacatggat gatatctacc gagtgatagg caaggcggtg 60

tcacgactga tagcatcagg aatacctgta gagaccgata atattatcgc ccagctcaaa 120

gattccgaag agcagacggt ggacggcacc cggcaaatat acgctgaggc gattcggtta 180

gtggcgagtg gcgtgggcgg cgacatgcaa caatggctgt cctga 225

<210>18

<211>468

<212>DNA

<213>Cronobacter dublinensis

<400>18

atgcagatgg aaaatcgtat tcgctcgctt cggctcgccc gcgcctggtc acaggagcaa 60

ctggccgaac tcgcctcggt cagcgtccgc accattcagc gcattgagaa aggcgagccg 120

ccgtcgctcg aaaccttaag cgcgctcgcc gccgtttttg aaacgcgtat tgaagcgctg 180

actggcgagc cgcttaatgc tgcaggccca ctggatgacg ccattacgct tgcacgccag 240

cggcttgatg aagaacgcct tttttaccgc agccttacca ctgcgcttat cgtttgcggg 300

gcgcttttcc tgttgaaccg ttataccgcg ccgcaaagcc actggtcact gtgggtcatt 360

gctatctggg gtgcgttatt actgatgaaa gggattcgct tgtttttact gcgtgagtgg 420

attgcgcaat ggcagcagcg tcggctgcaa aagttactgc gtaagtaa 468

<210>19

<211>399

<212>DNA

<213>Cronobacter dublinensis

<400>19

atgttgatgt acacgacgat tggaacccac gatccagacc gtatgatcgc gttttatgac 60

gcgattttta aggtgttagg cgtgtctcgt ctcccttcct ggaccgaagg ctgggcaacc 120

tggggagagc ccattgaaac cggcttcagt ttttgcattt gtgagccgta taaccagcag 180

aaggcaacgg cgggtaacgg cacgatgttt tcattccggg cggcaagtgc cgcgcaggtc 240

cgagaattcc atgccgccgg cctcaggcat ggcggaagtg atgaaggacg ccccggcata 300

cgagaagcct acgggccaga tttctatgtc gcttatctca gagacccgga cggacataag 360

ctcgcctgcg tatgttatcc ctttcatcct gaaacgtaa 399

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<400>20

gttactgctg ctgataatgt 20

<210>21

<211>20

<212>DNA

<213> Artificial sequence

<400>21

taagtaaacg tcacctgtac 20

<210>22

<211>20

<212>DNA

<213> Artificial sequence

<400>22

ccggtagcct gttctttatg 20

<210>23

<211>20

<212>DNA

<213> Artificial sequence

<400>23

ggtgtcggca gtttgatagt 20

<210>24

<211>20

<212>DNA

<213> Artificial sequence

<400>24

cactggccga cgatattcat 20

<210>25

<211>20

<212>DNA

<213> Artificial sequence

<400>25

ccggtaagta aacgtcacct 20

<210>26

<211>20

<212>DNA

<213> Artificial sequence

<400>26

gcgcatgtac cgtcttgatg 20

<210>27

<211>20

<212>DNA

<213> Artificial sequence

<400>27

tacaggcgaa caacttctac 20

<210>28

<211>20

<212>DNA

<213> Artificial sequence

<400>28

cccgcgtggt atttgatggt 20

<210>29

<211>20

<212>DNA

<213> Artificial sequence

<400>29

gccagccatg ccaaaatcgt 20

<210>30

<211>20

<212>DNA

<213> Artificial sequence

<400>30

attaacacca gcgcggagat 20

<210>31

<211>20

<212>DNA

<213> Artificial sequence

<400>31

agatcgagaa gacgcatagt 20

<210>32

<211>20

<212>DNA

<213> Artificial sequence

<400>32

cagggctcgt gattaaagat 20

<210>33

<211>20

<212>DNA

<213> Artificial sequence

<400>33

ggaatgtaaa gacgctgcat 20

<210>34

<211>20

<212>DNA

<213> Artificial sequence

<400>34

gtgcctcatg tgaaatatct 20

<210>35

<211>20

<212>DNA

<213> Artificial sequence

<400>35

ttagcgaacc aaaacgacat 20

<210>36

<211>20

<212>DNA

<213> Artificial sequence

<400>36

taacgcgata gcagacaact 20

<210>37

<211>20

<212>DNA

<213> Artificial sequence

<400>37

atttatggtg tcctggctgt 20

<210>38

<211>20

<212>DNA

<213> Artificial sequence

<400>38

cgtgagttta ccgttgagtt 20

<210>39

<211>20

<212>DNA

<213> Artificial sequence

<400>39

acacaggcga aaatcagcat 20

<210>40

<211>20

<212>DNA

<213> Artificial sequence

<400>40

acagaacagg gagccacaat 20

<210>41

<211>20

<212>DNA

<213> Artificial sequence

<400>41

acgcgcataa aacggctgat 20

<210>42

<211>20

<212>DNA

<213> Artificial sequence

<400>42

cgggtggttg attctgtagt 20

<210>43

<211>20

<212>DNA

<213> Artificial sequence

<400>43

ggaacggaca gtagaggagt 20

<210>44

<211>20

<212>DNA

<213> Artificial sequence

<400>44

accggcgatt tatccagaat 20

<210>45

<211>20

<212>DNA

<213> Artificial sequence

<400>45

gtcgggaaga tgtgctctgt 20

<210>46

<211>20

<212>DNA

<213> Artificial sequence

<400>46

tacggtatgg atgtttggat 20

<210>47

<211>20

<212>DNA

<213> Artificial sequence

<400>47

ggaaggtaga gatactgcat 20

<210>48

<211>20

<212>DNA

<213> Artificial sequence

<400>48

tccatacgca cactgatcct 20

<210>49

<211>20

<212>DNA

<213> Artificial sequence

<400>49

gtcgtgcagc ggcggataat 20

<210>50

<211>20

<212>DNA

<213> Artificial sequence

<400>50

atgaaggcac tatccatgag 20

<210>51

<211>20

<212>DNA

<213> Artificial sequence

<400>51

agcgtatgat cgaccacaat 20

<210>52

<211>20

<212>DNA

<213> Artificial sequence

<400>52

gcattgttaa gccagaagac 20

<210>53

<211>20

<212>DNA

<213> Artificial sequence

<400>53

cactaaccga atcgcctcag 20

<210>54

<211>20

<212>DNA

<213> Artificial sequence

<400>54

gaaaatcgta ttcgctcgct 20

<210>55

<211>20

<212>DNA

<213> Artificial sequence

<400>55

cagtaacttt tgcagccgac 20

<210>56

<211>20

<212>DNA

<213> Artificial sequence

<400>56

gttgatgtac acgacgattg 20

<210>57

<211>20

<212>DNA

<213> Artificial sequence

<400>57

caggatgaaa gggataacat 20

Claims (9)

1. The application of the nucleotide sequence in identifying the Cronobacter sakazakii is characterized in that the nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO. 19; the Cronobacter sakazakii is Cronobacter sakazakii, Cronobacter malonate, Cronobacter zuchenii, or Cronobacter dubliniensis.

2. The use according to claim 1, wherein the sequences of seq id No.1 and seq id No.2 are used to identify cronobacter sakazakii; the sequence SEQ ID NO. 3-7 is used for identifying the malonates Cronobacter malonate; the sequence SEQID NO. 8-12 is used for identifying Cronobacter zurich; the sequence SEQ ID NO. 13-19 is used for identifying Cronobacter dublin.

3. The primer set for identifying Cronobacter sakazakii, wherein the primer set is designed based on the sequence of claim 1.

4. The primer set according to claim 3, wherein the sequence of the primer set is represented by SEQ ID NO.20 to 57 from 5 'to 3'; wherein:

SEQ ID NO.20 and SEQ ID NO.21 are primer sets corresponding to the sequence of SEQ ID NO. 1;

SEQ ID NO.22 and SEQ ID NO.23 are primer sets corresponding to the sequence of SEQ ID NO. 2;

SEQ ID NO.24 and SEQ ID NO.25 are primer sets corresponding to the sequence of SEQ ID NO. 3;

SEQ ID NO.26 and SEQ ID NO.27 are primer sets corresponding to the sequence of SEQ ID NO. 4;

SEQ ID NO.28 and SEQ ID NO.29 are primer sets corresponding to the sequence of SEQ ID NO. 5;

SEQ ID NO.30 and SEQ ID NO.31 are primer sets corresponding to the sequence of SEQ ID NO. 6;

SEQ ID NO.32 and SEQ ID NO.33 are primer sets corresponding to the sequence of SEQ ID NO. 7;

SEQ ID NO.34 and SEQ ID NO.35 are primer sets corresponding to the sequence of SEQ ID NO. 8;

SEQ ID NO.36 and SEQ ID NO.37 are primer sets corresponding to the sequence of SEQ ID NO. 9;

SEQ ID NO.38 and SEQ ID NO.39 are primer sets corresponding to the sequence of SEQ ID NO. 10;

SEQ ID NO.40 and SEQ ID NO.41 are primer sets corresponding to the sequence of SEQ ID NO. 11;

SEQ ID NO.42 and SEQ ID NO.43 are primer sets corresponding to the sequence of SEQ ID NO. 12;

SEQ ID NO.44 and SEQ ID NO.45 are primer sets corresponding to the sequence of SEQ ID NO. 13;

SEQ ID NO.46 and SEQ ID NO.47 are primer sets corresponding to the sequence of SEQ ID NO. 14;

SEQ ID NO.48 and SEQ ID NO.49 are primer sets corresponding to the sequence of SEQ ID NO. 15;

SEQ ID NO.50 and SEQ ID NO.51 are primer sets corresponding to the sequence of SEQ ID NO. 16;

SEQ ID NO.52 and SEQ ID NO.53 are primer sets corresponding to the sequence of SEQ ID NO. 17;

SEQ ID NO.54 and SEQ ID NO.55 are primer sets corresponding to the sequence of SEQ ID NO. 18;

SEQ ID NO.56 and SEQ ID NO.57 are primer sets corresponding to the sequence of SEQ ID NO. 19.

5. Use of the primer set according to claim 3 or 4 for identifying Cronobacter sakazakii, Cronobacter malonate, Cronobacter zuchenotii, or Cronobacter dubliniensis.

6. A method for identifying crohn's disease, comprising the steps of:

s1: performing PCR amplification on a sample DNA to be detected by using one of the primer sets according to claim 3 or 4;

s2: carrying out gel electrophoresis to detect the amplification product;

s3: observing whether the amplification product is in accordance with the expectation.

7. The method of claim 5, wherein the PCR amplification system in S1 comprises 1 XPCR Mix, template DNA, a primer set and sterile double distilled water.

8. The method of claim 7, wherein the PCR amplification system is: 2 XPCR Mix12.5. mu.L, template DNA100ng, 10. mu.M primers 1. mu.L each, sterile double distilled water to make up the volume to 25. mu.L.

9. The method of claim 5, wherein the PCR amplification procedure in S1 is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 50-62 ℃ for 30 s; extension at 72 ℃ for 30 s; 35 cycles of denaturation, annealing and extension are carried out; finally, extension is carried out for 10min at 72 ℃.

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