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Keywords:

  • milk;
  • pathogen detection;
  • PCR ;
  • Salmonella ;
  • total DNA preparation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

In this study, methodologies were developed for cost-effective, rapid and user-friendly culture-independent detection of Salmonella in milk by real-time PCR. The SYBR Green-based real-time PCR assay was standardized with primers targeting the Salmonella enterotoxin gene (stn) that have been earlier used for its detection by conventional PCR. Inclusivity tests generated the specific amplifications with a Tm corresponding to 81 ± 0·5°C. The specificity of the reaction was evaluated with a panel of 36 non-Salmonella strains. Standard curves generated, with different number of cells of this organism in milk, depicted the detection of five cells with a CT value of 37·17 (SD 0·43). To make the assays user-friendly and suitable for field applications, protocols were also established for the immobilization of the SYBR Green reaction mixes in the reaction tubes. The immobilized master mixes were stable at 25°C for 4 months and at 8°C for over 6 months. Total DNA was prepared from 150 samples of full-fat dairy milk and subjected to real-time PCR detection wherein 31 samples tested positive for Salmonella. The time of analysis was <5 h.

Significance and Impact of the Study

With the familiarity of the polymerase chain reaction (PCR) in diagnostic microbiology, it is becoming increasingly possible to use the nucleic acid amplification methodologies for rapid detection of pathogens under field conditions. However, it is imperative to develop assays that are culture-independent, cost-effective and user-friendly. The methodologies developed in this study, particularly the immobilization of the reaction components on the vessels and total DNA extraction from milk, may help to facilitate the use of this technology under field settings. The protocols may thus facilitate the routine culture-independent detection of pathogens, in particular Salmonella, in dairy milk.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Food-borne pathogens cause an estimated 9·4 million episodes of illness, 55 961 hospitalizations and 1351 deaths each year in the USA alone. Nontyphoidal salmonellosis is the second most frequent cause of such illnesses, and the first among bacterial pathogens, causing about 11% of the total episodes (Scallan et al. 2011).This indicates the extent of the problem worldwide, as no such estimates are available for most of the developing countries where food-borne illnesses are more prevalent owing to lesser strict food safety regulations (Ashbolt 2004; Cahill and Jouve 2004). In the recent past, Salmonella has been implicated in many episodes of food-borne outbreaks related to eggs, peanut butter, milk and other foods (Sheth et al. 2011; Brooks et al. 2012; Davies et al. 2013). Milk is an important food commodity with increasingly high chances of contamination by Salmonella (Guh et al. 2010; Riyaz-Ul-Hassan et al. 2009; Van Kessel et al. 2011; Jackson et al. 2012). Thus, it is important to have rapid and high-throughput methods available for pathogen detection in milk for routine quality control testing.

Advances in the techniques pertaining to molecular biology, particularly the polymerase chain reaction (PCR), have allowed for more reliable microbial identification and surveillance (Hoorfar 2011; Sibley et al. 2012). Methods based on PCR have also become valuable tools for investigating food-borne outbreaks and identifying the responsible aetiological agents. PCR-based methods for pathogen detection are rapid, sensitive, robust and reproducible (Cheng et al. 2008; Riyaz-Ul-Hassan et al. 2008; Hoorfar 2011). The use of real-time PCR has provided several advantages over the conventional PCR such as quantification, real-time and in situ analyses, in addition to automation (Moore and Feist 2007; Hoorfar 2011). With the costs of real-time PCR machines coming down, real-time analyses of food and clinical samples are going to be the method of choice for pathogen detection and enumeration in the near future (Min et al. 2011; Verdoy et al. 2012). Various methods are available for the detection of Salmonella by nucleic acid amplification (Riyaz-Ul-Hassan et al. 2004; Persson et al. 2012; Ravan and Yazdanparast 2012). However, development of culture-independent methods poses a real challenge in pathogen detection by molecular techniques. Further, the routine use of real-time PCR for microbial detection demands that the PCR kits are stable at room temperature or at the best at refrigeration and the whole process is made user-friendly, so that the technology finds its application in the field rather than remaining confined to the state-of-the-art laboratories. Thus, in this study, an effort was made to develop a SYBR Green-based real-time PCR assay for culture-independent detection of Salmonella in milk. Further, a method was established for the immobilization of PCR components to increase the stability of the kits and make the assay simple and user-friendly.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

To develop a SYBR Green–based real-time PCR assay for the detection of Salmonella, we adapted the primers QVR133-134 targeting a 200-bp fragment of Salmonella enterotoxin gene (Riyaz-Ul-Hassan et al. 2004). The reactions were initially standardized with DNA templates from Salmonella Typhimurium using different annealing temperatures. Optimum amplification plots and sharp dissociation curves were generated when reactions were run with an annealing temperature of 60°C. Inclusivity tests were carried out with templates from fifteen strains of Salmonella, consisting of nine serotypes (Table 1). The amplification plots were consistent without any significant differences between CT, and the dissociation curves displayed a Tm of 81 ± 0·5°C without any strain-dependent variations. To evaluate the specificity of the assay, 36 non-Salmonella strains, consisting of 18 different genera and 25 different species, were analysed. None of the strains, including the other enteric pathogens, produced any amplification, as depicted by the linear lines generated in the amplification plots and dissociation curves, under the reaction conditions described. A protocol for the extraction of total DNA from milk was standardized. To overcome the effects of fat on DNA preparation, a combination of ammonium sulfate precipitation and centrifugation was used to remove the fat and pellet down the microbial cells. The pellet was again washed with ammonium sulfate to get rid of any leftover fat. This process improved the DNA extraction protocol as depicted in Fig. 1; the amount of DNA obtained in the ammonium sulfate-treated samples was found to be much higher than the untreated samples. The ratio of absorbance, A260/A280, was found to range from 1·7 to 2·0, thus indicating that the DNA was of good quality and suitable for PCR amplification. DNA was extracted from pasteurized full-fat milk samples seeded with known number of Salmonella cells and subjected to the SYBR Green qRT-PCR assay to generate a standard curve. The standard curve with an R2 of 0·989 was generated depicting the detection of five cells of Salmonella with a CT value of 37·17 (SD 0·43) (Fig. 2). A SYBR Green reaction mix was prepared in house, and the concentration of different components was standardized to obtain optimum results. The best results, depicted by amplification plots with CT values comparable with the commercial master mix, were obtained with 2× concentration of SYBR Green dye, 1× PCR buffer, 200 μmol l−1 dNTP mix, 1·5 mmol l−1 MgCl2, 400 nmol l−1 each primer and 1 U of Taq. Concentrations lower than 2× decreased the sensitivity of the assay and also decreased the fluorescence, relatively. To make the assays user-friendly and suitable for field applications, immobilization of the commercial as well as the in-house master mix was carried out in PCR tubes using various substances as matrices. Among the matrices, silica, starch, α-cellulose, DEAE-cellulose, carboxymethyl cellulose (CMC) or agarose, evaluated for the immobilization of the reaction components including the Taq DNA polymerase, encouraging results were obtained with CMC and low-melting agarose. Firm and consistent pellets of the reaction mixes were obtained with both the matrices after freeze-drying. However, all the concentrations of agarose (0·1–0·5%) and concentrations of CMC higher than 1% were found to interfere significantly with the fluorescence collection, thus shifting the CT values by 0·5–1 cycle. A concentration of 0·1% CMC in the reaction mix was least interfering in fluorescence collection, and no significant shift in the CT was observed in comparison with the normal control reaction (Fig. 3). The kits thus produced by immobilization on CMC (0·1%) were found stable for over 6 months at 8°C and up to 4 months at 25°C. Hundred and fifty samples of full-fat raw milk were analysed for the detection of Salmonella with the assay developed in this study. The total DNA was prepared by the (NH4)2SO4-STE method, developed in this study, and the assays were run using the immobilized master mix. A total of 31 samples (20·7%) were found positive with the number of organisms ranging from 25 to 1·6 × 104 (Figs 4 and 5). However, only one sample showed very high numbers of Salmonella (i.e. 1·6 × 104), while the number of Salmonella cells was in the range of 25–500 in the rest of the samples.

Table 1. List of strains used in the inclusivity and exclusivity test reactions. The assays were run with all these bacterial strains to test for selectivity and specificity
Salmonella typhimurium MTCC 98Alcaligenes faecalissubsp.faecalis MTCC126
Salmonella brunnei MTCC 1168Bacillus cereus IIIM25
Salmonella virchow MTCC 1163Bacillus thuringiensis subsp. israelensis MTCC 869
Salmonella infantis MTCC 1162Campylobacter coli MTCC 1126
Salmonella bovismorficans MTCC 1162Clostridium pasteurianum MTCC 116
Salmonella cholerasius MTCC 660Enterobacter aerogenes MTCC 111
Salmonella typhimurium MTCC 733Enterococcus faecalis MTCC 439
Salmonella weltevreden MTCCErwinia herbicola MTCC3 609
Salmonella typhimurium AIIMS 1Escherichia coli MTCC 1678
Salmonella typhimurium AIIMS 2Escherichia coli MTCC 452
Salmonella paratyphi A AIIMS 2Escherichia coli MTCC 901
Salmonella paratyphi B AIIMS 1Escherichia coli P8
Salmonella typhi AIIMS 3Escherichia coli STEC 5
Salmonella typhi AIIMS 4Escherichia coli STEC 6
Salmonella paratyphi B AIIMS 2Lactobacillus fermentum MTCC 903
 Lactococcus lactis subsp. lactis MTCC 440
 Listeria monocytogenes MTCC 657
 Proteus vulgaris MTCC 426
 Pseudomonas fluorescens MTCC103
 Pseudomonas fluorescens IIIM
 Pseudomonas putida MTCC 102
 Serratia marcescens MTCC 7298
 Shigella boydii NCTC 09351 Escherichia coli O157: H7
 Shigella dysenteriae NCTC 9952
 Shigella flexneriAIIMS9
 Shigella flexneri ATCC
 Shigella flexneri NCTC 9989
 Shigella sonnei AIIMS10
 Staphylococcus aureus ATCC
 Staphylococcus aureus MTCC 1144
 Staphylococcus epidermidis MTCC 435
 Staphylococcus hominis MTCC 4435
 Staphylococcus warneri MTCC 4436
 Streptococcus pyogenes MTCC 442
 Xanthobacter flavus MTCC 132
 Yersinia enterocolitica MTCC 840
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Figure 1. Of 0·7% agarose gel showing the total DNA preparation from milk samples showing the comparison of DNA preparations by the STE method and the (NH4)2SO4-STE method, developed in this study. Lanes 1 and 2: DNA prepared by the (NH4)2SO4-STE method from two different samples, A and B, respectively. Lanes 3 and 4: DNA samples prepared by the normal STE method, without the treatment of ammonium sulfate, from samples A and B, respectively.

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Figure 2. Standard curve constructed by plotting log numbers of cells (5 to 5 × 105) of Salmonella seeded in 1 ml of milk against the corresponding CT values. The template DNA was prepared by the (NH4)2SO4-STE method and subjected to the specific qualitative real-time PCR assay. The experiment was carried out in triplicate, and CT values represent mean of three independent experiments.

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Figure 3. Amplification plots depicting the performance of the qPCR master mixes (MM) immobilized on different matrices to amplify Salmonella DNA: (a) normal reaction run with the in-house MM, (b) reaction run with the MM immobilized on 0·1% carboxymethyl cellulose, (c) reaction run with the MM immobilized on 0·1% low-melting agarose, (d) reaction run with the MM immobilized on 0·1% soluble starch. 0·1% CMC interfered least with the fluorescence collection as no significant variations in the CT values were observed.

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Figure 4. Amplification plots showing the detection of Salmonella in different raw milk samples by direct DNA preparation and subsequent specific real-time PCR assay. The flat lines show absence of Salmonella in the samples, as also in the negative control (NTC).

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Figure 5. Dissociation curves of the amplified products from different raw milk samples. The samples that show amplification generate a specific dissociation curve with a Tm of 81 ± 0·5°C. The flat lines show the absence of Salmonella in the samples.

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The methodologies developed in this study address several issues in addition to development of a real-time PCR assay for the detection of Salmonella in milk. The sensitivity, specificity, selectivity, automation and online analyses make real-time PCR a method of choice for the detection of pathogens in food and clinical samples. However, several issues hinder the wide application of such methods under field settings, namely (i) higher costs and portability issues related to the real-time PCR equipment, (ii) lack of stability of reagents required for a real-time PCR assay, particularly at room temperature (which is important for transportation and storage at field sites lacking the modern facilities), (iii) lack of methods for template DNA preparation, (iv) difficulty in setting up the real-time PCR assay (that small variations in the concentrations of different reagents or improper handling may produce false-positive or false-negative results). In this piece of research, in addition to establishing a real-time PCR assay for the detection of Salmonella, we made an effort to address the last three issues for detection of Salmonella in milk, owing to its recognition as an important source for possible salmonellosis (Guh et al. 2010; Riyaz-Ul-Hassan et al. 2009; Van Kessel et al. 2011). The development of low-cost and portable PCR systems (Min et al. 2011; Verdoy et al. 2012) has already addressed the first issue with a great extent, and other issues need immediate attention to take advantage of this highly useful technology for the routine detection of pathogens in food, right at the field sites or the factory settings. Salmonella enterotoxin gene (stn) is a well-established marker for the detection of Salmonella (Riyaz-Ul-Hassan et al. 2004; Moore and Feist 2007). The assay developed in this study was found specific for the detection of Salmonella, in addition to being very sensitive. All the samples spiked with Salmonella cells generated the specific amplification, thus indicating that PCR inhibitors from milk were efficiently removed by the DNA extraction procedure. The template preparation method from milk, developed in this study, was very effective for obtaining high yields of DNA, suitable for the real-time PCR assay and removed the need for any enrichment procedures. Further, this method is cost-effective and rapid. The overall process, from DNA preparation to real-time PCR analyses, takes <5 h to complete, thus reducing the time of analyses significantly. Immobilization of the master mixes increased the stability of the PCR reagents and also made the assays user-friendly, with a requirement to just add the template DNA and make up the reaction volume with sterile water prior to loading on the real-time PCR equipment. Klatser et al. (1998) have described a conventional PCR for the detection of mycobacteria using trehalose-based immobilized master mix. However, no study has been carried out to find out whether it is suitable for the real-time PCR. In our study, we found that as we increased the concentration of CMC in the master mix, there was more interference with the fluorescence collection resulting in increased CT values. Thus, the kits were made with a final concentration of 0·1% (w/v) CMC. The study on raw dairy milk samples presented a grim situation pertaining to its microbiological quality in India, with about 20% samples showing the presence of Salmonella and some of them with very high counts. Rest of the samples that showed negative results did not produce any amplifications and the dissociation curves were flat, indicating the absence of any amplification. However, in the absence of an enrichment step, dead cells or residual DNA from Salmonella may also be detected. Nonetheless, the presence of Salmonella DNA indicates improper handling of milk. Earlier, we have reported high incidence of Escherichia coli and Shiga-toxic E. coli in raw milk samples (Riyaz-Ul-Hassan et al. 2009) in the same region. In a nationwide survey in the USA, 21·96–57·94% of the milk samples were contaminated with Salmonella, with a count reaching up to 60 CFU ml−1 (Jackson et al. 2012). Thus, the presence of Salmonella in raw milk is a worldwide problem. The risk of infection is, however, reduced in India, as raw milk is routinely boiled before consumption. Nonetheless, there is an immediate need to maintain high levels of hygiene in the dairies to prevent the contamination of milk by high-risk pathogens.

The real-time PCR assay, based on SYBR Green chemistry, developed in this study was found rapid, specific, sensitive and reproducible for the detection of Salmonella. In addition, the assay offers a provision of quantification. The (NH4)2SO4-STE method reported in this study offers an efficient and cost-effective protocol to prepare total DNA from milk samples suitable for PCR analyses. Immobilization of the reaction components may be helpful in bringing the technology from scientific laboratories to the field sites. Thus, the methodologies developed in this study may be applicable for the routine detection of Salmonella in milk as was demonstrated by the analyses of 150 raw milk samples from the local dairies.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

Bacterial cultures and template preparation

A total of 51 bacterial cultures were used in inclusivity and exclusivity test reactions. All strains were grown on trypticase soy agar (Difco) at 37°C. Inclusivity tests were carried out with 15 strains of Salmonella enterica consisting of nine serotypes, whereas 36 cultures were used in the exclusivity test reactions (Table 1). DNA templates were prepared from bacteria by resuspending approx. 108 cells in 200 μl deionized water and incubating the suspension at 100°C for 5 min in a dry block heater (Labtech, East Sussex, UK). The resulting cell lysates were centrifuged at 10 000 g for 5 min, and 2 μl supernatant was used as template DNA for the assay.

Primers

Oligonucleotide primers, QVR133-134, 5′-GAAGCAGCGCCTGTAAAATC-3′ and 5′-TGGCTGTGGTGCAAAATATC-3′, previously described by us (Riyaz-Ul-Hassan et al. 2004) for the detection of Salmonella by conventional PCR were adapted for SYBR Green real-time PCR assay. The primers target the Salmonella enterotoxin gene (stn) (GenBank Acc. No. L16014), from positions 268 to 467, generating an amplification of 200 bp. PAGE-purified primers were procured from Sigma, New Delhi, India, and reconstituted in sterile deionized water to a stock concentration of 100 picomoles per microlitre.

SYBR Green reaction assays

The PCR assays were performed on an Mx3000P real-time PCR machine (Stratagene, Santa Clara, CA, USA). A 25-μl reaction assay usually consisted of 1× concentration of SYBR Green QPCR Master Mix (Stratagene), 10 pmol of each primer and 2 μl template DNA. The thermal profile was initial denaturation and activation of hot-start Taq polymerase at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s. Fluorescence measurements were obtained online at the end of each cycle and analysed with the MxPro software (version 4·10; Stratagene). Amplification plots were obtained by plotting dR (baseline-corrected raw fluorescence) against CT (threshold cycle). The dissociation curves were obtained by collecting the fluorescence from 60 to 95°C after completion of amplification and plotting –R′(T) (the first derivative of the raw fluorescence reading multiplied by −1) against temperature (°C). A sharp peak was obtained at the Tm of the amplified product. Each run included at least one positive control (template DNA from Salmonella) and a nontemplate control (NTC).

Specificity testing

Fifteen strains of Salmonella, consisting of nine serotypes, were used for inclusivity, and 36 strains of non-Salmonella strains were used in the exclusivity test reactions. The reactions were carried out at least thrice to check the reproducibility for specificity and selectivity.

In-house master mix

An in-house SYBR Green master mix was prepared consisting of 1× PCR buffer, 200 μmol l−1 dNTP mix (Fermentas), 1·5 mmol l−1 MgCl2, 400 nmol l−1 each primer and 1 U of Taq DNA polymerase (B. Genei, Bangalore, India). The assay was standardized using different concentrations of SYBR Green I dye (Sigma) with templates prepared from Salmonella Typhimurium and Salmonella Typhi. We found that the 2× concentration produced results comparable with the commercial kits.

Immobilization of qPCR master mix

The SYBR Green PCR master mix was immobilized in thin-walled PCR tubes by employing freeze-drying in the presence of different matrices. The PCR assays were set up with all the components except water and template DNA, and different concentrations of matrices, ranging from 0·1 to 0·5% (w/v), like silica, soluble starch, α-cellulose, DEAE-cellulose, carboxymethyl cellulose (CMC) or agarose, were added. The components were freeze-dried for 16 h at −90°C in a Freeze dryer (Delvac, Chennai, India). The tubes were stored in the dark at different temperatures for stability testing.

Total DNA preparation from milk by the (NH4)2SO4-STE method

Raw milk samples were collected on several different occasions from the local dairies located in the different locations of the city of Jammu. 50 μl of 4 mol l−1 ammonium sulfate was added to 250-μl aliquot milk. The contents were vortexed vigorously and centrifuged at 10 000 g for 5 min. The upper fat layer was removed with a sterilized needle and the supernatant was discarded. The pellet was washed with 100 μl ammonium sulfate (4 mol l−1) and sterilized deionized water, once each. The pellet, thus obtained, was suspended in 300 μl STE buffer (400 mmol l−1 Sucrose, 100 mmol l−1 TrisCl (pH 8·0), 20 mmol l−1 EDTA–Na) containing 5% SDS and 0·2% β-mercaptoethanol. The contents were agitated vigorously, and 250 μl of chilled 7·5 mol l−1 ammonium acetate was added. After incubation on ice bath for 5 min, the samples were twice extracted with 500 μl of chloroform. The DNA in the supernatant was precipitated with two volumes of ethanol and one-tenth volume of 7·5 mol l−1 ammonium acetate and pelleted at 18 000 g for 20 min. The resulting pellet was air-dried and dissolved in 10 μl of sterile deionized water. The whole amount of DNA was used in the reaction assay. Quality of DNA was assessed spectrophotometrically by obtaining the ratio of absorbance at 260 and 280 nm.

Standard curve and sensitivity

A suspension of Salmonella (Salmonella typhimurium or Salmonella typhi, in separate experiments), containing approx. 5 × 108 cells per ml was prepared by comparing the turbidity with Mac Farland Standard of 2. Accurate numbers of cells were estimated by standard plate count (CFU ml−1). The suspension was diluted serially, and aliquots of dilutions containing 5 to 5 × 105 cells were added to samples of 1 ml sterile whole milk. DNA was extracted as above and dissolved in 10 μl of sterile deionized water. Whole volume of DNA was subjected to the assay. A standard curve was generated by plotting the mean CT, of three individual experiments, against log number of cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References

This research was supported by the Council of Scientific and Industrial Research (CSIR, India) via Grant No. 5/258/45/2005-NMITLI.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and discussion
  5. Materials and methods
  6. Acknowledgements
  7. References
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