A quadruple Taqman qPCR assay for detecting Cronobacter cloacae.

Abstract

This invention discloses a quadruple Taqman qPCR detection reagent for detecting Cronobacter sakazakii, Cronobacter malondialdehyde, Cronobacter zurense, and Cronobacter dublin. The reagent includes specific primers and Taqman probes corresponding to these four Cronobacter sakazakii, Cronobacter malondialdehyde, Cronobacter zurense, and Cronobacter dublin. The primer-probe combination targeting Cronobacter sakazakii, Cronobacter malondialdehyde, Cronobacter zurense, and Cronobacter dublin provided by this invention has extremely high specificity and sensitivity, providing a reliable basis for the control of important Cronobacter species, effectively reducing the workload of single-pair qPCR detection, significantly improving work efficiency, and reducing economic costs.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection of foodborne bacteria, specifically relating to a quadruple Taqman qPCR detection reagent for detecting Cronobacter clonorchiasis. Background Technology

[0002] *Cronobacter*, formerly known as *Enterobacter sakazakii*, is a genus of foodborne opportunistic pathogens belonging to the family Enterobacteriaceae. Infection with *Cronobacter* can easily cause neonatal meningitis, sepsis, and necrotizing enterocolitis, with a mortality rate as high as 40% to 80%. Even after effective treatment and recovery, infected individuals often suffer from severe neurological sequelae. To date, seven species of *Cronobacter* have been identified: *Cronobacter sakazakii*, *Cronobacter malondiamide*, *Cronobacter zurichensis*, *Cronobacter univoskensis*, *Cronobacter moginskaensis*, *Cronobacter dublinum* (including three subspecies: *Cronobacter dublinum*, *Cronobacter natriureticum*, and *Cronobacter laushamensis*), and *Cronobacter monte*. Different species of *Cronobacter* exhibit varying virulence and pathogenicity due to differences in their genomic composition and biological characteristics. Currently, although all seven species have been retrospectively linked to clinical cases of infection in infants or adults, *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, and *Cronobacter zuriensis* have shown greater clinical importance. Therefore, precise identification of *Cronobacter* species, especially *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, and *Cronobacter zuriensis*, is crucial for promoting food safety and significantly reducing health risks to vulnerable populations with weakened immune systems.

[0003] Currently, traditional biochemical typing remains the "gold standard" for detecting Cronobacter spp. The national standard GB4789.40—2016, "National Food Safety Standard for Microbiological Examination of Food: Cronobacter spp. (Cronobacter sakazakii)," specifies the testing method for Cronobacter spp. in food. The sample undergoes enrichment, isolation, and purification steps, followed by final identification using biochemical analysis. This technique is not only cumbersome and time-consuming (up to 7 days), but it also cannot effectively distinguish between "species" and "subspecies" levels of Cronobacter, only identifying the test strain to the "genus" level. In recent years, molecular biology methods, such as polymerase chain reaction (PCR), have emerged. By directly targeting specific DNA fragments for species identification, they eliminate the complex pathogen isolation and culture steps, significantly shortening the pathogen detection cycle and making them the best alternative to traditional detection methods. Compared to conventional PCR, real-time quantitative PCR (qPCR) has gained wider attention and application due to its simplified process, real-time sample quantification, sample transfer and amplification band visualization without additional steps, and significant advantages such as high specificity and high sensitivity. Currently, many mature qPCR kits and detection methods for the detection of *Cronobacter* have been reported both domestically and internationally, including *Cronobacter* genus (level) identification based on 16S rRNA and rpoB gene, *Cronobacter sakazakii* identification based on ITS gene, dual identification of *Cronobacter sakazakii* and *Cronobacter dublini* based on gyrB gene, dual identification of *Cronobacter sakazakii* and *Cronobacter malondialdehyde* based on 16S rRNA and fusA gene, and species identification based on ompA gene. These detection methods have all demonstrated excellent performance in *Cronobacter* detection: high sensitivity and high specificity. However, to meet the need for species identification of *Cronobacter*, distinguishing only two species within the genus is clearly insufficient. While qPCR based on the ompA gene can achieve species classification within a genus, the high-resolution melting curve analysis at the back end further challenges the equipment and instruments used. Therefore, developing a multiplex qPCR kit capable of high-resolution, high-specificity species identification of the genus *Cronobacter* remains a significant application gap.

[0004] Due to the high similarity of genome sequences among various species of *Cronobacter* and the diversity of strains within the same species, using primers with insufficient resolution or poor specificity can lead to false positives (false detections) or false negatives (false negatives). Therefore, this multiplex qPCR kit designs highly specific primers and TaqMan probes based on specific gene fragments from four *Cronobacter* species identified at the whole-genome level: *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, and *Cronobacter dublinis*. Corresponding multiplex qPCR methods have been established to meet the current needs for identification and detection of important *Cronobacter* species, fill the gap in the market for molecular typing of *Cronobacter* species, promote the development of my country's food hygiene inspection industry, and safeguard food safety for the entire nation. Summary of the Invention

[0005] The purpose of this invention is to overcome at least one deficiency of the prior art and provide a quadruple Taqman qPCR detection reagent for detecting Cronobacter clonorchiasis.

[0006] The technical solution adopted in this invention is:

[0007] In one aspect, the present invention provides a quadruple Taqman qPCR detection reagent for detecting Cronobacterium sakazakii, Cronobacter malondialdehyde, Cronobacter zurophila, and Cronobacter dublin; the reagent comprises specific primers and Taqman probes corresponding to these four Cronobacterium sakazakii.

[0008] The primer sequences and Taqman probe sequences used for detecting Cronobacter sakazakii are: upstream primer Csk-F, downstream primer Csk-R, and Taqman probe Csk-P;

[0009] The Csk-F has a sequence as shown in SEQ ID NO.1;

[0010] The Csk-R has a sequence as shown in SEQ ID NO.2;

[0011] The Csk-P has a sequence as shown in SEQ ID NO.3;

[0012] The primer sequences and Taqman probe sequences used for detecting Cronobacter malonate are: upstream primer Cma-F, downstream primer Cma-R, and Taqman probe Cma-P;

[0013] The Cma-F has a sequence as shown in SEQ ID NO.4;

[0014] The Cma-R has a sequence as shown in SEQ ID NO.5;

[0015] The Cma-P has a sequence as shown in SEQ ID NO.6;

[0016] The primer sequences and Taqman probe sequences used for detecting Kronobacter dublin are: upstream primer Cdu-F, downstream primer Cdu-R, and Taqman probe Cdu-P.

[0017] The Cdu-F has a sequence as shown in SEQ ID NO.7;

[0018] The Cdu-R has a sequence as shown in SEQ ID NO.8;

[0019] The Cdu-P has a sequence as shown in SEQ ID NO.9;

[0020] The primer sequences and Taqman probe sequences used for detecting Cronobacter zosteri are as follows: upstream primer Ctu-F, downstream primer Ctu-R, Taqman probe Ctu-P, upstream primer Ctu-F1, downstream primer Ctu-R1, and Taqman probe Ctu-P1.

[0021] The Ctu-F has a sequence as shown in SEQ ID NO.10;

[0022] The Ctu-R has a sequence as shown in SEQ ID NO.11;

[0023] The Ctu-P has a sequence as shown in SEQ ID NO.12;

[0024] The Ctu-F1 has a sequence as shown in SEQ ID NO.13;

[0025] The Ctu-R1 has the sequence shown in SEQ ID NO.14;

[0026] The Ctu-P1 has a sequence as shown in SEQ ID NO.15.

[0027] In some instances, probes from different bacterial species were labeled with different fluorescent quenching groups.

[0028] In some instances, the probe Csk-P is labeled with the fluorescent reporter dye FAM at its 5' end and with the fluorescent quencher group BHQ1 at its 3' end.

[0029] The probe Cma-P is labeled with the fluorescent reporter dye JOE at its 5' end and with the fluorescent quencher group BHQ2 at its 3' end.

[0030] The probe Cdu-P is labeled with the fluorescent reporter dye ROX at its 5' end and with the fluorescent quencher group BHQ2 at its 3' end.

[0031] The probes Ctu-P and Ctu-P1 are labeled with NED fluorescent reporter dye at their 5' ends and BHQ1 fluorescent quencher group at their 3' ends.

[0032] In a second aspect, the present invention provides a quadruple Taqman qPCR kit for detecting Cronobacter spp., the quadruple Taqman qPCR kit comprising the quadruple Taqman qPCR detection reagent described in the first aspect.

[0033] In some instances, the pathogenic bacteria are selected from at least one of *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, or *Cronobacter dublinum*.

[0034] In some instances, the kit also includes a negative control, a positive control, and a lysis buffer.

[0035] In some instances, the positive control is a mixture of genomic DNA corresponding to *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurich*, and *Cronobacter dublin*.

[0036] In some instances, the negative control is DEPC water.

[0037] In some instances, the lysis buffer is composed of 4.9–5.1 mmol / L tris(hydroxymethylaminomethane) and 0.019–0.121% sodium dodecyl sulfate solution.

[0038] Thirdly, this invention provides a detection method using a quadruple Taqman qPCR reagent for detecting *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, and *Cronobacter dublinum*, comprising the following steps:

[0039] 1) Extract genomic DNA from the sample to be tested for later use;

[0040] 2) The genomic DNA of the sample to be tested is added as a template to a quadruple qPCR reaction system for amplification. The reaction system includes the quadruple Taqman qPCR kit described in the second aspect.

[0041] 3) Set up the qPCR program and collect the fluorescence signal during the amplification process, and determine the results.

[0042] The beneficial effects of this invention are:

[0043] 1. This invention enables the simultaneous detection of *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurichensis*, and *Cronobacter dublinensis* in a single reaction tube, providing a simple, rapid, efficient, and low-cost method for the species identification of *Cronobacter*.

[0044] 2. The primer-probe combination for targeting Cronobacter sakazakii, Cronobacter malondiamide, Cronobacter zurich, and Cronobacter dublin provided by this invention has extremely high specificity. Fluorescent signals can only be observed in the corresponding fluorescence detection channel when the target Cronobacter is present in the sample to be tested, effectively avoiding false positives or false negatives and making the detection results more accurate.

[0045] 3. The quadruple Taqman probe qPCR detection method established in this invention has high sensitivity, with a detection sensitivity of 1.5 × 10⁻⁶ for *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, and *Cronobacter dublinii*. 2 The results were CFU / mL and showed excellent reproducibility (CV% < 2%).

[0046] 4. The detection method established in this invention provides a reliable basis for the control of important Cronobacter species, significantly reducing the workload of singleton qPCR detection, greatly improving efficiency, and also reducing economic costs to some extent. Furthermore, compared to traditional biochemical identification relying on pure cultures (7 days), this detection method can complete the entire sample detection process within 6 hours (including a 4-hour pre-enrichment process), with the qPCR reaction time being 1 hour. This significant advantage of being economical and efficient makes it particularly suitable for large-scale sample screening and actual testing. Attached Figure Description

[0047] Figure 1 This is a singlet qPCR amplification curve for Cronobacter sakazakii.

[0048] Figure 2 This is a singlet qPCR amplification curve for Cronobacter malonate.

[0049] Figure 3 This is an amplification curve of Cronobacter zoster using the singlet qPCR method in Zurich.

[0050] Figure 4 This is an amplification curve for singlet qPCR detection of Cronobacter dublinii.

[0051] Figure 5 The results of a sensitivity test for detecting a single pathogen, Cronobacter sakazakii, using a quadruple Taqman qPCR reaction system.

[0052] Figure 6 The results of a sensitivity test for detecting a single pathogen, Cronobacter malondiamide, using a quadruple Taqman qPCR reaction system.

[0053] Figure 7 The results of a sensitivity test for detecting a single pathogen, Cronobacter zureri, using a quadruple Taqman qPCR reaction system.

[0054] Figure 8 The results of a sensitivity test for detecting a single pathogen, *Cronobacter dublinis*, using a quadruple Taqman qPCR reaction system.

[0055] Figure 9 The standard curve is the corresponding sensitivity test curve for detecting a single pathogen using a quadruple Taqman qPCR reaction system.

[0056] Figure 10 An example of multiplex qPCR detection using a mixture of four Cronobacter positive samples.

[0057] Figure 11 The amplification curve of Cronobacter sakazakii in a sensitivity test of simultaneous detection of four pathogens using a quadruple Taqman qPCR reaction system.

[0058] Figure 12 The amplification curve of Cronobacter malonate in a sensitivity test for simultaneous detection of four pathogens using a quadruple Taqman qPCR reaction system.

[0059] Figure 13 The amplification curve of Cronobacter zosteri in the sensitivity test of a quadruple Taqman qPCR system for the simultaneous detection of four pathogens is shown.

[0060] Figure 14 The amplification curve of Cronobacter dublin in a sensitivity test of a quadruple Taqman qPCR system for the simultaneous detection of four pathogens is shown.

[0061] Figure 15 This is a standard curve for the sensitivity test of a quadruple Taqman qPCR reaction system for the simultaneous detection of four pathogens.

[0062] Figure 16 Figure 1 shows the anti-interference ability of the quadruple Taqman qPCR reaction system. Figure 2 shows the actual amplification curves of the four target bacteria after the addition of high-density interfering bacteria, and Figure 3 shows the Ct value line graph corresponding to Figure 4. Detailed Implementation

[0063] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0064] Example 1

[0065] Bioinformatics analysis was performed on the whole genome DNA sequences of *Cronobacter* strains obtained from the GenBank database and our team's own sequencing, resulting in the identification of four specific gene fragments for *Cronobacter* strains. Primers and probes were designed for each of the four strains using Primer 6 and Oligo7, and the specificity of the designed primers and probes across all bacterial species was confirmed using the NCBI Primer BLAST tool. While adhering to primer and probe design principles, the amplified fragments were ensured to be within 200 bp. After a series of preliminary screenings and validations, the optimal primers and probes were determined, as detailed in the table below. Two pairs of primers and probes targeting *Cronobacter zurophila* (SEQ ID NO. 10-SEQ ID NO. 15) were selected.

[0066] Table 1. Specific qPCR detection primers and corresponding Taqman probe sequences for four types of Cronobacter.

[0067]

[0068] Example 2

[0069] The quadruple Taqman qPCR detection method based on the detection reagent described in Example 1 is as follows:

[0070] (1) Crude extraction of DNA from the sample to be tested. After picking suspected colonies and culturing them continuously for 14 h, take 1 mL of the enrichment broth and transfer it to a 1.5 mL sterile centrifuge tube. Centrifuge at 6000 r / min for 5 min to completely remove the supernatant. Add 30 μL of lysis buffer prepared with 5 mmol / L sodium dodecyl sulfate (SDS) and 0.02% (m / v) tris-base. Dissolve the precipitate at the bottom of the tube thoroughly with the lysis buffer, gently tap the tube wall to remove air bubbles, and heat at 99 °C for 10 min. Centrifuge at 12000 r / min for 15 min. The supernatant is the DNA of the sample to be tested. Place it on ice. If it is not used for a long time, centrifuge again. All obtained DNA samples are stored at -20 °C for later use.

[0071] (2) Four sets of qPCR primers and their corresponding probes were used for detection. The DNA obtained in (1) was used as a template, and qPCR amplification was performed using the primers and probes described in Example 1. Fluorescent signals were collected.

[0072] The qPCR reaction system consisted of 12.5 μL of 2×qPCR Mix, with initial concentrations of 10 μmol / L for Csk-F, Csk-R, Cma-F, Cma-R, Ctu-F / Ctu-F1, Ctu-R / Ctu-R1, Cdu-F, and Cdu-R at concentrations of 0.4, 0.4, 0.5, 0.5, 0.3, 0.3, and 0.25 μL respectively, and initial concentrations of 10 μmol / L for Csk-P, Cma-P, Ctu-P / Ctu-P1, and Cdu-P at concentrations of 0.4, 0.3, 0.5, and 0.25 μL respectively, plus 2.5 μL of template. The total volume was then made up to 25 μL using DEPC water.

[0073] The reaction program for qPCR amplification is as follows: the fluorescence channel is set to temperature control, and the program is set to 94℃ for 60s; 40 cycles include 95℃ for 20s and 59℃ for 30s; the fluorescence channels of the qPCR instrument are set as follows: Channel 1: FAM; Channel 2: JOE; Channel 3: NED; Channel 4: ROX; the qPCR instrument automatically collects fluorescence signals in the set fluorescence channels after each cycle.

[0074] (3) Result determination: Based on the fluorescence signal and Ct value collected in (2), determine whether the sample contains one or more of the four target Cronobacter species. When the Ct value is >35 or the system determines it to be negative, the result is determined to be negative.

[0075] Example 3

[0076] The construction and verification of the reagents described in Example 1 and the detection method described in Example 2 are as follows:

[0077] 1. Construction and optimization of multiplex qPCR system

[0078] First, using the extracted genomic DNA of the target strain as a positive control template, and DEPC water as a blank control, with four Cronobacter species serving as negative controls for each other, singlet qPCR reactions were performed using the corresponding primers and probes to preliminarily demonstrate the specificity of the designed primer-probe set. The results are as follows: Figures 1-4 As shown, the primer-probe set designed for *Cronobacter sakazakii*, *Cronobacter malondiol*, *Cronobacter zurichensis*, and *Cronobacter dublinensis* can distinguish each other within the same species. The amplification signal can only be observed in the corresponding fluorescence channel when the corresponding target bacterial DNA is present in the reaction system.

[0079] Subsequently, primers and probes with different final concentrations (setting concentration gradients of 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, and 0.5 μM) were selected for single-factor optimization experiments. Taking into account the cycle threshold (Ct) and fluorescence signal value (ΔRn) of the amplification curve, the final singlet TaqMan qPCR reaction system for the four Cronobacter species was determined. Then, using the extracted genomic DNA of the target strains as a positive control template, orthogonal experiments were conducted with appropriate primer concentrations to optimize the multiplex qPCR reaction system based on the singlet qPCR method. The optimal conditions for the multiplex reaction were selected based on the Ct value, fluorescence intensity change, and sensitivity of the amplification curve, allowing simultaneous detection of the four Cronobacter species in a single-tube system.

[0080] Optimization results of the multiplex qPCR reaction system showed that the concentrations of upstream and downstream primers and probes for *Cronobacter sakazakii* were 0.4 / 0.4 / 0.4 μmol / L, for *Cronobacter malondiamide* they were 0.5 / 0.5 / 0.5 μmol / L, for *Cronobacter dublinum* they were 0.25 / 0.25 / 0.25 μmol / L, and for *Cronobacter zuriensis* they were 0.3 / 0.3 / 0.5 μmol / L, which was designated as System 1.

[0081] For the second pair of upstream and downstream primers and probes (SEQ ID NO.13-SEQ ID NO.15) designed for *Cronobacter zurichensis*, the inventors found during the optimization of the multiplex qPCR reaction system (System 2) that this primer and probe pair could not achieve sensitive detection of *Cronobacter zurichensis*: with the same amount of template input, the Ct value would increase by 2-3 (Table 2), meaning that the subsequent detection sensitivity would be reduced by approximately one order of magnitude. Therefore, this primer and probe pair was removed from the experiment and will not be considered again in the future.

[0082] Table 2. Optimal selection of two multiplex qPCR reaction systems and comparison of mean Ct values.

[0083]

[0084] Note: The inoculation density for all four target bacteria was 1.5 × 10⁴. 7 CFU / mL

[0085] 2. Specificity validation of the multiplex qPCR system

[0086] To verify the specificity of the multiplex qPCR kit detection system constructed in this invention, genomic DNA was extracted from the target strain and other common non-target bacteria from other genera as templates. A 25 μL detection system was established according to the optimization results in Example 2 for multiplex qPCR detection, with each template subjected to three replicate reactions. A total of 84 strains were verified, including 45 strains of *Cronobacter* and 39 strains of other common non-target bacteria. Specifically, the *Cronobacter* strains included: *Cronobacter sakazakii* (1 standard strain, 9 isolates), *Cronobacter malondialdehyde* (10 isolates), *Cronobacter Zurich* (10 isolates), and *Cronobacter Dublin* (10 isolates). *Cronobacter sakazakii* (ATCC 25944), *Cronobacter malondialdehyde* (cro3316B3), *Cronobacter Zurich* (cro3866w), and *Cronobacter Dublin* (FSCC(I)145273) were used as positive controls, and DEPC water as a negative control. DNA extraction was performed in the same manner as in Example 2. All isolates used and their numbers are detailed in the appendix.

[0087] The specificity verification results of the multiplex qPCR kit are shown in Table 3. Of the total 84 bacterial strains (including the target bacteria and other common species), only *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, and *Cronobacter malondialdehyde* showed positive amplification results in their corresponding fluorescence channels. This result demonstrates the high specificity of the multiplex qPCR kit detection system constructed in this invention.

[0088] Table 3. Strains used for specificity validation of the multiplex qPCR kit detection system.

[0089]

[0090]

[0091]

[0092] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0093] Example 4: Quadruple Taqman qPCR Sensitivity Assay

[0094] Overnight cultures of *Cronobacter sakazakii* (ATCC 29544), *Cronobacter malondialdehyde* (cro3316B3), *Cronobacter zurichensis* (cro3866w), and *Cronobacter dublinensis* (FSCC(I)145273) were performed, and the bacterial density of these four pathogens was quantified using the plate count method. The quantitative results showed that the initial bacterial densities of *Cronobacter sakazakii* (ATCC 29544), *Cronobacter malondialdehyde* (cro3316B3), *Cronobacter zurichensis* (cro3866w), and *Cronobacter dublinensis* (FSCC(I)145273) were 6.40 × 10⁻⁶. 8 CFU / mL, 4.95×10 8 CFU / mL, 4.2×10 8 CFU / mL, 5.68×10 8 CFU / mL.

[0095] 1. Single infection experiment: Take 1 mL of each original bacterial culture and adjust the bacterial density of each culture to 1.5 × 10⁻⁶ by adding sterile physiological saline. 8 After reaching CFU / mL, seven 10-fold serial dilutions were performed (dilution factors: 0, -1, -2, -3, -4, -5, -6, -7). Subsequently, 1 mL of each bacterial genomic DNA was extracted from each serial dilution using the method described in Example 2. Multiplex qPCR amplification and detection were then performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2 to investigate the sensitivity of the detection method. Each reaction was repeated three times.

[0096] 2. Co-infection experiment: A certain volume of each target bacterium's original bacterial suspension and sterile physiological saline were taken and mixed to normalize the bacterial suspension density of each target bacterium in the mixture to 1.5 × 10⁻⁶. 8 CFU / mL. This normalized mixture was then serially diluted 10-fold in seven different gradients. Subsequently, 1 mL of each of these gradient dilutions was taken to obtain genomic DNA according to the method described in Example 2, and multiplex qPCR amplification and detection were performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2 to investigate the sensitivity of the detection method. Each reaction was repeated three times.

[0097] The results showed that this kit was effective at inoculation densities of 1.5 × 10⁻⁶. 7 Up to 1.5×10 2 When genomic DNA of *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, and *Cronobacter dublinis*, or mixtures thereof, at CFU / mL (6 gradients) was detected, good linearity (R0) was observed in all samples. 2>0.99); and the minimum detection density can be as low as 1.5×10 2 CFU / mL. Standard curves for single and multiple pathogen detection were constructed based on the corresponding mean Ct values ​​of this multiplex qPCR kit. For single pathogen detection, the slopes of the standard curves for *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, and *Cronobacter dublin* were -3.223, -3.845, -3.669, and -3.383, respectively. For multiple pathogen detection, the slopes of the standard curves for *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, and *Cronobacter dublin* were -3.172, -3.416, -3.258, and -3.164, respectively. This clearly demonstrates that the quadruple Taqman qPCR method possesses good amplification performance. It also shows that this kit can detect both single and multiple infected samples. Detailed results are shown in Table 4 and below. Figures 5-10 (Single pathogen infection) and Table 5 and Figures 11-15 (Multi-pathogen infection).

[0098] Table 4. Sensitivity test results of the quadruple Taqman qPCR system for detecting single pathogens.

[0099]

[0100] Note: "–" indicates a negative result, and the same applies below.

[0101] Table 5. Sensitivity test results of the quadruple Taqman qPCR assay for simultaneous detection of four target Cronobacter species.

[0102]

[0103] Example 5: Stability Test

[0104] Using the normalized concentration gradient premix from Example 4 as the input DNA template, a repeatability experiment (repeated 8 times) was conducted to test the "co-infection sensitivity" in Example 4, based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2. The stability of the method was assessed using the coefficient of variation (CV). Coefficient of variation = standard deviation / mean.

[0105] The results are shown in Table 6. When the normalized concentration gradient premix was detected by this quadruple qPCR, the CV% value between the eight replicates of each detection gradient was less than 2%, which met the precision threshold requirement (<15%) of the in vitro diagnostic kit, indicating that the system has good stability.

[0106] Table 6. Stability evaluation of simultaneous detection of four target Cronobacter species using quadruple Taqman qPCR.

[0107]

[0108]

[0109] Example 6: Anti-interference experiment

[0110] Salmonella typhimurium (ATCC 14028), Escherichia coli (ATCC 25922), Yersinia enterica (ATCC 52204), Staphylococcus aureus (ATCC 29213), and beta-hemolytic streptococci (CMCC(B)32210) were selected as interfering bacteria. The above five interfering bacteria and four target Cronobacteria were cultured overnight: Cronobacter sakazakii (ATCC 29544), Cronobacter malondialdehyde (cro3316B3), Cronobacter Zurich (cro3866w), and Cronobacter Dublin (FSCC(I)145273). The bacterial density of these nine pathogens was quantified by plate counting after culture. Quantitative results showed that the original bacterial cultures of Salmonella Typhimurium, Escherichia coli, Yersinia enterica, Staphylococcus aureus, and beta-hemolytic streptococci had densities of 2.92 × 10⁻⁶. 8 1.43×10 8 5.51×10 7 2.4×10 8 8.60×10 8 CFU / mL; the original bacterial cultures of *Cronobacter sakazakii*, *Cronobacter malondialdehyde*, *Cronobacter zurigensis*, and *Cronobacter dublinis* had densities of 3.26 × 10⁻⁶ CFU / mL, respectively. 8 2.45×10 8 2.88×10 8 3.51×10 8 CFU / mL. Then, the four target bacteria and five non-target bacteria solutions were serially diluted 10-fold with seven dilution factors (0, -1, -2, -3, -4, -5, -6, -7) and two dilution factors (0, -1, -2), respectively, and 5 mL of each of the four target bacteria dilutions (dilution factor -3) was mixed. Subsequently, 1 mL of each of the five interfering bacteria dilutions (dilution factor -2) was added to 1 mL of the prepared target bacteria dilution mixture and mixed in equal volumes. Then, 1 mL of each mixture was taken to extract DNA according to the method described in Example 2, and multiplex qPCR amplification and detection were performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2.

[0111] The results are shown in Table 7 and... Figure 16As shown, the addition of high-density interfering bacteria does not significantly interfere with the detection of the target bacteria, and the Ct values ​​remain basically consistent. This further demonstrates the high specificity of the designed primer-probe set and shows the potential of the quadruple Taqman qPCR detection reagent, kit, and detection method provided by this invention for the identification and detection of four Cronobacter species in food.

[0112] Table 7 Evaluation of the anti-interference ability of the quadruple qPCR reaction system

[0113]

[0114] Note: The positive control is the template DNA obtained by further diluting the target bacteria dilution with a dilution factor of -3 by 2 times.

[0115] Example 7: Artificially Contaminated Sample Test

[0116] Two mL of each of the four target bacterial gradient dilutions (eight gradients) prepared in Example 6 was mixed to form eight gradient premixes. One mL of each gradient premix was then added to eight vials of modified lauryl sulfate tryptone (mLST) broth enrichment solution containing formula milk powder (2.5 g milk powder + 22.5 mL mLST). After thorough shaking, one mL of each of the eight gradient mixtures was extracted for DNA extraction. The last five gradient dilution mixtures were then incubated sequentially at 37°C on a shaker (150 rpm) for 2, 3, and 4 hours, with one mL of DNA extracted from each gradient sample strictly following the time settings. After obtaining the DNA samples, multiplex qPCR amplification and detection were performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2. Here, the milk powder was strictly confirmed to be free of *Cronobacter* according to the labeling procedure in GB4789.40-2024.

[0117] When the dilution factor is -7, the bacterial concentration in the contaminated sample is approximately 1–10 CFU / mL, equivalent to a bacterial load of 1–10 CFU / g. Amplification results for artificially contaminated samples are detailed in Table 8. Without enrichment culture, the detection limits for *Cronobacter sakazakii* and *Cronobacter dublinii* in the contaminated samples are 10⁻⁶. 2 CFU / mL (dilution factor -5), the detection limit for *Cronobacter malonic acid* and *Cronobacter zurophila* in the contaminated sample was 10. 3 CFU / mL (dilution factor -4). The limit of positive detection reached 10 after 2 hours of enrichment. 0CFU / mL (dilution factor -7). However, appropriately extending the enrichment time to 4 hours, resulting in an amplified Ct value below 30 (Ct≤30), makes the results easier to interpret. Overall, compared to the approximately 7-day detection cycle of traditional methods, this method can complete the detection within 6 hours, including 4 hours of enrichment, 1 hour of qPCR, and 1 hour of manual operation, demonstrating significant time and cost-effectiveness.

[0118] Table 8. Results of multiplex qPCR detection after enrichment of artificially contaminated samples.

[0119]

[0120]

[0121] Example 8: Actual Sample Testing

[0122] The established multiplex qPCR method was used to test 17 food samples purchased from local supermarkets and farmers' markets, including vegetables (choy sum, Shanghai bok choy, Chinese cabbage, baby bok choy, lettuce, cucumber, scallions, cilantro, garlic sprouts, enoki mushrooms), aquatic products (salmon, tilapia, prawns), and processed products (corn and pork dumplings, dumpling wrappers, fried chicken strips, and ham sausages, one sample each). Specific methods: 25g of each sample was weighed and added to mL of ST enrichment broth. After homogenization, the broth was enriched at 37℃ for 4 hours. 1mL of the broth was then used to extract DNA as a template for amplification. Multiplex qPCR amplification and detection were performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2. Simultaneously, these 17 samples were tested for Cronobacter spp. according to the procedure outlined in GB4789.40-2024. For strains identified as Cronobacter, fusA sequencing was performed according to SN / T 5361-2021 to distinguish the species.

[0123] The results showed that, using this multiplex qPCR kit, *Cronobacter* was detected in 7 out of 17 food samples. Specifically, 6 samples tested positive for *Cronobacter sakazakii*, 5 for *Cronobacter malondialdehyde*, 0 for *Cronobacter zuriensis*, and 2 for *Cronobacter dublinum* (Table 9). According to the national standard identification method, *Cronobacter* was detected in 6 out of 17 samples. Further sequencing results showed that *Cronobacter sakazakii* was detected in 5 of the 17 samples, *Cronobacter malondialdehyde* in 3, *Cronobacter zuriensis* in 0, and *Cronobacter dublinum* in 2. In general, the results of positive samples detected by traditional methods were consistent with those of qPCR, but the positive detection rate of foodborne pathogens using qPCR was slightly higher than that of traditional methods. The inconsistency in the results can be attributed to the fact that traditional detection methods rely on obtaining pure bacterial cultures, which may result in the omission of target bacteria during the purification process.

[0124] Table 9. Comparison of detection results of four target bacteria in 17 food samples using quadruple Taqman qPCR, national standard identification methods, and sequencing technology.

[0125]

[0126] In summary, the above embodiments demonstrate and describe the basic principles and features of the present invention. The results show that the kit of the present invention has the characteristics of good accuracy and repeatability, and high sensitivity, and is suitable for rapid detection and identification of four important Cronobacter species, and has strong market application and promotion potential.

[0127] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A quadruple Taqman qPCR detection reagent for detecting Cronobacter, characterized by, The Cronobacter pathogen is Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter zurichensis and Cronobacter dublinensis; the reagent includes specific primers and Taqman probes corresponding to the four Cronobacter pathogen; The primer sequence and Taqman probe sequence for detecting Cronobacter sakazakii are: upstream primer Csk-F, downstream primer Csk-R, Taqman probe Csk-P; The Csk-F has the sequence shown in SEQ ID NO. 1; The Csk-R has the sequence shown in SEQ ID NO. 2; The Csk-P has the sequence shown in SEQ ID NO. 3; The primer sequence and Taqman probe sequence for detecting Cronobacter malonaticus are: upstream primer Cma-F, downstream primer Cma-R, Taqman probe Cma-P; The Cma-F has the sequence shown in SEQ ID NO. 4; The Cma-R has the sequence shown in SEQ ID NO. 5; The Cma-P has the sequence shown in SEQ ID NO. 6; The primer sequence and Taqman probe sequence for detecting Cronobacter dublinensis are: upstream primer Cdu-F, downstream primer Cdu-R, Taqman probe Cdu-P; The Cdu-F has the sequence shown in SEQ ID NO. 7; The Cdu-R has the sequence shown in SEQ ID NO. 8; The Cdu-P has the sequence shown in SEQ ID NO. 9; The primer sequence and Taqman probe sequence for detecting Cronobacter zurichensis are: upstream primer Ctu-F, downstream primer Ctu-R, Taqman probe Ctu-P, upstream primer Ctu-F1, downstream primer Ctu-R1, Taqman probe Ctu-P1; The Ctu-F has the sequence shown in SEQ ID NO. 10; The Ctu-R has the sequence shown in SEQ ID NO. 11; The Ctu-P has the sequence shown in SEQ ID NO. 12; The Ctu-F1 has the sequence shown in SEQ ID NO. 13; The Ctu-R1 has the sequence shown in SEQ ID NO. 14; The Ctu-P1 has the sequence shown in SEQ ID NO. 15; The probes of different bacteria are labeled with different fluorescence-quenching groups.

2. The quadruplex Taqman qPCR reagent according to claim 1, characterized in that, The 5' end of the probe Csk-P is labeled with a fluorescent reporter dye FAM, and the 3' end is labeled with a fluorescent quenching group BHQ1; The 5' end of the probe Cma-P is labeled with a fluorescent reporter dye JOE, and the 3' end is labeled with a fluorescent quenching group BHQ2; The 5' end of the probe Cdu-P is labeled with a fluorescent reporter dye ROX, and the 3' end is labeled with a fluorescent quenching group BHQ2; The 5' end of the probe Ctu-P and Ctu-P1 is labeled with a fluorescent reporter dye NED, and the 3' end is labeled with a fluorescent quenching group BHQ1.

3. A quadruple Taqman qPCR kit for detecting Cronobacter, characterized by, The quadruplex Taqman qPCR kit comprises the quadruplex Taqman qPCR detection reagent of claim 1 or 2.

4. The quadruplex Taqman qPCR kit according to claim 3, characterized in that, The kit further comprises a negative control, a positive control and a lysis solution.

5. The quadruplex Taqman qPCR kit according to claim 4, characterized in that, The positive control is a mixture of genomic DNA corresponding to C. sakazakii, C. malonaticus, C. zuhrii and C. dubliniensis.

6. The quadruplex Taqman qPCR kit according to claim 4, characterized in that, The negative control is DEPC water.

7. The quadruplex Taqman qPCR kit according to claim 4, characterized in that, The lysis solution comprises 4.9-5.1 mmol / L tris (hydroxymethyl aminomethane) and 0.019-0.121% sodium dodecyl sulfate solution.

8. A detection method of a quadruple Taqman qPCR detection reagent for detecting Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter zurichensis and Cronobacter dublinensis, which is not used for diagnosis or treatment of a disease, characterized in that, The method comprises the following steps: 1) extracting genomic DNA of the sample to be tested for standby; 2) adding the genomic DNA of the sample to be tested as a template into a quadruplex qPCR reaction system for amplification, the reaction system comprising the quadruplex Taqman qPCR kit of any one of claims 3-7; 3) setting a qPCR program and collecting fluorescence signals during the amplification process, and making a result determination.

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