Loop-Mediated Isothermal Amplification vs. Guobiao Standards Method for Detection of Salmonella in Yoghurt and Yoghurt-Based Drinks

Huo, Jianwei; Huang, Yan; Rajagopal, Raj

ABSTRACT

Background and Objective: Salmonella has emerged as an important pathogen in several products beyond traditional poultry products. Salmonella is not considered a risk in fermented foods due to low pH and the presence of probiotic organisms. However, Salmonella can survive in these products and grow given appropriate conditions. It is essential to detect rapidly and accurately contamination of Salmonella in these products. The objective of this study was to optimize growth conditions for Salmonella in yoghurt and yoghurt-based drinks and compare the Loop-mediated Isothermal Amplification (LAMP) assay to the traditional Guobiao standards (GB) 4789.4-2016 method for Salmonella detection. Materials and Methods: Twenty-five grams of different types of yoghurt were inoculated with various levels of Salmonella and enriched in Buffered Peptone Water (BPW) ISO (1:10 and 1:20 dilution) at 41.5°C for 24 hrs. For control, uninoculated samples were enriched similarly. All the samples were analyzed with a Salmonella LAMP-bioluminescent assay and culture-confirmed using GB 4789.4-2016 method. Results: Salmonella failed to grow to detectable levels in yoghurt samples with 1:10 enrichments in BPW ISO even at high levels of artificial contamination (1000 CFU/25 g). The pH was reduced to 4.2-4.3 after enrichment. However, with 1:20 BPW ISO enrichments, Salmonella grew to detectable levels at low spike levels (about 3 CFU/25 g) and was detected by both methods. Conclusion: The alternative LAMP assay enabled reliable and rapid detection of Salmonella in yoghurt and yoghurt-based drinks providing next-day results compared to 3 to 5 days for the GB method.

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INTRODUCTION

Salmonella, a Gram-negative bacterium, is globally recognized as a major cause of foodborne infection in humans. CDC estimates that about 1.35 million illnesses occur each year in the US due to salmonellosis and food is the source of most of these illnesses¹. Acute Gastrointestinal Illness (AGI) is a significant burden in China with about 748 million cases of AGI and 420 million healthcare visits each year². It was estimated that about 209 million cases of the foodborne disease occurred in China in 2010-2011². The most common foodborne pathogens involved in outbreaks in China are Salmonella species, Vibrio parahaemolyticus, Staphylococcus aureus and diarrheagenic Escherichia coli³, with Salmonella being the main target pathogen detected through microbiological food safety surveillance⁴.

Though Salmonella is primarily associated with poultry, it can contaminate a variety of food such as meat, eggs, milk, seafood, vegetables, fruits and even chocolate, ice cream and peanut butter¹. However, there are limited studies on Salmonella growth and detection in fermented products. It is generally believed that Salmonella and other foodborne pathogens do not survive the fermentation process and do not pose a threat to human health. However, Salmonella has been shown to survive in low acid foods such as juices and fermented products such as yoghurt^5-10. Although no outbreak of Salmonella has been linked to yoghurt, there is a need for rapid and cost-effective methods to detect Salmonella in fermented products such as yoghurt and yoghurt-based drinks to ensure the safety of these products.

China is a growing market for both food production and consumption and the dairy industry has been growing over the years. China yoghurt segment revenue accounted for more than USD 37 million in 2020 and CAGR is expected at 5.1% between 2020-2025¹¹. With the enactment of the 2015 Food Safety Law of the People’s Republic of China, prepackaged foods including general food and infant food need to comply with the quality and hygienic test requirements in the applicable Chinese National Food Safety (Guobiao, GB) Standards¹². Yoghurt belongs to the category of fermented milk in China with a minimum level of 1×10⁶ (CFU g^–¹) of lactic acid bacteria and titratable acidity of >70°T¹³. According to GB standard (GB 19302-2010), yoghurt is a product made of raw cow milk or goat milk or dry milk through a procedure of pasteurization and fermentation with Streptococcus thermophilus and Lactobacillus bulgaricus¹³. Per GB standard (GB 19302-2010) for fermented milk, the yoghurt samples have zero tolerance for Salmonella in 25 g samples¹³. The traditional GB 4789.4-2016 culture method for Salmonella detection requires 3-5 days¹⁴. Although fermented products such as yoghurt pose a low risk to consumers, effective control measures are critical to prevent foodborne infections and rapid detection methods enable quicker action to prevent foodborne outbreaks.

Loop-mediated Isothermal Amplification (LAMP) can amplify DNA under isothermal conditions (60-65°C) with high specificity and sensitivity in 60 min or less^15-19. 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^15-19. An extension of LAMP, LAMP-bioluminescent assay, utilizes LAMP for DNA amplification and bioluminescence for the detection of amplified products²⁰. Both amplification and detection occur simultaneously and continuously during the exponential phase providing real-time results and a short run time. The Salmonella LAMP-bioluminescent assay, 3M Molecular Detection Assay 2-Salmonella (MDA2SAL) has been used for the detection of Salmonella in a variety of food matrices^21-25and is equivalent to standard culture methods.

The objective of this study was to evaluate the performance of a Salmonella LAMP-bioluminescent assay for the detection of Salmonella in yoghurt and yoghurt-based drinks manufactured in China as compared to culture confirmation by the GB 4789.4-2016 method.

MATERIALS AND METHODS

Study area: The study was conducted at 3M China Research and Development Center in Shanghai, China in 2019 and 2020.

Inoculum preparation: Salmonella enterica serovar Paratyphi Type B (CMCC 50094, National Center for Medical Culture Collection, Beijing, China) and E. coli (ATCC 25922, American Type Culture Collection, Manassas, VA, USA) isolates were used in this study. The strains obtained were streaked onto nutrient agar and incubated for 24 hrs at 37°C. To prepare Salmonella or E. coli inoculum, an isolated colony from nutrient agar plate was inoculated into 100 mL of brain heart infusion broth (Beijing Land Bridge Technology Co. Ltd., Beijing, China) using a sterile inoculating loop and incubated for 24 hrs at 37°C. After incubation, serial 10-fold dilutions of cultures were prepared in buffered peptone water (BPW, 3M Food Safety, St. Paul, MN) and plated on 3M Petrifilm Aerobic Count Plate (3M Food Safety) and incubated at 37°C for 24 hrs. The colonies on the plates were counted and an average count of each dilution was used to determine the appropriate amount of inoculum added to each sample.

Enrichment of samples: A variety of yoghurt samples (low-fat avocado, green lemon, kiwi fruit yoghurt, caramel yoghurt, strawberry and mulberry flavoured yoghurt, pure yoghurt with no sugar, and yoghurt-based drinks) were collected from a local supermarket. Labels on all samples indicated >10⁷ CFU mL^–¹ of lactic acid bacteria. Samples were equilibrated to room temperature and 25 g samples were weighed into a sample enrichment bag (650 mL 3M Plain Sample bag, 3M Food Safety). In the initial trial, 225 mL of BPW ISO (3M Food Safety) was added to each of the samples (1:10 dilution) and inoculated with about 100-1000 CFU of Salmonella per sample. The samples were thoroughly mixed and incubated at 41.5°C for 30 hrs. Samples were tested by the LAMP assay at 24, 26, 28 and 30 hrs of incubation and culture-confirmed by GB 4789.4-2016 method at 30 hrs of incubation. In subsequent experiments, 25 g samples were tested with 1:20 dilution (475 mL of BPW ISO, 3M Food Safety). Several uninoculated and inoculated (about 3 and 78 CFU/25 g) samples were tested by the LAMP assay after incubation at 41.5°C for 24 hrs. Also, for some of the samples, E. coli at about 100 CFU/25 g was used as an interferent organism.

Lactic acid bacteria enumeration: The lactic acid bacteria count and the pH for each of the matrices was determined beforehand after enrichment. GB 4789.35-2016 requires enumeration of lactic acid bacteria on De Man Rogosa Sharpe (MRS) agar (anaerobic incubation) and S. thermophilus on Modified Chalmers (MC) agar (aerobic incubation)²⁶. For lactic acid bacteria enumeration, samples from enrichment bags were serially diluted in Butterfield’s buffer (3M Food Safety) and plated on 3M Petrifilm Lactic Acid Bacteria Count plates (3M Food Safety) and incubated at 36°C for 48 hrs. For S. thermophilus enumeration, serially diluted samples were plated on MC agar and incubated at 36°C for 72 hrs. According to GB 4789.35-2016, the lactic acid bacteria count includes a total of lactic acid bacteria counts from anaerobic incubation and S. thermophilus counts on MC agar²⁶.

Salmonella detection: All enriched samples were tested with a Salmonella LAMP assay, MDA2SAL²¹ (3M Food Safety). A 20 μL of the sample after enrichment was collected and processed for detection following manufacturer’s instructions²¹. All primary enrichments were confirmed following GB 4789.4-2016 method¹⁴. The flow chart for the detection of Salmonella in yoghurt samples is shown in Fig. 1. All bacterial culture media for the GB method were obtained from Beijing Land Bridge Technology Co. Ltd. Biochemical confirmation of isolated colonies was done using API 20E strips (bio Mérieux China Limited, Beijing, China).

Image for - Loop-Mediated Isothermal Amplification vs. Guobiao Standards Method for Detection of Salmonella in Yoghurt and Yoghurt-Based Drinks

Fig. 1:

Flow chart for detection of Salmonella in yoghurt samples by the LAMP method²¹ and the GB 4789.4-2016 reference method¹⁴

Analysis of results: Presumptive results obtained for Salmonella detection with the LAMP assay were compared with the culture-confirmed results. Probability of Detection (POD) was computed for the LAMP method (POD alternate, POD_a) and the culture confirmation by GB method (POD reference, POD_r) and used as a statistical model to compare the LAMP method to the GB method²⁷. The difference between POD_a and POD_r, dPOD was computed and a 95% confidence interval for POD was calculated.

RESULTS AND DISCUSSION

In initial experiments, Salmonella failed to grow to detectable levels in 1:10 enrichments with the yoghurt samples tested even after 30 hrs of incubation at 41.5°C. Neither the LAMP assay nor the GB method was able to detect Salmonella with 1:10 enrichments in 16 out of 20 samples tested (Table 1). Salmonella was detected only in yoghurt-based drinks. Salmonella failed to grow in yoghurt samples even at high levels of artificial contamination (about 1000 CFU/25 g). The pH of BPW ISO was reduced to 4.2-4.3 after enrichment for all the yoghurt samples except yoghurt-based drinks which had a pH of 5.0 (Table 2). As the pH of yoghurt-based drinks was about 5.0, it probably allowed Salmonella growth.

In subsequent experiments, samples were tested with 1:20 dilution and Salmonella grew to detectable levels in 24 hrs of incubation at 41.5°C (Table 1). The pH of BPW ISO was reduced to 4.6-5.6 after enrichment for samples tested (Table 2). Samples did not show any natural contamination and inoculated samples were detected by the LAMP assay (Table 1). All the presumptive results were confirmed by the GB 4789.4-2016 culture method¹⁴. Based on these results, 1:20 dilution of samples enabled LAMP assay to provide next-day results for Salmonella detection. The presence of interferent organisms (E. coli) in Salmonella inoculated samples did not affect the detection of Salmonella and the LAMP assay did not detect E. coli in samples inoculated with E. coli alone.

The yoghurt samples had at least 10⁷ CFU g^–¹ of lactic acid bacteria based on the label and the actual counts varied from 10⁶-10⁹ CFU g^–¹ (Table 2). The lactic acid bacteria count for the yoghurt samples did not change appreciably between 1:10 and 1:20 enrichments (Table 2). The initial pH of the matrices was in the range of 6.7-6.9. After enrichment for 24 hrs, the pH of 1:10 dilution samples was reduced to 4.2-4.3 indicating that the amount of BPW ISO was not enough to neutralize the pH to enable Salmonella growth. With 1:20 dilution, the pH of the enrichment after 24 hrs of incubation was 4.6-5.6 and Salmonella was able to grow at this level of pH. In additional experiments, vancomycin at 10 μg mL^–¹ was added to 1:10 enrichments to suppress lactic acid bacteria growth and this also enabled Salmonella growth (data not shown). However, 1:20 dilution is a better approach as it avoids the unnecessary use of antibiotics in testing.

Analysis of dPOD computed for the yoghurt samples (1:20 dilution) showed that the detection of Salmonella with the LAMP assay was not significantly different (95% confidence interval) from the GB culture confirmation method (Table 3).

LAMP uses a unique DNA polymerase for continuous DNA amplification that is resistant to matrix interference and inhibitors^{15-19,21-25,28-31}. LAMP assays have been reported to have the same or higher sensitivity compared to PCR and culture-based assays in detecting foodborne pathogens such as Salmonella, Listeria spp., Listeria monocytogenes, Campylobacter, from various food matrices^{17,21-25,28-37}.

Molecular methods based on the amplification of specific DNA targets in pathogenic microorganisms are more specific than the traditional methods that are based on the use of selective agents or biochemical reactions. 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 3-5 days for traditional testing^17,29,38-41.

The China food safety laws require accurate detection of foodborne pathogens in a variety of food samples. Yoghurt is considered fermented milk with zero tolerance for Salmonella¹³. The lactic acid bacteria in yoghurt, the low pH and antimicrobial compounds in yoghurt is thought to prevent the growth of foodborne pathogens. However, pathogens such as Salmonella, E. coli O157, Listeria monocytogenes have been shown to survive and grow in yoghurt samples^5-10. A rapid method with optimized protocols for Salmonella detection in yoghurt samples will enable Chinese producers to assess the safety of these products quickly and release the product on time. In addition, the rapid method needs to be better or equivalent to the standard culture GB method.

Table 1: Detection of Salmonella with the 3M Molecular Detection Assay 2-Salmonella and the GB 4789.4-2016 method
Sample^{a, b}	Number of samples	Presumptive result (MDA2SAL)^c	Culture confirmed result^d
1:10 enrichment
Salmonella (about 87 CFU/25 g)^e	20	4	4
Salmonella (about 78 CFU/25 g)	12	0	0
Salmonella (about 1000 CFU/25 g)	7	0	0
1:20 enrichment
Uninoculated	13	0	0
Salmonella (about 3 CFU/25 g)	10	7	7
Salmonella (about 78 CFU/25 g)	17	17	17
^aSamples included low-fat avocado, green lemon, kiwi fruit yoghurt, caramel yoghurt, strawberry and mulberry flavoured yoghurt, pure yoghurt with no sugar, and yoghurt-based drinks. The label on all samples indicated >10⁷ CFU g^–¹ of lactic acid bacteria, ^bTwenty-five-gram samples were enriched in 225 mL (1:10) or 475 mL (1:20) BPW ISO and incubated at 41.5°C for 24 hrs, ^cNumber of positive samples detected by the LAMP assay, MDA2SAL²¹, ^dNumber of samples detected through culture. All samples were culturally confirmed regardless of presumptive results using GB 4789.4-2016 method¹⁴ and ^eSamples were tested at 24, 26, 28 and 30 hrs of incubation by the LAMP assay and the results were similar at all time points

Table 2: pH and lactic acid bacteria count in yoghurt samples before and after enrichment in BPW ISO
	pH of 1:10 enrichment^a		pH of 1:20 enrichment^a		Lactic acid bacteria count^b (log CFU g^–¹)			Modified Chalmers agar count^c (log CFU g^–¹)
	Before	After	Before	After	Before	After 1:10	After 1:20	Before	After 1:10	After 1:20
Yoghurt sample	enrichment	enrichment	enrichment	enrichment	enrichment	enrichment	enrichment	enrichment	enrichment	enrichment
Low-fat avocado, green	6.7	4.2	6.9	4.6	6.6	7.2	6.5	9.0	6.5	8.1
lemon, kiwi fruit yoghurt
Caramel yoghurt	6.8	4.2	6.9	4.9	5.7	5.5	5.3	8.9	6.0	5.8
Strawberry and mulberry	6.8	4.3	6.9	4.9	5.6	5.0	4.4	8.8	6.9	7.2
flavored yoghurt
Pureyoghurt with no sugar	6.8	4.2	6.9	4.6	6.7	6.5	7.5	8.9	7.3	8.1
Yoghurt-based drinks	6.8	5	6.9	5.6	9	7.7	6.5	0.0	0.0	0.0
^aTwenty-five-gram samples were enriched in 225 mL (1:10) or 475 mL (1:20) BPW ISO and incubated at 41.5°C for 24 hrs, ^bLactic acid bacteria count was determined using 3M Lactic Acid Bacteria Petrifilm Count plates and ^cMC agar was used per GB 4789.35-2016 to estimate the counts of S. thermophilus. According to GB 4789.35-2016, the total lactic acid bacteria count includes lactic acid bacteria counts under anaerobic conditions plus counts on MC agar²⁶

Table 3: Probability of detection for the Salmonella LAMP assay and the GB 4789.4-2016 culture confirmation method for the detection of Salmonella in 1:20 BPW ISO enriched yoghurt samples
				Presumptive	Confirmed				95% CI^e
				positives	positives
Matrix		Inoculation level	N^a	(alternative)	(reference)	POD_a^b	POD_r^c	dPOD^d	LCL	UCL
		Uninoculated	13	0	0	0	0	0	-0.23	0.23
Yoghurt and yoghurt-based drinks	Inoculated (about 3 CFU/25 g)	10	7	7	1	1	0	0	-0.36	0.36
		Inoculated (about 78 CFU/25 g)	17	17	17	1	1	0	-0.18	0.108
^aN: Total number of samples analyzed, ^bPOD_a: Probability of Detection for the alternative LAMP assay, ^cPOD_r: Probability of Detection for the GB 4789.4-2016 culture reference method, ^ddPOD: Differential between the POD_a and the POD_r and ^e95% CI: LCL is the lower confidence level, UCL is the upper confidence level. If the Confidence Interval (CI) of a dPOD contains zero, then the difference is not statistically significant at the 5% level

In this study, a Salmonella LAMP assay was compared to the culture confirmation by the GB method for the detection of Salmonella in yoghurt samples from China. The LAMP method with optimized protocols had equivalent sensitivity to the GB method. Also, the LAMP method provided next-day results compared to GB4789.4-2016 culture reference method requiring 3-5 days¹⁴. Hence, the LAMP method used in this study offered an easy-to-use analytical tool to assess the prevalence of Salmonella in yoghurt samples.

SIGNIFICANCE STATEMENTS

Salmonella is generally not considered a significant pathogen in fermented products. However, it can survive under low pH conditions and grow given appropriate conditions. Hence, dairy processors need effective control measures to assess contamination risks and rapid detection methods enable quicker action to ensure product safety. With the alternative LAMP assay, Salmonella can be detected next-day compared to 3-5 days for the culture-based GB method allowing dairy processors to quickly assess the safety of fermented products.

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International Journal of Dairy Science

Research Article