Application of Nucleic Acid Reference Material for Rapid Detection of Cronobacter sakazakii (Cronobacter spp.) in Flammulina velutipes

Cronobacter sakazakii (C. sakazakii) widely exists in the environment and is a common foodborne pathogenic microorganism. It can cause meningitis, bacteremia, and necrotizing enterocolitis in infants and elderly people with low immunity. Therefore, the development of the rapid detection method for foodborne pathogen C. sakazakii is very important for food safety control. This study has developed a kind of genomic nucleic acid reference material as a positive reference for the rapid detection of C. sakazakii in the edible fungus Flammulina velutipes. First, the genomic nucleic acids were extracted from the standard strain of C. sakazakii, and the nucleic acids were lyophilized. The uniformity test result of the lyophilized powder showed that there were no significant differences between samples. In short-term stability tests at 4°C and 37°C for 14 days, the properties of the nucleic acid reference material were good, and in long-term stability tests at 4°C and −20°C for 8 months, the samples are still stable. To verify the application of this nucleic acid reference material, it was used as a positive control template for PCR detection and applied to the rapid detection of C. sakazakii in F. velutipes. It was found that 13 of the 15 F. velutipes samples were positive for C. sakazakii. As far as we know, this is the first attempt to detect C. sakazakii in F. velutipes using the PCR method with nucleic acid reference material as the positive reference. This study is helpful in promoting the extensive and in-depth application of nucleic acid reference materials in foodborne pathogens detection.


Introduction
Foodborne pathogens are pathogenic bacteria that can cause food poisoning or use food as a medium [1]. ey threaten human health and can cause serious food safety problems [2].
ere are many types of food pathogens, such as diarrheagenic Escherichia coli, Staphylococcus aureus, Salmonella spp., Listeria monocytogenes, and Enterobacter sakazakii. Cronobacter sakazakii, previously called Enterobacter sakazakii, is an facultative anaerobic Gram-negative Bacillus. is pathogenic bacterium could be isolated from a wide variety of foods and has been detected in infant milk powder, meat, water, vegetables, and other foods [3,4]. Infants and young children are high-risk groups of Cronobacter infections [3,5]. e infections mainly cause bacteremia, meningitis, and necrotizing enterocolitis, and the fatality rate is as high as 50%-80% [6]. Edible fungi may be contaminated by foodborne pathogens during planting, harvesting, packaging, and transportation. It is reported that pathogenic bacteria, such as L. monocytogenes, have been detected in edible fungi [7,8]. Cronobacter spp. also could be detected in dried edible macrofungi samples [9]. A recent study showed that the detection rate of Cronobacter spp. is 17.54% (37 positive in 211 samples) in Flammulina velutipes from 44 cities in China [10], indicating that the detection of F. velutipes contamination should be strengthened to avoid food safety issues. e traditional detection method of food pathogenic microorganisms is to conduct biochemical identification after enrichment and cultivation of bacteria in the tested sample [11]. However, this detection method has cumbersome steps and a long time-consuming process, which cannot meet the requirements of rapid foodborne pathogen detection. erefore, the establishment of accurate, sensitive, rapid, and specific detection methods for the detection of food pathogenic microorganisms could play an important role in the prevention and control of food poisoning events. At present, a variety of rapid detection methods have been developed, these methods are mainly polymerase chain reaction (PCR) method and loop-mediated isothermal amplification (LAMP) method based on molecular biology [12], and enzyme-linked immunosorbent assay (ELISA) based on immunology [13]. Among them, the most commonly used methods based on molecular biology are ordinary PCR and fluorescent quantitative PCR [1,14]. And ordinary PCR is widely used in food inspection department because of its easiness to operate with simple steps, short time-consuming, and easy to conduct [1]. e common point of these detection methods is that they require DNA reference materials as reaction templates. e template can be genomic DNA of pathogenic bacteria or plasmid carrying characteristic virulence genes. So the preparation of nucleic acid reference materials of standard strains of foodborne pathogens as positive control substances could play an important role in rapid detection of foodborne pathogens.
In order to provide a positive reference material for the rapid detection of C. sakazakii in food inspection, the study of ready-to-use C. sakazakii nucleic acid reference materials was conducted in this paper, and its application in detection of F. velutipes was also performed. e research results not only provides a positive reference material but also greatly shortens the detection time, saves the cost, and improves the detection accuracy.

Strains and Culture
Condition. Cronobacter sakazakii FC4146 (CMCC 45404) was obtained from National Institute for Food and Drug Control of China. For genome extraction, the bacterial strains were cultured in BHI, and for enrichment of C. sakazakii, the samples to be detected were cultured in modified lauryl sulfate tryptose broth-vancomycin medium (mLST-Vm). , and the genomic DNA were used as templates to amplify the 16S-23S with the same method above, and sterilized water was used as the negative control. e PCR products were analyzed by 1.0% of agarose gel electrophoresis.

Preparation of the
For sensitivity evaluation, the genomic DNA of C. sakazakii were diluted with sterilized water with gradients of 10 − 1 , 10 − 2 , 10 − 3 , 10 − 4 , 10 − 5 , and 10 − 6 . And the diluted samples were applied as templates to amplify 16S-23S. e negative control used sterilized water as template in the reaction. e assessment of PCR products was conducted by 1.0% of agarose gel electrophoresis.

Preparation of Lyophilized Powder of Genomic DNA Reference Material of C. sakazakii.
e genomic samples of C. sakazakii were extracted and diluted to 20 ng/μL; then, the samples were sent to Chinese Academy of Inspection and Quarantine for lyophilization, with 300 μL of genomic DNA each ampoule. e lyophilized powder was stored at − 20°C.

Homogeneity Determination of Genomic DNA Reference
Material of C. sakazakii. Among the 200 bottles of genomic DNA reference materials, 12 bottles were randomly selected, and 300 μL of sterile ddH 2 O was added to hydrate the lyophilized powder; then, 1 μL sample was used to determine the concentration, and each sample was tested under the same condition twice. e test data were processed by SPSS with single factor statistical analysis. And the characteristic sequence 16S-23S was determined for each sample.

Short-Term Stability Test of Genomic DNA Reference Material of C. sakazakii.
e short-term storage stability tests were conducted under the condition of 4°C and 37°C. Forty-two bottles are randomly selected and tested three samples on 1, 3, 5, 7, 9, 11, and 14 days at 4°C and 37°C, respectively. e concentration and the characteristic sequence 16S-23S were determined.

Long-Term Stability Test of Genomic DNA Reference Material of C. sakazakii.
irty bottles of genomic DNA samples were selected randomly from the lyophilized powder samples, and 15 samples were stored at 4°C, and the other samples were stored at − 20°C. e concentration and characteristic 16S-23S sequence amplification tests were conducted at 7 time points in the first, second, fourth, sixth, eighth, and tenth months, respectively. irty samples of fresh Flammulina velutipes were collected from supermarkets and traditional retail markets in July 2020 from Jinan, China. While the samples were obtained, the analyses were managed immediately.

Determination of C. sakazakii in the Edible Fungus Flammulina velutipes.
e needle mushroom (Flammulina velutipes) was obtained from the market and put into the sterile sealed bag in the low temperature insulation box. As the sample arrived at the laboratory, the detection was started to avoid additional pollution. e needle mushroom was taken out from the bag in the aseptic operation table, cut into pieces, and placed into the mLST-Vm enrichment medium.
e enrichment of C. sakazakii was conducted according to the National Food Safety Standard of China Food microbiological examination: Enterobacter sakazakii for powdered infant formula [15]. After enrichment of bacteria, the culture was treated by boiling waterbath for 10 min to release the genomic DNA. en, the genomic DNA was taken as the template to amplify the characteristic 16S-23S sequence. e PCR products were analyzed by 1% agarose gel electrophoresis.

Verification of the Detection of C. sakazakii in the Edible Fungus Flammulina velutipes.
e Flammulina velutipes samples that were positive for the 16S-23S sequence of C. sakazakii detected by PCR were identified according to the method of the National Food Safety Standard of China Food microbiological examination: Enterobacter sakazakii for powdered infant formula [15]. First, 1 loop of the enriched culture was taken and streaked into two chromogenic C. sakazakii agar plates and cultivated at 37°C for 24 h. en, at least 5 suspicious colonies were chosen; if there were less than 5, all the suspicious colonies were taken, inoculated them on the TSA plate, and incubated at 25°C for 48 h. en, the yellow suspicious colonies on the TSA plates were conducted by biochemical identification in the C. sakazakii identification reagent strip.

Preparation of Genomic DNA of C. sakazakii.
e genomic DNA of C. sakazakii was extracted following the steps in the kit instructions, and the concentration and purity of DNA samples were determined using an ultraviolet spectrophotometer (Table 1), and the integrity was analyzed by agarose gel electrophoresis (Figure 1). e concentration of the genomic DNA of C. sakazakii was approximately 50 ng/ μL, and the purity index A260/A280 was more than 1.7, indicating that the sample was not contaminated by protein.
Also on the agarose gel, it could be observed that the genomic DNA band is single and bright, no protein and RNA contamination, consistent with the purity test results, showing that the purity and concentration of the extracted DNA are good, meeting the requirements of subsequent experiments.

Specificity Analysis of Primers of 16S-23S.
e specificity verification of the primers of the characteristic sequence 16S-23S was performed, and the result is shown in Figure 2.
ere was a bright band (282 bp) on the gel while using the genomic DNA of C. sakazakii FC4146 as the template and applying primers of 16S-23S, but no bands could be observed when using other bacterial genomic DNA detected, indicating that the primers for amplifying the sequence of 16S-23S of C. sakazakii is specific.

Sensitive Analysis of Primers of 16S-23S Amplification of C. sakazakii.
e sensitive analysis of the primers for characteristic sequence 16S-23S was detected. e genomic DNA was diluted by gradient dilution, and PCR reaction was conducted using the diluted DNA as template and applying the primers of 16S-23S, and each gradient concentration was detected twice. As shown in Figure 3, the bands of PCR products were observed when the template content was greater than or equal to 0.004 ng, illustrating that the detection sensitivity of primers of 16S-23S was as low as 0.004 ng, also showing that the detection limit of the primers applied was at the 4 pg level using genomic DNA of C. sakazakii as the template.

Uniformity Analysis of Genomic DNA Reference
Materials of C. sakazakii. After the genomic samples were extracted, the samples were divided into 300 μL each bottle, 6 μg in total for lyophilization, and the total number of samples was 200 bottles. Each bottle is an indivisible whole unit, so 15 samples were randomly selected for uniformity inspection. e contents were detected using a UV spectrophotometer, and the statistical analysis of the detection result was conducted by the variance analysis method of one-way ANOVA. ree individual tests were performed for each sample. As shown in Table 2, the average content for 15 samples was 6.00 μg, and the variance of the determination results showed that with 95% confidence probability, F value, 0.289, was less than the F critical value, 2.04, indicating that there is no significant difference between samples. And the characteristic sequence 16S-23S amplification test showed that the PCR products were positive and had a single band on the agarose gel for each test (Figures 4 and 5). Both the results indicate that the uniformity of the sample meets the requirements of the reference material.

Short-Term Stability Analysis of Genomic DNA Reference
Material of C. sakazakii. e genomic DNA reference   Journal of Food Quality materials were stored at 4°C and 37°C for a period of 1, 3, 5, 7, 9, 11, and 14 days, respectively; then, the concentration of each sample was detected, and the contents of genomic DNA were calculated, and statistical analysis by one-way ANOVA was conducted. e F value (0.485) was less than the critical value (1.53) at 95% confidence, and the contents changes of nucleic acid reference material are shown in Figure 6, showing that the genomic nucleic acid reference material was stable for 14 days stored at 4°C or 37°C. Also, PCR result displays a single bright specific band on the agarose gel using the genomic DNA as the template to amplify 16S-23S sequence after stored at 4°C and 37°C, respectively (Figure 7), indicating that the property of genomic DNA reference materials is good.

Long-Term Stability Analysis of Genomic DNA Reference
Material of C. sakazakii. After the genomic DNA reference materials were lyophilized, they were stored at − 20°C. For each test of the long-term stability test, 3 bottles of samples were randomly taken out of the lyophilized samples. At the 1st, 2nd, 4th, 6th, and 8th month, a total of 5 time points were sampled and tested. e content changes among the samples at different time points are shown in Figure 8. e F value of 0.696 was less than the critical value of 1.66 at 95% confidence, showing that the genomic DNA reference material was stable for 8 months stored at 4°C or − 20°C. Figure 9 shows the results of PCR of the characteristic sequence 16S-23S of each sample. e characteristic gene test is positive, and the bands of PCR products were single, indicating that the sample is stable for 8 months.

Detection of Foodborne Pathogen C. sakazakii in Flammulina velutipes.
Fifteen batches of flesh Flammulina velutipes were purchased from supermarkets and traditional retail markets spread over Jinan. e samples were detected immediately while they arrived in the laboratory. e results showed that 16S-23S fragments of C. sakazakii were positive in all tested samples from 8 supermarkets ( Figure 10) and 5 positive of 7 samples from traditional retail markets (Figure 11). In total, the detection rate was 86.7%.  Figure 12, the colonies of no. 1 bacterium isolated from Flammulina velutipes on TSA plate were yellow and on the chromogenic Enterobacter sakazakii agar plate were blue-green (other samples were not shown). e biochemical identification results are shown in Table 3, which were consistent with the positive control of C. sakazakii FC4146. All the results indicated that the bacterium detected in Flammulina velutipes is C. sakazakii.

Discussion
In this study, genomic DNA reference materials were prepared by genomic DNA extraction and lyophilization.
e uniformity analysis showed that the samples have no significant differences among samples, and they were stable at 4°C or 37°C for 14 days, which could meet the requirements of long-distance transportation. e samples keep good as long as 8 months while stored at 4°C or − 20°C. e reference materials were used as the positive control in the detection of Cronobacter sakazakii of edible fungus Flammulina velutipes. is reference material is suitable for the verification, confirmation, and evaluation of PCR detection methods of Cronobacter spp. in food and other samples, quality control of the detection process, personnel assessment, evaluation of laboratory detection capabilities, and interlaboratory comparisons. Also, this reference material could be used as a quantitative tool for Cronobacter spp. using other detective methods. For example, a standard curve could be formed based on this reference material using fluorescence quantitative PCR analysis, and the pathogenic bacteria content of the sample to be tested     Data Availability e data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.