Journal of Food: Microbiology, Safety & Hygiene
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Introduction

Foodborne diseases are global public health issue caused by foodborne pathogens. According to World Health Report, about 1 in 10 people around the world are affected by food contamination resulting in about 420,000 deaths annually by foodborne diseases [1]. Consumers with damaged or weak immune systems and children, especially infants and neonates are more vulnerable to foodborne diseases from contaminated food.

Contamination of Powdered Infant Formula (PIF) with Cronobacter and Salmonella and the management of risk to consumers is a major concern to public health and regulatory officials and manufacturers [2-6]. Salmonella and Cronobacter have been linked to several outbreaks and clear evidence of causality has been established for PIF.  Cronobacter   multi-species complex (previously Enterobacter sakazakii) is a conditional pathogen that can affect infants causing infant meningitis, necrotizing enterocolitis, bacteremia, and neonate deaths [6]. Salmonella infection typically causes diarrhea and, in some infants, bacteremia and meningitis. Several serotypes of Salmonella (Kedougou, Derby, Tennessee, Bredeney, Ailing, Virchow, Anatum and Agona) have been linked to outbreaks with PIF [2,4,7].

The contamination of finished products can occur through raw materials and the processing environment [2,6-8]. The primary source of Cronobacter has been found to be PIF residues, fluid beds, drying areas, floors, and soil adjacent to the production facilities [6,8-10]. The drying tower has been identified as one of the sources of Salmonella [2,4,7].

Mainland China has a birth rate of about 1.14% leading to almost 16 million births/year and about 85% of the newborn are formula-fed [11]. Therefore, the microbial safety and quality of the PIF is very important for China consumers. In 2019, China had over 100 factories producing 730,000 tons of PIFs [9,10]. Current international regulations require zero tolerance for Cronobacter spp. and Salmonella spp. in PIF [4-6]. With the enactment of the 2015 Food Safety Law of the People’s Republic of China, prepackaged foods including general and infant food need to comply with the quality and hygienic test requirements in the applicable Chinese National Food Safety (Guobiao, GB) Standards [12]. The traditional GB 4789.4-2016 (Salmonella) and GB 4789.40-2016 (Cronobacter) methods require 3 to 5 days to provide results [13,14].

While the advances in rapid methods such as immunoassays and PCR have enabled accurate detection of foodborne pathogens [15-17], there is still a need for faster and simpler technology for foodborne pathogen detection to enable PIF producers to assess risks in a timely manner. With the advance of new molecular methods Loop-mediated Isothermal Amplification (LAMP) has emerged as an alternative method to PCR. LAMP can amplify DNA under isothermal conditions (60 to 65°C) with high specificity and sensitivity in 60 min or less [16,18,19]. The DNA amplification is driven by Bst polymerase, a unique enzyme with DNA strand- displacement activity that enables the continuous, rapid isothermal amplification of DNA. LAMP uses multiple primers to recognize distinct regions of the genome and Bst DNA polymerase to provide continuous and rapid amplification of genetic material. An extension of LAMP, LAMP-bioluminescent assay, utilizes LAMP for DNA amplification and bioluminescence for the detection of amplified products [20]. Both amplification and detection occur simultaneously and continuously during the exponential phase providing real-time results and a short run time.

The LAMP-bioluminescent assays, 3M Molecular Detection System Assay 2-Cronobacter and 3M Molecular Detection Assay 2-Salmonella have been used for detection in a variety of food matrices and environmental samples and have been shown to be equivalent to standard culture methods [21,22]. The objective of this study was to determine the specificity and sensitivity of the respective LAMP assays to detect Salmonella and Cronobacterin PIF, related raw materials and environmental samples as compared to the respective traditional GB method (GB 4789.4-2016 and GB 4789.402016).

Materials and Methods

Inoculum preparation

Cronobacter sakazakii (ATCC 29544, American Type Culture Collection, Manassas, VA, USA), Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 14028) and Escherichia coli (ATCC 25922) were used for inoculation of matrices in different experiments. The strains obtained were streaked onto nutrient agar and incubated for 24 hours at 37°C. To prepare inoculum, an isolated colony from nutrient agar plate was inoculated into 100 mL of brain heart infusion broth using a sterile inoculating loop and incubated for 24 hours at 37°C. After incubation, serial 10- fold dilutions of cultures were prepared in Butterfield’s phosphate buffer (pH 6.8), plated on 3M Petrifilm Aerobic Count Plate (3M Food Safety, St. Paul, MN, USA) and incubated at 37°C for 24 hours. The colonies on the plates were counted, and an average count of each dilution was used to determine the appropriate amount of inoculum to add to each sample.

Method comparison study

In a paired study, 185 samples including raw materials (n=50), PIF (n=125), and environmental (n=10) samples were used for detection of Cronobacter. The samples were analyzed by the LAMP method and compared with the reference GB method for the detection of Cronobacter spp. For Salmonella detection, 74 samples including raw materials (n=20), PIF (n=44), and environmental (n=10) samples were used for detection of Salmonella. Out of these samples, 14 PIF samples and 10 environmental samples had dual inoculation of Cronobacter and Salmonella. In addition, 10 PIF samples had E. coli as an interferent organism.

Enrichment of samples

For uninoculated samples, 100 g of the raw materials (n=21) and PIF (n=37) samples were weighted into a stomacher bag and enriched in 900 ml pre-warmed BPW ISO for 18 hours at 37â??. For inoculated samples, 100 g of the raw materials (n=29) and PIF (n=64) samples were weighted into a stomacher bag and inoculated with 100 microlitre inoculum to obtain about 0.1-10 CFU/raw materials sample and 1-10 CFU/PIF sample. In separate experiments, PIF and environmental samples were inoculated with both C. sakazakii and S. Typhimurium (10 or 100 CFU of each/sample). In addition, E. coli (about 100 CFU) was used as an interferent with some of the samples. The PIF samples were enriched in 900 mL pre-warmed BPW ISO for 18 hours at 37â??, Environmental samples were collected from a processing facility using 3M Hydrated Sponge with neutralizing buffer (3M Food Safety). The environmental samples were enriched in 225 mL of BPW ISO for 18 hours at 37°C. After enrichment, samples were analyzed by the LAMP assays and GB method.

Cronobacter and Salmonella detection

The enriched samples were tested with the Cronobacter LAMP assay (MDA2CRO) and Salmonella LAMP assay (MDA2SAL) obtained from 3M Food Safety. A 20 µL of sample after enrichment was collected and processed for detection following manufacturer’s instructions [21,22]. All samples were culture-confirmed following the GB 4789.40-2016 (Cronobacter) (Figure 1) and GB 4789.4-2016 (Salmonella) (Figure 2). All bacterial culture media for the GB method were obtained from Beijing Land Bridge Technology Co. Ltd., Beijing, China. Biochemical confirmation of isolated colonies was done using API 20E strips (bioMérieux China Limited, Beijing, China) [13,14].

Figure 1: Paired analysis with the LAMP assay, 3M Molecular Detection Assay 2 - Cronobacter and the GB 4789.40-2016 method.

Figure 2: Paired analysis with the LAMP assay, 3M Molecular Detection Assay 2 - Salmonella and the GB 4789.4-2016 method.

Analysis of results

Presumptive results obtained for Cronobacter and Salmonella detection with the LAMP assays were compared with the culture- confirmed results. Probability of Detection (POD) was computed for the LAMP method (POD alternate, PODa) and the culture confirmation by GB method (POD reference, PODr) and used as a statistical model to compare the LAMP method to reference method [23]. The difference between PODa and PODr, dPOD was computed and 95% confidence interval for POD (paired analysis) was calculated. The specificity and sensitivity of each LAMP method was calculated according to ISO 16140-2: 2016 [24].

Results

Cronobacter detection

For the raw material samples, two samples out of 21 uninoculated and 9 samples out of 29 inoculated were positive by the LAMP assay. For the PIF samples, three samples out of 37 uninoculated and 62 samples out of 63 inoculated samples were positive by the LAMP assay. The presumptive positives (two from each of uninoculated raw materials and PIF samples and one from each of inoculated raw material and PIF samples) were not confirmed by the GB culture method .With dual inoculated PIF and environmental samples, presumptive results from the LAMP assay for Cronobacter were in complete agreement with the GB culture method. For dual inoculated PIF samples with E. coli as an interferent organism, three presumptive positive samples could not be confirmed by the culture method . The specificity and sensitivity of the Cronobacter LAMP assay for the matrices tested was 91% and 100%, respectively. The raw material samples inoculated with Cronobacter had low positive rate (31%) compared to PIF (96%). This could be due to the inhibitory effect of raw materials on growth of Cronobacter (Table 1).

Table 1: Paired comparison between the LAMP assay and the GB method for the detection of Cronobacter in PIF and related matrices.

Cronobacter LAMP assay had few false-positives (9 out of 185 samples) with the samples tested as some of the presumptive positive results could not be confirmed by the culture method. The LAMP assa had no false-negatives with any of the samples tested. It is possible that the samples had non-viable cells or free DNA from dead cells, and this may have contributed to false-positive results. Molecular methods including LAMP are not able to distinguish non-viable from viable cells leading to false-positive results [16,17,25]. PIF are not necessarily sterile and spray-drying used does not act as a kill step [6,17,26-28]. Cronobacter can survive the drying process and the cells may be damaged and not culturable. Also, nucleic acids are relatively stable even after cell death and may be present in food matrices after heat treatment [6,28]. Hence, it is possible that the samples had free DNA or non-culturable cells leading to false-positive results. Methods have been developed using DNase I treatment before extraction of DNA from viable cells to prevent false-positive results [29]. DNase I treatment was not evaluated in this study.

Salmonella detection

Two samples out of 20 inoculated raw material samples and all 20 inoculated PIF samples were positive by the LAMP assay and all the presumptives results were confirmed by the GB culture method. With dual inoculated PIF and environmental samples and PIF samples with E. coli as an interferent organism, presumptive results from the Salmonella LAMP assay were in complete agreement with the GB culture method. Both specificity and sensitivity of the Salmonella LAMP assay was 100%. The raw material samples inoculated with Salmonella had low positive rate (10%) compared to PIF (100%). This could be due to the inhibitory effect of raw materials on growth of Salmonella (Table 2).

Table 2: Paired comparison between the LAMP assays and the GB method for the detection of Salmonella in PIF and related matrices.

Data analysis

Analysis of dPOD for PIF, raw materials and environmental samples showed that the detection of Cronobacter spp. with the Cronobacter LAMP assay was not significantly different from the GB 4789.40-2016 reference method (Table 3). Similarly, the detection of Salmonella in PIF, raw materials and environmental samples by the Salmonella LAMP assay was not significantly different from the GB reference method (Table 4).

Table 3: POD analysis for paired comparison of Cronobacter detection in PIF and related matrices.

Table 4: POD analysis for paired comparison of Salmonella detection in PIF and related matrices.

Discussion

LAMP is recognized throughout the scientific literature as a highly robust, efficient, sensitive, specific, and simple nucleic acid amplification technique [16,18,19]. LAMP uses a unique DNA polymerase for continuous DNA amplification that is resistant to matrix interference and inhibitors [7,16,18,19,30]. LAMP assays have the same or higher sensitivity compared to PCR assays and traditional culture methods in detecting foodborne pathogens, such as Salmonella spp., Campylobacter spp., Listeria spp., and Listeria monocytogenes, from various food matrices [16,21,22,30- 33]. There have been limited studies on the comparison of rapid detection methods to GB standard methods. China’s PIF industry is growing, and consumers are concerned about the potential risk of these pathogens in PIF. The PIF producers need rapid, easy to use and specific detection of Cronobacter and Salmonella for monitoring of raw materials, process environment and finished products for implementing effective control measures to prevent contamination [34,35].

Conclusion

This study compared the LAMP assays against the GB method for detection of Cronobacter and Salmonella in PIF and related matrices. The study also evaluated the detection of both target organisms in the same enrichment and the LAMP assays detected both organisms equally well without any interference. The results of LAMP assays were similar to the GB method and provided next- day results compared to the GB method requiring 3 to 5 days. In addition, interference has been observed, especially for Cronobacter on typical agars used for isolation.

Bacteria, like Franconibacter spp. and Siccibacter spp. show typical Cronobacter phenotype on chromogenic agar and need further biochemical confirmation. While colony confirmation is still relevant to laboratory testing, it is also important to recognize the higher specificity of molecular detection methods for pathogen testing which allow next-day results as compared to 3 to 5 days for traditional testing. DNA-based assays target specific genes of the target bacterium offering sensitive and specific detection. The LAMP assays used in this study offered an easy-to-use analytical tool to assess the prevalence of Cronobacter and Salmonella in PIF, raw material and environmental samples.

Author's Contribution

Conceptualization, Yan Huang, Jianwei Huo, Zhiyong Dai, Chenyan Niu, Xiqing Wang, Can Yi, Jichao Liu, Jun Zhou, Feng Liu, Qing Tao; methodology, Jianwei Huo, Yan Huang, and Raj Rajagopal; data generation, Jianwei Huo, Yan Huang, Qing Tao; data analysis, Yan Huang, Raj Rajagopal; Writing-original draft, Raj Rajagopal; writing - review and editing, Yan Huang and Raj Rajagopal. All authors have read and agreed to the submitted version of the manuscript.

Conflict of Interest

The authors, Jianwei Huo, Yan Huang, and Raj Rajagopal are employees of 3M Food Safety which offers multiple commercial solutions, including 3M Molecular Detection system and 3M Molecular Detection Assays to the food industry. Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Ausnutria Dairy (China) Co., Ltd Beijing Sanyuan Foods Co., Ltd., and Synutra Nutritional Food Co., Ltd.

References

1. Angulo F J, Cahill S M, Kaye W I, Lourdes C M D and Ben E P K (2008) Powdered infant formula as a source of Salmonella infection in infants. Clinical Inf Di. 2008; 2 : 268-273.
2. Bird P, Benzinger J, Bastin B, Crowley E, Agin J, Goins D and Monteroso L (2019) Evaluation of the 3M™ Molecular Detection Assay (MDA) 2 – Cronobacter for the detection of Cronobacter species in select foods and environmental surfaces: collaborative study, first action 01. J AOAC Int. 2018; 102:108-117.
3. Bird P, Flannery J, Crowley E, Agin J R, Goins D and Monteroso L . Evaluation of the 3M™ Molecular Detection Assay (MDA) 2 - Salmonella for the detection of Salmonella spp. in select foods and environmental surfaces: collaborative study, first action J AOAC Int. 2016 ;99(4):980-997
4. Ceuppens S, Li D, Uyttendaele M, Renault P, Ross P, Ranst M V, et al. Molecular methods in food safety microbiology: interpretation and implications of nucleic acid detection. Compr Rev Food Sci Food Saf. 2014;13(4):551-577
5. Chap J, Jackson P, Siqueira R, Gaspar N , Forsythe S J .International survey of Cronobacter sakazakii and other Cronobacter spp. in follow up formulas and infant foods. Int J Food Microbiol. 2009;136(2):185-188.
6. Du X J, Wang X Y, Dong X, Li P and Wang S (2018) Characterization of the desiccation tolerance of Cronobacter sakazakii strains. Front Microbiol .2018;9:2867
7. Fei P, Jiang Y, Feng J, Forsythe S J, Li R, Zhou Y and Man C (2017) Antibiotic and desiccation resistance of Cronobacter sakazakii and C. malonaticus isolates from powdered infant formula and processing environments. Front Microbiol. 2017 2;8:316.
8. Food and Agriculture Organization, World Health Organization, Enterobacter sakazakii and Salmonella in powdered infant formula. Food and Agriculture Organization, World Health Organization Microbiological Risk Assessment Series No. 10, World Health Organization, Geneva, Switzerland (2006).
9. Food and Agriculture Organization, World Health Organization , Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formulae. Food and Agriculture Organization, World Health Organization Microbiological Risk Assessment Series No. 15, World Health Organization, Geneva, Switzerland (2008).
10. Forsythe S J . Updates on the Cronobacter Genus. Annu Rev Food Sci Technol .2018;9:923-944.
11. Gandelman O A, Church V L, Moore C A, Kiddle G, Carne C A, Parmar S, et al .Novel bioluminescent quantitative detection of nucleic acid amplification in real-time. 2010;5: e14155.
12. Guobiao Standard . National Standard of the People’s Republic of China. National food safety standard - food microbiological examination - Salmonella. GB 4789.4-2016.
13. Guobiao Standard. National Standard of the People’s Republic of China. National food safety standard - food microbiological examination - examination of Cronobacter (Enterobacter sakazakii). GB 4789.40-2016.
14. Hu L, Deng X, Brown E W, Hammack T S, Ma L M and Zhang G . Evaluation of Roka Atlas Salmonella method for the detection of Salmonella in egg products in comparison with culture method, real-time PCR and isothermal amplification assays. Food Control. 2018:94;123-131.
15. International Organization for Standardization, Microbiology of the food chain — method validation — Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method. International Organization for Standardization, Geneva. ISO 16140-2.  (2016).
16. Iversen C, Druggan P and Forsythe S. A selective differential medium for Enterobacter Sakazakii, a preliminary study. Int J Food Microbiol. 2004;96(2):133-139.
17. Jacobs C, Braun P and Hammer P . Reservoir and routes of transmission of Enterobacter sakazakii (Cronobacter spp.) in a milk powder-producing plant. J Dairy Sci
18. 2011;94(8):3801-3810
19. Ling N, Jiang Y, Zeng H, Ding Y and Forsythe S.  Advances in our understanding and distribution of the Cronobacter genus in China. J Food Sci. 2021;86(2):276-283.
20. Lu Y, Liu P, Li C, Sha M, Fang J, Gao J, et al., Prevalence and genetic diversity of Cronobacter species isolated from four infant formula production factories in china Front Microbiol. 2019;10:1938.
21. Mangal M, Bansal S, Sharma S K and Gupta R K (2016) Molecular detection of foodborne pathogens: A rapid and accurate answer to food safety. Crit Rev Food Sci Nutr.2016;56(9):1568-1584
22. Mori Y, Kanda H and Notomi T (2013) Loop-mediated isothermal amplification (LAMP): Recent progress in research and development. J Infect Chemother. 2013 ;19(3):404-11
23. Niessen L, Luo J, Denschlag C and Vogel R F (2013) The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food Microbiol. 2013 ;36(2):191-206.
24. Notomi T, Mori Y, Tomita N and Kanda H . Loop-mediated isothermal amplification (LAMP): Principle, features, and future prospects. J Microbiol .2015;53(1):1-5.
25. Osaili T and Forsythe S . Desiccation resistance and persistence of Cronobacter species in infant formula. Int J Food Microbiol. 2009 ;136(2):214-220.
26. Svobodová B, Vlach J, Junková P, Karamonová L, Blažková M and Fukal L. Novel method for the reliable identification of Siccibacter and Franconibacter strains: from 'pseudo-Cronobacter' to new Enterobacteriaceae genera. Applied and Environmental Microbiology. 2017; 83: e00234-17.
27. Rajagopal R, Barnes C A, David J M, Goseland J. and Goseland J . Evaluation of a commercial loop-mediated isothermal amplification assay, 3M Molecular Detection Assay 2 - Campylobacter, for the detection of Campylobacter from poultry matrices. Br Poult Sci. 2021;62(3):404-413
28. Tang K, Liu Y, Meng K, Jiang L and Chen J. Breastfeeding duration of different age groups and its associated factors among Chinese women: A cross-sectional study. Int Breastfeed . 2019 ;14:19
29. Wang Z, Fan Y, Zhang Z and Godefroy S (2017) Food safety standards,. In Food safety in China: Science, technology, management and regulation, pp. 363-380.
30. Wehling P, Labudde R A, Brunelle S L and Nelson M T (2011) Probability of detection (POD) as a statistical model for the validation of qualitative methods. J AOAC Int.2011;94(1):335-347
31. Villarreal J V, Jungfer C, Obst U and Schwartz T. DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses. J Microbiol Methods. 2013;94(3):161-169
32. World Health Organization (2015) World Health Organization estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group 2007-2015. https://apps.who.int/iris/bitstream/handle/10665/199350/9789241565165_eng.pdf. Accessed July 1, 2021.
33. Wiedmann M, Wang S, Post L and Nightingale K (2014) Assessment criteria and approaches for rapid detection methods to be used in the food industry. J Food Prot . 2014 ;77(4):670-690
34. Yang B, Zhao H, Cui S, Wang Y, Xia X and Xi M . Prevalence and characterization of Salmonella enterica in dried milk-related infant foods in Shaanxi, China. J Dairy Sci 2014 ;97(11):6754-6760
35. Yang Q, Domesle K J and Ge B (2018) Loop-mediated isothermal amplification for Salmonella detection in food and feed: current applications and future directions. Foodborne Pathog Dis.2018 ;15(6):309-331

References

1. Angulo F J, Cahill S M, Kaye W I, Lourdes C M D and Ben E P K (2008) Powdered infant formula as a source of Salmonella infection in infants. Clinical Inf Di. 2008; 2 : 268-273.
2. Bird P, Benzinger J, Bastin B, Crowley E, Agin J, Goins D and Monteroso L (2019) Evaluation of the 3M™ Molecular Detection Assay (MDA) 2 – Cronobacter for the detection of Cronobacter species in select foods and environmental surfaces: collaborative study, first action 01. J AOAC Int. 2018; 102:108-117.
3. Bird P, Flannery J, Crowley E, Agin J R, Goins D and Monteroso L . Evaluation of the 3M™ Molecular Detection Assay (MDA) 2 - Salmonella for the detection of Salmonella spp. in select foods and environmental surfaces: collaborative study, first action J AOAC Int. 2016 ;99(4):980-997
4. Ceuppens S, Li D, Uyttendaele M, Renault P, Ross P, Ranst M V, et al. Molecular methods in food safety microbiology: interpretation and implications of nucleic acid detection. Compr Rev Food Sci Food Saf. 2014;13(4):551-577
5. Chap J, Jackson P, Siqueira R, Gaspar N , Forsythe S J .International survey of Cronobacter sakazakii and other Cronobacter spp. in follow up formulas and infant foods. Int J Food Microbiol. 2009;136(2):185-188.
6. Du X J, Wang X Y, Dong X, Li P and Wang S (2018) Characterization of the desiccation tolerance of Cronobacter sakazakii strains. Front Microbiol .2018;9:2867
7. Fei P, Jiang Y, Feng J, Forsythe S J, Li R, Zhou Y and Man C (2017) Antibiotic and desiccation resistance of Cronobacter sakazakii and C. malonaticus isolates from powdered infant formula and processing environments. Front Microbiol. 2017 2;8:316.
8. Food and Agriculture Organization, World Health Organization, Enterobacter sakazakii and Salmonella in powdered infant formula. Food and Agriculture Organization, World Health Organization Microbiological Risk Assessment Series No. 10, World Health Organization, Geneva, Switzerland (2006).
9. Food and Agriculture Organization, World Health Organization , Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formulae. Food and Agriculture Organization, World Health Organization Microbiological Risk Assessment Series No. 15, World Health Organization, Geneva, Switzerland (2008).
10. Forsythe S J . Updates on the Cronobacter Genus. Annu Rev Food Sci Technol .2018;9:923-944.
11. Gandelman O A, Church V L, Moore C A, Kiddle G, Carne C A, Parmar S, et al .Novel bioluminescent quantitative detection of nucleic acid amplification in real-time. 2010;5: e14155.
12. Guobiao Standard . National Standard of the People’s Republic of China. National food safety standard - food microbiological examination - Salmonella. GB 4789.4-2016.
13. Guobiao Standard. National Standard of the People’s Republic of China. National food safety standard - food microbiological examination - examination of Cronobacter (Enterobacter sakazakii). GB 4789.40-2016.
14. Hu L, Deng X, Brown E W, Hammack T S, Ma L M and Zhang G . Evaluation of Roka Atlas Salmonella method for the detection of Salmonella in egg products in comparison with culture method, real-time PCR and isothermal amplification assays. Food Control. 2018:94;123-131.
15. International Organization for Standardization, Microbiology of the food chain — method validation — Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method. International Organization for Standardization, Geneva. ISO 16140-2.  (2016).
16. Iversen C, Druggan P and Forsythe S. A selective differential medium for Enterobacter Sakazakii, a preliminary study. Int J Food Microbiol. 2004;96(2):133-139.
17. Jacobs C, Braun P and Hammer P . Reservoir and routes of transmission of Enterobacter sakazakii (Cronobacter spp.) in a milk powder-producing plant. J Dairy Sci
18. Ling N, Jiang Y, Zeng H, Ding Y and Forsythe S.  Advances in our understanding and distribution of the Cronobacter genus in China. J Food Sci. 2021;86(2):276-283.
19. Lu Y, Liu P, Li C, Sha M, Fang J, Gao J, et al., Prevalence and genetic diversity of Cronobacter species isolated from four infant formula production factories in china Front Microbiol. 2019;10:1938.
20. Mangal M, Bansal S, Sharma S K and Gupta R K (2016) Molecular detection of foodborne pathogens: A rapid and accurate answer to food safety. Crit Rev Food Sci Nutr.2016;56(9):1568-1584
21. Mori Y, Kanda H and Notomi T (2013) Loop-mediated isothermal amplification (LAMP): Recent progress in research and development. J Infect Chemother. 2013 ;19(3):404-11
22. Niessen L, Luo J, Denschlag C and Vogel R F (2013) The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food Microbiol. 2013 ;36(2):191-206.
23. Notomi T, Mori Y, Tomita N and Kanda H . Loop-mediated isothermal amplification (LAMP): Principle, features, and future prospects. J Microbiol .2015;53(1):1-5.
24. Osaili T and Forsythe S . Desiccation resistance and persistence of Cronobacter species in infant formula. Int J Food Microbiol. 2009 ;136(2):214-220.
25. Svobodová B, Vlach J, Junková P, Karamonová L, Blažková M and Fukal L. Novel method for the reliable identification of Siccibacter and Franconibacter strains: from 'pseudo-Cronobacter' to new Enterobacteriaceae genera. Applied and Environmental Microbiology. 2017; 83: e00234-17.
26. Rajagopal R, Barnes C A, David J M, Goseland J. and Goseland J . Evaluation of a commercial loop-mediated isothermal amplification assay, 3M Molecular Detection Assay 2 - Campylobacter, for the detection of Campylobacter from poultry matrices. Br Poult Sci. 2021;62(3):404-413
27. Tang K, Liu Y, Meng K, Jiang L and Chen J. Breastfeeding duration of different age groups and its associated factors among Chinese women: A cross-sectional study. Int Breastfeed . 2019 ;14:19
28. Wang Z, Fan Y, Zhang Z and Godefroy S (2017) Food safety standards,. In Food safety in China: Science, technology, management and regulation, pp. 363-380.
29. Wehling P, Labudde R A, Brunelle S L and Nelson M T (2011) Probability of detection (POD) as a statistical model for the validation of qualitative methods. J AOAC Int.2011;94(1):335-347
30. Villarreal J V, Jungfer C, Obst U and Schwartz T. DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses. J Microbiol Methods. 2013;94(3):161-169
31. World Health Organization (2015) World Health Organization estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group 2007-2015. https://apps.who.int/iris/bitstream/handle/10665/199350/9789241565165_eng.pdf. Accessed July 1, 2021.
32. Wiedmann M, Wang S, Post L and Nightingale K (2014) Assessment criteria and approaches for rapid detection methods to be used in the food industry. J Food Prot . 2014 ;77(4):670-690
33. Yang B, Zhao H, Cui S, Wang Y, Xia X and Xi M . Prevalence and characterization of Salmonella enterica in dried milk-related infant foods in Shaanxi, China. J Dairy Sci 2014 ;97(11):6754-6760
34. Yang Q, Domesle K J and Ge B (2018) Loop-mediated isothermal amplification for Salmonella detection in food and feed: current applications and future directions. Foodborne Pathog Dis.2018 ;15(6):309-331