KR20230068483A - A biomarker specific for Cronobacter sakazakii, a kit and a method for ditection Cronobacter sakazakii using the biomarker - Google Patents

Abstract

The present invention relates to a biomarker specific for Cronobacter sakazakii, a food poisoning-causing bacterium, and a detection kit for Cronobacter sakazakii using the biomarker and a method for detecting the same.

Description

A biomarker specific for Cronobacter sakazakii, a kit and a method for ditection Cronobacter sakazakii using the biomarker}

The present invention relates to a biomarker specific for Cronobacter sakazakii, a food poisoning-causing bacterium, and a detection kit for Cronobacter sakazakii using the biomarker and a method for detecting the same.

-Serra, Ja

n-Luchoro, Skovbjerg, et al., 2020; Kwon, Lee, Park, & Kim, 2021). Comparative genomics is the comparison of biological information of different species. Originally used to identify evolutionary characteristics of species, it has recently been applied to various fields such as public health, medical and food industries (Makarova, Slesarev, Wolf, Sorokin, Mirkin, Kunin et al., 2006). Publicly available whole-genome sequence data from various bacterial strains have been registered, and this information can be used in a variety of research fields (Makarova, Slesarev, Wolf, Sorokin, Mirkin, Koonin, et al., 2006). For these studies, pan genome analysis has been widely applied to identify a core genome shared by all variants and an accessory genome shared by one or more but not all variants (Tettelin, Riley, Cattuto, & Medini, 2008). The analyzed genomic data is further visualized to identify whether three or more core genomes are clearly divided or not. From these advantages, comparative genomics-based pan genome analysis has infinite potential for application in the food industry, especially in the development of prevention and treatment strategies for foodborne pathogens (Kim, Gu, Kim, & Lee, 2020). For example, it can be applied to the polymerase chain reaction (PCR) assay, which has been widely used for rapid detection of foodborne pathogens. Primer design can be the most important part of a PCR assay since specific targets of DNA are amplified during the PCR assay. In this regard, several recent attempts to develop primers based on pan genome analysis have been reported (Gonzales-Siles, Karlsson, Schmidt, Salv.

—Serra, Ja

n-Luchoro, Skovbjerg, et al., 2020; Kwon, Lee, Park, & Kim, 2021).

Cronobacter sakazakii is a Gram-negative, selective anaerobic, non-spore forming bacterium. Previously classified as Enterobacter sakazaki for a long time, the name was changed to Chronobacter sakazaki. The pathogen is highly resistant to osmotic pressure and can survive and proliferate for several years in a water-poor environment (0.3-0.85 aw) (Du, Wang, Dong, Li, & Wang, 2018). In addition, C. sakazakii can form biofilms on stainless or polyvinyl chloride (PVC) surfaces, making them more resistant to antimicrobial intervention (Ling, Forsyte, Wu, Ding, Zhang, & Zeng, 2020). In addition, cross-contamination of other materials, manufacturing facilities, packaging materials, and hands can transmit C. Sakazakii (Lou, Si, Yu, Qi, Liu, & Fang, 2014). In this regard, powdered infant formula (PIF) has long been considered a common vector of C. Sakazakii infection (Ha & Kang, 2014). PIF is pasteurized during processing, but minimal processing is preferred in view of high nutritional quality. In addition, some components vulnerable to heat are added after heat treatment, which can become a source of contamination of C. Sakazakii . In this regard, outbreaks related to Sakazaki infection have been reported in many countries from 1958 to the present (Henry & Fouladkhah, 2019). It is well known that neonates and infants are highly susceptible to C. sakazakii infection, resulting in a mortality rate of 30-80%. Neonatal meningitis and necrotizing enterocolitis are serious diseases caused by Sakazaki infection that cause death in some patients (Hunter & Bean, 2013). Therefore, it is important to rapidly and selectively detect Sakazakii in food samples containing PIF.

Various types of C. Sakazakii detection methods have been reported. Several enrichment broths can be used for the concentration step, including Enterobacteriaceae concentrate (EE), E. Sakazakii selection broth (ESSB), and modified lauryl sulfate broth (mLST) (Iversen & Forsyte, 2007). Then, selective media such as Druggan-Forsythe-Iversen Agar (DFI), R&F Agar (R&F Agar), and CCI (Chromogenic Cronobacter Isolation) can be used for differentiation (Iversen, Druggan, & Forsy, 2004). Concentration followed by cultivation in a selective medium along with biochemical tests such as API kits or Vitek assays have been commonly used, but these methods have been used to detect C. sakazakii and Enterobacter spp. Because of the similar biochemical properties between the two, labor is high as well as low selectivity. Originally, the US Food and Drug Administration (US FDA) proposed a method of using broth and selective agar media to detect Sakazakii in PIF, which took 5-7 days, but this was time consuming and labor intensive. In addition, due to the limitations of long detection period and low specificity, the US FDA revised the protocol to incorporate real-time PCR-based testing and chromogenic agar (US FDA, 2021). In the revised protocol, real-time PCR is used for confirmation as well as screening. Lampel & Chen (2009) confirmed that this new FDA method is applicable for high- and low-level pathogen detection. Therefore, real-time PCR assay has been widely used to detect C. sakazakii in PIF. As mentioned above, primer design is the most important part of PCR analysis, making detection sensitive and selective (Sun, Wang, Wang, Cao, He, Li, et al., 2012). However, primer design for C. sakazakii based on comparative genetics has not been reported to date.

Accordingly, the present inventors tried to develop primer sequences useful for detecting C. sakazakii in powdered infant formula (PIF) by applying comparative genomics.

In this regard, the primer sequence proposed by Seo & Bracket, 2005 was used to detect E. sakazakii in PIF. They designed a primer with a specific target sequence within the macromolecular synthesis (MMS) operon, and this sequence was registered as a Bacterial Analysis Manual (BAM) method by the US Food and Drug Administration (FDA). In a study by Seo & Bracket, 2005, the sensitivity and specificity of the primer sequence based on the MMS operon was confirmed in various strains, but the test for Chronobacter species was not performed. This is likely to have focused on Enterobacter species rather than Chronobacter species, as the target pathogen was originally classified as E. sakazakii (Wang, Zhu, Xu, & Zhou, 2012). Also, primers based on the MMS operon with an internal amplification control (IAC) were used for the detection of E. sakazakii in food samples. The sensitivity of the designed primer sequence was confirmed in various strains of C. sakazakii, and the selectivity of the primer was confirmed in several pathogens. However, it was not confirmed whether the primer sequences were present in closely related pathogens.

The main advantage of the comparative genomics approach is that it ensures the specificity of the primers in a proactive manner through large amounts of data. Typically, primers are designed to target specific genes specific to a pathogen and have been identified by testing various strains of the pathogen. For example, the 16S rRNA gene has been widely used as a target for primer sequences. However, Healy et al. (2009) reported that 16S rRNA gene sequencing did not distinguish C. sakazakii from its closely related strains. Therefore, comparative genomics-based primer design could be an alternative method, and recently several researchers investigated the applicability of comparative genomics to primer design for the detection of foodborne pathogens. Previously, Dr. Kwon, Lee, Park, and Kim (2021) revealed that Bacillus cereus and Bacillus subtilis could be simultaneously detected with high sensitivity and selectivity by applying a comparative design. Ye, Shang, Chen, Pang, Li, Xiang et al. (2021) reported that comparative genomics using pan genome analysis was an effective method for designing targets for PCR used for rapid detection of Salmonella serobacteria. Similarly, Shang, Ye, Wu, Pang, Shang, Wang et al. (2020) investigated novel specific targets of PCR serotypes of Salmonella based on pan genome analysis. Therefore, comparative genomes based on pan genome analysis will be widely used to design primer sequences for rapid and accurate detection of foodborne pathogens, and we are the first approach to design primers for C. sakazakii based on pan genome analysis .

Republic of Korea Patent Application Publication No. 10-2021-0041355 Korean Patent Application Publication No. 10-2009-0118407

Accordingly, the present invention intends to provide a kit for detecting Chronobacter sakazaki that can detect Chronobacter sakazaki with very high accuracy using a biomarker specific to Chronobacter sakazaki. In addition, by using the primer or kit of the present invention, it is intended to provide a method for accurately and conveniently identifying or isolating Chronobacter sakazaki bacteria from foods such as powdered milk susceptible to it.

One aspect of the present invention includes a primer capable of detecting Cronobacter sakazakii , wherein the primer is a forward primer composed of the nucleic acid sequence shown in SEQ ID NO: 1, and a reverse primer composed of the nucleic acid sequence shown in SEQ ID NO: 2 It relates to a primer set for detecting Chronobacter Sakazaki, characterized in that it includes a primer.

Another aspect of the present invention relates to a primer set for detecting Cronobacter sakazakii, which clones all or part of the type 1 ciliary protein ( fimp1 ) gene of a Cronobacter sakazakii strain.

Another aspect of the present invention relates to a polymerase chain reaction kit for detecting Chronobacter sakazaki, including the primer set described herein.

In another aspect of the present invention, a polymerase chain reaction is performed on a target sample using the primer set described in the present invention,

The present invention relates to a method for detecting a Chronobacter Sakazaki strain, which includes determining the presence or absence of the Chronobacter Sakazaki strain by checking the size of a product of the polymerase chain reaction or analyzing its nucleotide sequence.

The present invention relates to a primer set and a polymerase chain reaction kit for detecting Cronobacter sakazakii , and the primer set of the present invention has a high specificity for Cronobacter sakazakii compared to biomarkers used in other polymerase chain reaction kits, resulting in very high accuracy It can be detected with this, and also has the advantage of being able to detect only a very small amount of DNA concentration.

1 is a diagram showing the analysis of the pan genome of C. sakazakii .
Figure 2 is a diagram confirming that the target gene of C. sakazakii ATCC 29544 was well amplified using the primer and probe set of an embodiment of the present invention regardless of the initial inoculation level (threshold: 100 RFU).
Figure 3 is a standard curve of C. sakazakii ATCC 29544 at various thresholds of the present invention. 3A shows a relative fluorescence unit (RFU) threshold of 100, FIG. 3B a threshold of 150, and FIG. 3C a threshold of 200.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

Unless otherwise indicated, nucleic acids are written in 5′→3′ orientation from left to right. Numerical ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are incorporated herein by reference.

One aspect of the present invention includes a primer capable of detecting Cronobacter sakazakii , wherein the primer is a forward primer composed of the nucleic acid sequence shown in SEQ ID NO: 1, and a reverse primer composed of the nucleic acid sequence shown in SEQ ID NO: 2 It relates to a primer set for detecting Chronobacter Sakazaki, characterized in that it includes a primer.

SEQ ID NO: 1: GAD GGW AAT GCA GTT AAC A

SEQ ID NO: 2: TAC CGT TAG GCG TAA TCA CA

(D= A or G or T; W= A or T; Y= C or T)

In the present invention, comparative genomics was applied to design primer sequences for detecting C. sakazakii . Genomic information associated with several Chronobacter strains and Enterobacter strains was used in the application. In the analysis of the pan genome, C. sakazakii was fortunately identified as having a unique gene cluster clearly distinguishable from closely related species (see Fig. 1).

When selecting a gene cluster as a target for primer design, gene duplication, diversity, length, and similarity with other variants were mainly considered. In this way, the primers were designed to target fimp1 , and the primers and probes targeting fimp1 The sequence of is:

Oligonucleotide primer and probe sequences used in the present invention

The fimp1 has high specificity from closely related species and may also be a virulence factor. Also, because of its low diversity, it is suitable for beginners.

The primer set may further include a probe composed of the nucleic acid sequence of SEQ ID NO: 3:

SEQ ID NO: 3: ATG CCA ACG CCT AAT CCT GTC GCY (Where Y=C or Y)

When PCR analysis is performed using the above primer set and/or probe set, high sensitivity and selectivity for C. Sakazakii detection are exhibited.

Historically, C. Sakazakii (formerly E. sakazaki ) was named the yellow-pigmented Enterobacter cloacae , indicating that C. Sakazakii has very similar characteristics to E. cloacae (Farmer Iii, Asbury). , Hickman, Brenner, & Group, 1980). Biochemical tests that differentiate between these two species are limited. The primers of SEQ ID NO: 1 or 2 can selectively detect C. Sakazakii from strains of E. cloacae , thereby distinguishing between two similar species.

In addition, the primer of SEQ ID NO: 1 or 2 of the present invention can detect C. Sakazakii from Chronobacter strains including non-Sakazaki strains, showing high selectivity and specificity.

Another aspect of the present invention relates to a primer set for detecting Cronobacter sakazakii, which clones all or part of the type 1 ciliary protein ( fimp1 ) gene of a Cronobacter sakazakii strain. The type 1 ciliary protein ( fimp1 ) gene has the sequence of SEQ ID NO: 4. A primer set for cloning all or part of the type 1 ciliary protein ( fimp1 ) gene of the Cronobacter sakazakii strain may include the primers of SEQ ID NO: 1 and/or 2. The primer set of the above embodiment may further include a probe composed of the nucleic acid sequence of SEQ ID NO: 3. The probe may include a fluorescent material at the 5' end of the nucleotide sequence of SEQ ID NO: 3 and a quenching material at the 3' end. A probe refers to a nucleic acid fragment such as RNA or DNA corresponding to several to hundreds of bases capable of specific binding to a nucleic acid sequence, and is labeled so that the presence or absence of a specific mRNA can be confirmed. The probe may be produced in the form of a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or an oligonucleotide probe. Conditions for hybridizing the probe with the cDNA can be determined in a series of steps by an optimization procedure. For example, temperature, concentration of components, hybridization time, washing time, buffer components, etc. may vary depending on factors such as the length of the probe or the target nucleotide sequence.

The primer set of the present invention can be used in a conventional polymerase chain reaction. The polymerase chain reaction is a method selected from the group consisting of RT (real-time)-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blot, or DNA chip. It may be, and specifically, real-time PCR may be used.

The primer set of the present invention may further include a fluorescent, phosphorescent or radioactive material. Specifically, the material may include VIC, NED, FAM, BHQ ₁ or PET. When PCR is performed by labeling VIC, NED, FAM or PET at the 5'-end of the primer, the target sequence can be labeled with a detectable fluorescent labeling material. In addition, when a radioactive material is used, when a radioactive isotope such as ³² P or ³⁵ S is added to the PCR reaction solution during PCR, radioactivity may be incorporated into the amplification product.

Another aspect of the present invention relates to a kit for detecting Chronobacter Sakazaki strain, including the primer set for detecting Chronobacter Sakazaki strain of the present invention. The kit may include conventional components of a microorganism detection kit using the primer set and polymerase chain reaction of the present invention, such as a reaction buffer, a label, and Taq DNA polymerase, and may further include reagents necessary for the reaction. can include Reagents required for the polymerase chain reaction may include, for example, a dNTP mixture, a PCR buffer containing KCl, Tris-HCl, and MgCl ₂ , and water.

The kit may further include instructions. The guide is a printout explaining how to use the kit, eg, how to prepare the PCR buffer, reaction conditions, and the like. The guide may be provided through an electronic medium such as the Internet.

Another aspect of the present invention, a polymerase chain reaction is performed on a target sample using the primer set of the present invention,

It relates to a method for detecting the Chronobacter Sakazaki strain, comprising determining the presence or absence of the Chronobacter Sakazaki strain by checking the size of the product of the polymerase chain reaction or analyzing its nucleotide sequence. The presence or absence of the strain can be detected by electrophoresis of the DNA product amplified by polymerase chain reaction and checking the size of the amplified DNA product. The sample may be food, a microorganism isolated from food, or a nucleic acid sample extracted therefrom. The food may be powdered infant formula.

The detection method takes a sample suspected of Chronobacter sakazaki contamination, suspends it in sterile water or physiological saline, treats it with proteinase K, recovers the pellet, extracts it with hot water, prepares a PCR reaction, and then PCR may include performing PCR is performed, for example, by methods such as electrophoresis. Alternatively, a sample suspected of Chronobacter sakazaki contamination may be taken, and DNA may be extracted using a commercially available bacterial DNA extraction kit and used in a PCR reaction. The hot water extraction is a conventional DNA separation method. After adding sterile water and treating a phenol/chloroform mixture, a supernatant is obtained, and ethanol is added to the supernatant, followed by centrifugation.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.

Example

1. Pan Genome Analysis: Construction of Pan genome and Core genome of Chronobacter and Enterobacter strains

In this experiment, genome collections of Chronobacter and Enterobacter strains available in the NCBI Genebank (accessed February 9, 2021) were used for pan X analysis. While all 7 Chronobacter species were used in the analysis, 9 of the 22 Enterobacter species ( E. dykesii, E. vonholyi, E. quasihormaechei, E. wuhouensis, E. chuandaensis, E. huaxiensis, E. genomosp) O, E. genomosp. S, E. timonensis ) were not used for analysis due to lack of whole genome sequence data. As a result of the analysis, the total number of gene clusters and the number of core gene clusters were 31,793 and 104, respectively, when a cutoff of 1.00 was used. After PanX analysis, single nucleotide polymorphisms (SNPs) extracted from all core genes were used to construct the core genome phylogenetic tree. As shown in the core genome phylogenetic tree topology (Fig. 1), 10 strains of Chronobacter sakazaki were clearly separated from 201 Enterobacter strains and 7 non-Sakazaki Chronobacter strains. These results show that Sakazaki bacteria have evolutionary characteristics that are distinct from closely related microorganisms. Several candidates can be found that can be used as targets for primer design. Specifically, primers and probes using the panX assay were designed.

Specifically, 17 Chronobacter strains (10 C. sakzakii , 1 C. condiment , 1 C. dublinensis , 2 C. malonaticus , 1 C. muytijensii , 1 C. turicensis , 1 C. universalis ) and 201 Enterobacter strains (14 E. asburiae , 4 E. bugandensis , 3 E. cancerogenus , 1 E. chengduensis , 26 E. cloacae , 107 E. hormaechei , 11 E. kobei , 10 E. ludwigii , 1 E. mori , 1 E. oligotrophicus , 21 E. roggenkampii , 1 E. sichuanensis , and 1 E. soli ) were collected from NCBI RefSeq and NCBI RefSeq. Obtained from the Genebank database (accessed September 2, 2021).

[Table 1]

Comparative genome analysis was performed using the default option, pan X (Ding, Baumdicker, & Neher, 2018), for core and pan genome analysis of Chronobacter and Enterobacter strains. The core genome-single nucleotide polymorphism (SNP) based phylogenetic tree was visualized using FigTree version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree) (Fig. 1). In the defined ortholog, 16 gene clusters unique to C. sakazakii were identified (Table 2).

[Table 2]

Candidate gene clusters for C. sakazakii primers analyzed by pan genome sequence and its characterization

2. Primer and Probe Design Based on Pan Genome Analysis

The next step is to decide which gene clusters to select for primer design. Gene duplication, diversity, length, and similarity to other strains were considered important factors when selecting gene clusters in this study. Of the 31,793 gene clusters, 16 gene clusters were selected. Of the 16 candidates, two showed gene duplication and the other 5 candidates were non-C. It showed similarities with the Sakazaki strain. Therefore, these gene clusters were excluded from primer design. Among the remaining 9 candidates, the gene named type 1 ciliary protein was determined as a primer target because it is known as a virulence factor of C. sakazaki with a relatively low diversity.

Specifically, among the candidates shown in Table 2 above, four cilia-related genes were compressed. Among the four candidates, a gene called sthA was excluded due to similarities with other strains such as Cronobacter malonaticus and Thermomonospora amylolytica . Although the remaining three candidates did not show similarities with other variants in gene duplication, the gene attached to the type 1 cilia protein was selected as a primer target through low diversity and was named fimp1 and used for further experiments. Primer and probe sets targeting fimp1 were designed as follows using the AlleID7 program (Table 3).

[Table 3]

Oligonucleotide Primer and Probe Sequences Used in the Present Invention

Carboxyfluorescein (FAM) and black hole quencher-1 (BHQ ₁ ) fluorescent dyes were used for probes. It was confirmed that the designed primer probe covers all 10 strains of C. sakazakii . The newly designed primer-probe set was used for sensitivity, specificity testing and food application experiments.

3. Evaluation of sensitivity and specificity of developed primers

(1) Bacterial culture and cell suspension

의 농축 완충액에서 배양되었다.For sensitivity and selectivity testing of PCR assays, Enterobacter spp., Cronobacter spp. and other pathogenic non-pathogenic microorganisms. Microorganisms, except for the isolated Bacillus sakazakia, were supplied by the Bacterial Culture Room of Seoul National University (Seoul, Korea). The two C. sakazakii strains were isolated from raw milk and named C. sakazakii IS1 and IS2. The list of microorganisms used in the sensitivity and selectivity test is shown in Table 6. Each strain was cultured at 37 °C for 24 h before DNA extraction.

were incubated in concentrated buffer.

(2) Real-time PCR analysis

In order to confirm the effectiveness of the primer set prepared above, real-time polymerase chain reaction was performed with the composition shown in Table 4.

[Table 4]

DNA was extracted from the medium using the Genelix ^TM bacterial extraction kit (Bead type, Sanigen Co., Ltd., Anyang, Korea) according to the manufacturer's instructions. The concentration of purified DNA extracted from the microbial strain was 10 ng/μL. 5 μL of DNA, 200 nM of each forward and reverse primer, 200 nM of probe, 10 μL of master mix (BioFACT ^TM 2X Multi-Star Real Time PCR Master mix), and appropriate amounts of ultrapure water (Weljin, Gyeongsangbuk-do, Korea) were added to make a final volume of 20 μL. PCR analysis was performed using the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). After pretreatment at 50 °C for 2 min and initial denaturation at 95 °C for 10 min, 40 cycles for denaturation and annealing were replicated at 95 °C for 15 sec and 60 °C for 1 min, respectively. The PCR analysis result was detected as an increase in fluorescence signal and expressed as a quantitative cycle (Cq) value. The DNA standard curve of C. sakazakii ATCC 29544 was expressed in relative fluorescence units (RFU) with thresholds of 100, 150 and 200 and the slope, R2 and efficiency were indicated (Table 5).

[Table 5]

Validation of fimp1 test based on a standard curve of relative fluorescence units (RFU) with thresholds of 100, 150 and 200 using the CFX96 (Bio-Rad) PCR system

(3) Interference test

High-concentration (6 log CFU/ml) E. coli nutrient media (TSB, EE), PBS, and DNA were selected as interfering substances. Each interfering material was mixed with 5 μl of 2 log CFU/ml of C. sakazakii to 25% or 50% of the DNA volume. The coefficient of variation (CV) was calculated by comparing the Cq values of the interfering sample with the Cq value of the non-interfering sample. The CV value is obtained by dividing the standard deviation amount by the mean amount of the replication group. CV is a measure of precision that indicates the same value if it is close to zero.

(4) Food application

Powdered infant formula (PIF) (Namyang, Gongju, Korea), PIF containing lactic acid bacteria (Pasteur Co., Ltd., Seoul), and milk (Seoul Milk Co., Ltd., Seoul, Korea) were purchased from a grocery store (Seoul, Korea). . Detection of C. sakazakii ATCC 29544 inoculated into powdered infant formula was tested by a conventional selective method (Cromogenic Cronobacter Isolation (CCI) agar) and quantitative PCR (qPCR) assays. 5 ml of 2 log CFU/ml C. sakazaki was inoculated into 25 g/ml of each sample. PIF and PIF containing lactic acid bacteria were dried for 2 hours on a clean bench. Samples were then homogenized in 225 mL of concentrated buffer (Tryptic Bean Broth; TSB; Difco, Franklin Lakes, NJ, USA). After concentrating at 37°C for 24 hours, pathogens were detected using qPCR and CCI agar. Experimental conditions and procedures for qPCR analysis are not different from the methods described in 2-2) above. To detect Sakazaki bacteria in CCI agar, 100 μL of each homogenate was taken and spread on CCI agar. The agar plates were incubated at 37° C. for 24 hours, and cells representing the blue-green population were measured.

(5) Statistical analysis

Data represent the average of at least 3 independent experiments. Results are expressed as mean ± standard deviation (SD). Food application values were analyzed by the variance analysis procedure of the Statistical Analysis System (SAS Institute, Cary, NC) and mean values were separated using Duncan's multirange test. Significant differences were determined at the significant p = 0.05 level.

(6) Results

Regardless of the initial inoculation level, it was confirmed that the target gene of C. sakazakii was well amplified using the designed primer and probe set (FIG. 2). Second, the standard curve of C. sakazakii with various thresholds was checked to select the threshold level in further experiments (Fig. 3). In addition, the slope of the graph, R2, and efficiency were also analyzed (Table 5). When using the CFX96 real-time PCR amplification system, analytical efficiencies of 94.8, 94.5, and 94.7 were observed for relative fluorescence units (RFU) of 100, 150, and 200, respectively. Then, the sensitivity and selectivity of the primers developed for detecting C. sakazakii were confirmed (Table 6). In the sensitivity and specificity test at 10 ng/μl DNA concentration, Enterobacter spp. 23 species and Chronobacter spp. A total of 59 strains including 12 strains (7 C. sakazaki ) were used. Most of the microorganisms tested were pathogens.

Historically, C. sakazakii (formerly E. sakazakii ) was named the yellow-pigmented Enterobacter cloacae , indicating that C. sakazakii has very similar characteristics to E. cloacae (Farmer Iii, Asbury). , Hickman, Brenner, & Group, 1980). Biochemical tests distinguishing these two species were limited, so we included five strains of E. cloacae to confirm that the proposed primers selectively detected C. Sakazaki. Our results shown in Table 6 verify that the designed primers selectively detect C. sakazakii from E. cloacae . All C. sakazakii strains showed positive results, whereas all strains except C. sakazakii showed negative results. This means that no false positive or negative results were observed. These results indicate that the primer sequences developed based on the Pan X-analysis have high sensitivity and selectivity. In particular, all of the five strains, other than the Sakazaki strain, showed negative results, which means that there is high selectivity for closely related species.

[Table 6]

C. Sensitivity and selectivity test of fimp1 for detecting Sakazaki

4. Application to food samples

Several types of compounds such as tryptic soy broth (TSB), phosphate buffered saline (PBS), enterobacteriaceae enriched (EE), and E. coli DNA can interfere during PCR analysis. Therefore, the effect of these compounds on the detection of C. sakazakii was confirmed before food application (Table 7).

[Table 7]

Effect of Interfering Compounds on C. sakazakii Detection by Developed Primer-Probe Sequences

Compounds other than 50% EE did not significantly affect the detection of C. sakazakii , whereas C. sakazakii was not detected when 50% EE was used as an interferent.

High concentrations of EE may affect detection sensitivity, but other substances such as TSB, PBS and E. coli DNA do not significantly interfere with the detection of C. sakazakii . Therefore, an accurate detection result can be obtained using the primer sequence of the present invention, or when the previously used primer sequence gives an inaccurate result, the newly proposed primer sequence of the present invention can be used. Additionally, this sequence would be a good option for performing double or multiplex real-time PCR tests of interest recently (Xie & Liu, 2021).

PCR analysis using the developed primers when C. sakazakii ATCC 29544 was inoculated into powdered infant formula showed accurate results (p > 0.05) that were not significantly different from those of the previously used selective media (Table 8).

[Table 8]

Application of CCI and PCR to Detect C. sakazaki ATCC 29544 Inoculated in Powdered Infant Milk Powder (w or w/o Lactobacillus) and Milk ^a,b

The above experimental results validated the high sensitivity and specificity of the proposed C. sakazakii detection sequence using real-time PCR of food samples including buffer and PIF, Lactobacillus -containing PIF and milk. Therefore, it is expected that the designed sequences can be applied in various fields to ensure safety from C. sakazakii infection.

Likewise, the results of PCR analysis from infant formula and milk samples of powders containing lactobacilli were not significantly different from those of the selective media ( p > 0.05). These results show that C. sakazakii can be effectively detected in various types of food samples using the primer sequences developed in the present invention.

References

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Farmer III, J., Asbury, M., Hickman, F., Brenner, DJ, & Group, ES (1980). Enterobacter sakazakii : a new species of "Enterobacteriaceae" isolated from clinical specimens. International Journal of Systematic and Evolutionary Microbiology, 30(3), 569-584.

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<110> Seoul National University Industry Foundation <120> A biomarker specific for Cronobacter sakazakii, a kit and a method for detection Cronobacter sakazakii using the biomarker <130> P21-0369 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> artificial sequence <220> <223> A primer targeting a fimp1, wherein d = A or G or T; w = A or T, y = C or T <400> 1 gadggwaatg cagttaaca 19 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> A primer targeting a fimp1, wherein d = A or G or T; w = A or T, y = C or T <400> 2 taccgttagg cgtaatcaca 20 <210> 3 <211> 24 <212> DNA <213> artificial sequence <220> <223> A probe targeting a fimp1, wherein y= c or y <400> 3 atgccaacgc ctaatcctgt cgcy 24 <210> 4 <211> 537 <212> DNA <213> artificial sequence <220> <223> fimp1 of Cronobacter sakazakii <400> 4 atgaaaaaat atgcattagt gcttgcgctg aatgccattc tcgcagcggg agtggttcag 60 gctgcagaaa atgatgtttc agcgacgctt tctgtatcag gcacagttaa agatgcgtcc 120 gtgggttgta caattgaatt tgttcagccg actgtcgata tgggaagccg tgatatcgat 180 tctttgccat tgcaagggca tcacgttgat ggagctcaac tcaccgcggt cacagcaaac 240 tttatcggca actgcaccaa agatggccag ttagctccga acctcttatt taccggcatc 300 gttgatgatg cagaaggtaa tgcactggtt aacactgcaa ccggttctac cgcagcgaca 360 ggattaggcg ttggcattta ctcttatgca ggcaatgtga ttacgcctaa cggtagtccg 420 gttataggta ccggtaaaga gagcaccatg ttttatatag gcatggtgaa acttaacggt 480 gctaccccga caccgggcca ggttcagtca tcattaacca tgcagataga atcactg 537

Claims (13)

Includes primers capable of detecting Cronobacter sakazakii ,
The primer is a forward primer composed of the nucleic acid sequence shown in SEQ ID NO: 1, and
A primer set for detecting Chronobacter Sakazaki characterized by comprising a reverse primer composed of the nucleic acid sequence shown in SEQ ID NO: 2. The primer set according to claim 1, wherein the primer is capable of amplifying type 1 ciliary protein ( fimp1 ) of Chronobacter sakazaki strain. The primer set according to claim 1, wherein the primer set further comprises a probe composed of the nucleic acid sequence of SEQ ID NO: 3. A primer set for detecting Cronobacter sakazakii , which clones all or part of a type 1 ciliary protein ( fimp1 ) gene of a Cronobacter sakazakii strain. The primer set according to claim 4, wherein the type 1 ciliary protein ( fimp1 ) gene has the sequence of SEQ ID NO: 4. The primer set according to claim 4, wherein the primers include a forward primer composed of the nucleic acid sequence of SEQ ID NO: 1 and a reverse primer composed of the nucleic acid sequence of SEQ ID NO: 2. The polymerase chain reaction kit for detecting Chronobacter Sakazaki according to any one of claims 1 to 6, comprising the primer set. The method of claim 7, wherein the polymerase chain reaction is performed by RT (real-time) -PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blot or DNA chip. Polymerase chain reaction kit for detecting Chronobacter Sakazaki, carried out by a method selected from the group consisting of: A polymerase chain reaction is performed on the target sample using the primer set of claim 1 or claim 4,
A method for detecting the Chronobacter Sakazaki strain, comprising determining the presence or absence of the Chronobacter Sakazaki strain by checking the size of the product of the polymerase chain reaction or analyzing its nucleotide sequence. The detection method according to claim 9, wherein the primer set further comprises a probe composed of the nucleic acid sequence of SEQ ID NO: 3. 10. The method of claim 9, wherein the polymerase chain reaction is a group consisting of real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, northern blot, or DNA chip. , Detection method carried out by a method selected from. The method of claim 9, wherein the target sample is a food, a microorganism isolated from food, or a nucleic acid sample extracted therefrom. The detection method according to claim 12, wherein the food is powdered infant formula.

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